Sourdough Biotechnology-Springer US (2013)

Handbook on Sourdough Biotechnology
Marco Gobbetti • Michael Gänzle

Editors
Handbook on Sourdough
Biotechnology
Editors
Marco Gobbetti Michael Gänzle
Department of Soil, Plant Department of Agricultural,
and Food Science Food, and Nutritional Science
University of Bari Aldo Moro University of Alberta
Bari, Italy Edmonton, Canada
ISBN 978-1-4614-5424-3 ISBN 978-1-4614-5425-0 (eBook)

DOI 10.1007/978-1-4614-5425-0
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2012951618
© Springer Science+Business Media New York 2013

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Contents
1 History and Social Aspects of Sourdough ........................................... 1

Stefan Cappelle, Lacaze Guylaine, M. Gänzle,
and M. Gobbetti
2 Chemistry of Cereal Grains ................................................................. 11
Peter Koehler and Herbert Wieser
3 Technology of Baked Goods ................................................................. 47
Maria Ambrogina Pagani, Gabriella Bottega,
and Manuela Mariotti
4 Technology of Sourdough Fermentation
and Sourdough Applications ................................................................ 85
Aldo Corsetti
5 Taxonomy and Biodiversity of Sourdough
Yeasts and Lactic Acid Bacteria........................................................... 105
Geert Huys, Heide-Marie Daniel, and Luc De Vuyst
6 Physiology and Biochemistry of Sourdough Yeasts ........................... 155
M. Elisabetta Guerzoni, Diana I. Serrazanetti,
Pamela Vernocchi, and Andrea Gianotti
7 Physiology and Biochemistry of Lactic Acid Bacteria ....................... 183
Michael Gänzle and Marco Gobbetti
8 Sourdough: A Tool to Improve Bread Structure ................................ 217
Sandra Galle
9 Nutritional Aspects of Cereal Fermentation
with Lactic Acid Bacteria and Yeast .................................................... 229
Kati Katina and Kaisa Poutanen
10 Sourdough and Gluten-Free Products ................................................ 245
Elke K. Arendt and Alice V. Moroni
v
vi Contents
11 Sourdough and Cereal Beverages ........................................................ 265

Jussi Loponen and Juhani Sibakov
12 Perspectives ........................................................................................... 279
Index ............................................................................................................... 287
Chapter 1
History and Social Aspects of Sourdough
Stefan Cappelle, Lacaze Guylaine, M. Gänzle, and M. Gobbetti
1.1 Sourdough: The Ferment of Life
The history of sourdough and related baked goods follows the entire arc of the
development of human civilization, from the beginning of agriculture to the present.
Sourdough bread and other sourdough baked goods made from cereals are examples
of foods that summarize different types of knowledge, from agricultural practices
and technological processes through to cultural heritage. Bread is closely linked to
human subsistence and intimately connected to tradition, the practices of civil soci-
ety and religion. Christian prayer says “Give us this day our daily bread” and the
Gospels report that Jesus, breaking bread at the Last Supper, gave it to the Apostles
to eat, saying, “This is my body given as a sacrifice for you”. Language also retains
expressions that recall the close bond between life and bread: “to earn his bread”
and “remove bread from his mouth” are just some of the most common idioms, not
to mention the etymology of words in current use: “companion” is derived from
cum panis, which means someone with whom you share your bread; “lord”, is
derived from the Old English vocabulary hlaford, which translates as guardian of
the bread [1]. The symbolic assimilation between bread and life is not just a template
that has its heritage in the collective unconscious, but it is probably a precipitate of the
history of culture and traditions. Throughout development of the human civiliza-
tion, (sourdough) bread was preferred over unleavened cereal products, supporting
S. Cappelle (*) • L. Guylaine

Puratos Group, Industrialaan 25, Groot-Bijgaarden, Belgium
e-mail: [email protected]
M. Gänzle
Department of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, Canada
M. Gobbetti
Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari, Italy
M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, 1

DOI 10.1007/978-1-4614-5425-0_1, © Springer Science+Business Media New York 2013
2 S. Cappelle et al.
the hypothesis of a precise symbolism between the idea of elaborate and stylish, and
that of sourdough. Fermentation and leavening makes bread something different
from the raw cereals, i.e. an artifact, in the sense of “made art”. Besides symbolism,
sourdough bread has acquired a central social position over time. Bread, and espe-
cially sourdough bread, has become central in the diet of peasant societies. This
suggests that the rural population empirically perceived sensory and nutritional
transformations, which are also implemented through sourdough fermentation. In
other words, the eating of bread, and especially of sourdough bread, was often a
choice of civilization.
The oldest leavened and acidified bread is over 5,000 years old and was discovered
in an excavation in Switzerland [2]. The first documented production and consumption
of sourdough bread can be traced back to the second millennium B.C. [3]. Egyptians
discovered that a mixture of flour and water, left for a bit of time to ferment, increased
in volume and, after baking along with other fresh dough, it produced soft and light
breads. Much later, microscopic observations of yeast as well as measurements of
the acidity of bread from early Egypt demonstrate that the fermentation of bread
dough involved yeasts and lactic acid bacteria – the leavening of dough with sour-
dough had been discovered [4]. Eventually, the environmental contamination of
dough was deliberately carried out by starting the fermentation with material from
the previous fermentation process. Egyptians also made use of the foam of beer for
bread making. At the same time, Egyptians also selected the best variety of wheat
flour, adopted innovative tools for making bread, and used high-temperature ovens.
The Jewish people learned the art of baking in Egypt. As the Bible says, the Jews
fleeing Egypt took with them unleavened dough.
In Greece, bread was a food solely for consumption in wealthy homes. Its prepa-
ration was reserved for women. Only in a later period, does the literature mention
evidence of bakers, perhaps meeting in corporations, which prepared the bread for
retail sale. The use of sourdough was adopted from Egypt about 800 B.C. [4]. Greek
gastronomy had over 70 varieties of breads, including sweet and savoury types,
those made with grains, and different preparation processes. The Greeks used to
make votive offerings with flour, cereal grains or toasted breads and cakes mixed
with oil and wine. For instance, during the rites dedicated to Dionysus, the god of
fertility, but also of euphoria and unbridled passion, the priestesses offered large
loaves of bread. The step from the use of sacrificial bread to the use of curative
bread was quick. Patients, who visited temples dedicated to Asclepius (the god of
medicine and healing), left breads, and, upon leaving the holy place, received a part
of the breads back imbued with the healing power attributed to the god [5, 6].
The use of sourdough is also part of the history of North America. The use of
sourdough as a leavening agent was essential whenever pioneers or gold prospectors
left behind the infrastructure that would provide alternative means of dough leavening.
Examples include the Oregon Trail of 1848, the California gold rush of 1849, and
the Klondike gold rush in the Yukon Territories, Canada, in 1898. During the 1849
gold rush, San Francisco was invaded by tens of thousands of men and women in
the grip of gold fever. Following the gold rush, sourdough bread remained an ele-
ment that distinguishes the local tradition until today. Some bakeries in San Francisco
claim to use sourdough that has been propagated for over 150 years. The predominant
1 History and Social Aspects of Sourdough 3
yeast in San Francisco sourdoughs is not brewer’s yeast but Kazachstania exigua
(formerly Saccharomyces exiguus), which is tolerant to more acidic environments.
Lactobacillus sanfranciscensis (formerly Lactobacillus brevis subsp. lindneri and
sanfrancisco) was first described as a new species in San Francisco sourdough [7].
The use of sourdough during the Klondike gold rush in 1898 resulted in the use of
“sourdough” to designate inhabitants of Alaska and the Yukon Territories and is
even in use today. The Yukon definition of sourdough is “someone who has seen the
Yukon River freeze and thaw”, i.e. a long-term resident of the area.
From antiquity to most recent times, the mystery of leavening has also been
unveiled from a scientific point of view. The definitive explanation of microbial
leavening was given in 1857 by Louis Pasteur. The scientific research also verified
an assumption that the Greeks had already advanced: sourdough bread has greater
nutritional value. Pliny the elder wrote that it gave strength to the body. The history
and social significance of the use of sourdough is further described below for coun-
tries such as France, Italy and Germany where this traditional biotechnology is
widely used, and where its use is well documented.
1.2 History and Social Aspects of Sourdough in France
The history of sourdough usage in France was linked to socio-cultural and socio-
economic factors. There is little information about sourdough usage and bakery
industries (it seems to be more appropriated than baking), in general, in France
before the eighteenth century. It seems as if sourdough bread was introduced in
Gaul by the Greeks living in Marseille in the fourth century B.C. In 200 B.C., the
Gauls removed water from the bread recipe and replaced it with cervoise, a drink
based on fermented cereal comparable to beer. They noticed that the cloudier the
cervoise, the more the dough leavened. Thus, they started to use the foam of
cervoise to leaven the bread dough. The bread obtained was particularly light.
During the Middle Ages (400–1400 A.D.), bread making did not progress much
and remained a family activity. In the cities, the profession of the baker appeared.
The history of bread making in France was mainly linked to Parisian bakers because
of the geographic localization of Paris. The regions with the biggest wheat production
were near Paris, and Paris had major importance in terms of inhabitants. In that
period, the production of bread was exclusively carried out using sourdough
fermentation, the only method known at that time. Furthermore, the use of sour-
dough, thanks to its acidity, permitted baking without salt, an expensive and taxed
(Gabelle) raw material, and allowed one to produce breads appropriate for eating
habits in the Middle Ages [8].
The seventeenth century marked a turning point in the history of French bakery.
Until then, sourdough was used alone to ensure fermentation of the dough even if in
some French regions wine, vinegar or rennet was added. Toward 1600 A.D., French
bakers rediscovered the use of brewer’s yeast for bread making. The yeast came
from Picardie and Flanders in winter and from Paris breweries in summer. The breads
obtained with this technique were named pain mollet because of the texture of the
dough, which was softer than the bread produced up to that point (pain brie). Two
French queens, Catherine de Medicis (Henri II’s wife) and Marie de Medicis (Henri
IV’s wife) contributed to the success and development of these yeast-fermented
breads. In 1666, the use of brewer’s yeast was authorized for bread making but, after
a great deal of debate, in 1668, the use of brewers’ yeast was prohibited. Following
the request of Louis XIV, the Faculty of Medicine of the Paris University studied the
consequences of yeast usage on public health. According to the doctors, yeast was
harmful to human health, because of its bitterness, coming from barley and rotting
water. Despite this negative conclusion by the Faculty, Parliament, in its decision of
21st March 1670, authorized the use of brewer’s yeast for bread making in combina-
tion with sourdough. Besides the apparition of yeast in bread making, during that
period, eating habits evolved towards less acidic foods. Thus, back-slopping tech-
niques were adapted in order to reduce bread acidity [9].
The seventeenth century was also a period of development of the French philo-
sophic and encyclopaedic mind and, fortunately, bread making did not escape this
movement. Two books detail the art of bread making and provide information on
bread-making techniques and knowledge of that period: “L’Art de la Boulangerie”
[10] and “Le Parfait Boulanger” [11]. We have already learned that sourdough was
obtained from a part of the leavened dough prepared on the day in question. The
volume of this dough piece is progressively increased through addition of flour and
water (back slopping) to prepare a sourdough that is ready to be used to ferment the
dough. The original piece of dough, called levain-chef, must not be too old or too
sour. The weight of the levain-chef is doubled or tripled by addition of water and
flour leading to the levain de première. After 6 or 7 hours of fermentation, water and
flour are added to give the levain de seconde, which is fermented for 4 or 5 hours.
Again, water and flour are added. The dough obtained is called levain tout point and
after 1 or 2 hours of fermentation is added to the bread dough. This technique called
travail sur 3 levains was recommended by Parmentier [11], who imputed the bad
quality of Anjou bread to bread making based only on one sourdough. Bread mak-
ing based on two or three sourdoughs was predominantly used in that period. In
addition, it was understood that outside Paris, bread was mainly produced at home
by women. It is interesting to note that Malouin had already made the distinction
between sourdough and artificial sourdough in 1779 [10]. Artificial sourdough
refers to sourdough obtained from a dough that may contain yeast. This distinction
between sourdough and artificial sourdoughs remained in the nineteenth century.
Until 1840, the yeast was always used in association with sourdough to initiate
fermentation. On this date, an Austrian baker introduced a bread-making process in
France based on yeast fermentation alone. This technique was called poolish. The
bread obtained, called pain viennois, had much success but use of this method remained
limited. In the middle of the nineteenth century, bread making based on three sour-
doughs progressively disappeared and was replaced by bread making based on two
sourdoughs. Indeed, the back slopping, necessary to maintain the fermentative activity
of sourdoughs, imposed a hard working rhythm on the bakers. In 1872, the opening of
the first factory for the production of yeast from grain fermentation in France by
Fould-Springer facilitated the development of bread making based on yeast to the

detriment of sourdough bread making. This yeast was more active, more constant, with
a nice flavour and most of all had a longer shelf life than brewer’s yeast. As a conse-
quence, from 1885, bread making based on polish fermentation was becoming more
wide spread. Sourdough bread was, from that time on, called French bread.
In 1910, a bill that prohibited night work and, in 1920, the reduction of working
hours, necessitated modification within fermentation processes. Sourdough bread
making regressed to a greater and greater extent in the cities when bread making
based on three sourdoughs totally disappeared even though, in 1914, the first fer-
mentôlevain appeared. After the First World War, the use of yeast was extended
from Paris to the provinces. Indeed, yeast that was produced on molasses from 1922
had a better shelf life and was thus easier to distribute over long distances. However,
homemade loaves were still produced, even though they no longer existed in the
cities, in the country until 1930 in the form of the levain chef, kept in stone jugs, and
passed on from one family to another. The return of war in 1939 led to a further
reduction in the use of homemade sourdough bread. In 1964, Raymond Calvel [12]
wrote that “sourdough bread making does not exist anymore”. Indeed, baker’s yeast
was systematically added to promote dough leavening, which permitted one to
obtain lighter breads. In addition, the use of baker’s yeast permitted one to better
manage bread quality and to reduce quality variations. Two sourdough bread-
making methods remained in this period. The first was a method based on two
sourdoughs, which was mainly used in West and South Loire, and the second, more
commonly used, method was based on one sourdough with a high level of baker’s
yeast. Between 1957 and 1960, the sensory qualities of bread decreased as a conse-
quence of cost reduction. Fermentation time was reduced to a minimum. Sourdough
bread was no longer produced. It was only during the 1980s that sourdough bread
making gained popularity again thanks to consumer requests for authentic and tasty
breads. Since 1990, the availability of starter cultures facilitated the re-introduction
of sourdough in bread-making processes. Indeed, these starters permit one to obtain
a levain tout-point with a single step and simplify the bread-making process. A regu-
lation issued on 13th September 1993 [13] defined sourdough and sourdough bread.
According to Article 4, sourdough is “dough made from wheat or rye, or just one of
these, with water added and salt (optional), and which undergoes a naturally acidi-
fying fermentation, whose purpose is to ensure that the dough will rise. The sour-
dough contains acidifying microbiota made up primarily of lactic bacteria and
yeasts. Adding baker’s yeast (Saccharomyces cerevisiae) is allowed when the dough
reaches its last phase of kneading, to a maximum amount of 0.2% relative to the
weight of flour used up to this point”. This definition allowed one to dehydrate sour-
dough with the flora remaining active (amounts of bacteria and yeast are indicated).
Sourdough can also be obtained by addition of starter to flour and water. Article 3
of the same regulation declares that “Breads sold under the category of pain au
levain must be made from a starter as defined by Article 4, just have a potential
maximum pH of 4.3 and an acetic acid content of at least 900 ppm”. The syndicat
national des fabricants de produits intermédiaires pour boulangerie, patisserie et
biscuiterie is working on a new definition of sourdough in order to be closer to the
reality of sourdough bread.
1.3 History and Social Aspects of Sourdough in Italy
The people in early Italy mainly cultivated barley, millet, emmer and other grains,
which were used for preparation of non-fermented focacce and polenta. Emmer was
not only used for making foods, but also performed as a vehicle of transmission in
sacred rituals. At first, the Romans mainly consumed roasted or boiled cereals, sea-
soned with olive oil and combined with vegetables. After contact with Greek civili-
zation, the Romans learned the process of baking and the technique of building
bread ovens. Numa Pompilius sanctioned this gastronomic revolution with the
introduction of celebrations dedicated to Fornace, the ancient divinity who was the
guardian for proper functioning of the bread oven. The Romans gave a great boost
to improvements in the techniques of kneading and baking of leavened products,
and regulated manufacture and distribution by bakers (pistores). Cato the Elder
described many varieties of bread in De agri coltura (160 B.C.), which by then had
already spread to Rome: the libum or votive bread, the placenta, a loaf of wheat
flour, barley and honey, the erneum, a kind of pandoro, and the mustaceus, bread
made with grape must. In the first century A.D., Pliny the Elder [14] refers to several
alternative methods of dough leavening, including sourdough that was air-dried
after 3 days of fermentation, the use of dried grapes as a starter culture, and particu-
larly the use of back-slopping of dough as the most common method to achieve
dough leavening. Pliny the Elder specifically refers to sourdough in his indication
that “it is an acid substance carrying out the fermentation”. According to Pliny the
Elder, it was generally acknowledged that “consumption of fermented bread
improves health” [14].
After the triumph of classical baking, there were no novel developments in this
field throughout the Middle Ages. Finding bread and flour in these centuries was
difficult, because of involution of agriculture and the famine and epidemics raging
at this time. The bread was divided into two categories: black bread, made from
flours of different cereals, of little value and reserved for the most humble people,
and white bread, made from refined flour, which was more expensive and present on
the tables of the rich. A special bread, whose tradition has been preserved to this day
in different national or regional varieties, is the Brezel, originating from the South
of Germany. It has a characteristic shape of a knotted and dark red crust, which is
generated by application of alkali prior to baking, and is sprinkled with coarse salt
crystals. According to legend, it was invented by a German court baker in Urach in
South West Germany, who, to avoid the loss of his job, was asked by the Duke of
Württemberg to develop a bread that allows the sun to shine through three times.
This special bread requires 2 days of working: the first to prepare the sourdough
with wheat flour, and the second to mix it with water, flour, salt, lard and malt.
During the Renaissance, the practice of holding banquets in the courts of the
nobles was a triumph for bread, which was presented in various forms in support of
the different dishes. In Venice “fugassa” was prepared for the Easter holidays, a
sweet bread made with sugar, eggs and butter. In Tuscany, they used to prepare
“pane impepato”, while in Milan it appeared as “panettone”. Only towards the end
of the 1600s was the use of yeast re-introduced for the distribution of luxury bread,
which was salty and had added milk. In 1700, a very important innovation in the art
of bread making was disseminated: the millstones in mills were replaced with a
series of steel rollers. This allowed cheaper refining of flour. Also, pioneering mix-
ers were set up. With the advances brought by the industrial revolution, bread was
increasingly emerging as a staple food for workers. Rather than making the bread at
home, people preferred to buy it from bakers. This change was criticized as distort-
ing traditional values. At the same time, a health movement that originated in
America started a battle against leavened bread, stating it was deleterious to health.
Baker’s yeast was considered a toxic element, perhaps because it was derived from
beer, while the sourdough gave a bad taste to the bread, which was remediated by
the addition of potash, equally harmful. When Louis Pasteur discovered that micro-
organisms caused the fermentation, the concern over the toxicity of biological
agents was amplified. Pasteur’s discovery eventually benefitted the supporters of the
bread, as they stated that the use of selected yeast and related techniques was helpful
in the manufacture of bread with a longer shelf life. The education of taste in differ-
ent food cultures explains, however, the different relationship that has existed
between the perception of the quality of bread and its level of acidity.
During the First World War, the so-called “military bread” was used in Europe,
which was a loaf of 700 g weight with a hard crust. It was initially distributed to
soldiers and then also passed on to the civilian population. In the post-war period,
thanks to the much-discussed Battle of Wheat, strongly supported by Mussolini, the
production of wheat was plentiful and the bread was brought to the table of the
general population. The Second World War again resulted in an insufficient supply
of bread. With the arrival of the American allies, the bread of liberation – a square
white bread – became disseminated. Today, bread is regaining some importance.
With a turnaround in the culinary habits of Westerners, bread made with unrefined
flour, so-called black bread, is more widely consumed.
A brief mention should be made, finally, of the various breads that are currently
made with modern baking practices. Typical breads, with PDO (Denomination of
Protected Origin) or PGI (Protected Geographical Indication) status, are the
Altamura bread, the bread of Dittaino, the Coppia Ferrasese, the bread of Genzano
and the Cornetto of Matera. The manufacture of these breads is based on new pro-
cesses, but still at an artisanal level [15].
1.4 History and Social Aspects of Sourdough in Germany
Acidified and leavened bread has been consistently produced in Central Europe
(contemporary Austria, Germany, and Switzerland) for over 5,000 years. Leavened
and acidified bread dating from 3,600 B.C. was excavated near Bern, Switzerland
[2]; comparable findings of bread or acidified flat bread were made in Austria (dating
from 1800 B.C.) and Quedlinburg, Germany (dating from 800 B.C.) [16]. It remains
unknown whether these breads represent temporary and local traditions or a permanent
and widespread production of leavened and acidified bread; however, these

archaeological findings indicate that the use of sourdough for production of leavened
breads developed independently in Central Europe and the Mediterranean.
Paralleling the use of leavening agents in France, sourdough was used as the sole
leavening agent in Germany until the use of brewer’s yeast became common in the
fifteenth and sixteenth centuries [4, 16]. In many medieval monasteries, brewing
and baking were carried out in the same facility to employ the heat of the baking
ovens to dry the malt, and to use the spent brewer’s yeast to leaven the dough. The
close connection between brewing and baking is also documented in the medieval
guilds. In Germany, bakers and brewers were often organized in the same guild. In
many cities, bakers also enjoyed the right to brew beer [17].
Baker’s yeast has been produced for use as a leavening agent in baking since the
second half of the nineteenth century [4, 16, 18]. Baker’s yeast was initially pro-
duced with cereal substrates, but the shortage of grains in Germany in the First
World War forced the use of molasses as a substrate for baker’s yeast production [4].
Although artisanal bread production relied on the use of sourdough as the main
leavening agent until the twentieth century, the use of baker’s yeast widely replaced
sourdough as the leavening agent. Maurizio indicates in 1917 that baker’s yeast was
the predominant leavening agent for white wheat bread, whereas whole grain and
rye products continued to be leavened with sourdough [19]. In 1954, Neuman and
Pelshenke referred to baker’s yeast as the main or sole leavening agent for wheat
bread and as an alternative leavening agent in rye bread [20]. The industrial produc-
tion of baker’s yeast to achieve leavening in straight dough processes was followed
by the commercial production of sourdough starter cultures in Germany from
1910.
The continued use of sourdough in Germany throughout the twentieth century
particularly relates to the use of rye flour in bread production. Rye flour requires
acidification to achieve optimal bread quality. Acidification inhibits amylase activ-
ity and prevents starch degradation during baking. Moreover, the solubilisation of
pentosans during sourdough fermentation improves water binding and gas retention
in the dough stage. Following the introduction of baker’s yeast as a leavening agent,
the aim of sourdough fermentation in rye baking shifted from its use as a leavening
agent to its use as an acidifying agent [18]. This use of sourdough for acidification
of rye dough in Germany is paralleled in other countries where rye bread has a
major share of the bread market, including Sweden, Finland, the Baltic countries,
and Russia. For example, the industrialization of bread production in the Soviet
Union in the 1920s led to the development of fermentation equipment for the large
scale and partially automated production of rye sourdough bread [21].
Chemical acidulants for the purpose of dough acidification became commercially
available in the twentieth century as alternatives to sourdough fermentation.
However, artisanal as well as industrial bakeries continued to use sourdough
fermentation owing to the substantial difference in product quality. To differentiate
between chemical and the more labour-intensive and expensive biological
acidification, German food law provided a definition of sourdough as dough con-
taining viable and metabolically active lactic acid bacteria, and defines sourdough
bread as bread where acidity is exclusively derived from biological acidification.

Sourdough is thus one of very few intermediates of food production that is regulated
by legislation, and recognized by many consumers [4]. The consumer perception as
well as the regulatory protection of the term “sourdough” in Germany and other
European countries facilitated the recent renaissance of sourdough use in baking. In
comparison, the term “sourdough” is not protected in the United States and the
widespread labelling of chemically acidified bread as “sourdough bread” resulted in
a widespread consumer perception of sourdough bread as highly acidic bread, and
the use of alternative terminology to label bread produced with biological
acidification.
The commercialization of dried sourdough with high titratable acidity consti-
tuted a compromise between economic bread production based on convenient use of
baking improvers, and the use of sourdough fermentation for improved bread qual-
ity. These products were introduced in the 1970s [18]. Their economic importance
rapidly surpassed the importance of sourdough starter cultures. Dried or stabilized
sourdoughs produced for acidification provided the conceptual template for the
increased use of sourdough products as baking improvers over the last 20 years.
Sourdough fermentation was thus no longer confined to small-scale, artisanal fer-
mentation to achieve dough leavening and/or acidification. Sourdough fermentation
is also carried out in industrial bakeries at a large scale matching large-scale bread
production, and in specialized ingredient companies for production of baking
improvers specifically aimed at influencing the storage life as well as the sensory
and nutritional quality of bread.
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–geschichte. In: Eiselen H (ed) Deutsches Brotmuseum Ulm, Germany
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Sauerteig, 6th edn. Behr’s Verlag, Hamburg, pp 1–5
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Sillabe, Livorno, pp 18–24
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II. Isolation and characterization of undescribed bacterial species responsible for the souring
activity. Appl Microbiol 21:459–465
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Technologie des Métiers de Bouche. Maé-Erti, Vezoul
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forums/bnweb/dt/lecturelevain/lecturelevainacc.php, consultée le 07/06/2012 à 14h42
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pour fabriquer les différentes sortes de pastes et de pains, 2nd edn. Paris
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du pain. Imprimerie royale, Paris
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Paris
13. Décret n°93-1074 du 13 septembre 1993 pris pour l’application de la loi du 1er août 1905 en
ce qui concerne certaines catégories de pains
14. Pliny the Elder G (1972) Naturalis Historia XVIII, 102–104, edition of Le Biniec H; Pline
L’Ancien, Historie Naturelle, Livre XVIII, Societé D’Editions le Belles Lettres, Paris
15. Buonassisi V (1981) Storia del pane e del forno. SIDALM, Milano
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18. Brandt MJ (2007) Sourdough products for convenient use in baking. Food Microbiol
24:161–164
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20. Neumann MP, Pelshenke PF (1954) Brotgetreide und Brot, 5th edn. Parey, Berlin
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Handbuch sauerteig, 6th edn. Behr’s Verlag, Hamburg, pp 329–352
Chapter 2
Chemistry of Cereal Grains
Peter Koehler and Herbert Wieser
2.1 Introductory Remarks
Cereals are the most important staple foods for mankind worldwide and represent
the main constituent of animal feed. Most recently, cereals have been additionally
used for energy production, for example by fermentation yielding biogas or bioetha-
nol. The major cereals are wheat, corn, rice, barley, sorghum, millet, oats, and rye.
They are grown on nearly 60% of the cultivated land in the world. Wheat, corn, and
rice take up the greatest part of the land cultivated by cereals and produce the largest
quantities of cereal grains (Table 2.1) [1]. Botanically, cereals are grasses and belong
to the monocot family Poaceae. Wheat, rye, and barley are closely related as mem-
bers of the subfamily Pooideae and the tribus Triticeae. Oats are a distant relative of
the Triticeae within the subfamily Pooideae, whereas rice, corn, sorghum, and mil-
let show separate evolutionary lines. Cultivated wheat comprises five species: the
hexaploid common (bread) wheat and spelt wheat (genome AABBDD), the tetra-
ploid durum wheat and emmer (AABB), and the diploid einkorn (AA). Triticale is
a man-made hybrid of durum wheat and rye (AABBRR). Within each cereal species
numerous varieties exist produced by breeding in order to optimize agronomical,
technological, and nutritional properties.
The farming of all cereals is, in principle, similar. They are annual plants and
consequently, one planting yields one harvest. The demands on climate, however,
are different. “Warm-season” cereals (corn, rice, sorghum, millet) are grown in
tropical lowlands throughout the year and in temperate climates during the frost-
free season. Rice is mainly grown in flooded fields, and sorghum and millet are
adapted to arid conditions. “Cool-season” cereals (wheat, rye, barley, and oats)
grow best in a moderate climate. Wheat, rye, and barley can be differentiated into
P. Koehler (*) • H. Wieser

German Research Center for Food Chemistry,
Lise-Meitner-Strasse 34, 85354 Freising, Germany

12 P. Koehler and H. Wieser
Table 2.1 Cereal production in 2010 [1]

Cultivated area Grain production
Species (million ha) (million tons)
Corn 162 844
Rice 154 672
Wheat 217 651
Barley 48 123
Sorghum + millet 76 85
Oats 9 20
Triticale 4 13
Rye 5 12
winter or spring varieties. The winter type requires vernalization by low temperatures;
it is sown in autumn and matures in early summer. Spring cereals are sensitive to
frost temperatures and are sown in springtime and mature in midsummer; they
require more irrigation and give lower yields than winter cereals.
Cereals produce dry, one-seeded fruits, called the “kernel” or “grain”, in the form
of a caryopsis, in which the fruit coat (pericarp) is strongly bound to the seed coat
(testa). Grain size and weight vary widely from rather big corn grains (~350 mg) to
small millet grains (~9 mg). The anatomy of cereal grains is fairly uniform: fruit and
seed coats (bran) enclose the germ and the endosperm, the latter consisting of the
starchy endosperm and the aleurone layer. In oats, barley, and rice the husk is fused
together with the fruit coat and cannot be simply removed by threshing as can be
done with common wheat and rye (naked cereals).
The chemical composition of cereal grains (moisture 11–14%) is characterized by
the high content of carbohydrates (Table 2.2) [2, 3]. Available carbohydrates, mainly
starch deposited in the endosperm, amount to 56–74% and fiber, mainly located in
the bran, to 2–13%. The second important group of constituents is the proteins which
fall within an average range of about 8–11%. With the exception of oats (~7%),
cereal lipids belong to the minor constituents (2–4%) along with minerals (1–3%).
The relatively high content of B-vitamins is, in particular, of nutritional relevance.
With respect to structures and quantities of chemical constituents, notable differ-
ences exist between cereals and even between species and varieties within each
cereal. These differences strongly affect the quality of products made from cereal
grains. Because of the importance of the constituents, in the following we provide an
insight into the detailed chemical composition of cereal grains including carbohy-
drates, proteins, lipids, and the minor components (minerals and vitamins).
2.2 Carbohydrates
Cereal grains contain 66–76% carbohydrates (Table 2.2), thus, this is by far the
most abundant group of constituents. The major carbohydrate is starch (55–70%)
followed by minor constituents such as arabinoxylans (1.5–8%), b-glucans (0.5–
7%), sugars (~3%), cellulose (~2.5%), and glucofructans (~1%).
2 Chemistry of Cereal Grains 13
Table 2.2 Chemical composition of cereal grains (average values) [2, 3]

Wheat Rye Corn Barley Oats Rice Millet
(g/100 g)
Moisture 12.6 13.6 11.3 12.1 13.1 13.0 12.0
Protein (N × 6.25) 11.3 9.4 8.8 11.1 10.8 7.7 10.5
Lipids 1.8 1.7 3.8 2.1 7.2 2.2 3.9
Available carbohydrates 59.4 60.3 65.0 62.7 56.2 73.7 68.2
Fiber 13.2 13.1 9.8 9.7 9.8 2.2 3.8
Minerals 1.7 1.9 1.3 2.3 2.9 1.2 1.6
(mg/kg)
Vitamin B1 (thiamine) 4.6 3.7 3.6 4.3 6.7 4.1 4.3
Vitamin B2 (riboflavin) 0.9 1.7 2.0 1.8 1.7 0.9 1.1
Nicotinamide 51.0 18.0 15.0 48.0 24.0 52.0 18.0
Panthothenic acid 12.0 15.0 6.5 6.8 7.1 17.0 14.0
Vitamin B6 2.7 2.3 4.0 5.6 9.6 2.8 5.2
Folic acid 0.9 1.4 0.3 0.7 0.3 0.2 0.4
Total tocopherols 41.0 40.0 66.0 22.0 18.0 19.0 40.0
2.2.1 Starch
Starch is the major storage carbohydrate of cereals and an important part of our
nutrition. Because of its unique properties starch is important for the textural prop-
erties of many foods, in particular bread and other baked goods. Finally, starch is
nowadays also an important feedstock for bioethanol or biogas production (for
reviews see [4, 5]).
2.2.1.1 Amylose and Amylopectin
Starch occurs only in the endosperm and is present in granular form. It consists of the
two water-insoluble homoglucans amylose and amylopectin. Cereal starches are typi-
cally composed of 25–28% amylose and 72–75% amylopectin [6]. Mutant genotypes
may have an altered amylose/amylopectin ratio. “Waxy” cultivars have a very high
amylopectin level (up to 100%), whereas “high amylose” or “amylostarch” cultivars
may contain up to 70% amylose. This altered ratio of amylose/amylopectin affects
the technological properties of these cultivars [7, 8]. High-amylose wheat has been
suggested as a raw material for the production of enzyme-resistant starch [9].
Amylose consists of a-(1,4)-linked d-glucopyranosyl units and is almost linear.
Parts of the molecules also have a-(1,6)-linkages providing slightly branched struc-
tures [10, 11]. The degree of polymerization ranges from 500 to 6,000 glucose units
giving a molecular weight (MW) of 8 × 104 to 106. Amylopectin is responsible for
the granular nature of starch. It contains 30,000–3,000,000 glucose units and, there-
fore, it has a considerably higher MW (107–109) than amylose [12]. Amylopectin is
a highly branched polysaccharide consisting of a-(1,4)-linked d-glucopyranosyl
chains, which are interconnected via a-(1,6)-glycosidic linkages, also called branch
points [13]. The a-(1,4)-linked chains have variable length of 6 to more than 100
glucose units depending on the molecular site at which they are located. The
unbranched A- or outer chains can be distinguished from the branched B- or inner
chains, which can be subdivided into B1-, B2-, B3-, and B4-chains [14]. The molecules
are “terminated” by a single C-chain containing the reducing glucose residue [15].
Amylopectin has a tree-like structure, in which clusters of chains occur at regular
intervals along the axis of the molecule [16]. Short A- and B1-chains of 12–15 glu-
cose residues form the clusters which have double-helical structures. The longer,
less abundant B2-, B3-, and B4-chains interconnect 2, 3 or 4 clusters, respectively.
B2-chains contain approximately 35–40, B3-chains 70–80, and B4-chains up to
more than 100 glucose residues [12, 17].
2.2.1.2 Starch Granules
In the endosperm starch is present as intracellular granules of different sizes and

shapes, depending on the cereal species. In contrast to most plant starches, wheat,
rye, and barley starches usually have two granule populations differing in size.
Small spherical B-granules with an average size of 5 mm can be distinguished
from large ellipsoid A-granules with mean diameters around 20 mm [18]. In the
polarization microscope native starch granules are birefringent indicating that
ordered, partially crystalline structures are present in the granule. The degree of
crystallinity ranges from 20 to 40% [19] and is primarily caused by the structural
features of amylopectin. It is thought that the macromolecules are oriented per-
pendicularly to the granule surface [12, 16] with the nonreducing ends of the
molecules pointing to the surface.
A model of starch granule organization from the microscopic to the nanoscopic
level has been suggested [12]. At the microscopic level alternating concentric
“growth rings” with periodicities of several hundreds of nanometers can be observed.
They reflect alternating semicrystalline and amorphous shells [12]. The latter are
less dense, enriched in amylose, and contain noncrystalline amylopectin. They fur-
ther consist of alternating amorphous and crystalline lamellae of about 9–10 nm [20].
Crystalline regions contain amylopectin double helices of A- and B1-chains ori-
ented in parallel fashion and possibly 18 nm-wide, left-handed superhelices formed
from double helices. Amorphous regions represent the amylopectin branching sites,
which may also contain a few amylose molecules. The lamellae are organized into
larger spherical blocklets, which vary periodically in diameter between 20 and
500 nm [21]. The amylopectin double helices may be packed into different crystal
types. The very densely packed A-type is found in most cereal starches, while the
more hydrated tube-like B-type is found in some tuber starches, high amylose cereal
starches, and retrograded starch [12, 19]. Mixtures of A- and B-types are designated
C-type.
2.2.1.3 Changes in Starch Structure During Processing
In many cereal manufacturing processes flour and also starch is usually dispersed in
water and finally heated. In particular heating induces a series of structural changes.
This process has been termed gelatinization [22]. Depending on water content, water
distribution, and intensity of heat treatment the molecular order of the starch granules
can be completely transformed from the semicrystalline to an amorphous state.
The mixing of starch and excess water at room temperature leads to a starch sus-
pension. During mixing starch absorbs water up to 50% of its dry weight (1) because
of physical immobilization of water in the void space between the granules, and (2)
because of water uptake due to swelling. The latter process increases with tempera-
ture. If the temperature is below the gelatinization temperature, the described changes
are reversible. As the temperature increases, more water permeates into the starch
granules and initiates hydration reactions. Firstly, the amorphous regions are hydrated
thereby increasing molecular mobility. This also affects the crystalline regions, in
which amylopectin double helices dissociate and the crystallites melt [23, 24]. These
reactions are endothermic and irreversible. They are accompanied by the loss of
birefringence, which can be observed under the polarization microscope. Endothermic
melting of crystallites can also be followed by differential scanning calorimetry
(DSC). Viscosity measurements, for example in an amylograph or a rapid visco ana-
lyzer, also allow one to monitor the gelatinization process. Characteristic points are
the onset temperature (To; ca. 45 °C), which reflects the initiation of the process, as
well as the peak (Tp; ca. 60 °C) and conclusion (Tc; ca. 75 °C) temperatures. These
temperatures are subject to change depending on the botanical source of the starch
and the water content of the suspension. The loss of molecular order and crystallinity
during gelatinization is accompanied by further granule swelling due to increased
water uptake and a limited starch solubilization. Mainly amylose is dissolved in
water, which strongly increases the viscosity of the starch suspension. This phenom-
enon has been termed “amylose leaching,” and it is caused by a phase separation
between amylose and amylopectin, which are immiscible [25]. During further heating
beyond the conclusion temperature of gelatinization swelling and leaching continue
and a starch paste consisting of solubilized amylose and swollen, amorphous starch
granules is formed. The shapes of the starch granules can still be observed unless
shear force or higher temperatures are applied [23, 26].
Upon cooling with mixing the viscosity of a starch paste increases, whereas a
starch gel is formed on cooling without mixing at concentrations above 6%. The
second process is relevant in cereal baked goods. The changes that occur during
cooling and storage of a starch paste have been summarized as “retrogradation” [22].
Generally, the amorphous system reassociates to a more ordered, crystalline state.
Retrogradation processes can be divided into two subprocesses. The first is related
to amylose and occurs in a time range of minutes to hours, the second is caused by
amylopectin and takes place within hours or days. Therefore, amylose retrograda-
tion is responsible for the initial hardness of a starch gel or bread, whereas amylo-
pectin retrogradation determines the long-term gel structure, crystallinity, and
hardness of a starch-containing food [27].
On cooling granule remnants that are enriched in amorphous amylopectin

become incorporated into a continuous amylose matrix. Amylose molecules that are
dissolved during gelatinization reassociate to local double helices interconnected by
hydrated parts of the molecules, and a continuous network (gel) forms [27]. As
amylose retrogradation proceeds, double helix formation increases and, finally, very
stable crystalline structures are formed, which cannot be melted again by heating.
Amylopectin retrogradation takes several hours or days and occurs in the granule
remnants embedded in the initial amylose gel [27]. Crystallization mainly occurs
within the short-chain outer A- and B1-chains of the molecules. The amylopectin
crystallites melt at ca. 60 °C and, therefore, aged bread can partly be “refreshed” by
heating. This so-called “staling endotherm” can be measured by DSC to evaluate
amylopectin retrogradation. Amylopectin retrogradation is strongly influenced by a
number of conditions and substances, including pH and the presence of low-molecular-
weight (LMW) compounds such as salts, sugars, and lipids [26].
2.2.1.4 Interaction with Lipids
Amylose is able to form helical inclusion complexes in particular with polar lipids
and this can occur in native (starch lipids; see below) as well as in gelatinized starch [28].
During gelatinization amylose forms a left-handed single helix and the nonpolar moi-
ety of the polar lipid is located in the central cavity [16]. The inclusion complexes
give rise to a V-type X-ray diffraction pattern. The presence of polar lipids strongly
affects the retrogradation characteristics of the starch, because amylose-lipid com-
plexes do not participate in the recrystallization process [26]. Complex formation is,
however, strongly affected by the structure of the polar lipid [29]. For example,
monoglycerides are more active than diglycerides and saturated fatty acids more
active than unsaturated ones, because inclusion complexes are preferably formed
with linear hydrocarbon chains and with compounds having one fatty acid residue.
In addition, lipids, in particular lysophospholipids (lysolecithin), are minor con-
stituents of cereal starches in amounts of 0.8–1.2% [30]. As so-called starch lipids
they are associated with amylose as well as with the outer branches of amylopectin
[28]. These lipid complexes lead to a delay of the onset of gelatinization and affect
the properties of the starch especially in baking applications.
2.2.2 Nonstarch Polysaccharides (NSP)
Polysaccharides other than starch are primarily constituents of the cell walls and are
much more abundant in the outer than in the inner layers of the grains. Therefore, a
higher extraction rate is associated with a higher content of NSP. From a nutritional
point of view NSP are dietary fiber, which has been associated with positive health
effects. For example, cereal dietary fiber has been related to a reduced risk of chronic
life style diseases such as cardiovascular diseases, type II diabetes, and gastrointestinal
cancer [31–36]. In addition, technological functionalities have been described for
the arabinoxylans (AX) of wheat (reviewed by [4]) and rye.
2.2.2.1 Arabinoxylans
AX are the major fraction (85–90%) of the so-called pentosans. Different cereal
species contain different amounts of AX. The highest contents are present in rye
(6–8%), whereas wheat contains only 1.5–2% AX. On the basis of solubility AX
can be subdivided into a water-extractable (WEAX) and a water-unextractable
fraction (WUAX). The former makes up 25–30% of total AX in wheat and
15–25% in rye [37]. In particular WEAX has considerable functionality in
breadmaking.
AX consist of linear b-(1,4)-d-xylopyranosyl-chains, which can be substituted
at the O-2 and/or O-3-positions with a-l-arabinofuranose [38, 39]. A particular
minor component of AX is ferulic acid, which is bound to arabinose as an ester at
the O-5 position [40]. AX of different cereals may vary substantially in content,
substitutional pattern and molecular weight [41–43]. WEAX mainly consist of two
populations of alternating open and highly branched regions, which can be distin-
guished by their characteristic arabinose/xylose ratios, ranging between 0.3 and
1.1 depending on the specific structural region [44]. WUAX can be solubilized by
mild alkaline treatment yielding structures that are comparable to those of WEAX
[37, 45–48].
The unique technological properties of AX are attributable to the fact that AX
are able to absorb 15–20 times more water than their own weight and, thus, form
highly viscous solutions, which may increase gas holding capacity of wheat doughs
via stabilization of the gas bubbles [49]. In total, WEAX bind up to 25% of the
added water in wheat doughs [50]. Under oxidizing conditions, in particular under
acidic pH, the so-called “oxidative gelation” [51] leads to AX gel formation by
inducing di- and oligoferulic acid cross-links [52, 53]. This is thought to be one
major structure-forming reaction in rye sourdoughs. Because of covalent cross-
links to the cell wall structure WUAX do not dissolve in water. Although they have
high water-holding capacity and assist in water binding during dough mixing they
are considered to have a negative impact on wheat breadmaking as they form physi-
cal barriers against the gluten network and, thus, destabilize the gas bubbles.
However, the baking performance can be affected by adding endoxylanases, which
preferentially hydrolyze WUAX. This produces solubilized WUAX, which have
techno-functional effects comparable to WEAX [54, 55].
Beside AX the pentosan fraction contains a small part of a water-soluble, highly
branched arabinogalactan peptide [41]. It consists of b-(1,3) and b-(1,6) linked
galactopyranose units with a-glycosidically bound arabinofuranose residues. The
peptide is attached by 4-trans-hydroxyproline. Unlike AX, arabinogalactan
peptides have no significant effects in cereal processing.
2.2.2.2 b-Glucans
b-Glucans are also called lichenins and are present particularly in barley (3–7%)
and oats (3.5–5%), whereas less than 2% b-glucans are found in other cereals. The
chemical structure of these NSP is made up of linear D-glucose chains linked via
mixed b-(1,3)- and b-(1,4)-glycosidic linkages. b-Glucans show a higher water
solubility than AX (38–69% in barley, 65–90% in oats) and form viscous solutions,
which in the case of barley may interfere in wort filtration during the production of
beer.
2.3 Proteins
The average protein content of cereal grains covers a relatively narrow range
(8–11%, Table 2.2), variations, however, are quite noticeable. Wheat grains, for
instance, may vary from less than 6% to more than 20%. The content depends on the
genotype (cereal, species, variety) and the growing conditions (soil, climate,
fertilization); amount and time of nitrogen fertilization are of particular importance.
Proteins are distributed over the whole grain, their concentration within each
compartment, however, is remarkably different. The germ and aleurone layer of
wheat grains, for instance, contain more than 30% proteins, the starchy endosperm
~13%, and the bran ~7% [3]. Regarding the different proportions of these compart-
ments, most proteins of grains are located in the starchy endosperm, which is the
source of white flours obtained by milling the grains and sieving.
White flours are the most important grain products. Therefore, the predominant
part of the literature on cereal proteins deals with white flour proteins. The amino
acid compositions of flour proteins from various cereals are shown in Table 2.3.
Typical of all flours is the fact that glutamic acid almost entirely occurs in its amidated
form as glutamine [56]. This amino acid generally predominates (15–31%), fol-
lowed by proline in the case of wheat, rye, and barley (12–14%). Further major
amino acids are leucine (7–14%) and alanine (4–11%). The nutritionally essential
amino acids tryptophan (0.2–1.0%), methionine (1.3–2.9%), histidine (1.8–2.2%),
and lysine (1.4–3.3%) are present only at very low levels. Through breeding and
genetic engineering, attempts are being made to improve the content of essential
amino acids. These approaches have been successful in the case of high-lysine
barley and corn.
2.3.1 Osborne Fractions
Traditionally, cereal flour proteins have been classified into four fractions (albumins,
globulins, prolamins, and glutelins) according to their different solubility and based
on the fractionation procedure of Osborne [57]. Albumins are soluble in water,
Table 2.3 Amino acid composition (mol-%) of the total proteins of flours from various cereals [56]
Amino acid Wheat Rye Barley Oats Rice Millet Corn
Asxa 4.2 6.9 4.9 8.1 8.8 7.7 5.9
Thr 3.2 4.0 3.8 3.9 4.1 4.5 3.7
Ser 6.6 6.4 6.0 6.6 6.8 6.6 6.4
Glxa 31.1 23.6 24.8 19.5 15.4 17.1 17.7
Pro 12.6 12.2 14.3 6.2 5.2 7.5 10.8
Gly 6.1 7.0 6.0 8.2 7.8 5.7 4.9
Ala 4.3 6.0 5.1 6.7 8.1 11.2 11.2
Cys 1.8 1.6 1.5 2.6 1.6 1.2 1.6
Val 4.9 5.5 6.1 6.2 6.7 6.7 5.0
Met 1.4 1.3 1.6 1.7 2.6 2.9 1.8
Ile 3.8 3.6 3.7 4.0 4.2 3.9 3.6
Leu 6.8 6.6 6.8 7.6 8.1 9.6 14.1
Tyr 2.3 2.2 2.7 2.8 3.8 2.7 3.1
Phe 3.8 3.9 4.3 4.4 4.1 4.0 4.0
His 1.8 1.9 1.8 2.0 2.2 2.1 2.2
Lys 1.8 3.1 2.6 3.3 3.3 2.5 1.4
Arg 2.8 3.7 3.3 5.4 6.4 3.1 2.4
Trp 0.7 0.5 0.7 0.8 0.8 1.0 0.2
Amide group 31.0 24.4 26.1 19.2 15.7 22.8 19.8
a
Asx Asp+Asn, Glx Glu+Gln
while globulins are insoluble in pure water but soluble in dilute salt solutions.
Prolamins are classically defined as cereal proteins soluble in aqueous alcohols, for
example 60–70% ethanol. Originally, glutelins were described as proteins that were
insoluble in water, salt solution, aqueous alcohols and soluble in dilute acids or
bases. Later, it was ascertained that notable portions of glutelins are insoluble in
dilute acids such as acetic acid, and that extraction with strong bases destroys the
primary structure of proteins. Nowadays, complete solubility of glutelins is achieved
by solvents containing a mixture of aqueous alcohols (e.g., 50% propanol), reduc-
ing agents (e.g., dithiothreitol), and disaggregating compounds (e.g., urea).
Regarding their functions, most of the albumins and globulins are metabolic
proteins, for example enzymes or enzyme inhibitors (see Sect. 2.3.4). Oats are an
exception containing considerable amounts of legume-like globulins such as 12S
globulin [58]. Albumins and globulins are concentrated in the aleurone layer, bran,
and germ, whereas their concentration in the starchy endosperm is relatively low.
Predominantly, prolamins and glutelins are the storage proteins of cereal grains (see
Sects. 2.3.2 and 2.3.3). Their only biological function is to supply the seedling with
nitrogen and amino acids during germination. They are located only in the starchy
endosperm; in white flours, their proportions based on total proteins amount to
70–90%. In general, none of the Osborne fractions consists of a single protein, but
of a complex mixture of different proteins. A small portion of proteins does not fall
into any of the four solubility fractions. Together with starch, they remain in the
insoluble residue after Osborne fractionation and mainly belong to the class of lipo
(membrane) proteins.
The prolamin fractions of the different cereals have been given trivial names:
gliadin (wheat), secalin (rye), hordein (barley), avenin (oats), zein (corn), kafirin
(millet, sorghum), and oryzin (rice). The glutelin fraction of wheat has been termed
glutenin. Terms for the other glutelin fractions such as secalinin (rye), hordenin
(barley), and zeanin (corn) are scarcely used today. Gliadin and glutenin fractions of
wheat have been combined in the terms gluten or gluten proteins.
The content of the Osborne fractions varies considerably and depends on geno-
type and growing conditions. Moreover, the results of Osborne fractionation are
strongly influenced by experimental conditions, and the fractions obtained are not
clear-cut. Therefore, data from the literature on the qualitative and quantitative com-
position of Osborne fractions is differing and, in parts, contradictory. On average,
the smallest proportion of total protein is present in the globulin fraction, followed
by the albumin fraction. An exception is oat globulins amounting to more than 50%
of total proteins. In most cereal flours, prolamins are the dominating fractions,
oat prolamins, however, are minor protein components and rice flour is almost free
of prolamins. Beside quantitative aspects the Osborne procedure is still useful for
the preparation and characterization of flour proteins and the enrichment of differ-
ent protein types.
2.3.2 Storage Proteins of Wheat Rye, Barley, and Oats
2.3.2.1 Classification and Primary Structures
Storage proteins (prolamins and glutelins) have been extensively investigated by the
analysis of amino acid compositions, amino acid sequences, MW, and intra- and
interchain disulfide linkages. The results indicated that, in accordance with phylogeny
(see Sect. 2.1), the storage proteins of wheat, rye, and barley are closely related,
whereas those of oats, in particular their glutelins, are structurally divergent.
According to common structures storage proteins have been classified into three
groups by two different principles. Shewry and coworkers [59] defined all storage
proteins as prolamins and grouped them into the high-molecular-weight (HMW),
sulfur-poor (S-poor) and sulfur-rich (S-rich) prolamins based on differences in MW
and sulfur (cysteine, methionine) content. To prevent confusion, however, the term
“prolamin” is not used for total storage proteins in the present paper, since classi-
cally the term prolamins comprises only the alcohol-soluble portions of storage
proteins and does not include glutelins. We classified storage proteins according to
related amino acid sequences and molecular masses into the following groups [60, 61]:
(1) a HMW group; (2) a medium-molecular-weight (MMW) group; and (3) a
LMW group. The proteins of these groups can be divided into different types on
the basis of structural homologies (Table 2.4). Each type contains numerous
closely related proteins; the small differences in their amino acid sequences can
be traced back to substitutions, insertions, and deletions of single amino acids and
short peptides.
Table 2.4 Characterization of storage protein types from wheat, rye, barley, and oats [61, 63]
Partial amino acid
composition
Repetitive unitb,c (mol-%)b
Group/type Code Residues Statea (frequency) Q P F +Y G L V
HMW group
HMW-GS x Q6R2V1 815 a QQPGQG (72×) 36 13 5.8 20 4.4 1.7
HMW-GS y Q52JL3 637 a QQPGQG (50×) 32 11 5.5 18 3.8 2.3
HMW-secalin x Q94IK6 760 a QQPGQG (66×) 34 15 6.7 20 3.7 1.5
HMW-secalin y Q94IL4 716 a QQPGQG (60×) 34 12 5.0 18 3.2 1.8
D-hordein Q40054 686 a QQPGQG (26×) 26 11 5.5 16 4.1 4.1
MMW group
w5-gliadin Q402I5 420 m (Q)QQQFP (65×) 53 20 10.0 0.7 3.1 0.2
w1,2-gliadin Q6DLC7 373 m (QP)QQPFP (42×) 42 29 9.9 0.8 4.0 0.5
w-secalin O04365 338 m (Q)QPQQPFP (32×) 40 29 8.6 0.6 4.4 1.8
C-hordein Q40055 328 m (Q)QPQQPFP (36×) 37 29 9.4 0.6 8.6 0.3
LMW group
a/ß-gliadin Q9M4M5 273 m QPQPFPPQQPYP 36 15 7.4 2.6 8.1 5.1
(5×)
g-gliadin Q94G91 308 m (Q)QPQQPFP (15×) 36 18 5.2 2.9 7.2 4.6
LMW-GS Q52NZ4 282 a (Q)QQPPFS (11×) 32 13 5.7 3.2 8.2 5.3
g-40 k-secalind – – m QPQQPFP 34 18 5.5 2.4 7.4 4.7
g-75 k-secalin Q9FR41 436 a QQPQQPFP (32×) 38 22 6.1 1.6 4.8 5.3
g-hordein P17990 286 m QPQQPFP (15×) 28 17 7.7 3.1 7.0 7.3
B-hordein P06470 274 a QQPFPQ (13×) 30 19 7.3 2.9 8.0 6.2
avenin Q09072 203 m PFVQQQQ (3×) 33 11 8.4 2.0 8.9 8.3
a
a aggregative, m monomeric
b
One-letter-code for amino acids
c
Basic unit frequently modified by substitution, insertion, and deletion of single amino acid
residues
d
Gellrich et al. [65]
The nomenclature of types is rather confusing and inconsequential. On the one

hand prolamins have been termed according to their electrophoretic mobility in acid
polyacrylamide gel electrophoresis (PAGE) with band regions designated as w
(lowest mobility), g (medium mobility), and a/b (highest mobility). On the other
hand, the nomenclature is based on their apparent sizes (after reduction of disulfide
bonds) as indicated by sodiumdodecyl sulfate (SDS-) PAGE; examples are HMW-
and LMW-glutenin subunits (GS), HMW-secalins, D-, C-, and B-hordeins. Because
of the different importance of HMW-GS for the bread-making quality of wheat,
single subunits have been numbered according to their mobility on SDS-gel
electrophoresis (original nos. 1–12), the genome (1A, 1B, or 1D), and the type (x or y);
examples of nomenclature are HMW-GS 1Ax1, 1Bx7, and 1Dy10 [62].
The HMW group contains HMW-GS of wheat, HMW-secalins of rye, and
D-hordeins of barley (Table 2.4); this type is missing in oats. HMW-GS and HMW-
secalins can be subdivided into the x-type and the y-type. The proteins comprise
around 600–800 amino acid residues corresponding to MW of 70,000–90,000.
Fig. 2.1 Schematic structure

and disulfide bonds of
a/b-gliadins, g-gliadins,
LMW-, and HMW-GS
(Adapted from [64])
The amino acid compositions are characterized by high contents of glutamine, glycine,
and proline. The amino acid sequences [63] can be separated into three structural
domains: a nonrepetitive N-terminal domain A of ~100 residues, a repetitive central
domain B of 400–700 residues, and a nonrepetitive C-terminal domain C with ~40
residues (Fig. 2.1) [64, 65]. Domain B is dominated by repetitive sequences such as
QQPGQG (one-letter-code for amino acids) as a backbone with inserted sequences
like YYPTSL, QQG, and QPG with remarkable differences between x- and y-types
(Table 2.5). Domains A and C have a more balanced amino acid composition and
more amino acid residues with charged side chains. In a native state, the proteins of
the HMW group are aggregated through interchain disulfide bonds (Fig. 2.1).
The MMW group consists of the homologous w1,2-gliadins of wheat, w-secalins
of rye, and C-hordeins of barley including amino acid residues between 300 and
400 and MW around 40,000 (Table 2.4). Additionally, wheat contains unique w5-
gliadins with more than 400 residues and MW around 50,000. This group, likewise,
is not present in oats. The proteins of the MMW group have extremely unbalanced
amino acid compositions characterized by high contents of glutamine, proline, and
phenylalanine, which together account for ~80% of total residues. Most sections of
the amino acid sequences are composed of repetitive units such as QPQQPFP or
QQQFP. This type of protein occurs as monomers and is readily soluble in aqueous
alcohols and, in parts, even in water.
Table 2.5 Partial amino acid sequences of domain B of HMW-GS 1Dx2 (positions 93–338)
and of HMW-GS 1Dy10 (positions 106–380) [63]
Position Sequencea Position Sequencea
93 YYPSVTSPQQVS 106 YYPGVTSPRQGS
105 YYPGQASPQRPGQG 118 YYPGQASPQQPGQG
119 QQPGQG 132 QQPGKW
125 QQSGQGQQG 138 QEPGQGQQW
134 YYP--TSPQQPGQW 147 YYP--TSLQQPGQG
146 QQPEQGQPG 159 QQIGKGQQG
155 YYP--TSPQQPGQL 168 YYP--TSLQQPGQGQQG
167 QQPAQG 183 YYP--TSLQHTGQR
173 QQPGQGQQG 195 QQPVQG
182 QQPGQGQPG 201 QQPEQG
191 YYP-TSSQLQPGQL 207 QQPGQWQQG
204 QQPAQGQQG 216 YYP--TSPQQLGQG
213 QQPGQGQQG 228 QQPRQW
222 QQPGQG 234 QQSGQGQQG
228 QQPGQGQQG 243 HYP--TSLQQPGQGQQG
237 QQPGQG 258 HYL--ASQQQPGQGQQG
243 QQPGQGQQG 273 HYP--ASQQQPGQGQQG
252 QQLGQGQQG 288 HYP--ASQQQPGQGQQG
261 YYP--TSLQQSGQGQPG 303 HYP--ASQQEPGQGQQG
276 YYP--TSLQQLGQGQSG 318 QIPASQ
291 YYP--TSPQQPGQG 324 QQPGQGQQG
303 QQPGQL 333 HYP--ASLQQPGQGQQG
309 QQPAQG 348 HYP--TSLQQLGQGQQT
315 QQPGQGQQG 363 QQPGQK
324 QQPGQGQQG 369 QQPGQG
333 QQPGQG 375 QQTGQG
a
One-letter-code for amino acids; - deletion
The LMW group consists of monomeric proteins including a/b- and g-gliadins
of wheat, g-40 k-secalins of rye, g-hordeins of barley, and avenins of oats, and of
aggregative proteins including LMW-GS of wheat, g-75 k-secalins of rye, and
B-hordeins of barley (Table 2.4). They have around 300 amino acid residues and
MW ranging from 28,000–35,000, besides g-75 k-secalins (~430 residues, MW
~50,000) and avenins (~200 residues, MW ~23,000). The amino acid compositions
of the LMW group proteins are characterized by relatively high contents of hydro-
phobic amino acids besides glutamine and proline. The amino acid sequences consist
of four (a/b-gliadins five) different sequence sections (Fig. 2.1). The N-terminal
section I is rich in glutamine, proline, and phenylalanine forming repetitive units
such as QPQPFPPQQPY (a/b-gliadins), QQPQQPFP (g-gliadins), QQPPFS
(LMW-GS), or PFVQQQQ (avenins). Section I of g-75 k-secalins is prolonged by
around 130 residues as compared to g-40 k-secalins and that of avenins is shortened
to around 40 residues. Section II is unique to a/b-gliadins and consists of a polyglu-
tamine sequence (up to 18 Q-residues). Sections III, IV, and V possess more balanced
amino acid compositions and most of the cysteine residues that form only intrachain
disulfide bonds (monomeric proteins) or both intra- and interchain disulfide bonds
(aggregative proteins). The comparison of the amino acid sequences demonstrates
that sections III and V contain homologous sequences, whereas section IV is, in part,
unique to each type (Table 2.6). g-Type proteins (g-gliadins, g-40 k-secalins [66],
g-75 k-secalins, g-hordeins) show the highest conformity; a/b-gliadins, LMW-GS, and
avenins have the lowest degree of homology within the LMW group. Most oat glu-
telins are globulin-like proteins and do not show any structural relationship with the
HMW-, MMW-, and LMW-type proteins described above [67]. The reasons as to
why they are not extractable with a salt solution are not yet clear.
As mentioned earlier, the quantitative composition of storage protein is strongly
dependent on both genotype and growing conditions. Nevertheless, some constant
data can be observed (Table 2.7) [68–70]: Proteins of the LMW group belong, by far,
to the major components. Within this group, monomeric proteins (55–77% of total
storage proteins) exceed aggregative proteins (10–25%) in the case of wheat species,
whereas rye and barley are characterized by more aggregative (34–48%) than mono-
meric proteins (~25%). Proteins of the MMW and HMW groups belong to the minor
components except w-secalins (18%) and C-hordeins (36%) or are missing (oats).
Within wheat species significant differences can be observed. Common wheat is
characterized by the highest values for aggregative proteins (HMW-, LMW-GS) and
a low monomeric/aggregated (m/a) ratio, and the “old” wheat species emmer and
einkorn by low proportions of HMW-GS and high m/a ratios.
2.3.2.2 Disulfide Bonds
Disulfide bonds play an important role in determining the structure and properties
of storage proteins. They are formed between sulfhydryl groups of cysteine residues,
either within a single protein (intrachain) or between proteins (interchain). Most
information on disulfide bonds is available for wheat gliadins and glutenins. With a
few exceptions, w-type gliadins are free of cysteine and, consequently occur as
monomers. Most a/b- and g-gliadins contain six and eight cysteine residues, respec-
tively, and form three or four homologous intrachain disulfide bonds, present within
or between sections III and V (Fig. 2.1) [64]. A small portion of gliadins is different
from most gliadins and contains an odd number of cysteine residues. They may be
linked to each other or to glutenins by an interchain disulfide bond. Homologous to
g-gliadins, g-40 k- and g-75 k-secalins, g- and B-hordeins as well as avenins contain
eight cysteine residues at comparable positions within sections III–V (Table 2.6).
Probably they form four intrachain disulfide bonds homologous to those of g-glia-
dins (Fig. 2.2). LMW-GS include eight cysteine residues, six of which form three
intrachain disulfide bonds homologous to those of a/b- and g-gliadins [64, 65]. Two
cysteine residues located in sections I and IV are unique to LMW-GS; they are obvi-
ously not able to form intrachain bonds for steric reasons. They are involved in
interchain bonds with residues of different proteins (LMW-GS, modified gliadins,
y-type HMW-GS).
Table 2.6 Amino acid sequences of sections III, IV, and V of the LMW group [63]
Type Positions Sequences
(a) Section III
a/b-gliadin 119–186 ILQQILQQQLIPCMDVVLQQHNIVHGRSQVLQQSTY-----QLLQELCCQHLWQIPEQSQCQAIHNVVHAIIL
g-gliadin 153–224 FIQPSLQQQLNPCKNILLQQCKPASLVSSL-WSIIWPQSDCQVMRQQCCQQLAQIPQQLQCAAIHSVVHSIIM
LMW-GS 101–173 IVQPSVLQQLNPCKVFLQQQCSPVAMPQRLARSQMWQQSRCHVMQQQCCQQLSQIPEQSRYDAIRAITYSIIL
g-40 k-secalina – SIQLSLQQQLNPCKNVLLQQCSPVALVSSL-RSKIFPQSECQVMQQQCCQQLAQIPHHLQCAAIHSVVHAIIM
g-75 k-secalin 285–356 SIQLSLQQQLNPCKNVLLQQCSPVALVSSL-RSKIFPQSECQVMQQQCCQQLAQIPQQLQCAAIHSVVHAIIM
g-hordein 135–206 TIQLYLQQQLNPCKEFLLQQCRPVSLLSYI-WSKIVQQSSCRVMQQQCCLQLAQIPEQYKCTAIDSIVHAIFM
2 Chemistry of Cereal Grains
B-hordein 112–184 YVHPSILQQLNPCKVFLQQQCSPVPVPQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQFRHEAIRAIVYSIFL

Avenin 43–114 FLQPLLQQQLNPCKQFLVQQCSPVAAVPFL-RSQILRQAICQVTRQQCCRQLAQIPEQLRCPAIHSVVQSIIL
(b) Section IV
a/b-gliadin 187–222 HQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQN
g-gliadin 225–253 QQQQQQQQQQGMHIFLPLSQQQQVGQGSL
LMW-GS 174–228 QEQQQGFVQAQQQQPQQSGQGVSQSQQQSQQQLGQCSFQQPQQQLGQQPQQQQQQ
g-40 k-secalina – QQEQREGVQILLPQSHQQLVGQGAL
g-75 k-secalin 357–381 QQEQREGVQILLPQSHQQHVGQGAL
g-hordein 207–231 QQGQRQGVQIVQQQPQPQQVGQCVL
B-hordein 185–225 QEQPQQLVEGVSQPQQQLWPQQVGQCSFQQPQPQQVGQQQQ
Avenin 115–155 QQQQQQQQFIQPQLQQQVFQPQLQLQQQVFQPQLQQQVFQP
(c) Section V
a/b-gliadin 223–273 PQAQGSVQPQQLPQF-EEIRNLALQTLPAMCNVYIPPYCTI--APFGIFGTNYR
g-gliadin 254–308 VQGQGIIQPQQPAQL-EAIRSLVLQTLPSMCNVYVPPECSIMRAPFASIVAGIGGQ
LMW-GS 229–282 VLQGTFLQPHQIAHL-EAVTSIALRTLPTMCSVNVPLYSATTSVPFAVGTGVSAY
g-40 k-secalina – AQVQGIIQPQQLSQFNVGIVLQMLQNLPTMCNVYVPRQCPPSRRHLHAMSLVCGH
g-75 k-secalin 382–436 AQVQGIIQPQQLSQL-EVVRSLVLQNLPTMCNVYVPRQCSTIQAPFASIVTGIVGH
g-hordein 232–286 VQGQGVVQPQQLAQM-EAIRTLVLQSVPSMCNFNVPPNCSTIKAPFVGVVTGVGGQ
B-hordein 226–274 VPQSAFLQPHQIAQL-EATTSIALRTLPMMCSVNVPLYRILRGVGPSVGV
Avenin 156–203 QLQQVFNQPQMQGQI-EGMRAFALQALPAMCDVYVPPQCPVATAPLGGF
a
25
Gellrich et al. [65]

Table 2.7 Proportions (%) of storage protein types of different wheat species, rye,
and barley [68–70]
Group
HMW MMW LMW
Cereal Variety a m a m m/aa
Common wheat Rektor 9.1 10.4 25.1 55.4 1.9
Spelt wheat Schwabenkorn 6.6 10.4 17.7 65.3 3.1
Durum wheat Biodur 5.0 6.7 19.3 69.0 3.1
Emmer Unknown 2.6 10.8 10.0 76.6 6.9
Einkorn Unknown 3.5 12.8 19.3 64.5 3.4
Rye Halo 9.0 17.6 48.4 25.0 0.7
Barley Golden promise 5.0 35.8 34.1 25.1 1.6
a
a aggregative, m monomeric
Fig. 2.2 Schematic two-dimensional structures of the C-terminal domain (sections III–V) of
g-gliadins (Taken from [94])
Because HMW-GS do not occur as monomers, it is generally assumed that they

form interchain disulfide bonds. The x-type subunits, except subunit 1Dx5, have
three cysteine residues in domain A and one in domain C (Fig. 2.1). Cysteines Ca
and Cb were found to be linked by an intrachain bond, thus, the others (Cd, Cz) are
available for interchain bonds. Subunit 1Dx5 has an additional cysteine residue at
the beginning of domain B, and it has been suggested that this might form another
interchain bond. Recently, a so-called head-to-tail disulfide bond between HMW-GS
has been identified [71]. The y-type subunits have five cysteine residues in domain
A and one in each of domains B and C. At present, interchain linkages have only
been found for adjacent cysteine residues of domain A (Cc1, Cc2), which are con-
nected in parallel with the corresponding residues of another y-type subunit, and for
cysteine Cy of domain B, which is linked to Cx of section IV of LMW-GS. Thus,
HMW- and LMW-GS fulfill the requirement that at least two cysteines forming
interchain disulfide bonds are necessary to participate in a growing polymer; they
act as “chain extenders.” The most recent glutenin model suggests a backbone
formed by HMW-GS linked by end-to-end, probably head-to-tail interchain disulfide
bonds [65]. LMW-GS form also linear polymers via cysteine residues of sections I
and IV; they are linked to domain B of y-type HMW-GS. y-Type HMW-secalins of
rye have a second cysteine in domain C, which opens the possibility that an intrachain
disulfide bond within domain C is formed inhibiting an interchain bond for polym-
erization [72]. As far as is known, D-hordeins possess ten (!) cysteine residues [63];
the formation of a regular polymer backbone appears to be impossible.
2.3.2.3 Molecular Weight Distribution
Most information on the quantitative MW distribution (MWD) of native storage

(gluten) proteins is available for wheat, because MWD of gluten proteins has been
recognized as one of the main determinants of the rheological properties of wheat
dough. Native gluten proteins consist of monomeric a/b- and g-gliadins with MW
around 30,000 and monomeric w5- and w1,2-gliadins with MW between 40,000 and
55,000. They are alcohol-soluble and amount to ~50% of gluten proteins (Fig. 2.3).
Besides monomers the alcohol-soluble fraction contains oligomers with MW
roughly ranging between 60,000 and 600,000. They are formed by modified gliadins
with an odd number of cysteine residues and LMW-GS via interchain disulfide
bonds and account for ~15% of gluten proteins. Composition and quantity of the
oligomeric fraction are strongly determined by the conditions of alcohol extraction,
for example by temperature and duration. The remaining proteins (~35%) are alco-
hol-insoluble and mainly composed of LMW-GS and HMW-GS linked by disulfide
bonds. Their MW ranges approximately from 600,000 to more than 10 million. The
largest polymers termed “glutenin macropolymers” (GMP) are insoluble in SDS
solutions and have MW well into the multimillions indicating that they may belong
to the largest proteins in nature [73, 74]. Their amounts in flour (20–40 mg/g) are
strongly correlated with dough strength and bread volume. GMP is characterized
by higher ratios of HMW-GS to LMW-GS and x-type to y-type HMW-GS in
Fig. 2.3 Molecular weight

distribution of native wheat
storage (gluten) proteins
(Modified from [73])
comparison with total glutenins; the HMW-GS combination 1Dx5 + 1Dy10

appears to produce higher GMP concentrations than 1Dx2 + 1Dy12 [73].
Rye storage proteins have a strongly different MWD as compared to wheat.
Although rye shows higher proportions of aggregative to monomeric storage pro-
teins than wheat (Table 2.7), the proportions of polymers is much lower (~23%) and
the amount of GMP (~5 mg/g flour) strongly reduced [75, 76]. The deficiency of
polymeric proteins is balanced by the higher proportion of oligomers (~30%), whereas
the proportion of rye monomers (~47%) is similar to that of wheat. Obviously rye
storage proteins consist of many more chain terminations (e.g., g-75 k-secalins,
y-type HMW-secalins) and less chain extenders than wheat, which apparently
prevents gluten formation during dough mixing. Information about the MWD of
native barley and oat proteins is not yet available.
2.3.2.4 Influence of External Parameters
Many studies have substantiated that both structures and quantities of storage pro-
teins are exposed to a continuous change from the growing period of plants to the
processing of end products. Because of the importance of wheat as a unique “bread
cereal,” most investigations have been focused on gluten proteins. In principle, how-
ever, the effect of external parameters is similar for all cereal proteins.
Fertilization
The supply with minerals during growing is essential for optimal plant develop-
ment. Nitrogen (N) fertilization is, in particular, important for common wheat,
because a high N supply provides a high flour protein content and thus, increased
bread volume. Fertilization with different N amounts demonstrated that the quantities
of albumins/globulins are scarcely influenced, whereas those of gluten proteins

increase with higher N supply [77]. The effects on gliadins are more pronounced
than on glutenins resulting in an elevated gliadin/glutenin ratio. Particularly, the
proportions of w-type gliadins are strongly enhanced by high N supply.
In the past, sulfur (S)-containing fertilizers were not widely used for cereal crops,
because air pollution from industry and traffic provided sufficient amounts of S in the
soil. The massive decrease in the input of S from atmospheric deposition over the last
decades reduced S availability in soils dramatically, and has led to a severe S deficiency
in cereals, which exerts a large influence on protein composition and technological
properties. In the case of wheat, S deficiency provokes a drastic increase of S-free
w-type gliadins and a decrease of S-rich g-gliadins and LMW-GS [78]. Moreover, S
deficiency has been reported to impair dough and bread properties [79].
Infections
The infection of cereal plants with Fusarium strains induces a premature fading of
individual spikelets and then, a fading of the whole ears. An outbreak of this infec-
tion is often accompanied by mycotoxin contamination of grains and flours, for
example by the trichothecene deoxinivalenol. Studies on wheat demonstrated that
infection with Fusarium caused a distinct reduction in the content of both total glu-
tenins and HMW-GS and impaired dough and bread quality [80]. Glutenins of com-
mon wheat have been shown to be more strongly affected than those of emmer and
storage proteins of naked barley [81, 82].
Germination
Proteins as well as other constituents are stable in dry grains. Water supply, how-
ever, induces germination of grains accompanied by the activation of enzymes, in
particular, amylases and peptidases. The latter have been shown, for instance, to
cause a fast degradation of prolamins of wheat, rye, barley, and oats during germi-
nation [83]. Studies on the Osborne fractions of wheat demonstrated that both
monomeric gliadins and polymeric glutenins were strongly degraded during germi-
nation for 168 h at 15–30 °C, whereas albumins/globulins were scarcely affected [84].
The degradation of gluten proteins has a drastic negative effect on the bread-making
quality of wheat.
Oxidation
Grains contain a considerable amount of LMW thiols such as glutathione, which are
known to affect the structures and functional properties of polymeric storage proteins
by thiol/disulfide interchange reactions during dough preparation [64]. To prevent
this deleterious effect on the bread-making quality of wheat, week-long storage of
flour under air (direct oxidation of LMW thiols) and treatment with L-ascorbic acid
(indirect oxidation catalyzed by glutathione dehydrogenase) are recommended.
Enzymes
Breads prepared from rye and wheat sourdoughs are of increasing consumer interest
due to the improvement of sensorial and nutritional quality, the prolongation of shelf
life, and the delay in staling. Wheat storage proteins, however, which are responsible
for the viscoelastic and gas-holding properties of dough and for the texture of the
bread crumb, are profoundly degraded [85]. Protein degradation during fermentation
is primarily due to acidic peptidases present in flour and activated by the lowered pH
caused by Lactobacillus strains. The strongest decrease was found for the glutenin
macropolymer and total glutenins. The extent of the decrease of monomeric gliadins
was lower and more pronounced for the g-type than for the a/b- and w-types.
Heat
The baking process involves a drastic heat-treatment of proteins, with temperatures

of more than 200 °C on the outer layer (crust) and near 100 °C in the interior (crumb)
of bread. HPLC analysis of crust proteins from wheat bread indicated serious struc-
tural damage of both gliadins and glutenins [86]. With respect to crumb, the extract-
ability of total gliadins with 60% ethanol is strongly reduced compared with those
from flours. The single gliadin types are affected differently, w-type gliadins less
and a/b- and g-types much more. Most gliadins can be recovered in the glutenin
fraction after reduction of disulfide bonds suggesting that major heat-induced cross-
links of gliadins to glutenins are disulfide bonds.
High Pressure
The effect of hydrostatic pressure is similar to that of heat. Treatment of gluten with
pressure in the range of 300–600 MPa at 60 °C for 10 min provokes a strong reduc-
tion of gliadin extractability [87]. Within gliadin types, cysteine containing a/b- and
g-type gliadins, but not cysteine-free w-type gliadins, are sensitive to pressure and
are transferred to the ethanol-insoluble glutenin fraction. Cleavage and rearrange-
ment of disulfide bonds have been proposed as being responsible for pressure-
induced aggregation.
2.3.2.5 Wheat Gluten
Wheat is unique among cereals in its ability to form a cohesive, viscoelastic dough,
when flour is mixed with water. Wheat dough retains the gas produced during
fermentation and this results in a leavened loaf of bread after baking. It is commonly
accepted that gluten proteins (gliadins and glutenins) decisively account for the physi-
cal properties of wheat dough. Both protein fractions are important contributors to
these properties, but their functions are divergent. Hydrated monomeric and oligo-
meric proteins of the gliadin fraction have little elasticity and are less cohesive than
glutenins; they contribute mainly to the viscosity and extensibility of dough. In con-
trast, hydrated polymeric glutenins are both cohesive and elastic, and are responsible
for dough strength and elasticity. Thus, gluten is a “two-component glue,” in which
gliadins can be understood as a “plasticizer” or “solvent” for glutenins [65]. A proper
mixture (~2:1) of the two is essential to give desirable dough and bread properties.
Native gluten proteins are amongst the most complex protein networks in nature
due to the presence of several hundred different protein components. Even small
differences in the qualitative and quantitative protein composition decide on the
end-use quality of wheat varieties. Numerous studies demonstrated that the total
amounts of gluten proteins (highly correlated with the protein content of flour), the
ratio of gliadins to glutenins, the ratio of HMW-GS to LMW-GS, the amount of
GMP, and the presence of specific HMG-GS determine dough and bread quality.
Amongst chemical bonds disulfide linkages (Fig. 2.1) play a key role in deter-
mining the structure and properties of gluten proteins. Intrachain bonds stabilize the
steric structure of both monomeric and aggregative proteins; interchain bonds pro-
voke the formation of large glutenin polymers. The disulfide structure is not in a
stable state, but undergoes a continuous change from the maturing grain to the end
product (e.g., bread), and is chiefly influenced by redox reactions. These include (1)
the oxidation of free SH groups to S-S linkages, which supports the formation of
large aggregates, (2) the presence of chain terminators (e.g., glutathione and glia-
dins with an odd number of cysteine), which stop polymerization, and (3) SH-SS
interchange reactions, which affect the degree of polymerization of glutenins.
Consequently, oxygen is known to be essential for optimal dough development and
oxidizing agents, for example potassium bromide, azodicarbonimide, and dehy-
droascorbic acid (the oxidation product of ascorbic acid) have been found to be
useful as bread improvers [88].
Conversely, reducing agents such as cysteine and sodium metabisulfite are used
to soften strong doughs, accompanied by decreased dough development and resis-
tance and increased extensibility. They are specifically in use as dough softeners for
biscuits. The overall effect is to reduce the average MW of glutenin aggregates by
SH/SS interchange.
Beside disulfide bonds, dityrosine and isopeptide bonds have been described as
further covalent cross-links between gluten proteins. Compared with the concentra-
tion of disulfide bonds (~10 mmol per g flour) tyrosine-tyrosine cross-links
(~0.7 nmol per g flour) appear to be only of marginal importance [89]. Interchain
cross-links between lysine and glutamine residues (isopeptide bonds) are catalyzed
by the enzyme transglutaminase (TG). Addition of TG to flour results in a decrease
in the quantity of extractable gliadins and an increase of the glutenin fraction and
the nonextractable fraction [90]. Thereby, dough properties and bread-making qual-
ity can be positively influenced, similar to the actions of chemical oxidants.
Fig. 2.4 A model double unit for the interchain disulfide structure of LMW-GS and HMW-GS of
glutenin polymers (Adapted from [65])
The covalent structure of gluten proteins is complemented by noncovalent bonds

(hydrogen bonds, ionic bonds, hydrophobic bonds). Glutamine, predestinated for
hydrogen bonds, is the most abundant amino acid in gluten proteins (Table 2.4) and
chiefly responsible for the water-binding capacity of gluten. In fact, dry gluten
absorbs about twice its own weight of water. Moreover, glutamine residues are
involved in frequent protein-protein hydrogen bonds. Though the number of ioniz-
able side chains is relatively low, ionic bonds are of importance for the interactions
between gluten proteins. For example, salts such as NaCl are known to strengthen
dough, obviously via ionic bonds with glutenins [91]. Hydrophobic bonds can also
contribute to the properties of gluten. Because the energy of hydrophobic bonds
increase with increased temperature, this type of noncovalent bonds is particularly
important for protein interactions during the oven phase.
Both covalent and noncovalent bonds determine the native steric structures
(conformation) of gliadins and glutenins. Studies on the secondary structure have
indicated that the repetitive sequences of gliadins and LMW-GS are characterized
by b-turn conformation, whereas the nonrepetitive sections contain considerable
proportions of a-helix and b-sheet structures [92]. The nonrepetitive sections of
a/b-, g-gliadins, and LMW-GS include intrachain disulfide bonds, which are con-
centrated in a relatively small area and form compact structures including two or
three small rings and a big ring (Fig. 2.2) [93]. The nonrepetitive sections A and C
of HMW-GS are dominated by a-helix and b-sheet structures, whereas the repeti-
tive section B is characterized by regularly repeated b-turns [94]. They form a loose
b-spiral similar to that of mammalian connective tissue elastin; b-spirals have been
proposed to transfer elasticity to gluten.
A range of models has been developed to explain the structure and functionality
of glutenins. Most recently, the experimental findings on disulfide bonds were trans-
formed into a two-dimensional model [65] (Fig. 2.4). HMW-GS and LMW-GS
polymerize separately, both forming linear backbone polymers. Both polymers are
cross-linked by a disulfide bond between section IV of LMW-GS and section B of
y-type HMW-GS. The backbone of HMW-GS is established by end-to-end, probably
head-to-tail linkages. LMW-GS polymers are linked between two sections I and
between sections I and IV. The polymerization of HMW-GS and LMW-GS is termi-
nated by chain terminators, either by modified gliadins or LMW thiol compounds.
2.3.3 Storage Proteins of Corn, Millet, Sorghum, and Rice
Overall, the storage proteins of corn, sorghum, millet, and rice are, in part, related
and differ significantly from those of wheat, rye, barley, and oats. According to the
amino acid composition they contain less glutamine and proline and more hydro-
phobic amino acids such as leucine [56]. Corn storage proteins, called zeins, can be
subgrouped into alcohol-soluble monomeric zeins and cross-linked zeins alcohol-
soluble only on heating or after reduction of disulfide bonds. With respect to differ-
ent structures zeins have been divided into four different subclasses [95]. a-Zeins
are the major subclass (71–85% of total zeins), followed by g- (10–20%), b- (1–5%)
and d-zeins (1–5%), respectively [96]. a-Zeins are monomeric proteins with appar-
ent MW of 19,000 and 22,000 determined by SDS-PAGE. Their amino acid
sequences contain up to ten tandem repeats [97]. Proteins of the other subclasses are
cross-linked by disulfide bonds and their subunits have apparent MW of 18,000 and
27,000 (g-zein), 18,000 (b-zein), and 10,000 (d-zein).
In many ways the storage proteins of sorghum and millet called kafirins are
similar to zeins. Sorghum kafirins have also been subdivided into a, b-, g- and
d-subclasses based on solubility, MW, and structure [98]. a-Kafirins are monomeric
proteins and represent the major subclass accounting for around 65–85% of total
kafirins. Proteins of the other subclasses are highly cross-linked and alcohol-soluble
only after reduction of disulfide bonds. On average, each of them accounts for less
than 10% of total kafirins [99, 100]. Within the numerous millet species and variet-
ies the proteins of foxtail millet were studied in detail [101]. SDS-PAGE of unre-
duced kafirins revealed bonds with apparent MW ranging from 11,000 to 150,000.
After the reduction of disulfide bonds two major bands with MW of 11,000 (subunit
A) and 16,000 (subunit B) were obtained. Unreduced proteins with higher MW
were formed by cross-links of A and/or B subunits. The storage proteins of rice are
characterized by the highly unbalanced ratio of prolamins to glutelins (~1:30) [102].
Both fractions show the lowest proline content (~5 mol-%) amongst cereal storage
proteins [56]. SDS-PAGE patterns of rice prolamins (oryzins) showed a major band
with MW 17,000 and a minor band with MW 23,000 [103]. The apparent MW of
glutelin subunits was in a range from 20,000 to 38,000.
2.3.4 Metabolic Proteins
Most proteins of the albumin and globulin fractions are metabolic proteins, mainly
enzymes and enzyme inhibitors. The corresponding extensive studies have been
summarized by Kruger and Reed [104] and recently by Delcour and Hoseney [105].
Many of these proteins are located in the embryo and aleurone layer; others are
distributed throughout the endosperm. They have nutritionally better amino acid
compositions than storage proteins, particularly because of their higher lysine
contents. Those enzymes that hydrolyze carbohydrates and proteins and, thereby,
provide the embryo with nutrients and energy during germination, are of most
significant importance.
2.3.4.1 Hydrolyzing Enzymes
Carbohydrate-Degrading Enzymes
The many carbohydrate-degrading enzymes include a-amylases, b-amylases,

debranching enzymes, cellulases, b-glucanases, and glucosidases. Amylases are
enzymes that hydrolyze the polysaccharides in starch granules. They can be
classified as endohydrolases, which attack glucosidic bonds within the polysac-
charide molecules and exohydrolases, which attack glucosidic bonds at or near
the end of chains. The most important enzyme of the endohydrolase type is
a-amylase. The enzyme hydrolyzes a-1,4-glucosidic bonds of amylose and amy-
lopectin and produces a mixture of dextrins together with smaller amounts of
maltose and oligosaccharides; the pH-optimum is about 5. The other major amy-
lase type is b-amylase, an exohydrolase, which hydrolyzes a-1,4-glucosidic bonds
near the nonreducing ends of amylose and amylopectin to produce maltose. Its pH-
optimum is similar to that of a-amylase. Both amylase types exist in multiple
forms or isoenzymes with different chemical and physical properties. Neither
a- nor b-amylase can break a-1,6-glucosidic bonds present in amylopectin. For
this kind of hydrolysis debranching enzymes are present in cereal grains. Along
with a-glucosidases they assist a- and b-amylases in a more complete conversion
of starch to simple sugars and small dextrins. A number of other carbohydrate degrad-
ing enzymes exist, their amounts, however, are very low compared to amylases.
Examples are a- and b-glucosidases, cellulases, and arabinoxylanases.
Proteolytic Enzymes
Enzymes that hydrolyze proteins are called proteinases, proteases, or peptidases.

They attack the peptide bond between amino acid residues and include both endo-
and exopeptidases. The latter are divided into carboxypeptidases, when acting from
the carboxy terminal and aminopeptidases, when acting from the amino terminal.
The most important proteolytic enzymes are acidic peptidases. They exist in multiple
forms having pH-optima between 4.2 and 5.5 and include both endo- and exotypes.
On the basis of their catalytic mechanism they can be classified as serine, metallo-,
aspartic, and serine peptidases. According to their biological function to provide the
embryo with amino acids, their activity is highest during the germination of grains.
Other Hydrolyzing Enzymes
Lipases are the most important enzymes that hydrolyze ester bonds. They attack
triacylglycerols yielding mono- and diacylglycerols and free fatty acids. Lipase
activity is important, because free fatty acids are more susceptible to oxidative ran-
cidity than fatty acids bound in triacylglycerols. The activity varies widely among
cereals with oats and millet having the highest activity. Exogenous lipases are in use
to improve the baking performance of wheat flour.
Phytase is an esterase that hydrolyzes phytic acid to inositol and free phosphoric
acid. Even partial hydrolysis of phytic acid by phytase is desirable from a nutritional
point of view, because the strong complexation of cations such as zinc, calcium, and
magnesium ions by phytic acid is significantly reduced.
2.3.4.2 Oxidizing Enzymes
Lipoxygenase is present in high levels in the germ. It catalyzes the peroxidation of

certain polyunsaturated fatty acids by molecular oxygen. Its typical substrate is
linoleic acid containing a methylene-interrupted, doubly unsaturated carbon chain
with double bonds in the cis-configuration.
Polyphenoloxidases preferably occur in the outer layers of the grains. They catalyze
the oxidation of phenols, such as catechol, pyrogallol, and gallic acid, to quinons by
molecular oxygen. Peroxidase and catalase may be classified as hydroperoxidases
catalyzing the oxidation of a number of aromatic amines and phenols, for example
ferulic acid in arabinoxylans, by hydrogen peroxide. Other oxidizing enzymes are
ascorbic acid oxidase and glutathione dehydrogenase.
2.3.4.3 Enzyme Inhibitors
Many investigators have isolated and characterized enzyme inhibitors from germ
and endosperm. Most important inhibitors are targeted on hydrolyzing enzymes to
prevent the extensive degradation of starch and storage proteins during grain
development and to defend plant tissues from animal (insect) or microbial enzymes.
Predominant classes are amylase and protease inhibitors concentrated in the albu-
min/globulin fractions. Amylase inhibitors can be directed towards both cereal and
noncereal amylases and protease inhibitors towards proteases from both cereals and
animals. Some inhibitors appear to be bifunctional inhibiting amylases as well as

proteases.
2.4 Lipids
2.4.1 Lipid Composition
Cereal lipids originate from membranes, organelles, and spherosomes and consist
of different chemical structures. Depending on cereal species average lipid contents
of 1.7–7% in the grains are present (Table 2.2). Lipids are mainly stored in the germ,
to a smaller extent in the aleurone layer and to the lesser extent in the endosperm. In
particular oats are rich in lipids (6–8%) in contrast to wheat and rye (1.7%). Cereal
lipids have similar fatty acid compositions, in which linoleic acid reaches contents
of 39–69%, while oleic acid and palmitic acid make up 11–36% and 18–28%,
respectively [106, 107]. Although wheat lipids are only a minor constituent of the
flour, they greatly impact the baking performance and have, therefore, been exten-
sively studied.
While triglycerides are the dominating lipid class in the germ and the aleurone
layer, phospho- and glycolipids are present in the endosperm (Fig. 2.5). Depending
on the extraction rate wheat flour contains 0.5–3% lipids [108]. Extraction with a
polar solvent at ambient temperature, i.e., water-saturated butanol, dissolves the
nonstarch lipids that make up approximately 75% of the total flour lipids [109]. The
residual 25% are the so-called starch lipids. The composition of the nonstarch lipids
is given in Table 2.8. They contain about 60% nonpolar lipids, 24% glycolipids, and
15% phospholipids. By extraction with solvents of different polarities they can be
further subdivided into a free and a bound fraction. The nonpolar lipids are mainly
present in the free lipid fraction, whereas glyco- and phospholipids are part of the
bound fraction, in which they can be associated, for example with proteins [106, 107].
The major glycolipid class is the digalactosyldiglycerides. Starch lipids are primar-
ily composed of lysophospholipids, which form inclusion complexes with amylose
helices already in native starch [28].
2.4.2 Effects of Lipids on the Baking Performance

of Wheat Flour
Only nonstarch lipids affect the rheological properties of wheat doughs. Interactions
between starch lipids and starch are sufficiently strong so that this lipid fraction is
not available before the starch gelatinizes. Studies with nonstarch lipids have shown
that only the polar lipids have a positive effect on baking performance, whereas the
Fig. 2.5 Polar lipids that

affect the baking performance
of wheat
Table 2.8 Composition of nonstarch lipids of wheat flour [107]. Content (g/100 g) based on total
lipid
Nonstarch lipids: 1.70–1.95 g/100 g flour
Polar 36–42 Nonpolar 58–64
Phospholipids 14–16 Sterol esters 1.9–4.2
Acylphosphatidyl ethanolamine 4.2–4.9 Triglycerides 39.5–49.4
Acyllysophosphatidyl ethanolamine 1.6–2.3 Diglycerides 3.3–5.4
Phosphatidyl ethanolamine/phosphatidyl 0.7–1.1 Esterified monogalactosyldi- 2.7–3.9
glycerol glycerides/monoglycerides
Phosphatidyl choline 3.8–4.9 Esterified sterolglycerides 0.8–4.2
Phosphatidyl serine/phosphatidyl inosit 0.4–0.7
Lysophosphatidyl ethanolamin 0.3–0.5
Lysophosphatidyl glycerol 0.2–0.3
Lysophosphatidyl choline 1.4–2.1
Glycolipids 22–26
Monogalactosyldiglycerides 5.0–5.9
Monogalactosylmonoglycerides 0.9–0.4
Digalactosyldiglycerides 12.6–16.5
Digalactosylmonoglycerides 0.6–3.4
nonpolar lipids have the opposite effect [110]. In particular glycolipids have been
shown to contribute to the high baking performance of wheat flour [29, 111–113],
whereas the functionality of the phospholipids has been found to be less important.
If the term “specific baking activity” would be defined, polar lipids would be found
to affect the baking performance of wheat flour to a considerably greater extent than
proteins. The addition of only 0.13% polar lipids would yield the same increase of
loaf volume as a protein content that would be increased by 1%. Polar lipids affect
dough properties in many ways, i.e., the dough handling properties are improved
and the gas-holding capacity during proofing is increased enabling a prolonged

oven spring, increased loaf volume, better crumb resilience, and, in some cases,
retardation of bread staling.
2.4.3 Modes of Action of Polar Lipids in Baking
The high baking activities of polar lipids, in particular of the glycolipids, might be
explained by modes of action based on the formation of liquid films at the dough
liquor/gas cell interface. Possible modes of action are the direct influence of the sur-
factants on the liquid film lamellae and gas cell interfaces through direct adsorption
resulting in an increase of surface activity as suggested by Gan et al. [49, 114] and
Sroan et al. [115] as the secondary stabilizing mechanism in the so-called dual film
theory. It suggests the presence of liquid lamellae, providing an independent mecha-
nism of gas cell stabilization. As shown recently, the effects of different surface active
components may be explained by the type of monolayer that they form [116].
However, in particular the positive effect of some polar lipids such as acylated
sterol glucosides and sterol glucosides cannot be explained with this mode of directly
stabilizing the liquid film lamellae. Here another mode of action could be the answer,
for example the indirect stabilization of the dough liquor/gas cell interface through
this type of surfactant [116, 117]. These polar lipid classes have a positive influence
on the phase behavior of the endogenous lipids present in the dough liquor in that
they lead to an increase in surface activity of the endogenous lipids and hence a bet-
ter availability and accumulation at the liquid film lamellae/gas cell interface, thus
increasing gas cell stabilization, and consequently the bread volume.
Inclusion complexes between amylose helices and polar lipids with one fatty acid
residue are responsible for two effects. Complexes present in native starch (starch
lipids) increase the temperature of gelatinization and, thus, prolong the oven spring.
Inclusion complexes between amylose helices and polar lipids with one fatty acid
residue may also form during and after the gelatinization process and are responsible
for the anti-staling effect of some polar lipids, for example monoglycerides.
2.5 Minor Constituents
2.5.1 Minerals
The mineral content of cereals ranges from ca. 1.0 to 2.5% (Table 2.2). Compared
to other foods this is an intermediate concentration with milk, meat, and vegetables
having somewhat lower mineral contents and pulses, which are extraordinarily rich
in minerals (mean mineral content ~3.5%). As cereals are among the most important
staple foods, and are consumed in high quantities, they are important sources of
minerals in the human diet. The major portion of the minerals (>90%) is located in
the outer layers of the grains, namely in the bran, the aleurone layer, and the germ.
Consequently, products made from whole grains should increasingly be introduced
into human nutrition to benefit from the mineral content of cereals.
2.5.2 Vitamins
Cereals contain vitamins in concentrations ranging from below 1 to ca. 50 mg/kg,

depending on the compound (Table 2.2). Thus, cereals are a good source of vita-
mins from the B-group, and, in industrial countries, they cover about 50–60% of
the daily requirement of B-vitamins. The most important fat-soluble vitamins are
the tocopherols, which are present in concentrations exceeding 20 mg/kg. Like the
minerals, vitamins are concentrated in the outer layers of the grains, in particular
in the aleurone layer as well as in the germ. Therefore, milling of cereals into white
flour will remove most of the vitamins. Consequently, the use of whole-grain prod-
ucts or products enriched in vitamin-containing tissues will be of nutritional benefit
for the consumer.
Abbreviations
AX Arabinoxylans
DSC Differential scanning calorimetry
GMP Glutenin macropolymer
GS Glutenin subunits
HMW High-molecular-weight
HPLC High-performance liquid chromatography
LMW Low-molecular-weight
m/a Monomeric/aggregated
MMW Medium-molecular-weight
MW Molecular weight
MWD Molecular weight distribution
NSP Nonstarch polysaccharides
PAGE Polyacrylamide gel electrophoresis
SDS Sodium dodecyl sulfate
TG Transglutaminase
WEAX Water-extractable arabinoxylans
WUAX Water-unextractable arabinoxylans
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Chapter 3
Technology of Baked Goods
Maria Ambrogina Pagani, Gabriella Bottega, and Manuela Mariotti
3.1 Introduction
Baked goods are a heterogeneous market category of products. The high

diversification that distinguishes these products makes it difficult to find one single,
general and satisfactory definition. It is, however, possible to identify baked goods
based on the common commodities matrix since they are all foods derived from
cereal flour [1]. Other similarities within this group are the basic ingredients used,
mainly wheat flour or, less commonly, rye flour, water and leavening agents.
Although the technological processes may differ, each one comprises mixing, leavening
and baking. These successive stages allow the transformation of flour, or a mixture
of different types of flour, into an appetizing and digestible food, which, at the
macroscopic level, has a complex structure differentiated by a friable crust and an
internal alveolar structure.
The classification of baked goods may be based on different criteria [2]. One
criterion, which is widely used in the baking trade but is not governed by legislation,
is the presence of sugar in the formula which can be perceived by taste and corre-
sponds to at least 10% of the weight of the flour. Baked products with more simple
formulas, therefore, constitute the various types of bread. Despite its importance,
not only sensorial but also nutritional and textural, the amount of sugar in the dough
is not sufficient for classifying this complex category in a satisfactory way. As pro-
posed by Cauvain and Young [1], the distinction of the basic products comprising
the formula may be made by considering the fat:flour ratio.
M.A. Pagani (*) • G. Bottega • M. Mariotti (*)

Department of Food, Environmental and Nutritional Sciences (DeFENS),
University of Milan, via Celoria 2, 20133 MILANO, Italy
e-mail: [email protected]; [email protected];
[email protected]

48 M.A. Pagani et al.
A further criterion for differentiating baked goods is the evaluation of their lightness
and softness, and so of the specific volume (correlated with lightness) and humidity
(which determines the softness). These two structural characteristics are the result
of complex phenomena that occur during each stage of the technological process.
Indeed, the final characteristics of the product not only depend on leavening but also
on mixing and baking (see Sect. 3.4). A light and soft baked good usually has a
specific volume higher than 2.5–3.0 mL/g and humidity higher than 15–18%. A dry
and friable baked good has a specific volume between 1.3 and 2.5 mL/g and humid-
ity values lower than 5–10% [2]. On the basis of these two criteria, four categories
of baked products can be identified. Moreover, each category can be further subdi-
vided according to the leavening method used (see Sect. 3.3.3).
3.2 Wheat, a Preferred Raw Material for Bread

and Baked Goods
The choice of ingredients is of fundamental importance for the production of leavened

baked goods that satisfy consumer tastes. Although barley and rye flours provide a
workable dough, bakers realized a long time ago that the best results in terms of vol-
ume development were always obtained with wheat flour. The superior quality of this
cereal, especially its most evolved species Triticum durum and T. aestivum, is of a
purely technological nature. Only wheat flour provides a cohesive and homogenous
dough, where the single and original particles of flour are no longer recognizable.
The property that makes wheat dough unique is its viscoelasticity. This rheologi-
cal property enables the mass to be stretched and deformed without rupturing. At
the same time, the dough is elastic and tenacious, capable of maintaining its shape
even when subjected to physical stress. After baking, gluten proteins denature and
loose viscoelasticity, ensuring the maintenance of the final shape of baked goods.
The reason for this versatile behaviour does not depend on differences in the quan-
tity of components. Indeed, the protein content of the numerous varieties of wheat
extends over quite a wide interval, from 9 to 16% of the weight of the grain [3]. This
variability coincides with that of other cereals. The technological superiority of
wheat is related to complex qualitative differences at the level of protein fractions.
Wheat storage proteins are generally classified based on molecular weight (MW),
solubility and conformation. Storage proteins, gliadins and glutenins, account for
ca. 80% of the entire protein fraction and have a particular amino acid composition.
Gluten proteins have a high percentage of glutamine, about one third of all amino
acids, and proline, and low levels of lysine (Chap. 2, [4]). This composition is
responsible for the low nutritional value of wheat protein. However, it also accounts
for the protein–protein interactions that lead to the formation of gluten, the three-
dimensional network which is continuous and homogenous throughout the mass.
Both non-covalent bonds, such as hydrophobic interactions and hydrogen bonds
(guaranteed by the large amounts of glutamine), and covalent bonds are involved,
the most significant of which are the disulfide bonds between cysteine residues.
3 Technology of Baked Goods 49
Gliadins and glutenins have different roles for the viscoelastic characteristics of
gluten [5–7]. Gliadins can be easily deformed and stretched, thanks to their viscous
properties that are typical of fluids, because of their globular shape and interconnec-
tions via disulfide bonds. In contrast, glutenins form long chains by means of inter-
protein disulfide bonds, which resist deformation and form an elastic and tenacious
mass. Although durum wheat semolina is used for bread making, especially in the
Mediterranean regions, the common wheat (T. aestivum) flour may be the optimal
raw material because it gives the best leavening results [8]. Soluble proteins in
wheat flour are mostly proteins with enzymatic activity. Most of these enzymes are
hydrolases (amylases, proteases, lipases), which specifically act on the reserve mac-
romolecules. Also oxidative enzymes (lipoxygenases, peroxidases) are present. For
many wheat processes, the enzymatic activities have a central and strategic role
which has to be carefully monitored (see also Chap. 2).
3.2.1 The Milling Process
About 70% of the worldwide wheat production is used for food [9], mainly by bakery
industries. The essential preliminary step is the milling process. The objective of
milling is twice. On the one hand, it separates the starchy endosperm, the area con-
taining gluten proteins, from germ, pericarp and seed coats, that form bran and other
by-products. On the other hand, it reduces the particle size of the endosperm to
values lower than 150–200 mm for common wheat flour and 500 mm for durum
wheat semolina. The fine granulometry of the flour gives an optimal workability to
the resulting dough and contributes to its processing into a palatable and appetizing
food. The removal of bran improves both the hygienic properties of the flour, since
the peripheral parts of the grain are often contaminated by chemical residues and
biotic pollutants, and the technological characteristics of the flour. In fact, non-
starch polysaccharides and enzymes, which are abundant in the bran, worsen the
rheology properties of the dough [10]. The separation of the oil-rich germ prevents
rancidity, which compromises the storage of flour [11]. However, the elimination of
these two parts, which are rich in several functional components, decreases the
nutritional value of the flour.
Overall, milling is much more complex than mere grinding. Because of the par-
ticular morphological structure of wheat grain, which is characterized by the crease
or ventral furrow, an introflexion along the length of the kernel, which hides a dou-
ble layer of teguments, the milling has to promote the extraction of the flour from
grains. Therefore, the first step consists of opening the grain. Then, proceeding from
the inside towards the outside, the endosperm is recovered via repeated sequences
of size reduction and separation stages, excluding the more external areas (bran,
aleurone layer, etc.), which are known as tailing products. This procedure fully
justifies the definition of “flour extraction yield”, defined as the quantity of flour
produced from 100 parts of cleaned and conditioned wheat grains [12]. This is the
only solution for preventing the passage of the bran layers from the furrow, in which
Table 3.1 Chemical composition of wheat regions (Adapted from [15]) (data expressed as
g/100 g dm)
Kernel Starch and soluble Proteins Lipids NSPa (cellulose,
Region (%) sugars (%) (%) (%) etc) (%) Ash (%)
Fruit coat 5 14 ÷ 16 10 ÷ 14 1÷3 60 ÷ 74 3÷5
(pericarp)
Seed coat 2 9 ÷ 11 13 ÷ 19 3÷5 53 ÷ 63 9 ÷ 15
Aleurone 8 10 ÷ 14 29 ÷ 35 7÷9 35 ÷ 41 5 ÷ 15
Endosperm 82 80 ÷ 85 8 ÷ 14 2÷3 1÷3 0.5 ÷ 1.5
Germ 3 19 ÷ 21 36 ÷ 40 13 ÷ 17 20 ÷ 24 4÷6
a
Non-starch polysaccharides
25–30% of total bran is hidden, into the flour of the bran layers [13]. Milling is
generally simpler for those cereals without furrow.
The physical separation of the various parts of the wheat grain is made possible
by the different composition of the three morphological areas of the kernel, that
determine different behaviour during processing (Table 3.1). The separation of the
endosperm from the bran is not quantitative, since parts of the endosperm are lost
into the milling by-products and small percentages of bran fragments are inevita-
bly present in the flour. Therefore, the milling process has to reach a compromise
between “extraction yield” and “grade of refinement” (accuracy of elimination of
bran) of the flour. The main criterion for flour classification is based on the accu-
racy of teguments separation. The approach, used by legislation in many countries,
is based on the threshold value of a number of parameters, especially ash and
proteins.
The current technology for milling considers the following four stages: (1)
receiving, pre-cleaning and storage of the incoming wheat; (2) cleaning and condi-
tioning; (3) milling; (4) storage of the flours. Wheat coming to the mill is usually
transported in bulk in trucks, trains or ships and it is unloaded into large hoppers
with a grilled opening to facilitate the elimination of large foreign matter. This oper-
ation is preceded by rapid analytical inspections of samples representative of the
entire batch, to assess quality parameters. Preliminary cleaning operations made
before loading the wheat into the silos are intended to remove mainly coarse foreign
materials, in order to provide for better storage. The cleaning of the wheat is carried
out immediately before the milling process. This involves a sequence of operations,
each performed by a special machine, with the aim of removing impurities, foreign
matters and powders. Differences in size, shape and density compared to the whole
and sound wheat grains are used to achieve this aim [14]. The conditioning or tem-
pering of kernels is decisive to achieve an optimal milling. This operation includes
grain humidification and the successive resting time, to increase the water content
of 2–4%. The conditioning step toughens the bran, favouring its break off in form of
large particles, and mellows the endosperm, thereby facilitating the separation
between these two parts. The conditioning time (from 6 to 24–36 h), the quantity of
water used, and the ways to add water (one or two tempering steps) depend on
the initial humidity and on the hardness and vitreousness of the wheat kernels.
Fig. 3.1 Simplified schema of wheat milling operations (Adapted from [15])
After this stage, caryopses have 15.5–17% humidity and are in the best state for
milling [14]. The milling process is complex in terms of both the type and number
of operations involved and the methods used (Fig. 3.1). The process is classified into
different systems: (a) break system, which separates the endosperm from bran and
germ; (b) sizing or purification system, which separates particles according to the
presence of bran pieces; and (c) reduction system, where the large particles of
endosperm are reduced to flour [15]. First, the kernel is subjected to breaking by
means of roller mills that are made up of pairs of cast iron cylinders, each at a set
distance from each other, with corrugated surfaces and turning at differential speeds.
The breaking system has the function to open, cut and flake the grains: it separates
the endosperm from teguments and leaves bran in the form of large and flat flakes to
facilitate their removal. Grains have to be ground gradually (four to five subsequent
breaking steps), thus limiting the formation of flour and the disintegration of bran
[12]. Each breaking step is followed by sieving, carried out by plansichters or sift-
ers. They are large machines, within which numerous sieves are stacked with a
mesh granulometry suitable for the material to be sifted (Fig. 3.2). The coarse par-
ticles of endosperm are called “semolina”. Some of them, referred to as middlings,
have various degrees of attached bran layers, whereas others are clean middlings,
composed of pure endosperm. These two fractions are separated according to their
specific gravity and size. The sizing rolls, formed by smooth or slightly corrugated
rolls, detach the bran pieces attached to the middlings. These operations are imme-
diately followed by classification through plansichters. The clean middlings are sent
Fig. 3.2 Sieving or grading section: plansichters (above) and middling fractions separated by
sieving action (below) (Courtesy of Bühler AG, Switzerland)
to the reduction system, the final stage of the milling process, with the objective to
reduce the size of clean middlings to flour. It consists of a sequence of several
smooth roll mills (up to eight to ten, according to the size and the expected starch
damage) and sifters. The milling diagram comprises a number of the above steps, to
ensure that the majority of the endosperm is converted into flour and that most of the
teguments are removed as by-products.
The flour extraction yield varies between 74 and 76%. Since the bran and the
germ together represent around 20% of the weight of the wheat grain, the flour
extraction yield is lower than the theoretical value. More refined flours have lower
extraction rates, since most of the external layers of the endosperm are eliminated
with teguments. Milling of durum wheat requires different diagrams, which are
characterized by a higher number of purifiers to improve the separation of bran
particles. Nevertheless, the yield is lower (68–72%), since semolina is mostly
formed by particles larger than 250–300 mm and contains only a minimum amount
of fine particles (lower than 200 mm).
The separation of the more external layers and germ of the caryopsis inevitably
causes a marked change of the chemical composition and, consequently, of the
nutritional value of the flour. It has a lower concentration of ash, proteins, vitamins
and soluble sugars than the caryopsis and a higher starch content. This difference
depends on the efficiency of the separation of the more external layers of the cary-
opsis from the endosperm. Consequently, refined flours, corresponding to an extrac-
tion yield of approximately 75%, contain only 5% of fibre, 45% of fats, 30% of
Fig. 3.3 Composition of flour according to the extraction rate: comparison with whole kernel
(dotted line refers to wheat kernel) (Adapted from [15])
minerals and between 15 and 40% of different vitamins in comparison to the native
caryopsis (Fig. 3.3).
3.2.1.1 Milling Optimization and Innovation
During the last decade, new food safety-related regulations, for example HACCP,
ISO Standards 9001; 22000 and 22005, traceability, labelling health claim and use
of GMO, (FAO/WHO, 1997) have supported the improvement of the process con-
trol in milling and bakery industries [16, 17]. In particular, technological solutions
that not only consider the production and economic aspects, but also the hygiene
and nutritional characteristics of flour were proposed.
Cleaning
The most recent innovation proposed for this stage is the use of an optical sorter
(www.buhlergroup.com), a device that efficiently removes all types of contaminants
and foreign materials in one step. The product stream is fed inside the sorting
machine and different high-resolution cameras detect and recognize defects based
on colour, shape and other optical properties. Specific sensors and high-speed ejectors
carry out a precise ejection, enabling the accurate separation of contaminants in a

very short time.
Debranning and Pearling
Debranning technology, also referred to as pre-processing or pearling, is currently

used in rice milling and barley processing for removing hulls that have no techno-
logical and nutritional value. Although this practice is necessary in the case of cov-
ered cereals, the sequential removal of the outer bran layers prior to milling is quite
uncommon in naked grain processing.
The original debranning systems for wheat are the Tkac process and the PeriTec
process marketed by Satake Corporation. In both processes, two distinct machines
assure the bran removal by friction (kernel to kernel) and abrasion (kernel to stone)
actions [18, 19]. New debranning equipments for wheat were developed to carry out
abrasion and friction on the same machine. The Vertical Debranner VCW (Satake
Corporation; www.satake-group.com) includes two separate working chambers in
the same equipment: the upper one has an abrasive zone where rotating abrasive
rings work the grain against a peripheral slotted screen to eliminate the outer bran
layers; partially debranned grains then enter the lower chamber, where friction com-
pletes the debranning process. Applying the Tkac system to several durum wheat
varieties, Dexter et al. [20] found an increase in semolina yield and a higher semo-
lina refinement. Consequently, the colour of pasta was improved. Other interesting
results were also found at laboratory and industrial scales [21]. Because of the posi-
tive results obtained with durum wheat, attention was shifted to common wheat.
Except for the decrease of the microbial contaminations [22] and bunt infections
[23], the results are still contradictory. Currently, the pre-milling treatment of com-
mon wheat consists of a peeling process carried out with a machine (DC-Peeler
Bühler AG – www.buhlergroup.com or the DHA Vertical Debranner from Ocrim
S.p.A.—www.ocrim.it) which promotes a mild removal of the outermost layer of
the kernel (maximum 1.5–2% of the grain weight) and, at the same time, enables
one to reduce contaminations (bacteria, mycotoxins and heavy metals) by 50%.
3.2.2 Improvement of Flour Performance
The natural aptitude of wheat to be processed into varieties of baked goods is further
improved by selective breeding research. These studies mainly concern changes of
the quality/quantity of seed storage proteins. Nonetheless, the performance of wheat
during processing is sometimes different from expectations due to characteristics
that are markedly influenced by environmental and agronomic parameters [24]. The
practice of improving the technological functionality of the flours by the addition of
improvers is, therefore, significantly widespread. Among the most commonly used
improvers there are enzymes (e.g. amylase, hemicellulase, lipase and protease) and
emulsifiers (e.g. mono- and di-glycerides), which increase the volume of the dough
and the gas retention and crumb softness of baked goods. It is interesting to note that
many of these improvers are naturally present in the wheat grain but they are removed
with the by-products (bran and germ) during milling.
3.3 Other Raw Materials
3.3.1 Water
Water plays a key role both during processing and for the shelf-life and sensory
properties of baked goods. On the basis of the definition used for studying poly-
mers, the adjective “plasticizing” is often used for water. This identifies a material
incorporated into a polymer to increase workability, flexibility and extensibility [25].
The water is subjected to significant changes during processing, both in terms of
absolute quantity (total humidity) and availability (relative humidity).
The amount of water added to convert flour to dough has to ensure the hydration
of all hydrophilic components, especially proteins. The addition of this solvent
determines the radical change of the three-dimensional conformation of proteins. In
1973, following their observations with an optic microscope, Bernardin and Kasarda
described an “explosion” of flour particles when in contact with excess water, and
the rapid formation of protein strings [26]. This spontaneous rearrangement is
caused by the immediate exposure to water of the hydrophilic areas of proteins
which are rich in polar amino acids, while the more hydrophobic areas are hidden
inside. The interaction among different protein chains is ensured through the forma-
tion of disulfide bonds and through stabilization via hydrogen bonds and hydropho-
bic interactions. Gluten proteins undergo a kind of glass transition when they absorb
water, passing from the hard and glassy state to the soft and rubbery state [27]. Cuq
et al. [28] described the changes that occur during the bread making using state
diagrams and phase changes. The glass transition is the period of marked increase
in molecular mobility that involves amorphous polymers (e.g. proteins) or the amor-
phous areas of semi-crystalline polymers (e.g. starch). Amorphous polymers are in
the glass state at low temperatures and/or with low water content. An increase of
temperature or of the water content induces amorphous polymers to become soft
and viscoelastic. When the water content is higher than 15–20%, the glass transition
of proteins occurs below environmental temperature [28]. During mechanical mix-
ing, the relative mobility of the protein molecules and their high reactivity cause the
formation of intermolecular covalent links that are responsible for the formation of
the continuous and homogenous gluten network.
The level of water needed to obtain an optimal consistency of the dough is not
always easy to quantify. Overall, hydration less than 35% does not give an optimal
and homogenous hydration of gluten [29]. The absorption of water varies according to
the degree of refinement, granulometry, level of damaged starch, quantity and quality
of proteins and humidity of the flour. The hydration capacity of the flour is usually
calculated based on the farinographic absorption index (see Sect. 3.5.1.1). Although it
is very practical for routine applications, this approach does not describe the distribution
of water within the components and the competition among the hydrophilic com-
pounds. The dough is a highly complex system where numerous aqueous stages coexist
and each one is variously rich in chemical components [30]. The dough is a sophisti-
cated metastable dispersed system, where water moves from one phase to another and
where thermodynamic incompatibility among different polymers occurs [31]. The
time needed to obtain homogeneous and uniform hydration of the flour particles is
strictly related to the dimension of the particles, their hardness and vitreosity, the pres-
ence of non-starch polysaccharides and intensity of mixing.
Water is not only necessary for the formation of gluten, but it also performs a
solvent action for the other ingredients present in the formula (e.g. salt and sugars)
and it allows the enzymatic activities to take place. Another function played indi-
rectly by water is the control of the dough temperature. After mixing, ca. 45% of the
total water in dough is associated with starch, 30% with proteins and 25% with non
starch polysaccharides (pentosans) [32]. Water allows the swelling of the starch
granules during baking and their gelatinization, a key phenomenon for the physical
and nutritional properties of baked goods [33].
3.3.2 Other Ingredients: Salt, Sugar and Fats
Flour, water and leavening agents are the indispensable ingredients for making baked
goods. Often the formula requires the addition of salt, which influences the sensory
and rheological properties of the dough, and additives or improvers. The use of salt
in leavened baked products generally refers to sodium chloride. Salt is an ingredient
that is almost always present in the formulation of bread and other bakery products.
The role of salt is related to its ability to enhance the aroma of the product and to
mask off-flavours such as a bitter and metallic taste, but salt addition also strengthens
the structure of the dough. This effect on dough structure is caused by the positive
effects on both hydrogen bonds and hydrophobic interactions between protein mac-
romolecules. Salt optimizes the mixing time and the kneading, and controls the speed
of yeast fermentation [34]. In dough without salt, the high speed of CO2 production
may be responsible for the deterioration in the product structure. On the contrary,
NaCl allows the leavening step to be controlled and optimized by slowing the rate of
gas production [35]. However, this positive effect is strongly influenced by the
amount of salt added [36]. In particular, a great increase in volume was observed in
bread prepared by adding 1.5–2% of NaCl, while quantities exceeding this threshold
were associated with a strong decrease in the volume of the product.
Other ingredients such as sugar or fats are optional. The addition of sugar or fat
over a certain value markedly changes the rheological properties of the dough. The
presence of sugars influences each stage of processing: it gives more consistency to
the dough, in some cases promotes the fermentation, facilitates the browning of the
crust during baking and ensures a soft crumb during storage. Fats such as olive oil,
lard or butter are also used. If the concentration of fat is lower than 5%, the main
technological role is as a lubricant: the gluten increases the extensibility before
rupture, favouring a higher dough volume [37]. If the formulation is very rich in fat,
the dough becomes short and loses its extensibility. This effect justifies the definition
of shortening for these components. Polar lipids such as mono- and di-glycerides
stabilize the air bubbles formed during mixing and provide a crumb with a finer and
more regular alveolar structure [38]. During storage, lipids prevent the interaction
among starch macromolecules, slowing down their reorganization into ordered and
crystalline structures (retrogradation), as well as the migration of water between
starch and proteins, slowing down the phenomena of staling and aging [39].
3.3.3 Leavening Agents
Essentially three types of leavening agents are used for making baked goods: bak-
er’s yeast, chemical agents and sourdough. Since sourdough will be covered in
detail in all the other chapters, this paragraph only aims at shortly describing the
main features of the other leavening agents.
3.3.3.1 Baker’s Yeast
Baker’s yeast refers to Saccharomyces cerevisiae strains. After a brief initial respi-
ratory activity due to the oxygen dispersed in the dough, baker’s yeast ferments
glucose, fructose, maltose and sucrose from the flour to CO2 and ethanol. Nowadays,
the types of baker’s yeast available on the market have different shelf life (yeast
cream, compressed yeast, dried yeast), osmotolerance features (suitable for baked
goods with elevated levels of sugars) and activity at low temperatures (frozen dough)
(see Chap. 6). The use of baker’s yeast as a leavening agent is very often an alterna-
tive to the use of sourdough, especially for industrial bakeries. During processing,
baker’s yeast mainly determines the leavening of the dough due to the production of
CO2; it also synthesizes some volatile compounds that positively affect the flavour
and taste of baked goods.
3.3.3.2 Chemical Agents
The production of CO2 within the dough can also be obtained by a reaction between
sodium bicarbonate and an acid (e.g. tartaric acid). Chemical agents are not used for
bread making but for sweet baked goods, both dry and light (such as biscuits, sponge-
cakes, etc.). The use of chemical agents for leavening is recommended when CO2 has
to be produced rapidly in doughs rich in sugars and fats, ingredients that slow down
and/or inhibit the metabolism of the biological agents. Sodium bicarbonate lacks
toxicity, is cheap and easy to use, and it has a high solubility at room temperature.
On the basis of the rapidity of the reaction, the chemical agents are classified as: fast
or immediate, slow or delayed, and double acting powders. The former develop gas
as soon as they are introduced into the dough. The delayed acting powders determine
the formation of negligible quantities of gas during mixing, which only develops at
high temperatures during baking in the oven. The double effect powders react in part
at room temperature and in part during baking, and in this case two acids are involved,
one soluble and one insoluble. The reactivity of a chemical agent is expressed as the
“neutralization value” (g of NaOH that are neutralized by 100 g of acid salt).
3.4 Baked Goods Making Process
A very large number of baked goods are manufactured worldwide. Breads are the
most diverse and several differences are found, for instance, between those manu-
factured in the Mediterranean areas and those from the Anglo-Saxon market [40].
Overall, in the Mediterranean areas no significant quantities of sugar or other high
hydrophilic substances are added and pans are generally not used in the leavening
and baking stages.
Apart from the large variety of baked goods, the technological process may be
summarized in a sequence of operations that require long periods of time and which
have the primary objective of aerating the dough and making it porous (Table 3.2).
3.4.1 Discontinuous Processes (Straight-Dough and Sponge-

and-Dough)
Bread making, both at artisan and industrial levels, is traditionally a discontinuous

process since the various stages of mixing, leavening and baking are carried out on
limited quantities of materials and in separated facilities. Discontinuous bread-making
processes are performed using the straight-dough or the sponge-and-dough meth-
ods. Bread making with sourdough could be considered as a particular sponge-and-
dough method. Bread characteristics are influenced not only by processing but also
by flour and formula. In the straight-dough method, the fastest and easiest to man-
age, all the ingredients are mixed together simultaneously to form a dough which is
then left to rise (Table 3.3). Fermentation has to be carried out at least in two stages.
The first leavening is generally made with large quantities of dough, and for variable
times (from 30 min to 3 h), depending on the process. The primary objective of this
operation is not to get a volume increase, but to induce changes in the rheological
properties of the dough [41]. The first leavening provides a greater workability to
the dough, which achieves the capacity to maintain shape during the second leavening
(or proofing). In this stage, individual pieces of dough, corresponding to the final size,
Table 3.2 Bread-making process: aim and modifications associated with the main operations
Step/phase/operation Aim Modifications
Mixing Homogeneous distribution of Hydration and solubilization of
ingredients (including minor the water compounds
components)
Formation of uniform and Formation of soluble gluten
“coherent” structure
Inclusion of air bubbles Inclusion of air microbubbles
Leavening/proofing Increase in volume of dough Formation of gas (CO2)
Development of typical flavour Production of fermentation
metabolites important for
developing flavour and able to
change the macromolecules
solubility
Shaping Giving shape to dough Subdivision of gas bubbles and
Division of dough into final pieces inclusion of new air
Cooking Giving the product its typical aspect Increase in volume due to
evaporation of gases:
20–30% of the volume is
obtained during baking
(oven-spring)
Formation of crust and crumb
Decrease of water content Protein denaturation
Stabilization of leavened and shaped Starch gelatinization
dough
Making product appetizing and Development of flavour
digestible
Completing leavening of the dough Evaporation of water and ethanol
Cooling Product packaging Change of solubility of sugars
Hardening of fats
Table 3.3 Characteristics of bread obtained according to the nature of the flour (straight-dough
process using 100 g flour)
Bread
Specific volume
Cereal flour bread Volume (mL) Height (mm) Weight (g) (mL/g)
100% wheat flour 900 121 140 6,4
100% einkorn flour 440 74 142 3,0
70% wheat flour + 30% 485 85 148 3,3
rye flour
70% wheat flour + 30% 440 75 146 3,0
maize flour
are maintained at controlled temperature and humidity conditions for approxi-

mately 1 h until the maximum volume is reached.
With the sponge-and-dough method, the ingredients are added at different times,
during the refreshments of the dough. Using baker’s yeast, the sponge starts by mix-
ing compressed yeast with part of the flour and water (from 50 to 70% of the total
dough) required for the formula. After a leavening phase (from 3 to 4 h up to 10 h
or more), the remaining parts of flour and water are added, together with any other
ingredient required. During the maturing stage, the yeast adapts to the environment
and reaches the optimal fermentative capacities when all the ingredients are added.
The final dough is cut into pieces, shaped and left to rise again for about 1–2 h,
before being baked. The long leavening time required to get the sponge ensures an
alveolar structure with a large number of bubbles in the product, some of them of
considerable size [42]. This structure guarantees extreme lightness, as indicated by
the high specific volume and porosity of the crumb (Table 3.4).
According to the consistency of the sponge, different definitions of the process
are given. Polish, Poolish or Viennese methods use highly fluid dough. This process
was initially introduced in Poland and subsequently widespread in Vienna at the
beginning of the last century [41]. The Poolish premix is generally prepared using
50–75% of the water required by the recipe, to which an equivalent amount of flour
is added. This ratio gives a mass of low consistency that is left to ferment from 3 to
8 h, according to the quantity of baker’s yeast added. This method gives sensory
advantages and allows the formation of a delicate aroma.
Leavening with sourdough requires the use of a starter, represented by a piece of
dough from a previous batch which is fermented and stored under controlled condi-
tions of temperature and humidity. Sourdough is generally used at 5–20% of the
formula. Refreshing is of fundamental importance for dough development and
maintains the microbial species typical of the sourdough [43, 44]. The intense
acidification markedly influences the sensory and shelf-life features of the baked
goods [43]. In particular, making bread with sponge and dough methods, and espe-
cially using sour dough, ensures a higher initial lightness compared to the straight
dough method (Table 3.4). The bread is characterized by a more highly developed
and irregular alveolar structure, since the longer fermentation times determine a
slow and progressive production of CO2, accompanied by coalescence phenomena.
The bread also has a characteristic taste and smell, caused by the formation of volatile
compounds that are mainly formed during baking, following the Maillard reaction
between glucose and free amino acids, which are released during fermentation.
In Anglo-Saxon countries, and in particular in Great Britain, the most wide-
spread bread-making process is the straight dough method known as the Chorleywood
Bread Process (CBP). It was developed in the 1960s by the Flour Milling and
Baking Research Association at Chorleywood in England [38, 45]. The leavening
period is substantially reduced thanks to the high-speed mixers used for mixing the
dough (see Sect. 3.4.3.1). CBP uses an intense mechanical mixing of the dough that
lasts just a few minutes and ensures the incorporation of large quantities of air
inside the mass. The decrease of time limits the cost of the process but the special
conditions used during mixing require enriched flours and formulas with elevated
concentration of baker’s yeast (higher than 2.5–3.0%). Emulsifiers and fats with a
high fusion point are also indispensable for the oxidizing properties and to stabilize
the numerous tiny alveoli that develop from the microscopic air bubbles included
during the mixing stage. Another characteristic of this process is the reduction of the
pressure during mixing to adjust the size of the alveoli.
3.4.2 Continuous Processes
These processes were introduced in the USA during 1950. The two best-known
types are the Do-Maker process, developed in 1954, and the Amflow process, devel-
oped in 1960 by AMF Incorporated [46]. These technologies took hold in the 1970s,
and were widespread especially in the United States and Great Britain.
Contrarily to the discontinuous process working in batches, the continuous pro-
cess is characterized by a substantial reduction of time, more compact machinery
and better durability of the characteristics of the baked goods [47]. These processes
are based on the possibility of eliminating the long leavening times by using yeast
cultures or pre-ferments propagated separately without or with small quantities of
flour. The subsequent high-speed mixing, with the simultaneous addition of all the
ingredients, favours volume development even without long leavening times. As for
the Chorleywood process, the intense mechanical stress during the high-speed mix-
ing can be “supported” by the dough only if strong oxidizing improvers are added;
emulsifying lipids are also indispensable.
3.4.3 The Main Stages of the Process
3.4.3.1 Mixing
As shown in Table 3.2, during mixing ingredients are distributed and blended within
the mass, and gluten is formed. These phenomena are described as dough develop-
ment. Many variables are involved. One of these is the quantity of water added to
the flour, which may be indicated as the “level of absorption” or “hydration”. In
some processes the level of absorption does not correspond to the optimal quantity
as determined by the farinograph but it is mainly related to the handling character-
istics of the dough. Stiff dough with hydration levels between 40 and 45% (dough
humidity: 38–41%) has reduced extensibility; consequently, the baked goods have a
limited porosity with a very fine alveolar structure. Soft or slack doughs have hydra-
tion levels higher than 60% (dough humidity: about 50%). They are difficult to
handle due to their low consistency, which is responsible for the long and irregular
shape such as the Italian Ciabatta [2]; the crumb presents large alveoli, often long in
shape, which result from the coalescence of smaller bubbles.
Table 3.4 Physical characteristics of flour, dough and bread according to the bread-making
process
Specific
volume Porosity
(mLg−1) (%)
Flour 1.5 –
Dough 0.85 –
Proofed dough 0.9–1.0 –
Wheat bread
– Straight 2.5 ÷ 3.0 20 ÷ 25
and dough
– Sponge 3.0 ÷ 10.0a 25 ÷ 75a

and dough
– Sourdough 3.2 ÷ 3.7 35 ÷ 40
a
According to the parameters of the technological process
Mixing during traditional processes is carried out at least in two different times
with different speeds. In the first stage (the French frasage), the low speed lasts
about 5 min. Water is distributed among ingredients to allow the hydration of the
protein macromolecules and the development of gluten. Air bubbles are entrapped
into the mass and their sizes vary between 30 and 100 mm [48]. This phenomenon
is completed during the high-speed stage (the French malaxage and soufflage). At
the microscopic level, the sequence of events is associated with significant rheologi-
cal changes and the mass is rather wet and sticky until hydration is completed dur-
ing the cleanup stage (Fig. 3.4). Development of the dough is completed when the
mass clears away from the walls and blades of the mixer bowl and starts to crackle
in the bowl. Furthermore, stretching deforms the dough without breaking until it
becomes a semi-transparent film [47], thanks to the viscoelastic properties of the gluten.
Fig. 3.4 Raw materials and “evolution” of dough aspect during dough-making
The air included at the end of the mixing stage represents ca. 8–10% of the volume
of the dough [48]. From this point onwards, any further mixing causes irreversible
changes of the rheological properties. The over-mixed dough becomes sticky and
loses elasticity.
The Mixers
The mixers are usually classified into two categories: vertical and horizontal mixers.
The former consist of machines with rotating bowls of various capacities ranging
from 100 to 3,000 kg and more. The first machines, known as Artofex, have two
reciprocating arms whose movement, both circular and vertical rotation at moderate
speed, simulates the movement of the baker’s arms. The action performed by the
dual-arms is very delicate. Consequently, the time necessary to completely develop
the dough is long and the productivity of the machine is low. Currently, they are
almost completely substituted by spiral mixers. The mechanical action against the
dough is usually completed by a rotating central post, a device whose function is to
hold and expose the dough to the action of the spiral tool, decreasing cutting effects
with a smoothly mixing action. The different types of mixer vary depending on the
capacity and rotation speed of the bowl, the arrangement and rotation speed of the
mixing tools, the possibility of cooling down the mass via insufflation of CO2 and
the possibility of extracting the bowl to guarantee the easy movement within the
working premises.
As mentioned previously, high-speed mixers, capable of completing the process
in a few minutes and ensure the retention of large volumes of air, were developed to
increase the productivity of the bread-making process. The horizontal bowl, nor-
mally made of stainless steel, presents a single mixing shaft with several transversal
bars, whose profile varies in function of the process involved. Auxiliary equipment
includes the microprocessor controls for monitoring all mixer functions. These
devices ensure constant and uniform characteristics in all dough batches. In the
Chorleywood method, the Tweedy mixers are preferred (www.bakerperkinsgroup.
com). These high-intensity mixers have an impact mixer plate at the bottom of the
bowl. Some baffles are present at the sidewalls of the bowl to direct the dough
towards the mixer plate. The rotation speed of the mixing device may exceed
300 rpm, while the bowl remains fixed. These conditions are suitable to fully develop
dough in only 4–5 min.
Fig. 3.5 Planetary mixer and examples of mixing tools (Courtesy of Sancassiano S.p.A., Italy)
The quantity of air retained in the dough varies based on the mixer characteristics,
flour strength and the addition of specific ingredients. To obtain batters (highly aer-
ated dough for sugar- and fat-based cakes), the planetary mixers are used. Their
mixing tool is similar to a whisk (Fig. 3.5) and performs a whipping action on the
mass. The presence of emulsifiers is indispensable to stabilize this very low density
foamy mass and to produce an extremely fine grain and uniform texture.
According to the mixing tool shape and the speed applied, the time needed to
obtain a well-developed dough or a light batter varies from 30 to just a few minutes.
Consequently, the temperature of the mass remains the same or just a little higher
Fig. 3.6 Carousel system (Courtesy of Sancassiano S.p.A., Italy)
than that of the ingredients (mixers with dual arms or spirals, 20–60 rpm) or may
increase even by 10–12°C in the case of ultra-high-speed mixers, working at
500–1,000 rpm.
The mixers previously described work discontinuously. The developed dough
must be removed and the bowl emptied ready for the next batch. This system has a
limited effect on the organization of the work at artisanal level but at industrial plants
it creates serious problems. One proposed solution consisted of the carousel, a modu-
lar system which, although it did not shorten the mixing times, guaranteed the avail-
ability of a well-developed dough at set times, with standardization and an almost
continuous feeding of the machines. As shown in Fig. 3.6, the carousel (www.
sancassiano.com) is formed by a number of stations that move automatically (con-
trolled by a Programmable Logic Control, PLC), rotating and occupying different
positions at set times. It passes from the first position (raw materials are dosed into
the first bowl) to intermediate mixing and kneading positions up to the last position
where the dough is discharged and poured out into the feeding hopper of the next
machine. The time between the first and the last position corresponds to that neces-
sary to develop a mass with the desired rheology and texture characteristics.
In recent years, the companies working in this sector have put forward solutions
for further improvement of the automation and versatility of this stage of the indus-
trial process. The different stages of dough development and leavening are con-
trolled by PLC in robotized plants. A variable number of bowls are handled and
moved by a robot shuttle which takes each bowl from a parking area and subjects it
to the various work stations, on the basis of the sequence of the production cycle.
The advantages of this plant are many, including high flexibility, capacity to satisfy
all types of technological cycles (direct, indirect, etc.), possibility of feeding several
lines in parallel (even with different types of dough) and high levels of hygiene and
cleaning.
3.4.3.2 Leavening
The production of any leavened baked goods concerns the transformation of the
semi-solid mass of the dough, a kind of emulsion with a continuous phase repre-
sented by hydrated gluten that surrounds the starch granules, and a dispersed phase,
consisting of microbubbles of air, into a foam, where the continuous phase retains
considerable volumes of gas (Table 3.2).
Leavening is the stage associated with the significant expansion of the original
volume of the mass. This is possible thanks to the viscoelastic properties of the
dough, and in some cases also to the presence of emulsifiers. The number of alveoli
retained in the mass upon completion of the mixing is estimated to be between 102
and 104 per mm3 [50]. Volume expansion can be obtained using biological and
chemical leavening agents and also through the physical approach. In this latter
case, the inclusion of air follows intense mechanical actions during mixing. Mixed
leavening is also considered. Microalveoli incorporated during mixing (physical
leavening) are further expanded following the chemical leavening in the oven.
Danish pastry, used for making particular sweet products, is obtained from mixed
leavening: the dough is first biologically leavened, then formed with a lamination
process to distribute the fat in thin and alternate layers within the dough. Although
CO2 is considered the major gas responsible for the development of dough volume,
other gases and low-boiling substances may interfere with the overall volume of the
dough. For instance, ethanol is solubilized in the aqueous stage of the mixture and
forms an azeotrope with a boiling point of 78°C and water vapour.
The most obvious phenomenon associated with leavening is the volume expan-
sion. The CO2 produced is solubilized firstly in the aqueous stage of the dough.
Once saturation is reached, the gas settles in the bubbles entrapped in the dough
gradually dilating and expanding them, without any breakages. The pressure inside
the alveoli increases but the dough reacts by stretching thanks to gluten viscoelastic-
ity. The high diameter of the bubble makes it possible to balance the overpressure
that is created. The film (ca. 1 mm) created by surfactants, soluble proteins, polar
lipids, or pentosans, on the surface of the alveolus plays the principal role in this
phenomenon [45, 50, 51]. The leavened dough is therefore a foam consisting of a
semi-solid aqueous phase where gas bubbles are distributed. The coalescence of
these gas bubbles is delayed as long as the lipoprotein film is able to expand, reduc-
ing its thickness. Its breakage is associated with the merger with adjacent bubbles.
Acidification during sourdough fermentation also influences the rheological
properties of the dough. As shown by extensographic analyses, the acidification
determines the full maturation of the dough. The extensibility of the dough is
modified so that it can better support the dividing and final moulding stages. A fully
maturated dough will break clean and sharp with minimum resistance to pull [47].
The reasons for this behaviour are numerous, complex, and only partly understood.
They are variously attributed to the continuous and progressive hydration of the
proteins, to the presence of metabolites (CO2, organic acids) that determine a new
organization of gluten, and to changes of the aqueous phases where polymers are
immersed. In some cases, the rheological changes are hidden by amylase and pro-
tease activities that occur simultaneously during leavening but with opposite
effects.
Fermentation and Proofing Rooms
At the artisanal level, fermentation is carried out in chambers or cabinets (for limited
daily production) where the temperature and humidity are kept constant. The rec-
ommended temperature ranges between 27°C and 37°C and the environmental
humidity has to be above 75–80% [47]. Lower relative humidity causes the forma-
tion of a surface skin that impedes dough development during baking. Optimal tem-
perature and humidity conditions are maintained thanks to air conditioning systems.
The baked goods are usually arranged on trays located on mobile trolleys that facilitate
movement. Long fermentation times, especially in the sponge-and-dough methods,
require one to work overnight. Today, equipment called the “retarder” or “sponge
conditioner” allows the optimization of the operations by controlling the kinetic of
fermentation at low temperatures [41]. These machines are thermostatic chambers
that allow one to control and monitor the temperature (from −10°C to +35°C),
through the presence of refrigerator groups and resistors, and the relative humidity.
After mixing, the dough is placed inside the chamber where the temperature is lowered
and then slowly raised to achieve the fermentation. This optimization of the process
reduces or eliminates the night shifts, and distributes the work during the day, guar-
antying fresh baked goods also in the evening.
At the industrial level, the proofing chambers usually consist of a tunnel with
dynamic transport and automated controls of the environmental variables. The
dough is placed on trays or belts and proceeds towards the exit, thanks to catenaries.
The tunnels are sized so that the leavening time coincides with the time needed for
the dough to travel inside.
3.4.3.3 Dough Makeup Operations
As briefly described before, the two leavening stages are alternated by dividing, and
rounding and moulding operations which were previously carried out manually.
Currently, these operations are performed by automated machines with different
working capacities.
Generally, dividing is carried out within the shortest possible time on a volumet-
ric basis. The fermented dough is inserted into a chamber with a piston whose course
is directly proportional to the volume to be divided. The individual piece of dough
is cut off by a knife, then ejected from the chamber and shaped. This operation is
usually alternated with a period of rest necessary to allow the dough to recover from
physical stresses associated with compression, followed by cutting and shearing.

The working of the divider is completed by rounding and moulding the individual
pieces with different machines. Generally, three steps are carried out: the sheeting
of dough pieces into a uniform layer, the rolling into a cylinder by means of a round-
ing or curling machine, and compression to obtain the desired shape.
3.4.3.4 Baking
Baking is considered the most important stage of the entire cycle. During baking,
the dough heats up and loses humidity. Heating occurs from the outside towards the
inside, water loss occurs in the opposite direction. These two phenomena cause
multiple changes, differing in their physical, chemical and biochemical nature and
intensity, according to the temperature and the area of the dough. The change from
the foam state to the sponge state [52], and the diversification between crust and
crumb are observed.
The sequence of changes (Table 3.5) is different according to bread area. The
temperature inside the dough is always below 100°C, while on the surface it reaches
180–200°C. As soon as the leavened dough is inserted into the oven, the fermentative
activity increases [38] until microbial death occurs at temperatures higher than
50°C. Heating causes a further significant volume expansion (oven spring) of about
40% of the volume compared to the leavened dough, corresponding to an increase
of the surface area of 10% [49]. The volume occupied by gases, CO2, ethanol
vapours, water vapour, increases as the temperature increases. Starting from 70°C,
the chemical and biochemical transformations of the macromolecules stabilize the
complex. A porous network of interconnected alveoli separated from each other by
a solid matrix with very fine walls is formed [52]. During this passage, proteins and
starch achieve new properties. The gluten is denatured, completely loses its extensi-
bility and achieves elasticity. The starch swells up and gelatinizes. The intensity of
these two phenomena depends on the distance from the geometric centre of the
dough. Baking is completed when, even in the most internal part of the dough, the
temperature has reached values that promoted the structural consolidation. The tem-
perature at the centre point has to be in the range of 90–95°C to prevent collapse due
to a non-rigid structure [52].
Because of the temperature gradient that is created in the dough during baking, the
surface, which is exposed to the oven temperature from the beginning, reaches the
sponge state much more quickly than the internal part. The surface areas, therefore,
become more and more dehydrated and permeable, facilitating evaporation and the
release of the water vapour that is generated and accumulated within. Once baking is
completed, the crust has a humidity of less than 5% [2]. This value ensures friability
and crispiness. The internal crumb retains higher humidity and remains soft and light.
The complex chemical reactions that occur during baking are of marked impor-
tance for the aroma and taste of baked goods. The starch in the crust is degraded into
dextrins between 110 and 140°C. Caramelization starts at 140–150°C and contin-
ues, producing pyrodextrins, at higher temperatures. Proteins irreversibly react with
Table 3.5 Changes during baking and storage of breada
During baking During storage
Macroscopic level Molecular level Macroscopic level Molecular level
3 Technology of Baked Goods
Crust Evaporation of gases Protein/sugar interaction Loss of crispness for increase Water migration from crumb to
Progressive drying Dextrinization/caramelization in crust moisture crust macromolecules
Non enzymatic browning Maillard reaction
Crumb Water migration towards Protein coagulation Increase in hardness Starch retrogradation
crust
Structure strengthening Starch gelatinization Crumbling tendency Water migration exchange at
inter and intra macromo-
lecular degree
Water retention by non-starch Loss of typical flavour Interaction among aromatic
polysaccharides compounds/macromolecules
Change of fat structure Appearance of “stale bread” Oxidative phenomena
flavour
a
According to bread size
69
sugars in the Maillard reaction, forming a number of compounds responsible for the
colour and typical aroma of the crust [53]. If the intensity of the Maillard reaction
does not exceed certain limits, these effects contribute to the sensory properties. On
the contrary, the protein–sugar interactions lead to unavailable lysine and, in
advanced stages, cause the synthesis of toxic compounds. The International Maillard
Reaction Society site (http://imars.case.edu) provides more information on the
nutritional and sensory properties of baked goods.
The intensity of the colour of the crust is strictly related to baking temperature,
while the thickness of the crust is influenced by baking time. Baked goods undergo
a loss of weight during baking, a key step of the process. Usually, bakers aim to
obtain the highest yield according to the values of humidity which are allowed by
national regulations. In that sense, the practice of including vapour in the oven dur-
ing the initial baking is justified. The vapour condenses on the surface of the dough,
accelerates heat transfer to the dough, slows down evaporation, and decreases
weight loss [45]. As a further positive effect, the viscoelastic properties are retained
for a longer time on the surface of the dough, allowing a higher development of the
baked goods.
In the oven, the heat transfer towards the dough occurs by radiation from the
walls and by air convection. Inside the dough, the transfer occurs by conduction.
Because the temperature is usually between 200 and 230°C, the kinetic of baking is
quite slow and provides the consolidation of the internal structure without unaccept-
able scorching of the crust.
Ovens
Regardless of the characteristics of the oven, the baking floor is called the sole or
deck and the upper part of the chamber the crown.
Ancient ovens, often made of stone or bricks, had a single chamber where com-
bustion and baking occurred. This type of baking is referred to as a “direct firing
system” because the combustion gases are in contact with the dough. The initial
temperature reaches 350–400°C and decreases during baking to 160–170°C. These
ovens, today used only for special traditional or typical breads (e.g. Italian Altamura
bread or Arabic bread), were replaced by ovens with an “indirect firing system”,
where the combustion area is separated from the baking chamber and the combus-
tion gases do not come in contact with the dough but circulate in tubes above and
below the baking surface [47]. This configuration provides a higher uniformity of
heating and guarantees hygiene. The heat transfer may be improved using forced air
systems inside the chamber (ventilated ovens) or forcing the circulation of the com-
bustion fumes into tubes via a ventilator (Cyclotherm ovens). This method also
improves the heat exchange thanks to the repeated passage of the combustion fumes
within the tubes positioned above and below the conveyor. Heat is transferred to the
Fig. 3.7 Modern rack oven (Courtesy of Rational AG, Germany)
dough mainly by radiation. Several oven doors are installed, which change the flow
of the fumes towards the upper or bottom area of the chamber to optimize baking
based on the dough characteristics. Systems that provide a better heat yield, with an
energy saving that may reach 30% compared to those from the conventional thermal
cycle ovens, are also available.
In artisanal bakeries, the most common ovens have fixed decks with separate
chambers which are arranged vertically or consist of a cabinet equipped with a
rotating rack carrying trays or frames (Fig. 3.7). In the former ovens, baked goods
have to be loaded and unloaded by hand using long peels or special loading devices
(Fig. 3.8). This operation requires time and skill. More recent solutions make it pos-
sible to vary baking conditions for each chamber. In the rack ovens, baked goods are
placed on the pans or trays located on the rack (often during the leavening stage)
that is inserted into the oven and is rotated to give a better uniformity during
baking.
In industrial bakeries, baking is a continuous operation performed in a long hori-
zontal tunnel with different sections or zones, each one having its own burner and
where the temperature is variable. Shutters control the evacuation of the water
vapour which accumulates in the chamber towards extraction flues. Baking time is
determined by the speed of the belt that transports baked goods and by the length of
the oven. According to the heating system, ovens are heated by gas, fuel oil or elec-
tricity. Microwave ovens combined with traditional ovens are also proposed, but
their application is suitable only for specific industrial purposes.
Fig. 3.8 Artisanal bakery (Courtesy of Wizard Bakery Company, Germany)
3.4.4 The Production Units
3.4.4.1 Artisanal Production
The equipment of a modern artisanal baking laboratory is shown in Fig. 3.8.

Although it covers a limited surface area, the solution is highly rational as it com-
bines machineries based on the different stages of the technology process. The mix-
ing areas (Fig. 3.8a) are adjacent to the shaping areas (Fig. 3.8b), while the leavening
section (Fig. 3.8c, d) is next to the oven area (Fig. 3.8e, f) to facilitate the movement
of the dough and its introduction into the oven.
3.4.4.2 Industrial Production
At the industrial level, processing is carried out continuously. All the different stages,
which are identical to those of the artisanal process, are connected via conveyor
belts that carry the semi-finished product to the next stage of the cycle. Rational solu-
tions (see www.wpib.de/; www.itecaspa.com/; www.esmach.it/) provide long horizon-
tal systems with linear transportation to avoid bends or turns. Modern plants consist
of completely automated systems of mixing area, controlled by a robotized centre that
exclude manual work for dosage of the ingredients, mixing and movement of the
bowls to the successive stages. After cutting and shaping, the dough comes to the
continuous proofing chamber, whose horizontal or vertical development is propor-
tional to the time needed to complete this stage. Baking under a continuous belt oven
is followed by a long cooling stage, which is controlled by circular net transporters.
3.5 Quality Assessment of Dough Baking Properties
Apart from information on the composition of the raw material, a complete knowl-
edge of the changes that occur during the whole technological process is necessary
to assess the quality of baked goods. Many instruments and techniques have been
developed for this purpose.
3.5.1 Rheology and Descriptive Empirical Measurements
Rheology is the study of the flow and deformation of materials in response to the appli-
cation of mechanical force. The force is usually defined in terms of stress, the amount
of force applied per unit area, with strain being the resulting deformation. The rheo-
logical features of the dough are important throughout the bread-making process and
determine the quality of baked goods [54]. Rheological measurements are carried out
to obtain a quantitative description of the mechanical properties of the materials as well
as information related to their molecular structure and composition [54]. Usually, rheo-
logical techniques are classified based on the type of strain imposed (e.g. compression,
extension, shear, torsion) and on the relative magnitude of the imposed deformation
(e.g. small or large deformation). The main techniques used for measuring the proper-
ties of cereals are descriptive empirical techniques and fundamental measurements.
3.5.1.1 Quality Assessment of Flour and Descriptive Rheology of Dough
After mixing, subjective manual assessments of the dough were used for a long time
to indicate whether it was suitable for processing and baking. Over time, a significant
use of descriptive empirical measurements of rheological properties was observed.

Devices such as the Penetrometer, Texturometer, Consistometer, Amylograph,
Farinograph, Mixograph, Extensigraph, Alveograph and Rheofermentometer were
developed [55] (Fig. 3.9). Different users may have different requirements. For
instance, plant breeders require rapid and automated tests which use small amounts
of sample, while millers and bakers require rapid and reliable tests to assay the bak-
ing quality [56]. All the tests mimic the complex responses (deformations) in the
dough when subjected to various stresses (mixing, overpressure during leavening
and baking, etc.). Nevertheless, the behaviour of the dough matrix is non-linear
since the deformation is not proportional to the strength that determines it. For
instance, the dough appears harder and stiffer when deformed slowly [57]. Therefore,
the results from these tests provide useful information only if the nature and intensity
of the deformations are similar to those that occur during in situ processing.
According to these considerations, the definition is of imitative or empirical or
descriptive rheology [45].
Empirical rheology is divided into two main classes [57]. The first class includes
instruments that measure the viscosity variation of the dough during mixing/torsion
at room temperature (e.g. Brabender Farinograph, Micro-DoughLAB, Mixograph)
or according to a gradient of temperature (e.g. Mixolab). The Farinograph (www.
brabender.com) measures the water absorption, dough development time, stability
and softness. The Mixograph (www.national-mfg.com) determines the mixing time,
maximum resistance and tolerance [57]. However, to determine the mixing properties
of the dough, mechanical parameters such as mixer speed, mixing bowl capacity,
and mixer geometry have to be precisely controlled. The Mixolab (www.chopin.fr)
is a relatively new instrument which resembles the Farinograph and Viscoamylograph
in terms of performance. It determines in real time the torque (Nm) produced by the
dough between the two blades and, once the dough is formed, the device measures
its behaviour as a function of time, mixing development and temperature. The second
class includes equipment with devices that record and quantify the elastic character-
istics of the dough, which are correlated to the resistance that the dough opposes to
stretching that is protracted until rupture occurs. The most used are the Alveograph
(www.chopin.fr) and the Extensograph (www.brabender.com). The Extensograph
performs an extensional test where a cylindrical dough sample is clamped horizon-
tally in a cradle and stretched by a hook which is placed in the middle of the sample
and moves downwards until the rupture occurs. The Alveograph uses air pressure to
inflate a thin sheet of dough and measures the resistance to expansion and the
extensibility of the dough by providing the measurement for maximum over pressure,
average abscissa at rupture, index of swelling and deformation energy of dough.
Another interesting instrument is the Rheofermentometer (www.chopin.fr). It
enables the measurement of gas production and retention, dough permeability, and
volume and tolerance during leavening. It is used to determine the quality of the
flour, the fermentative activity, the quality of the protein network, and the activities
of additives to be selected. Through height and pressure sensors, it measures the
fermentation and development of the dough under a weight imposed on it. During
recent years, the Texture Analyzer (www.stablemicrosystems.com) has found
Fig. 3.9 Representation of

the main tests used for the
quality assessment of dough
baking properties
increasing application. The instrument provides information about a long list of

textural properties: hardness, brittleness, elasticity, cohesiveness, stickiness, gum-
miness, springiness, consistency and fracturability. Kieffer et al. [59, 60] developed
a micro-method for extension tests. The Kieffer Extensibility Rig is a type of min-
iature of the Brabender Extensograph and is used in combination with various
mechanical testing machines. The cylindrically shaped dough is clamped at both
ends and is extended uniaxially by a hook that moves upwards. The Dough Inflation
System was introduced in the early 1990s and it was developed based on the con-
cept of the Alveograph. It measures the stress and strain relationships based on the
inflation of a sheet of dough through a biaxial extension test and operates at strain
rates lower than the Alveograph. The deformations involved in biaxial extension
tests are preferred as they are more relevant to the type of deformation of the dough
around an expanding gas bubble during proofing and baking.
All the above instruments provide a great deal of information on the quality
and performance of baked goods. Nevertheless, they are purely descriptive, fre-
quently disruptive, and they depend on the type of instrument used, the size and
geometry of the test sample and the specific conditions under which the test is
carried out [45, 61].
3.5.1.2 Fundamental Measurements
The study of the effective mechanical properties of dough concerns fundamental

rheology. It requires sophisticated equipment and considerable experience. It mea-
sures absolute and not relative parameters, providing results that are independent
from the particular instrument used. A material is considered solid when it does not
change shape continuously when subjected to stress. A material is considered liquid
when it changes shape continuously when subjected even to the slightest stress.
Dough cannot be classified univocally since it behaves as solid or liquid according
to the experimental conditions, and because it shows both solid and liquid features
simultaneously. These materials are defined as viscoelastic. The majority of foods
show a linear behaviour below a certain deformation value and a non-linear behav-
iour above. When this limit is exceeded the results depend on the experimental
conditions and do not describe the fundamental characteristics of the material [62].
Therefore, the measurements should be carried out during the linear viscoelastic
interval of the material. Nevertheless, studies on doughs have also recently focused
on non-linear and time-dependent behaviour [63].
Usually, the properties of viscoelastic materials are measured by creep and
recovery, stress relaxation or dynamic oscillatory tests. These measurements are
usually made on samples placed between two plates of a rheometer. In the creep
and recovery test, an instantaneous stress is applied to the sample at rest and the
change in strain (creep) is observed over time. When the stress is released, some
recovery may be observed as the material attempts to return to its original shape. In
the stress-relaxation test, the sample is subjected to an instantaneous strain and the
stress required to maintain the deformation is observed as a function of time [62].
In the oscillatory tests, samples are subjected to deformation or stress which varies
harmonically with time. Sinuosoidal and simple shear is typical [62]. This testing
procedure is the most common dynamic method for studying the viscoelastic
behaviour of food. Results are very sensitive to the chemical composition and phys-
ical structure [62]. Using a sinusoidally oscillating deformation of known magni-
tude and frequency, the phase lag angle between stress and strain is measured and
used to calculate the elastic (storage modulus or G¢) and viscous (loss modulus or
G″) components of a complex viscosity h*.
The rheological behaviour of the dough is determined by protein–protein inter-
actions at large deformations, while starch–starch interactions dominate at small
deformations. Therefore, empirical tests correlate well with the results of the baking
test [64], as the deformation that occurs is reasonably large compared with the
deformation applied during the creep and dynamic rheological tests. In contrast,
fundamental tests provide well-defined basic rheological information (viscosity and
elasticity) and provide better defined experimental conditions of stress and strain,
which allow results to be interpreted in fundamental units. Although various types
of tests and instruments have been developed to describe dough performance during
processing, it is fair to say that no single technique could completely describe its
rheological behaviour.
3.5.2 Innovative Approaches
3.5.2.1 Image Analysis
Recently, image analysis has been introduced to evaluate the quality of foods,
including baked goods. This technique uses protocols based on image digitalization
at the macro- and micro-structural level through different systems (e.g. scanners,
video cameras and microscopes). Image analysis provides a rapid and objective
definition of the morphological and densitometrical characteristics of single objects
or complex structures (Fig. 3.10). It makes it possible to study and model the phe-
nomena that occur during processing continuously or even on-line [65, 66].
The analysis of an image requires a number of passages: (1) image acquisition in
a digital format (a pixel image); (2) image pre-processing, to improve the image
while maintaining its original dimensions; (3) image segmentation, to divide the digi-
tal image into separate, non-overlapping areas (e.g. to better distinguish the objects
from the rest of the image, such as the alveoli in a slice of bread); (4) measurement of
objects, to determine their different characteristics (size, shape, colour, texture); and
(5) classification, to identify the objects by classifying them into different classes [67].
When used for baked good processing, image analysis allows the determination of
several parameters such as the increase of volume, changes of shape, time needed to
complete the dough development, extent and distribution of the alveolar structure
during leavening, initial increase and successive contraction of volume, and gelatini-
zation of the starch and surface browning during baking [68].
Fig. 3.10 The use of the image analysis approach in the evaluation of the leavening phase of
dough
3.5.2.2 Microscopy
The macroscopic behaviour of the dough depends on its microstructure. The latter
is affected by composition, spatial arrangements of the components and types of
bond existing [69].
Microscopy is often used to determine the optimal mixing time of the dough, the
extent of the development of the gluten and the nature of the gluten matrix [70–72].
Many details are explored via electronic microscopy, both by transmission (TEM,
transmission electron microscopy) and scanning (SEM, scanning electron micros-
copy). To minimize the influence of sample preparation, atomic force microscopy
(AFM) may be used. This technique provides high-resolution images of the surface
of the starch granules [73]. Confocal laser scanning microscopy (CLSM) has
recently found application in the analysis of foods. It offers the possibility of opti-
cally dissecting the material and reconstructing the 3D image [74], and to observe
dynamic processes such as the formation and growth of air bubbles in the dough
during leavening and baking [75]. The different components of the dough may also
be simultaneously identified and located, using specific fluorescent markers.
Electronic microscopy was also used to study the rupture of the gluten network fol-
lowing freezing and thawing [76]. A very useful instrument for the observation of
frozen matrices is the cryo-SEM. This instrument shows the ultrastructure of the
starch–protein associations and the state of the gluten fibrils forming the protein
network [77].
3.5.2.3 Spectroscopy
The request for quick, reliable and easy to use methods that provide automation or
on-line applications is increasingly frequent. Near-infrared spectroscopy (NIR)
satisfies these requirements. This technique is based on the acquisition of informa-
tion from a sample via the interactions that occur between its molecules and the
electromagnetic waves in the near infrared. NIR offers the possibility of analyzing
the matrix in a non-destructive way, does not require the use of reagents, and is
highly informative. NIR spectra allow the simultaneous quantification of the various
components or information regarding the mutual relations between them [78].
Recently, the NIR technique was used as a potential on-line sensor to monitor
processing [79, 80]. To completely exploit the potential of NIR, advanced chemomet-
ric techniques are needed for the interpretation of spectral data which are arranged
in wide bands with overlapping peaks that originate from the different components
present in the matrix [78]. NIR spectroscopy is largely used to quickly determine
the chemical composition of caryopses and flours. Other studies reported its appli-
cation to determine the technological quality of the flours [81–84], to evaluate the
molecular interactions between the dough components (water-protein-starch) [85,
86], and to monitor mixing [87], leavening, and staling [78].
Recent developments concerned the acquisition of information on dough via
interactions that occur between the molecules and the electromagnetic waves in the
infrared medium (MIR, mid-infrared spectroscopy). Nuclear magnetic resonance
(NMR) was also used for baked good processing. It was applied to monitor the dis-
tribution and mobility of the water, to investigate the structure of the product and
track the staling phenomenon [88–90].
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Chapter 4
Technology of Sourdough Fermentation
and Sourdough Applications
Aldo Corsetti
4.1 Definition of Sourdough
Sourdough technology is widely used in bread making and cake production as it

confers distinctive characteristics, high sensory properties and shelf life to the
resulting products (Table 1). Sourdough is “a mixture of wheat and/or rye flour
and water, possibly with added salt, fermented by spontaneous (from flour and
environment) lactic acid bacteria and yeasts which determine its acidifying and
leavening capability. These activities are obtained and optimized through con-
secutive refreshments (or re-buildings, replenishments, backslopping)” (3–5).
The term refreshment deals with the technique by which a dough made of flour,
water and possibly other ingredients ferments spontaneously for a certain time
(possibly at a defined temperature) and it is subsequently added as an inoculum to
start the fermentation of a new mixture of flour and water (and possibly other
ingredients).
When applied for a defined interval of time such a process provides a sourdough
with constant and repeatable leavening and acidifying performances reliant on the
growth of lactic acid bacteria and yeasts that are well adapted to the environment.
After the preparation of the sourdough, the refreshment technique is aimed at main-
taining the metabolic activity of the microbial communities at all times (6).
When the sourdough is added to a mixture of water and flour to start consecutive
propagations (refreshments) to obtain the final mass or full sour to be used as the
leavening agent, it can be designated the “mother sponge” (2, 4). Generally, a sour-
dough contains a variable number of lactic acid bacteria and yeasts, ranging from
107 to 109 cfu/g and 105 to 107 cfu/g, respectively, with a ratio of about 100:1 (7).
A. Corsetti (*)
Department of Food Science, University of Teramo, Teramo, Italy

86 A. Corsetti
Table 4.1 Characteristics of sourdough bread versus baker’s yeast bread (adapted from (1, 2))
Characteristics Sourdough bread Baker’s yeast bread
pH 3.8–4.6 5.3–5.8
Lactic acid 0.4–0.8% 0.005–0.04%
Acetic acid 0.10–0.40% 0.005–0.04%
Bread volume 0.22–0.30 £0.20
Flavour Complex aroma and flavour
Staling Slow Rapid
Shelf life Good protection against microbial High sensibility to bacteria and
contaminations mould spoilage
Some nutritional Optimal phytase activity and Low phytase activity, decalcifying
aspects hydrolysis of phytic acid effect
responsible for ion (Ca2+, Fe2+,
Mg2+ etc.) binding
Free amino acid concentration Free amino acid concentration
increase similar to that of flour
Decrease of glycemic index
The main role of lactic acid bacteria (mainly obligately and facultatively
heterofermentative lactobacilli) is in the acidification process while yeasts mainly
account for the leavening of the dough by releasing CO2 (4).
4.2 Sourdough Preparation and Storage
Sourdough (mother sponge) preparation can be fulfilled through many different

protocols. The main objective is to obtain a leavening agent that contains well-
adapted resident microorganisms. Such microorganisms have to produce sufficient
CO2 to leaven the dough, and organic acids and other metabolites to provide rye
or wheat bread with good texture and sensory properties, and extended shelf life
(3). Two classical procedures, for example the French and American systems, are
discussed below.
4.2.1 The French System
Mother sponge preparation for obtaining the French “pain au levain” begins
with a quite firm wheat flour dough (dough yield, DY of 150–152, see Sect. 4.6.2)
with addition of salt and malt. This dough undergoes a first fermentation step
lasting ca. 24 h. This corresponds to the early fermentative activity of flour-
resident yeast and lactic acid bacteria, which results in a low CO2 and organic
acid release (2). The decrease of pH induces the activity of flour endogenous
proteases which together with bacterial hydrolytic enzymes act on gluten and
4 Technology of Sourdough Fermentation and Sourdough Applications 87
lead to a lower dough firmness. The second step begins with the first refreshment
which is aimed at introducing oxygen and new fermentable carbohydrates into
the mixture to stimulate microbial growth and activity. The refreshment is
obtained by adding a quantity of flour corresponding to the weight of the
previous fermented dough and a quantity of water to bring the DY to a value
(e.g. 148) lower than the previous one. This dough ferments quickly and repre-
sents the starting dough for the next refreshment. By applying such a procedure
a sourdough with a steady fermentative and leavening capability is obtained.
During the last step, each refreshment is carried out at a regular interval of time
(e.g. 7–8 h), with the aim of maintaining an equilibrium in the ratio between
microbial communities (2). According to Calvel (2), when the dough volume
increases by three to fourfold with respect to the initial dough, a new refresh-
ment should be performed. The mother sponge (levain chef), which is obtained
following the above procedure, represents the dough used to prepare the full
sour needed to leaven the bread-dough.
4.2.2 The American System
The American system relies for the mother sponge preparation on a mix of water
and wheat and/or rye flours. In contrast to the French system, the value of DY
ranges from 225 (liquid dough with a ratio water:flour of 1.25:1) to 250 (liquid
dough with a ratio water:flour of 1.5:1). These values of DY remain unaltered
throughout the consecutive refreshments (8). The time and temperature of fer-
mentation are strictly controlled during each phase. In the first step, the water-
flour mixture ferments at 32–35°C for 24 h in order to acidify the dough. At the
end of this step, the first refreshment is obtained by adding flour and water to the
previous fermented dough, without changing the value of DY. After 8 h of fer-
mentation at 32–35°C, the second refreshment is carried out and the dough fer-
ments for another 16 h. After the above procedure is applied, refreshments are
carried out every 8–16 h between which the dough is allowed to ferment at
24–27°C. With the aim of calculating the amount of water and flour to be added
at each replenishment stage a multiplicative factor of 4 is considered. Under
these conditions, the weight of the fermented dough is multiplied by 4 every
two refreshments. This allows one to determine the quantity of water and flour
to be used for the next refreshment and the ratio between the two ingredients is
maintained to 1.25:1. By using this system the value of DY remains constant
until a sourdough with a pH value of 3.6–3.8 and TTA (total titratable acidity)
of 16–20 ml NaOH/20 g of dough (as calculated following the American sys-
tem) is obtained. A last fermentation of 8 h at 24–27°C is needed to confer to the
sourdough the sensory and leavening performances of the mother sponge.
Generally, a sourdough with the above characteristics is obtained in approxi-
mately 5 days, after which it is maintained in an active state by storage at low
temperature (e.g. 4°C) and subjected to refreshment at least once a day (8).
88 A. Corsetti
4.2.3 Sourdough Storage
The liquid sourdough (DY 225–250) that is frequently used by bread manufacturers
in the United States is stored at 1–2°C, after rapid refrigeration, or at 4–5°C. It is
used to start a new fermentation within 2–3 days, without the refreshment step. In
the case of prolonged storage (10 days) one or two refreshments are needed to
activate the metabolism of the lactic acid bacteria and yeasts. A prolonged storage
of the sourdough for a few months at 4–5°C is possible when the ratio between
water and flour is reduced by adding flour at ca. 0.43:1 (30% water: 70% flour).
In this case, a firm sourdough, with DY of 143, is produced. Such a storage type
necessarily requires sourdough reactivation (at least two refreshments) before use
(8). In many artisan bread preparations, different and often empirical storage
techniques are applied. Generally, as a consequence of the daily schedule of bread
manufacture, a portion of the sourdough is refreshed at least one time before its
use. Nevertheless, by applying a separate storage protocol, part of the mother
sponge can be stored at low temperature (4–6°C) for some weeks after putting it
in a cloth bag tied with string (9). Refreshments are needed before reusing such a
sourdough in a bread-making protocol. In some cases sourdough can be frozen
and reused after refreshment.
4.3 Classification of Sourdoughs
Sourdough bread making is an ancient biotechnological process and various proto-

cols for its use are applied in many countries. On the basis of the technology applied,
sourdoughs have been grouped into three types (10), to which a fourth type, named
sponge-dough, can be added.
4.3.1 Type I Sourdough
Traditional sourdoughs whose microorganisms are kept metabolically active through

daily refreshments are included in this group. Type I sourdoughs are generally suit-
able for achieving dough leavening without addition of baker’s yeast; the dough
propagation described above for French and U.S. sourdoughs are examples of Type
I sourdoughs. Generally, a three-stage protocol is applied relying on three refresh-
ments over 24 h in order to obtain the leavened dough to bake. Each step is charac-
terized by a given DY as well as fermentation temperature and time. At the end of
the last step of fermentation the sourdough is used as the leavening agent; thus it can
be considered as a natural starter culture containing many microbial strains (11). In
wheat and/or rye flour sourdoughs, dominating strains belong to the species
Lactobacillus sanfranciscensis which can co-exist with other obligately
heterofermentative lactic acid bacteria such as L. pontis, L. brevis, L. fermentum,

L. fructivorans and with the yeasts Candida milleri, C. holmii, Saccharomyces
cerevisiae and S. exiguus (recently renamed Kazachstania exigua).
4.3.2 Type II Sourdough
Sourdoughs obtained through a unique fermentation step of 15–20 h followed by

storage for many days belong to this group. Type II sourdoughs are generally not
suitable for achieving dough leavening but are used for dough acidification, and as
dough improvers. These sourdoughs are generally liquid (DY of ca. 200) and they
are produced at the industrial level using bioreactors or tanks at a controlled tem-
perature that exceeds 30°C. Such a protocol aims at shortening the fermentation
process (12). During storage, a portion of the mature sourdough can be used as the
inoculum with the aim of acidifying the dough and enriching it with aroma and
flavour compounds which are characteristics of sourdough baked goods. On the
basis of the long fermentation time, high DY, and temperature of fermentation, lac-
tobacilli such as L. panis, L. reuteri, L. johnsonii, and L. pontis, which are resistant
to low pH, dominate these sourdoughs (10, 13). Spontaneous flour yeasts are inhib-
ited and, consequently, the leavening of the final dough is obtained by adding com-
mercial baker’s yeast.
Through a combined approach consisting of culture-dependent and culture-inde-
pendent systems, Meroth et al. (14, 15), by using an experimental model based on a
laboratory scale-fermentation, showed a different prevalence of lactic acid bacteria
and yeasts in type I and type II rye-based sourdough, started with a mixture of com-
mercially available sourdough starters and baker’s yeast. In particular, L. sanfranci-
scensis, L. mindensis and C. humilis dominated the type I sourdough. L. crispatus,
L. pontis, L. fermentum and S. cerevisiae prevailed in the type II sourdough propa-
gated at 30°C; the same sourdough propagated at 40°C showed a dominance of
L. crispatus, L. panis and L. frumenti as well as the disappearance of all the yeasts
that had been added with the starter mixture. Finally, L. johnsonii, L. reuteri and
C. krusei characterized a type II sourdough fermented at high temperature (40°C),
but produced with rye bran instead of rye flour.
4.3.3 Type III Sourdough
Type II liquid sourdoughs, which are dried/stabilized after preparation, are named
type III sourdoughs (12). They are mainly used at the industrial level as their quality
is more constant compared to type I sourdough, and they are simpler to manage and
less time consuming. On the basis of the preparation method, type III sourdoughs
are dominated by drying-resistant lactic acid bacteria such as Pediococcus pentosa-
ceus, L. plantarum and L. brevis (10). A detailed description of this type of sour-
dough and its industrial application are reported in Sect. 4.8.
90 A. Corsetti
4.3.4 Sponge Dough
The sponge dough is aimed at acclimatizing baker’s yeast (S. cerevisiae) and
improving swelling of the flour components, loaf volume, taste and flavour of the
bread and its shelf life. It is an indirect process which, from a technological and
microbiological point of view, could be considered as an intermediate procedure
between straight-dough (or a direct process in which just baker’s yeast is used to
start the fermentation) and sourdough (an indirect process in which fermentation
starts without the addition of baker’s yeast). Sponge dough is obtained in two steps:
in the first dough (pre-dough) the baker’s yeast is mixed with a part of the flour and
water of the recipe, while the second dough is obtained by adding the rest of the
flour, water and possibly other ingredients to the fully fermented pre-dough.
Depending on the type of bread, the length of pre-dough fermentation can vary from
3 to 20 h and as a consequence, various percentages of flour and yeast, besides dif-
ferent combinations of DY and temperature can be applied in this step (16, 17). As
in the case of longer fermentations lactic acid bacteria present as contaminants from
either baker’s yeast or flour grow in the dough reaching typically more than 108 cfu/g
and contribute to the overall quality of baked goods (4), the sponge dough can be
included in the category of sourdoughs.
4.4 Examples of Sourdough Applications
Once produced, the sourdough is generally submitted to various refreshments either

to activate the microbial metabolism or to increase the dough mass (Fig. 4.1).
Different types of sourdough (e.g. type I, II or III) are used for the manufacture
of various leavened baked products, which range from traditional Italian or French
wheat bread, white pan bread, rye bread, San Francisco bread to traditional cakes
famous throughout the world such as Panettone. Examples of those sourdough
applications are given below.
4.4.1 A Traditional Italian Sourdough Bread:

The Altamura Bread
The Altamura bread is the first European bread that received the PDO (Protected
Denomination of Origin) status. It is manufactured in the Apulia region (Italy) using
specific cultivars of durum wheat (Triticum durum) flour and the technology is
based on type I sourdough. The full sour is obtained by a three-stage procedure in
order to gradually increase the amount of leavened dough. At each step, water and
durum wheat flour are mixed with a previously fermented dough, which is added at
the proportion of ca. 20% based on flour weight. On the basis of the ratio between
1st refreshment –
Water fermentation at 25°C Flour
for 5 - 6 h
2nd refreshment –
for 7 - 8 h
3rd refreshment –
for 2 - 3 h
Full sour
Leavening agent for

Mother sponge stored till using bread production
for the next bread-making
Fig. 4.1 Representative flow sheet to obtain a full sour from a mother sponge using a three-step
procedure. Fermentation time and temperature given in the example can change depending on
bread-making protocol (Adapted from (1))
the ingredients that are indicated in the original recipe (100 kg of durum wheat
flour + 20 kg of sourdough + 2 kg NaCl + 60 l of water) and assuming a full sour with
a DY of 160, which contains 12.5 kg of flour, the final dough should have a DY of
162. As in many traditional productions, the time and temperature used at each stage
are not defined but variously determined based on the bread-makers experience.
4.4.2 French Bread (Pain au levain)
Although various protocols based on one or two steps are used to obtain an adequate
quantity of full sour (levain tout point), a three-stage system is usually applied to
prepare the traditional wheat flour sourdough French bread, according to the type I
92 A. Corsetti
sourdough procedure. The first step consists of the mixing of a part of the mother
sponge (levain chef) with flour and water to achieve around four times the initial
mass. This dough (DY 160) ferments for 1.5–2 h at ca. 25°C and is named levain de
première (fresh sour). It is used as a starter to obtain a dough with a DY of ca. 185,
which ferments for 7–8 h at a temperature slightly higher than the previous one.
Those parameters, including the long mixing time to oxygenate the dough, stimu-
late the growth of yeasts and the leavening capability of the levain de seconde (basic
sour). By using this dough as the starter for the third refreshment, the last dough or
levain tout point (full sour) is obtained after a short fermentation time (ca. 2 h) with
the aim of controlling the hydrolytic activities of the dough and of preserving the
gas-retaining and bread-making capabilities. The levain tout point is used at the
proportion of 25% with respect to the mass of the final dough and the dough is ready
for baking after 30 min of leavening (2).
4.4.3 Rye Sourdough Bread
As reported for French bread, the rye or wheat/rye flour sourdough bread is obtained
using single or multi-step protocols, depending on the type of bread making and
product. The “three-stage sourdough process, basic-sour overnight” requires ca.
15–20 h and, as described by Spicher and Pomeranz (17), represents a good refer-
ence model for type I sourdough application in rye-flour-based bread making. The
first stage leads to the anfrischsauer (fresh sour), which, in turn, is started by the
anstellgut (mother sponge), a commercial starter or a part (ca. 10–15% of the total
dough weight) of a full sour (vollsauer) from a previous bread making. The
anfrischsauer has a DY of 200–250 and ferments for ca. 6 h at 25–26°C. Afterwards,
it contains a high number of yeasts and it is used to start a new dough based on rye
flour and water (DY of about 160–180). After 5–8 h of overnight fermentation at
26–30°C, lactic acid bacteria grow and the grundsauer (basic sour) is obtained.
A further refreshment (3 h at 30–33°C), using the basic sour as the starter for a mix
of rye flour and water (DY 180–200), leads to the vollsauer (full sour), which has
values of pH and TTA of 4.0 and 10.5 ml NaOH 0.1 N/10 g of dough, respectively.
A part (40%) of this sourdough represents the natural starter for the final dough (DY
170), which contains rye flour and an equivalent amount of wheat flour in the case
of a rye/wheat flour bread preparation. The dough is baked after dough resting of
10 min at 28°C, followed by proofing.
4.4.4 White Pan Bread
The white pan bread is one of the most diffused breads in the United States and
represents a good example of bread that is manufactured using type II sourdough.
As reported by Kulp (8), the manufacture of this bread relies on a “one-stage system”
where just one refreshment is needed to mix the liquid full sour with the other
ingredients of the recipe. The quite soft dough (DY 175) ferments in two separate
steps: the first at room temperature (ca. 25–26°C) for 25–30 min and the second at
35°C for ca. 75–100 min. Before baking, the dough shows values of pH of 4.3–4.4
and values of TTA of 9–11 ml NaOH 0.1 N/20 g of dough. Overall, the liquid starter
(full sour) represents 37% of the final dough but different percentages can be used,
which depend on the recipe and industrial or artisan levels. In the last case, a lower
quantity of starter is used, which requires a longer fermentation period and different
working schedule. Because of the high acidity of the sourdough starter, a short mix-
ing time is required due to the increased solubility of the gluten proteins, which
decreases the time for development of the gluten network.
4.4.5 Panettone Cake
Panettone is a traditional Italian cake consumed over Christmas and famous through-
out the world. Panettone has a soft structure, with regular holes, and characteristic
flavour, which is derived from dough ingredients (water, flour, butter, sugar, eggs,
salt, and others) and processing. Both at artisan and industrial levels, the sourdough
biotechnology is traditionally based on many refreshments (e.g. type I sourdough)
and an increased concentration of sugar is added during the last steps (9, 12).
Comparable to most other type I sourdoughs, sourdough microbiota are predomi-
nantly composed of L. sanfranciscensis. During traditional manufacture, a sour-
dough with a low fermentation quotient (FQ, see Sect. 4.6.3) is used and time and
temperature of fermentation are strictly controlled. In particular, the dough tem-
perature does not exceed 30°C and the temperature of water and other ingredients
has to be not lower than 20–22°C. Various studies have attributed the prolonged
softness and shelf life of Panettone cake (it is manufactured several months before
consumption) to the presence of dextran producing Leuconostoc mesenteroides in
the sourdough, which uses sucrose as an exo-polysaccharide (EPS) precursor (12).
On the basis of such a finding a new protocol relying on type III sourdough has
recently been proposed as an alternative to the traditional manufacture (see Sect. 4.8).
A sourdough containing 25% (on dry matter) of dextran, which is stabilized by
refrigeration, pasteurization or drying, has been used to shorten the time to obtain-
ing a product with similar characteristics to the traditional Panettone cake (18).
4.4.6 San Francisco Bread
San Francisco bread is traditionally manufactured using the very famous San
Francisco sourdough, which possesses a typical microbial community, mainly
consisting of L. sanfranciscensis and S. exiguus (renamed to K. exigua). Traditional
bread making relies on the type I sourdough. Various refreshments and long
94 A. Corsetti
fermentation times at low temperature are used to increase the concentration of

acetic acid synthesized by the obligately heterofermentative L. sanfranciscensis.
Recently, a liquid San Francisco sourdough, which is stabilized through pasteuriza-
tion, has been introduced to the market. Thus, also the type III sourdough is used as
an acidifying and flavouring agent for the manufacture of the San Francisco bread
in ca. 3 h (no-time-dough process) (see Sect. 4.7) (12).
4.5 Stability of the Sourdough Microbiota During

Propagation and Use
In order to keep microorganisms active, type I sourdoughs are, generally, propa-

gated daily. Thus, problems of microbiota stability during propagation and in the
use of such a type of “biological starter” arise.
In general, it is recognized that in a sourdough ready to be used (e.g. the mother
sponge—see Sect. 4.2) for bread and other baked goods production the following
basic characteristics occur:
– The prevalence of a microbial community different from that of the flour used as
raw material (3, 19, 20);
– A stable ratio among lactic acid bacteria and yeasts (to the order of 100:1 or
higher) (7);
– A predominance of facultatively and, especially, obligately heterofermentative
lactobacilli over other lactic acid bacteria (3).
As reported by Vrancken et al. (21), laboratory sourdoughs based on wheat,

rye, or spelt that are backslopped daily, reach equilibrium through a three-step
process: (1) prevalence of sourdough-atypical lactic acid bacteria, (2) prevalence
of sourdough-typical lactic acid bacteria, and (3) prevalence of highly adapted
sourdough-typical lactic acid bacteria (21). In a recent paper dealing with the
comparative analysis of seven mature sourdoughs of type I backslopped for
80 days at artisan and laboratory levels under constant technological parameters,
Minervini et al. (22) showed that, while the cell density of presumptive lactic
acid bacteria and related biochemical features were not affected by the environ-
ment of propagation, all sourdoughs harboured a certain number of species and
strains, which were dominant throughout time and, in several cases, varied
depending on the environment of propagation (21). Moreover, besides stable
species and strains, other lactic acid bacteria temporarily contaminated the sour-
doughs with differences between artisan and laboratory levels. Interestingly, this
occasional succession of strains and species only slightly affected the kinetic of
sourdough acidification; nevertheless, it could influence the sensory properties
of the resulting leavened baked product. Overall, as a stable microbiota during
sourdough propagation and use is essential in order to obtain standard and repeat-
able final products, the problem of microbial stability in terms of species and
Fig. 4.2 Persistence of strains of Lactobacillus plantarum (DB200, --♦--; 12H1, --l--; 2MF8,
--£--; G10C3, --D--) and Lactobacillus sanfranciscensis (LS6, -£-; LS41, –n–; LS48, —◊—;
LS3, —X—) after sourdough propagation at 30°C for 6 h during ten subsequent days (Adapted
from (24) and from (23))
strain composition in type I sourdough propagated by applying different endog-

enous (e.g. type of flour, quantity of water) and exogenous (e.g. temperature/time
of fermentation) parameters should be carefully addressed, both in terms of
identification and typing of dominant and sub-dominant microorganisms (see
Sect. 4.6.1) as well as to select robust, well-adapting and competitive starter strains
(23). The robustness of sourdough lactobacilli varies depending on the species
and on the strains. While the majority of L. sanfranciscensis strains showed quite
a low robustness during daily backslopping performed at the laboratory level (24),
selected strains of L. plantarum seemed to share several phenotypic traits that
determined the capacity to outcompete the contaminating lactic acid bacterium
biota (23) (Fig. 4.2).
4.6 Methods to Evaluate the Performance of the Sourdough
Both microbiological and physico-chemical parameters are used to evaluate the

performance of a sourdough. The level of complexity is different and depends on the
purpose of the analyses. The microbiological aspect essentially deals with the assess-
ment of the community of lactic acid bacteria and yeasts, as those microorganisms are
dominant in a good quality sourdough and are generally present at the ratio of approx.
100:1 (7). Nevertheless, an array of both phenotypic and genotypic methods is neces-
sary to identify the species/strain composition of the dominant and sub-dominant
microbiota of the sourdough. An overview of those systems is given below.
96 A. Corsetti
4.6.1 Determination of the Number of Lactic Acid Bacteria

and Yeasts
The standard plate count is used to estimate the cell density of both lactic acid bacteria
and yeasts. Suitable culture media to assess the number of sourdough lactic acid bacteria
include SDB (Sour Dough Bacteria) medium, as originally described by Kline and
Sugihara (25); or MRS (de Man, Rogosa, Sharpe) medium (51), with modifications
concerning the pH and the nutrient composition, particularly to include maltose and
fructose as carbon sources and electron acceptors, respectively, and cysteine as reducing
agent (26–28); Homohiochii medium, especially recommended to enumerate obligately
heterofermentative lactic acid bacteria (29); SFM (San Francisco medium), containing
wheat or rye bran especially useful for lactic acid bacteria as well as lactobacilli (30).
To all the above media cycloheximide (100 ppm) can be added to inhibit the
growth of yeast when requested.
Yeast are generally enumerated on WL medium (31); YPDA medium (32, 33);
Sabouraud dextrose agar (34, 35); YGC agar (14); and Malt extract agar (36).
To inhibit bacterial growth, all the above media generally contain 50–100 ppm of
chloramphenicol.
4.6.2 Phenotypic and Genetic Analyses to Identify and Type

Lactic Acid Bacteria and Yeasts
Sourdough lactic acid bacteria and yeasts are generally identified using a polyphasic
approach, combining both phenotypic and genotypic assays.
Besides morphological and physiological tests, specific biochemical assays are
available to identify at species level Weissella spp., Pediococcus spp. and
Enterococcus spp. as well as the predominant sourdough lactic acid bacteria,
which are often members of the genus Lactobacillus (19). In that case, the minia-
turized commercial test (e.g. API 50 CHL, Biomerieux, France) or automated
assay (e.g. Biolog system) are frequently applied after a preliminary evaluation
based on cell morphology, Gram stain, catalase test, growth at 15°C and 45°C,
CO2 production from glucose, and NH3 release from arginine (37). Nevertheless,
the high biochemical diversity in the above-mentioned genus, means that the
identification scheme must include some chemotaxonomic analysis (e.g. SDS-
PAGE, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis of total or
cell-wall associated proteins) (38, 39) or, mainly, genotypic tests such as 16S
rRNA gene sequencing (30). Moreover, on the basis of specifically designed prim-
ers targeting species-specific sequences, many rapid identification systems rely-
ing on PCR (e.g. multiplex-PCR, Intergenic Spacer Region-based PCR) have
been recently applied to sourdough lactic acid bacteria identification (27, 40, 41).
Among genotypic systems, PCR-RAPD (Random Amplified Polymorphic DNA)
as well as RFLP (Restriction Fragment Length Polymorphism) or PFGE (Pulsed
Field Gel Electrophoresis) have been successfully applied for the evaluation of
intra-specific differences (e.g. strain typing) (38, 42, 43).
On the basis of a similar identification scheme as described above, sourdough

yeasts can be preliminarily identified by a phenotypic approach consisting of cell
and colony morphology, presence and number of spores, carbohydrates, organic
acids, alcohols and nitrite fermentation/assimilation tests using commercial kits
(e.g. ID32C, API 20C AUX, RapID Yeast Plus System, Rapid Yeast Plus), growth at
different temperatures and salt concentrations (44). As for lactic acid bacteria,
chemotaxonomy based on fatty acids, proteins and polysaccharides can be success-
fully applied to yeast identification. The genotypic approach is often based on the
sequencing of various ribosomal genes (e.g. 18S, 5,8S, 26S, 5S, and spacer frag-
ments) (45), as well as on the PCR-RFLP of the rDNA 5,8S-ITS (Internal Transcribed
Spacers) (34). As for lactic acid bacteria, yeast strain typing mainly relies on the
PCR-RAPD technique.
Both lactic acid bacteria and yeasts can be identified and monitored during sour-
dough fermentation by culture-independent systems, for example by PCR-DGGE
(Denaturing Gradient Gel Electrophoresis), generally directed toward the
amplification of specific variable regions of 16S or 26S rRNA genes of lactic acid
bacteria and yeasts, respectively. A similar approach was applied by Meroth et al.
(14, 15) to evaluate the effect of type I and type II sourdough fermentation on micro-
bial population dynamics.
4.6.3 Determination of the Physico-Chemical Parameters
The balance between the communities of lactic acid bacteria and yeasts and the
composition of the microbial communities in terms of species and strains markedly
influences the sourdough performances and the overall quality of related leavened
baked goods. The microbial community is influenced by some sourdough
characteristics and, in turn, it modifies the chemical composition and physical
parameters of the sourdough, which is reflected in the overall quality of the prod-
ucts (Fig. 4.3). An overview of the physico-chemical characteristics of the sour-
dough, which are related to its performance, is determined through the evaluation
of the following parameters:
– Dough Yield (DY)
– Dough Acidity (pH and Total Titratable Acidity, TTA)
– Fermentation Quotient (FQ).
Dough Yield (DY). The ratio between water and flour in the dough is indicated as
dough yield and it deals with the dough consistency. Considering that different
flours have different capabilities to absorb water, doughs of various consistency are
obtained having the same DY. DY is calculated as follows: DY = (flour weight + water
weight) × 100/flour weight. To consider other ingredients in the formula, the expres-
sion is modified as follows: DY = dough weight × 100/flour weight.
Overall, a firm wheat flour sourdough has a DY of 150–160. A liquid sourdough
shows values close to 200. Intermediate values of DY indicate soft dough (12). It
has been observed that both DY and temperature of fermentation markedly influence
98 A. Corsetti
Endogenous factors
Carbohydrates
Nitrogen sources
Minerals
Lipids, free fatty acids
Enzymatic activities (e.g. proteases,
amylases) Lactic acid bacteria
Raw materials/ Environment
Microbiota Product quality
composition, flavour
ratio, texture
performances shelf-life
nutritional value
Inoculum Yeasts Contaminating microbiota
Exogenous factors
Temperature
Dough Yield (aw)
Oxygen (redox potential)
Fermentation time
Number of refreshments
Fig. 4.3 Endogenous and exogenous factors impacting on sourdough characteristics and
performances as well as on the overall quality of the final product (Adapted from (4))
the aroma of the sourdough and, especially, the molar ratio between lactic and acetic
acids (FQ). Overall, more acetic acid is present in firm dough fermented at 25–30°C,
while more lactic acid is found in soft dough fermented at 35–37°C (12, 46).
Obviously, on the basis of microbial adaptation to the various environmental fac-
tors, the combination of DY and temperature during refreshments markedly
influences sourdough microbiota and its performance.
Acetic acid plays an important role by influencing many bread properties (flavour,
rope inhibition, shelf life), and its content in sourdough can be increased by fructose
addition or by aeration in the presence of heterofermentative lactobacilli (47). In
many cases, the effect of temperature on the acetic acid content of type I sourdoughs
(generally propagated at a temperature of about 25°C), can strictly depend on the
yeast activity: at low temperature, yeasts grow and hydrolyze kestose and other
fructo-oligosaccharides, with the fructose being available as electron acceptors for
a higher production of acetic acid by L. sanfranciscensis via the acetate-kinase path-
way (47). At a high temperature (more than 32°C), growth of yeasts is inhibited,
fructo-oligosaccharides remain un-hydrolyzed, and L. sanfranciscensis has less
fructose available for acetate production (48).
pH. The final pH, which ranges from 3.5 to 4.3, is usually considered as an index of
a well-developed sourdough fermentation (50). The pH of the sourdough influences
the values of pH of the final dough and bread, depending on the amount of full sour
that is used as the inoculum. In the case of a standard inoculum of 20% (referred to
the dough weight), values of pH that range from 4.7 to 5.4 are usually found in the
final dough (50).
TTA (Total Titratable Acidity). The value of TTA is a measure of the total organic
acids synthesized during sourdough fermentation. TTA is expressed as ml of NaOH
0.1 N/10 g of dough. The values of TTA range from 30 to 150 ml NaOH 0.1 N/10 g
for liquid to 40–220 NaOH 0.1 N/10 g for dried sourdoughs. Nevertheless, the
optimal value of TTA for the sourdough depends on the type of bread. Overall,
sourdough with high TTA are preferred for bread making with rye flour (48).
Fermentation Quotient (FQ). FQ indicates the molar ratio between lactic and acetic
acids during sourdough fermentation. After the determination of the concentration
of lactic and acetic acids by enzymatic or chromatographic methods, the FQ is cal-
culated as follows: FQ = (g of lactic acid in 100 g of dough/molecular weight of
lactic acid): (g of acetic acid in 100 g of dough/molecular weight of acetic acid).
This parameter is strictly related to the type of lactic acid bacteria dominating
the fermentation and markedly varies depending on the balance between homo-
and hetero-fermentative lactobacilli. In turn, this balance depends on exogenous and
endogenous factors which prevail during fermentation (e.g. fermentable sugar and
oxygen concentration, DY, time and temperature, etc.).
4.7 The Use of Stabilized Sourdoughs Versus

Active Sourdoughs
On the basis of the research progress in the field of bread-making biotechnology,

novel types of sourdough have recently been developed with the main aim of
improving the flavour of leavened baked products without using traditional protocols
of manufacture based on active sourdough. Currently, various commercial
preparations of stabilized sourdough are available on the market. They are used for
making traditional breads and are based on a mix of tailor-made aroma compounds.
Different types of stabilized sourdough are obtained starting from an accurate
selection and mix of raw materials (e.g. flour from T. aestivum or T. durum wheat, rye
or other cereals) and microbial strains. Dried sourdough is a type of stabilized prod-
uct useful for this purpose. Different drying protocols can be applied, for example
freeze-drying, spray granulation, fluidized bed drying, spray drying and drum dry-
ing, among which the latter two can be cited here as the most common for type III
sourdough production (12, 48). In both cases, the higher the DY of the starting type
II sourdough the higher the resulting TTA value of the derived type III sourdough,
which also increases due to water evaporation. In the spray-drying process, the liquid
sourdough is dried by using a warm air flux that removes water until the humidity
becomes less than 10%. In the drum-drying system, the vapour of the warmed drums
removes water from the thin-layered liquid sourdough during contact. On the basis
of various combinations of time and temperature and on the extent of the Maillard
reaction, different type III sourdoughs are obtained, which show different degrees of
caramelization or toasting and, as a consequence different flavouring activities. Since
many volatile compounds (especially acetic acid) are missing due to the evaporation,
even if to a different extent depending on the drying technique, pasteurization, cool-
ing or salting can be applied to obtain a stabilized liquid or pasty sourdough (12, 48).
With the exception of cooling, all the other stabilization systems lead to microbiota
inactivation and stop gas and/or acid production (48), giving a sourdough which can
100 A. Corsetti
be classified as type III. Especially liquid sourdough represents an advantage for

industrial applications because it can be pumped and easily dosed, while showing
constant quality (12). Generally, the use of a stabilized sourdough is quite simple. It
can be stored at room temperature for a long time (30–60 days) and directly added to
the final dough at a proportion of 5–10%. Because of the low cell density of lactic
acid bacteria and yeasts, baker’s yeast is generally added to leaven the dough for
bread production. Examples of applications of type III sourdough in the modern
bakery industry are the use of a stabilized liquid rye sourdough for rye bread produc-
tion; dextran-containing sourdough (produced by the use of selected L. mesenteroi-
des strains) stabilized by cooling, pasteurization, or drying for the production of
Panettone, rye bread and toast bread; pasteurized San Francisco sourdough in liquid
form for the production of San Francisco bread (12).
The selection of microbial strains represents the current challenge for sourdough
bread-making industries and relies on several metabolic traits which are related to
the most important flavour compounds. Overall, strains isolated from raw materials
or sourdoughs are in vitro and in situ selected and evaluated mainly based on the
kinetics of acidification, proteolytic activity and nitrogen metabolism and synthesis
of aromatic compounds. Recently, fermentation processes based on selected lactic
acid bacteria and liquid sourdough have been evaluated with the aim of improving
the biosynthesis of specific chemical compounds (49).
4.8 Use of Pure Cultures of Sourdough Starters Versus

Stabilized Sourdoughs
As for other fermented food biotechnologies (dairy, oenology, meat) the interest in
using a commercial starter culture is rapidly increasing for sourdough breadmaking.
In this context, some researches have been devoted to the use of freeze-dried or
frozen preparations of lactic acid bacteria (4, 20). Nevertheless, only a few applica-
tions of selected lactic acid bacteria starters are currently documented both at arti-
san and industrial levels. Difficulties are mainly related to the possibility of obtaining
highly concentrated lactic acid bacteria preparations as well as to the performance
of the selected lactic acid bacteria in situ. From an applicative point of view starter
cultures should be pre-cultivated in a mix of flour and water to obtain metabolically
active strains before use as an inoculum. Nevertheless, it has been found that many
strains, which were in vitro selected for interesting traits, show some difficulties in
dominating the sourdough ecosystem and overcoming the endogenous lactic acid
bacteria throughout refreshments. Recently, Siragusa et al. (24) demonstrated that, after
ten consecutive refreshments, only three out of nine L. sanfranciscensis strains,
which were in vitro selected for the optimal rate of acidification, proteolytic activity
and release of flavour compounds, were able to persist during sourdough fermenta-
tion due to the dominance of autochthonous L. plantarum strains. Thus, the capabil-
ity to adapt to the sourdough ecosystem seems to be an essential trait for selecting
strains in order to obtain a sourdough with a constant microbial composition and
performance. Currently, this seems the main limitation in the use of pure cultures as
sourdough starters and the most innovative applications mainly deal with the use of
stabilized sourdoughs.
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Bacteriol 23:130–135
Chapter 5
Taxonomy and Biodiversity of Sourdough Yeasts
and Lactic Acid Bacteria
Geert Huys, Heide-Marie Daniel, and Luc De Vuyst
5.1 Taxonomy of Sourdough Yeasts and Lactic Acid Bacteria
5.1.1 Taxonomy of Sourdough Yeasts
Yeasts are microscopic fungi that undergo typical vegetative growth by budding or
fission resulting in an unicellular appearance and a sexual reproduction without a
within or upon which the resulting spores are formed [1]. From the agro-alimentary
and scientific point of view, yeasts are among the most important eukaryotes. Yeast
species found in sourdough microbial communities share an adaptation to the
specific and stressful environment created mainly by a low pH, high carbohydrate
concentrations and high cell densities of lactic acid bacteria (LAB). Such adapta-
tions can be found in species located mostly on one branch of the evolutionary tree
G. Huys
BCCM/LMG Bacteria Collection and Laboratory for Microbiology,
Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
H.-M. Daniel
Mycothèque de l’Université catholique de Louvain (MUCL),
Earth and Life Institute, Applied Microbiology, Mycology, Université catholique de Louvain,
Croix du Sud 3 bte 6, 1348 Louvain-la-Neuve, Belgium
L. De Vuyst (*)
Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences
and Bio-engineering Sciences, Vrije Universiteit Brussel,
Pleinlaan 2, 1050 Brussels, Belgium

106 G. Huys et al.
of fungi, which accommodates the ascomyceteous yeasts. Within this branch, recog-
nized by the current classification in the phylum Ascomycota, as the subphylum
Saccharomycotina, class Saccharomycetes, order Saccharomycetales [2], sourdough
yeasts belong to different genera. The major sourdough yeasts belong to genera that
are currently placed in the family Saccharomycetaceae, although family assignment of
yeast genera is still difficult because of a lack of informative data. Basidiomycetous
yeasts and dimorphic ascomycetes, also adapted to growth in liquid environments by
unicellular growth forms, lack the fermentative abilities that are common to the
Saccharomycetales and that are important for growth under oxygen limitations. The
taxonomy of the Saccharomycetales, classically based on morphology and physiol-
ogy, is in the process of being adapted to the increasing knowledge of evolutionary
relationships reconstructed from gene sequences, in other words, a phylogenetic sys-
tem of classification is being developed [2]. This implies a number of name changes.
The new genus names have the advantage of reflecting the common genetic back-
ground of related yeast species, hereby providing an informative classification in
contrast to the former largely artificial classification.
An overview of recent name changes restricted to species that have been obtained
from sourdough is given here. The name changes most relevant to the yeasts found
in sourdough concern the genera Saccharomyces Meyen ex Reess and Pichia E.C.
Hansen emend. Kurtzman. The genus Saccharomyces has been limited to the group
of species known as Saccharomyces sensu stricto, including the type species of the
genus, Saccharomyces cerevisiae, on the basis of multiple gene sequences [3]. The
group of species formerly often addressed as Saccharomyces sensu lato has been
divided into several genera. The new genus Kazachstania is accommodating the
former Saccharomyces exiguus, Saccharomyces unisporus, and Saccharomyces
barnettii as Kazachstania exigua, Kazachstania unispora, and Kazachstania bar-
nettii, respectively. Saccharomyces kluyveri has been assigned to the new genus
Lachancea as Lachancea kluyveri. The genus Pichia has been restricted to species
closely related to the generic type species Pichia membranifaciens, including Pichia
fermentans [4]. The former genus Issatchenkia has been integrated into the newly
defined genus Pichia as its species are located on the same branch as the type spe-
cies P. membranifaciens on the phylogenetic tree based on multiple gene sequences
used for the redefinition of genera. While the species epithet of Issatchenkia occi-
dentalis has been preserved in its new name Pichia occidentalis, a complete name
change of Issatchenkia orientalis to Pichia kudriavzevii was necessary as the com-
bination Pichia orientalis had been used for a different species before. The former
species Pichia anomala and Pichia subpelliculosa were found to be only distantly
related to the generic type species P. membranifaciens and have therefore been
assigned to the newly created genus Wickerhamomyces as Wickerhamomyces anom-
alus and Wickerhamomyces subpelliculosus. A review of the taxonomic consider-
ations including the earlier genus name Hansenula has been given by Kurtzman
[5, 6]. Other, only occasionally from sourdough isolated species from the former
genus Pichia have been reassigned to new genera, while preserving their species
epithet and include Kodamaea ohmeri, Meyerozyma guilliermondii, Millerozyma
farinosa, Ogataea polymorpha, Saturnispora saitoi, and Scheffersomyces stipitis.
5 Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria 107
To determine the entities, or taxa,1 that deserve attention in the sourdough context,
about 40 original publications were reviewed and the repeatedly reported species
are listed in Table 5.1. These publications span the time from the early 1970s until
present and it is obvious that not all of them are based on identification techniques
that are currently considered as the most accurate. However, most of the six regu-
larly (seven or more reports) encountered species S. cerevisiae (syn.2 S. fructuum);
Candida humilis (syn. Candida milleri); P. kudriavzevii (syn. I. orientalis, ana-
morph Candida krusei); K. exigua (syn. S. exiguus, anamorph Candida [Torulopsis]
holmii); Torulaspora delbrueckii, anamorph Candida colliculosa; and W. anomalus
(syn. P. anomala, Hansenula anomala, anamorph Candida pelliculosa) can be dis-
tinguished from each other reasonably well by classical methods based on morphol-
ogy and physiology as used in the reviewed literature up to the late 1990s. However,
comparing studies using phenotypic identification techniques (n = 19) with those
using DNA-based techniques (n = 23), the incidence of C. humilis has increased
markedly in the DNA-based studies, probably at the cost of a decreased frequency
of detecting K. exigua. These two phylogenetically closely related species may be
mistaken for each other if using phenotypical identification methods. For example,
originally S. exiguus was reported from San Francisco sourdough [7], while some
strains from this study that were preserved in culture collections, later served for the
description of C. milleri, currently a synonym of C. humilis. The sourdough isolate
M14 reported as S. exiguus and deposited also as CBS 7901 was suggested to belong
to C. humilis after DNA-based analyses [29, 50]. The regularity with which S. cer-
evisiae, C. humilis, P. kudriavzevii, K. exigua, T. delbrueckii, and W. anomalus are
encountered in sourdough is an indication of their common association with this
substrate. This is not necessarily an exclusive association with sourdoughs and
some of the species have to be considered as generalists, able to thrive in a wide
range of environmental conditions, as for example W. anomalus [51], while a
specific ecological niche of the most frequently encountered species S. cerevisiae
has been elucidated [52]. Other species are less frequently detected in sourdough,
namely Candida glabrata; P. membranifaciens (anamorph Candida valida);
Candida parapsilosis; Candida tropicalis; Candida stellata (syn. Torulopsis
stellata); K. unispora (syn. S. unisporus, Torulopsis unisporus); Kluyveromyces
marxianus (anamorph Candida kefyr); M. guilliermondii (syn. Pichia guilliermon-
dii, anamorph Candida guilliermondii); and Saccharomyces pastorianus. Finally,
14 species have been mentioned only in single reports and may be considered as
rather transient or present fortuitously in the sourdough ecosystem (Table 5.2).
1
A taxon (plural taxa) refers to a group of individuals that are judged to form a single unit. A taxon
may or may not be given name or rank (species, genus, family, etc.). Primarily, the term serves
communication about taxonomic units without the necessity or the possibility to be more specific
about them.
2
Synonymous names and those of asporogenic forms (asexual or anamorphic forms, not producing
ascospores) are only selectively mentioned if used in the sourdough literature. It is preferable to
use the name of the sporogenous form (sexual or teleomorphic form) if it exists and if no strong
reasons require the explicit referral to the asporogenous form.
Table 5.1 Overview of original studies that analyzed the yeast diversity of sourdoughs. Only species repeatedly obtained from sourdough are listed. Numbers
108
in species columns indicate the number of obtained isolates. These numbers depend on the isolation method (i.e., one or multiple representatives of the same
colony morphotype) and are mostly of value to judge species abundance within a given study. An “x” was used if the isolate number was not specified. Multiple
x’s indicate the relative abundance of a detected species if mentioned. Studies that report on more than one sourdough may include sourdoughs of differing
yeast species composition. However, a more detailed assignment of yeast species to single sourdoughs was not possible as not all studies reported the necessary
data. Synonyms or asporogenic forms (anamorphs) are listed only if used in the cited references or if resulting from recent taxonomic changes
Wheat (W), rye (R), sorghum (S), barley (B), maize

(M), spelt (Sp), buckwheat (Bw), teff (T)
Number of analyzed sourdoughs
Phenotypic (P) or molecular (M) identification
Saccharomyces cerevisiae
syn. S. fructuum
Candida humilis
Candida milleri
syn. of C. humilis
Pichia kudriavzevii
syn. Issatchenkia orientalis
Anamorph C. krusei
Kazachstania exigua
syn. S. exiguus
Anamorph C. (Torulopsis) holmii
Torulaspora delbrueckii
Anamorph C. colliculosa
Wickerhamomyces anomalus
syn. P. anomala, Hansenula anomala
Anamorph C. pelliculosa
Candida glabrata
Pichia membranifaciens
Anamorph C. valida
Candida parapsilosis
Candida stellata
syn. T. stellata
Candida tropicalis
Kazachstania unispora
syn. S. unisporus, T. unisporus
Kluyveromyces marxianus
Anamorph C. kefyr
Meyerozyma guilliermondii
syn. P. guilliermondii
Anamorph C. guilliermondii
Saccharomyces pastorianus
Reference Country
[7] W 5 P USA 148a
[8] W 3 P Italy x
[9] R 2 P Germany 11 27 4
[10] W 3 P Spain X
[11] R 20 P Finland 11 6 2 1 1
[12] W 2 P Spain 29 6
[13] W, R 10 P Italy X x x x
d
G. Huys et al.
[14] S 2 P Sudan x
5
[15] W 12 P France 220 25 25 27

[16] B, M 10 P Morocco 21d 23b
[17] W 24 P Italy 51 13 12 1
[18] M 10 P Ghana xd x x x
[19] W 7 P Italy 25
[20] R, M, W 33 P Portugal 27 11 13 5 12 1
[21] W 10 P Italy xd xx
[22] R 5 P, M Finland 3 10c
[23] R, M n.s. P Portugal 7 1 2
[24] W 5 P, M Greece 14 25
[25] R 2 P Denmark xd
[26] W 25 P Italy 17 1 1
[27] n.s. 14 P, M Italy 33d 2 7 8
d
[28] W 1 M Italy 403
[29] R 3 P, M Germany 48 36 50 19
[30] n.s. 35 P, M Italy 118 42 7 8 3
[31] W 13 P, M Italy 58d 3 5 2 3
[32] W 8 P, M Italy 6d 6 1
[33] W 1 M Italy 6d
[34] n.s. 35 P, M Italy 102 48 8 3
[35] W 1 P, M Italy 45 36
[36] W 1 P, M Italy 13 27
[37] n.s. 7 M Italy 23 36 2
Taxonomy and Biodiversity of Sourdough Yeasts and Lactic Acid Bacteria
[38] W 10 P, M Italy 37 15
[39] W 3 M Italy x xx
[40] W, R, Sp 33 P, M Belgium 102d 2 177 62 1
[41] W 4 M Italy 60d 18 2
(continued)
109
[46]
[45]
[44]
[43]
[42]
Reference
W
W
W
W
W
Wheat (W), rye (R), sorghum (S), barley (B), maize (M), spelt
(Sp), buckwheat (Bw), teff (T)
Table 5.1 (continued)
20
10
15
9
4
Number of analyzed sourdoughs

P
Phenotypic (P) or molecular (M) identification

P, M
P, M
P, M
P, M
Italy
Italy
Greece
Country
Pakistan
Thailand
x
4d
4d
33
49d
Saccharomyces cerevisiae
syn. S. fructuum
3
Candida humilis
10
Candida milleri
syn. of C. humilis
1
1
Pichia kudriavzevii
syn. Issatchenkia orientalis
Anamorph C. krusei
2
Kazachstania exigua
syn. S. exiguus
Anamorph C. (Torulopsis) holmii
5
Torulaspora delbrueckii
163
Anamorph C. colliculosa
Wickerhamomyces anomalus
syn. P. anomala, Hansenula anomala
Anamorph C. pelliculosa
Candida glabrata
Pichia membranifaciens
Anamorph C. valida
1
Candida parapsilosis
Candida stellata
syn. T. stellata
1
Candida tropicalis
Kazachstania unispora
syn. S. unisporus, T. unisporus
Kluyveromyces marxianus
Anamorph C. kefyr
Meyerozyma guilliermondii
syn. P. guilliermondii
Anamorph C. guilliermondii
Saccharomyces pastorianus
G. Huys et al. 110
5
[47] Bw, T 2 M Ireland xd x

[48] W 7 M China 43 22 2 2 6 3 2 1
Total occurrences 38 19 14 12 8 7 4 3 2 2 2 2 2 2 2
a
Originally designated as S. exiguus, but in culture collections deposited representative strains were reassigned to C. humilis. One of them served as the type
strain for the description of the currently not taxonomically recognized population C. milleri (NRRL Y-7244, NRRL Y-7245, NRRL Y-7246, NRRL Y-7248;
[49]). C. humilis and C. milleri isolates are listed here in partially separated columns as some studies distinguished them despite their taxonomic synonymy
b
Labeled as C. milleri by the authors, ITS sequence of one strain deposited as CBS 7541 shows intermediate similarities to C. humilis and C. milleri, while its
ITS RFLP HaeIII type is similar to C. humilis
c
Retrospective identification based on the presence of type and reference strains [22]
d
No baker’s yeast added as explicitly mentioned by the authors
n.s. Not specified by the original authors
111
112 G. Huys et al.
Table 5.2 Yeast species isolated from sourdough mentioned by single reports. None of these
reports refer to these species as the sole or dominating yeast found in sourdough. Species consid-
ered as rare contaminants (e.g., Schizosaccharomyces pombe, Rhodotorula glutinis, R. mucilagi-
nosa, Endomycopsis fibulinger) or reported as unidentified were not included
Species Synonymsa Reference
Candida boidinii [12]
Candida parapsilosis [42]
Hanseniaspora uvarum [38]
Kodamaea ohmeri Pichia ohmeri [23]
Lachancea kluyveri Saccharomyces kluyveri [20]
Meyerozyma guilliermondii P. guilliermondii, anamorph C. guilliermondii [12]
Ogataea polymorpha P. polymorpha [10]
Pichia fermentans Anamorph C. lambica [31]
Pichia occidentalis Issatchenkia occidentalis [20]
Saccharomyces bayanus Saccharomyces inusitatus [7]
Saturnispora saitoi P saitoi [9]
Scheffersomyces stipitis P. stipitis [42]
Wickerhamomyces P. subpelliculosa, Hansenula subpelliculosa [10]
subpelliculosus
Yarrowia lipolytica [24]
a
Synonyms or asporogenic forms (anamorphs) were listed only if used in the cited references or if
resulting from recent taxonomic changes
The species complex C. humilis/C. milleri frequently detected in sourdough

deserves a detailed elucidation. In terms of classification it was placed in the artificial
genus Candida, because no sexual reproduction could be observed. Phylogenetically,
C. humilis/C. milleri belongs to the same group as the genus Kazachstania [3].
It has, however, not yet been taxonomically placed in this genus, as the phyloge-
netic reclassification is treating sexually reproducing taxa with priority. The species
C. humilis has been described based on a yeast strain associated with South African
bantu beer, made from kaffir corn (Sorghum caffrorum) or finger millet (Eleusine
coracana) [53]. The species C. milleri has been described to accommodate yeast
strains isolated from San Francisco sourdough fermentations and initially assigned
to S. exiguus [7, 49]. The basis for this reassignment were significantly higher gua-
nine-plus-cytosine contents in selected San Franscisco sourdough strains compared
to the type and other reference strains of S. exiguus and growth stimulation of
C. milleri by calcium pantothenate. C. humilis and C. milleri were indicated to be
conspecific based on their identical D1/D2 region large subunit (LSU) ribosomal
DNA (rDNA) sequences, a locus that in rare cases may not suffice to resolve closely
related species [54, 55]. DNA-DNA reassociation of at least 90% between the
C. milleri type strain CBS 6897 and strain CBS 2664, the type strain of T. holmii
var. acidilactici, a synonym of C. milleri, are of interest in this situation and confirms
the conspecificity of these strains [54, 56]. Strain CBS 2664 shows ten substitutions
in the internal transcribed spacer (ITS) regions of the rDNA, if compared to the type
strain of C. milleri, indicating the degree of ITS divergence within this species.
In the sourdough context, C. humilis is often distinguished from its synonym

C. milleri by targeting the ITS rDNA region [27]. This is done by restriction frag-
ment length polymorphisms (RFLPs) of the ITS generated by the restriction enzyme
HaeIII that is recognizing and cutting the nucleotide sequence GGCC. This site is
made unrecognizable by a single nucleotide change from C to T (resulting in GGCT)
in a population represented by the C. milleri type strain. As a result of this single
nucleotide substitution the RFLP analysis shows two fragments, in contrast to three
fragments for strains with the intact recognition site. A comparison of relevant pub-
licly available ITS sequences (CBS 5658: AY046174, CBS 6897: AY188851, SY13:
DQ104399; CBS 2664 and CBS 7541: yeast database at www.cbs.knaw.nl/) shows
the transitional position of some strains between the type strain sequences of
C. humilis and C. milleri, especially CBS 7541, indicative of a continuum of ITS
sequence variants between both type strains. Therefore, these two taxa might be
best considered as populations of one species. Eventual heterogeneity of the species
C. humilis, especially of applied value, should be documented and accompanied by
the deposition of the isolates in public culture collections for further study.
5.1.2 Taxonomy of Sourdough Lactic Acid Bacteria
LAB comprise a heterogeneous group of Gram-positive, nonsporulating, strictly

fermentative lactic acid-producing bacteria that play an important role in the orga-
noleptic, health-promoting, technological, and safety aspects of various fermented
food products. As a result of natural contamination through the flour or the environ-
ment or by deliberate introduction via dough ingredients, a wide taxonomic range
of LAB has also been found in sourdoughs. In sourdough environments, LAB live
in association with yeasts and are generally considered to contribute most to the
process of dough acidification, while yeasts are primarily responsible for the leav-
ening. Although also obligately homofermentative LAB have been isolated from
sourdoughs, obligately or facultatively heterofermentative LAB species have the
best potential and competitiveness to survive and grow in this particular food envi-
ronment [57, 58].
Initially, classification of LAB was based on morphology, ecology, and physio-
logical characteristics [59]. At a later stage, also chemotaxonomical properties such
as cellular fatty acid and cell wall composition were included. In LAB as well as in
many other bacterial groups, phenotypic characters are often limited in their taxo-
nomic usefulness for discrimination of closely related species and suffer from poor
interlaboratory exchangeability. As a result, differentiation of LAB solely based on
phenotypic traits is generally only considered reliable at the genus level. The intro-
duction of DNA-based techniques such as genomic mol % GC, DNA-DNA hybrid-
ization, and sequencing of ribosomal RNA (rRNA) genes has brought significant
changes to LAB taxonomy [60–62]. Especially the use of rRNA gene sequences as
evolutionary chronometers has allowed the elucidation of phylogenetic relation-
ships between LAB species. As a result, comparison of 16S rRNA gene sequences
114 G. Huys et al.
with sequences in public online databases has become a standard approach for
identification of unknown LAB isolates. However, the low evolutionary rate of
ribosomal genes may compromise differentiation between LAB species exhibiting
identical or nearly identical 16S rRNA gene sequences [63–66]. Alternatively, the
use of multiple housekeeping genes encoding essential cellular functions has been
proposed for sequence-based identification of LAB. For instance, classification of
Lactobacillus species based on sequence analysis of the housekeeping genes pheS
and rpoA proved to be highly congruent with 16S rRNA gene phylogeny [67].
The LAB species diversity associated with sourdoughs has been reviewed by
several authors in recent years [57, 58, 68–70]. On the basis of these reviews, an
updated overview of the LAB species most commonly found in fermented sour-
dough is compiled in Table 5.3. As is the case for many food ecosystems, this over-
view again highlights that lactobacilli are by far the most frequently recovered LAB
species from sourdough ecosystems. The taxonomy of the genus Lactobacillus is
extremely complex; according to the April 2011 update, at least 171 species names
have so far been proposed in this genus (www.bacterio.cict.fr/l/lactobacillus.html).
However, as several of the proposed species names have meanwhile been synony-
mized, the actual number of phylogenetically unique species is lower. In sour-
doughs, more than 55 Lactobacillus species have been identified, of which the large
majority are obligately heterofermentative (Table 5.3). Given the taxonomic com-
plexity of this genus, accurate identification of unknown Lactobacillus isolates
requires specific expertise, for example the use of methods that offer sufficient taxo-
nomic resolution and the correct interpretation of identification results by compari-
son with complete and up-to-date databases. In most of the older studies, however,
identification of lactobacilli mainly or even exclusively relied on phenotypic
approaches with limited taxonomic resolution at species level. Therefore, it is safe
to assume that some of the Lactobacillus species previously reported in sourdough
environments may have been incorrectly identified at the species or even at the
genus level. A typical example is the taxonomic situation in the Lactobacillus plan-
tarum group where discrimination between the ubiquitous sourdough bacterium
Lb. plantarum and the phylogenetically highly related Lactobacillus paraplan-
tarum and Lactobacillus pentosus may be problematic when identification methods
with insufficient taxonomic resolution are used. In this regard, Torriani and col-
leagues [71] were among the first to suggest that sequences of housekeeping genes
such as recA rather than 16S rRNA gene sequences are recommended to distin-
guish between members of this phylogenetically tight species group. Likewise,
several sourdough isolates initially assigned to Lactobacillus alimentarius may in
fact belong to the later described and closely related Lactobacillus paralimentarius
due to phenotypic misidentification [72]. Also in this case, it has been shown that
molecular fingerprint- or sequence-based methods are required to differentiate
between both species [67, 73]. In Lactobacillus rossiae, a remarkable intraspecific
heterogeneity leading to the identification of several subspecific clusters based on
pheS gene sequencing may complicate unambiguous identification of Lb. rossiae
strains [77]. Finally, nomenclatural issues may also be a cause for taxonomic con-
fusion. Corrections of originally misspelled specific epithets, such as “Lactobacillus
Table 5.3 LAB species generally associated with sourdough fermentation or found in fermented
sourdoughsa
Obligately Facultatively Obligately
heterofermentativeb heterofermentative homofermentative
Lb. acidifarinae Lb. alimentarius E. casseliflavus
Lb. brevis Lb. casei/paracasei E. durans
Lb. buchneri Lb. coleohominis E. faecalis
Lb. cellobiosus Lb. kimchi E. faecium
Lb. collinoides Lb. paralimentarius Lb. acidophilus
Lb. crustorum Lb. pentosus Lb. amylolyticus
Lb. curvatus Lb. perolens Lb. amylovorus
Lb. fermentum Lb. plantarum Lb. crispatus
Lb. fructivorans Lb. sakei Lb. delbrueckii subsp.
delbrueckii
Lb. frumenti P. acidilactici Lb. farciminis
Lb. hammesii P. dextrinicus Lb. gallinarum
Lb. hilgardii P. pentosaceus Lb. gasseri
Lb. homohiochii Lb. helveticus
Lb. kefiri Lb. johnsonii
Lb. kunkeei Lb. mindensis
Lb. lindneri Lb. nagelii
Lb. mucosae Lb. salivarius
Lb. namurensis Lc. lactis subsp. lactis
Lb. nantensis S. constellatus
Lb. nodensis S. equinus
Lb. oris S. suis
Lb. panis
Lb. parabuchneri
Lb. pontis
Lb. reuteri
Lb. rossiae
Lb. sanfranciscensis
Lb. secaliphilus
Lb. siliginis
Lb. spicheri
Lb. vaginalis
Lb. zymae
Le. citreum
Le. gelidum
Le. mesenteroides subsp.
cremoris
dextranicum
mesenteroides
W. cibaria
W. confusa
(continued)
116 G. Huys et al.

Obligately Facultatively Obligately
heterofermentativeb heterofermentative homofermentative
W. hellenica
W . kandleri
W. paramesenteroides
W. viridescens
E. Enterococcus, Lb. Lactobacillus, Lc. Lactococcus, Le. Leuconostoc, P. Pediococcus,
S. Streptococcus, W. Weissella
a
Data compiled from [57, 58, 68, 70]
b
Classification in glucose fermentation types according to Felis and Dellaglio [59]
sanfrancisco” [75] (now Lactobacillus sanfranciscensis) and “Lactobacillus rossii”

[76] (now Lb. rossiae), and the synonymization of species, such as the recognition
of Lactobacillus suntoryeus as a synonym of Lactobacillus helveticus [77], can take
a while to be introduced in subsequent taxonomic literature.
Triggered by the introduction of molecular DNA-based taxonomic methods in
sourdough microbiology and the growing number of 16S rRNA gene sequences in
public databases, in recent years many new Lactobacillus species have been
described which were first isolated from a sourdough environment. Since 2000, 13
new Lactobacillus species originally isolated from sourdoughs have been proposed,
i.e., Lactobacillus frumenti [78], Lactobacillus mindensis [79], Lactobacillus spich-
eri [80], Lactobacillus acidifarinae [81], Lactobacillus zymae [81], Lactobacillus
hammesii [82], Lb. rossiae [76], Lactobacillus siliginis [83], Lactobacillus nanten-
sis [84], Lactobacillus secaliphilus [85], Lactobacillus crustorum [86], Lactobacillus
namurensis [87], and Lactobacillus nodensis [88] (Table 5.3). However, many of
these species have only been reported once or are rarely isolated from this type of
fermented food, and are represented by only a few strains. Therefore, it is not clear
which of these species are really typical for sourdough environments and if so, what
their geographical distribution is. In fact, only a few Lactobacillus species such as
the obligately heterofermentative Lb. sanfranciscensis and the facultatively hetero-
fermentative Lb. paralimentarius seem to be optimally adapted to this specific envi-
ronment and are rarely isolated from other sources. Other heterofermentative species
such as Lactobacillus brevis and the facultatively heterofermentative Lb. plantarum
are also frequently isolated from fermented sourdoughs, but have also been found
in many other food and nonfood environments [69].
Although the LAB microbiota of sourdoughs is clearly dominated by lactoba-
cilli, other less predominant or subdominant LAB species may also be found,
including members of the genera Weissella, Pediococcus, Leuconostoc, Lactococcus,
Enterococcus and Streptococcus (Table 5.3). Of these, specific species of Weissella,
Pediococcus, and Leuconostoc are particularly well adapted to survive and grow in
plant-derived materials [57, 89]. The taxonomy of the latter three genera is much
less complex than in Lactobacillus, and their presence in sourdoughs is restricted to
only a few species. Weissellas are obligately heterofermentative LAB of which a
number of species produce dextran, the best-documented exopolysaccharide formed

by heterofermentative LAB. In sourdoughs, the dextran-producing species Weissella
cibaria and Weissella confusa are most frequently found. Both taxa are positioned
together on one of the four phylogenetic branches in this genus based on 16S rRNA
gene analyses [90], but can be differentiated using restriction analysis of the
amplified 16S rDNA [91], randomly amplified polymorphic DNA-PCR (RAPD-
PCR) [92], and PCR targeting the ribosomal ITS [26]. Within the facultatively het-
erofermentative pediococci, the species Pediococcus acidilactici and Pediococcus
pentosaceus are most commonly found in sourdoughs. Differentiation between
these two biochemically and phylogenetically related species can be achieved by
fingerprinting methods such as ribotyping [93, 94], restriction analysis of the
amplified 16S rDNA (16S-ARDRA) [95], RAPD-PCR [96, 97], and by sequence
analyses of the 16S rRNA gene, the ribosomal ITS regions and the heat-shock pro-
tein 60 gene [98]. In the obligately heterofermentative genus Leuconostoc, the
majority of sourdough isolates so far identified belong to Leuconostoc mesenteroi-
des and Leuconostoc citreum. In the former species, further taxonomic distinction is
made at subspecies level between Le. mesenteroides subsp. mesenteroides, Le. mes-
enteroides subsp. dextranicum, and Le. mesenteroides subsp. cremoris (www.bacte-
rio.cict.fr/l/leuconostoc.html). The three subspecies can be separated by RAPD-PCR
fingerprinting [99].
5.2 Microbial Species Diversity of Sourdoughs
5.2.1 Influence of Geography
5.2.1.1 The Origin of Sourdough
Historically, sourdough production started as a conditio sine qua non to process

cereals for the production of baked goods [100]. Indeed, thousands of years ago the
first bread production must have been based on spontaneous wild lactic acid fer-
mentation whether or not associated with yeasts and with little or no leavening.
Leavening could not have been very pronounced because of the use of barley
(Hordeum vulgare) and ancient grains [such as spelt (Triticum aestivum subsp.
spelta), emmer (Triticum turgidum subsp. dicoccum), and kamut (T. turgidum subsp.
turanicum)] in early times and no addition of yeast for leavening. This form of flat
(sour) bread production is still daily practice in many countries of the world, in
particular in African countries and the Middle East. Leavening must have been an
accidental discovery when yeasts from the air or the flour were allowed to ferment
the cereal dough mixture extensively. However, it was only from the late nineteenth
century onwards that yeast starter cultures were introduced for bread production
from wheat (T. aestivum and Triticum durum) flour, first by using brewing yeasts as
remnants of beer production followed by intentionally produced commercial bak-
ers’ yeast, S. cerevisiae. Consequently, sourdough bread must have been consumed
118 G. Huys et al.
for a long time. Also afterwards, bread production in countries relying on cereals
other than wheat such as rye (Secale cereale) had still to be supported by lactic acid
fermentation. Rye bread baking requires dough acidification to inhibit the abundant
a-amylase in the rye flour and to make rye starch and pentosans more water-retain-
ing to form a good dough texture since not enough gluten is present in rye. Hence,
various (rye) breads from Germany, Central European countries, and Scandinavia
are based on sourdough. In the USA, sourdough was the main base of bread supply
in Northern California during the California Gold Rush, because of the easy way to
store it and to keep it active for daily bread production. Today, San Francisco sour-
dough bread is commercially produced in the San Francisco area and it remains a
part of the culture of the San Francisco bay area. In the early 1970s, the responsible
sourdough bacterium was identified as Lb. sanfrancisco, now Lb. sanfranciscensis,
named according to the area where it was discovered [75]. Notice that this LAB
species is actually identical to Lb. brevis var. lindneri (now Lb. sanfranciscensis),
which was found to be responsible for various sourdough breads produced in Europe
[17]. Also, it was shown that these sourdough LAB species occur in a stable asso-
ciation with the yeasts C. humilis (syn. C. milleri) and K. exigua (syn. S. exiguus),
respectively [7, 101]. Nowadays, sourdough is used for its technological (dough
processing and bread texture, flavor, and shelf life) and nutritional effects [57, 58,
69, 102, 103]. Moreover, sourdough products are appreciated for their traditional
value, gastronomic quality, and natural and healthy status [104].
5.2.1.2 The Origin of Sourdough Variation
Thanks to the fact that craftsmanship has determined bakery practice for a long
time, a huge variety of bakery products, in particular those based on sourdough,
exists, which may differ considerably from region to region. Most of these products,
including breads, cakes, snacks, and pizzas, originate from very old traditions. For
instance, in Italy, numerous different types of sourdough breads exist, often called
according to the name of the region, such as Altamura bread and Pugliese bread
[101]. Also, seasonal varieties exist, which are traditionally produced on the occa-
sion of religious festivities. For instance, Panettone cake in Milan and Pandoro in
Verona are made for Christmas, while Colomba is a Milanese cake made for Easter.
Alternatively, sourdough-based products such as crackers, French baguettes, and
Italian ciabattas are much more common, although the original sourdough recipe
has been replaced by the faster growing bakers’ yeast that is in only few cases
allowed to ferment longer to enable contaminating LAB to develop for flavor for-
mation (pre-doughs or type 0 sourdoughs). Fortunately, numerous bakery sour-
doughs have been kept alive for tens of years through backslopping procedures, i.e.,
repeated cyclic re-inoculation of a new batch of flour and water from a previous one
with a so-called “sour” during refreshment of the flour-water mixture by the baker,
thereby assuring the quality of the baked goods produced thereof. It turned out that
these traditional sourdoughs harbor a mixture of distinctive yeast and LAB strains,
which may be held responsible for the typical organoleptic quality of the breads
made thereof, as backslopping results in a prevalence of the best-adapted strains

[57, 68, 69]. This diversity of natural sourdough starters likely accounts for the
variety of artisan sourdoughs produced by bakeries, whether or not typical for a
certain geographical region. Alternatively, sourdough starter cultures, comprised
of one or more defined strains, are commercially available now. In addition to
Lb. sanfranciscensis strains, commercially available strains of other LAB species
include Lb. brevis, Lactobacillus delbrueckii, Lactobacillus fermentum, and
Lb. plantarum, albeit that not all strains in use are competitive enough to dominate
the sourdough fermentation processes that have to be started up [69]. These starter
cultures are used for rapid acidification of the raw materials and flavor formation
upon fermentation. Also, industrial manufacturers produce dried sourdough pow-
ders that are used as nonliving flavor ingredients in industrial bread production.
5.2.1.3 Region-Specific Sourdoughs and Their Associated Microbiota
Whereas it was initially thought that a relationship could be seen between the presence
of certain LAB species and the geographical origin of a particular sourdough, it turned
out through systematic and detailed taxonomic investigations that the species diversity
of both LAB and yeasts of local sourdoughs has nothing to do with the geography of
the sourdough production process (Tables 5.1 and 5.4; 57, 68, 69). For instance, the
typical sourdough bacterium of San Francisco sourdough bread of the San Francisco
bay area, Lb. sanfranciscensis, has been found in various wheat sourdoughs throughout
Europe, and hence its (unique) presence should be ascribed to other factors, which are
mainly based on the fermentation technology and practical conditions applied [121,
152–154]. In various countries, such as in Italy, several studies have been focused on
region-specific sourdoughs (Table 5.4). However, no clear-cut relationship could be
shown between for the region typical sourdoughs and their associated microbiota.
In contrast, Italian sourdoughs harbor simple to very complex communities of LAB
species depending on the final products examined, among which Lb. brevis, Lb. (par)
alimentarius, Lb. plantarum, Lb. sanfranciscensis, Lb. fermentum, P. pentosaceus, and
W. confusa are widespread. Similarly, Belgian bakery sourdoughs have been analyzed
extensively and are characterized by LAB consortia of Lb. brevis, Lb. hammesii,
Lb. nantensis, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. sanfranciscensis, and/or
P. pentosaceus [105, 106, 155]. Sourdoughs with both stable large and stable restricted
species diversities may occur [106]. Also, Lb. rossiae seems to have a wide distribution
in sourdoughs, as has been shown through its isolation from sourdoughs in Central and
Southern Italy, Belgium, and elsewhere [74, 105, 106, 156–158]. LAB species are
responsible for the acidification of the dough and contribute to flavor formation. Besides
LAB species, a large variety of yeast species are found in sourdough ecosystems
(Tables 5.1 and 5.2). S. cerevisiae has been found in almost every sourdough study (38
out of 42 reviewed) regardless as to whether or not bakers’ yeast is added (14 studies
mention S. cerevisiae, although they indicate that no bakers’ yeast was added). A single
sourdough usually harbors only one or two yeast species at a given time, among which
C. humilis (and K. exigua) and P. kudriavzevii occur most frequently [40]. Yeasts are
responsible for the leavening of the dough and also contribute to flavor formation.
120
Table 5.4 Nonexhaustive overview of the species diversity of LAB in sourdoughs made from various flours and of different geographical origins. The method
of identification of the LAB species is indicated
Cereal flour type
Method of
Country identification LAB species reported Reference(s)
Belgium Wheat Lb. acidifarinae, Lb. brevis, Lb. buchneri, Lb. crustorum, Lb. hammesii, Lb. [86, 87, 105,
Molecular helveticus, Lb. namurensis, Lb. nantensis, Lb. parabuchneri, Lb. paracasei, 106]
Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. rossiae, Lb. sakei, Lb.
sanfranciscensis, Lb. spicheri, Leuc. mesenteroides, P. pentosaceus, W.
cibaria, W. confusa
Rye Lb. brevis, Lb. hammesii, Lb. nantensis, Lb. paralimentarius, Lb. plantarum [105, 106]
Molecular
Wheat-rye E. mundtii, Lb. brevis, Lb. curvatus, Lb. fermentum, Lb. paracasei, Lb. [105, 106]
Molecular paralimentarius, Lb. plantarum, Lb. pontis, Lb. rossiae, Lb. sanfranciscen-
sis, P. acidilactici, P. pentosaceus, W. confusa
Spelt Lb. brevis, Lb. curvatus, Lb. hammesii, Lb. nantensis, Lb. paralimentarius, Lb. [105, 106]
Molecular plantarum, Lb. pontis, Lb. rossiae, Lb. sakei, Lb. sanfranciscensis, P.
pentosaceus, W. cibaria
Wheat (laboratory) Lb. fermentum, Lb. plantarum [107]
Molecular
Spelt (laboratory) Lb. brevis, Lb. paraplantarum, Lb. plantarum, Lb. rossiae [107]
Molecular
Wheat (laboratory) Lb. curvatus, Lb. fermentum, Lb. plantarum, P. pentosaceus [108]
Molecular + microarray
Spelt (laboratory) Lb. fermentum, Lb. plantarum, P. pentosaceus, W. confusa [108]
Molecular + microarray
Rye (laboratory) Lb. brevis, Lb. fermentum, Lb. plantarum, P. pentosaceus [109]
G. Huys et al.
Molecular
Cereal flour type
Method of
5
China Wheat Lb. brevis, Lb. crustorum, curvatus, Lb. fermentum, Lb. guizhouensis, Lb. [48]
Molecular helveticus, Lb. mindensis, Lb. paralimentarius, Lb. plantarum, Lb. rossiae,
Lb. sanfranciscensis, Lb. zeae, Lc. lactis, Leuc. citreum, W. cibaria, W.
confusa
Denmark Rye Lb. amylovorus, Lb. panis, Lb. reuteri [25]
Phenotypical
Ethiopia Teff (enjera) E. faecalis, Lb. brevis, Lb. fermentum, Lb. plantarum, Leuc. mesenteroides, P. [110]
Phenotypical cerevisiae
Finland Rye Lb. acidophilus, Lb. casei, Lb. plantarum [11]
Phenotypical
France Wheat Lb. acidophilus, Lb. brevis, Lb. casei, Lb. curvatus, Lb. delbrueckii subsp. [111]
Phenotypical delbrueckii, Lb. plantarum, Leuc. mesenteroides subsp. dextranicum, Leuc.
mesenteroides subsp. mesenteroides, P. pentosaceus
Wheat Lb. brevis, Lb. curvatus, Lb. plantarum, Lb. rhamnosus, Leuc. mesenteroides, P. [112]
Phenotypical pentosaceus
Wheat Lb. frumenti, Lb. hammesii, Lb. nantensis, Lb. panis, Lb. paralimentarius, Lb. [113]
Molecular pontis, Lb. sanfranciscensis, Lb. spicheri, Leuc. mesenteroides subsp.
mesenteroides
Wheat Lb. frumenti, Lb. hammesii, Lb. nantensis, Lb.panis, Lb. paralimentarius, Lb. [114]
Molecular sanfranciscensis, Lb. spicheri, Leuc. mensenteroides subsp. mensenteroides
Wheat E. hirae, Lb. brevis, Lb. curvatus, Lb. paracasei, Lb. paraplantarum, Lb. [115]
Molecular pentosus, Lb. plantarum, Lb. sakei, Lb. sanfranciscensis, Lc. lactis, Leuc.
citreum, Leuc. mesenteroides, P. pentosaceus, W. cibaria, W. confusa
Germany Wheat Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckii, Lb. fermentum, Lb. [116]
Phenotypical plantarums
(continued)
121
122

Cereal flour type
Method of
Rye Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. buchneri, Lb.casei, Lb. [9, 117]
Phenotypical farciminis, Lb. fermentum, Lb. fructivorans, Lb. plantarum, Lb.
sanfranciscensis
Rye – use of a starter Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. buchneri, Lb. casei, Lb. [118]
Phenotypical farciminis, Lb. fermentum, Lb. fructivorans, Lb. plantarum, Lb. sanfrancis-
censis, Pediococcus spp.
Wheat (Panettone, Lb. brevis, Lb. casei, Lb. farciminis, Lb. hilgardii, Lb. homohiochii, Lb. [119]
wheat bread from plantarum (spontaneous)
Germany, Italy, Lb. brevis, Lb. hilgardii, Lb. sanfranciscensis,W. viridescens (masa madre)
Sweden, and
Switzerland)
Phenotypical
Rye Lb. amylovorus, Lb. frumenti, Lb. pontis/panis, Lb. reuteri (type II) [120]
Molecular
Rye (bran) – use of a Lb. mindensis, Lb. sanfranciscensis (rye, type I) [29]
starter
Molecular Lb. crispatus, Lb. fermentum, Lb. frumenti, Lb. panis, Lb. pontis (rye, type II)
Lb. johnsonii, Lb. reuteri (rye bran, type II)
Rice Lb. paracasei, Lb. paralimentarius, Lb. spicheri [80]
Phenotypic + molecular
Wheat E. faecium, Lb. casei, Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis, P. [121]
Molecular pentosaceus, W. confusa
Rye Lb. pontis, Lb. sanfranciscensis [121]
Molecular
G. Huys et al.
Cereal flour type
Method of
5
Amaranth (from India, Lb. plantarum, Lb. sakei, P. pentosaceus [122]

Peru, Mexico,
Germany)
Molecular
Cereals and Lb. fermentum, Lb. helveticus, Lb. paralimentarius, Lb. plantarum, Lb. pontis, [123]
pseudocereals Lb. spicheri
Molecular
Greece Wheat Lb. brevis, Lb. paralimentarius, Lb. sanfranciscensis, Lb. zymae, W. cibaria [72]
Phenotypical + molecu-
lar
Italy Wheat (panettone) Lb. brevis, Lb. plantarum [8]
Phenotypical
Wheat (panettone, Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis, Leuc. mesenteroides, [13]
brioche) Pediococcus spp.
Phenotypical
Wheat (Umbria) Lb. farciminis, Lb. plantarum, Lb. sanfranciscensis [17]
Phenotypical
Pizza from Naples Lb. plantarum, Lb. sakei, Leuc. gelidum, Leuc. mesenteroides [124]
Phenotypical
Wheat (Molise, Lb. sanfranciscensis [125, 126]
Umbria, Venise)
Molecular
Mother sponges Lb. sanfranciscensis [21]
(Lombardy)
Molecular
Wheat (Apulia) Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. delbrueckii subsp. delbrueckii, [26]
Molecular Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis, Lc. lactis subsp. lactis,
Leuc. citreum, W. confusa
Wheat (Southern Italy) Lb. alimentarius, Lb. brevis, Lb. farciminis, Lb. fermentum, Lb. fructivorans, Lb. [127]
Molecular planta rum, Lb. sanfranciscensis, W. confusa
123
(continued)
124
Cereal flour type

Method of
Wheat (Molise) Lb. arizonensis/plantarum, Lb. brevis, Lb. kimchii/paralimentarius, Lb. [33]
Molecular paraplantarum, Lb. sanfranciscensis
Wheat (Sicily) Lb. casei, Lb. kimchii/alimentarius, Lb. plantarum, Lb. sanfranciscensis [128]
Molecular
Wheat (Molise) Lb. brevis, Lb. fermentum, Lb. plantarum, Lb. sanfranciscensis [129]
Molecular
Wheat (Altamura, Lb. brevis, Lb. casei, Lb. paracasei, Lb. plantarum, Leuc. mesenteroides [130]
Apulia)
Phenotypical + molecu-
lar
Wheat (Sardinia) Lb. brevis, Lb. pentosus, Lb. plantarum, Lb. sanfranciscensis, W. confusa [131]
Molecular
Wheat (Abruzzo) Lb. alimentarius, Lb. brevis, Lb. farciminis, Lb. fermentum, Lb. fructivorans, Lb. [133]
Physiological + molec- frumenti, Lb. hilgardii, Lb. mindensis, Lb. panis, Lb. paralimentarius, Lb.
ular paraplantarum, Lb. pentosus, Lb. plantarum, Lb. pontis, Lb. rossiae, Lb.
sanfranciscensis
Wheat (Abruzzo) E. faecium, P. pentosaceus [133]
Molecular
Wheat (Panettone from Lb. brevis, Lb. farciminis, Lb. plantarum, Lb. sanfranciscensis, Leuc. [39]
Central Italy) mesenteroides
Molecular
Wheat (Cornetto, Lb. curvatus, Lb. paraplantarum, Lb. pentosus, Lb. plantarum, Leuc. [134]
Basilicata) mensenteroides, W. cibaria
Wheat – use of a Lb. brevis, Lb. paralimentarius, Lb. plantarum, Lb. sanfranciscensis, Lc. lactis, [41]
starter Leuc. durionis, Leuc. fructosus, P. pentosaceus, W. cibaria
G. Huys et al.
Cereal flour type
Method of
5
Wheat (Marche) Lb. brevis, Lb. casei, Lb. curvatus, Lb. fermentum, Lb. paracasei, Lb. parali- [43]
Phenotypic + molecular mentarius, Lb. plantarum, Lb. rhamnosus, Lb. sakei, Lb. sanfranciscensis,
Leuc. citreum, Leuc. pseudomesenteroides, W. cibaria, W. confusa
Wheat – use of a Lb. paralimentarius, Lb. plantarum, Lb. rossiae, Lb. sanfranciscensis, Lc. lactis [135]
starter subsp. lactis, P. pentosaceus, W. cibaria, W. cibaria/confusa
Molecular
Barley – use of a Lb. alimentarius, Lb. brevis, Lb. paralimentarius, Lb. plantarum [136]
starter
Molecular
Emmer Lb. plantarum, Lb. rossiae, W. confusa [137]
Molecular
Spelt Lb. brevis, Lb. curvatus, Lb. plantarum, Lb. sanfranciscensis, P. pentosaceus/ [137]
Molecular acidilactici, W. confusa
Wheat Lb. plantarum, Lb. rossiae, P. pentosaceus [138]
Molecular
Wheat germ Lb. plantarum, Lb. rossiae, P. pentosaceus, W. confusa [139]
Molecular
Iran Sangak Lb. brevis, Lb. plantarum, Leuc. mesenteroides, P. cerevisiae [140]
Phenotypical
Ireland Oat Lb. coryniformis, Leuc. argentinum, P. pentosaceus, W. cibaria [141]
Molecular
Buckwheat Lb. crispatus, Lb. fermentum, Lb. gallinarum, Lb. graminis, Lb. plantarum, Lb. [47]
Molecular sakei, Lb. vaginalis, Leuc. holzapfelii, P. pentosaceus, W. cibaria
Buckwheat – use of a Lb. amylovorus, Lb. brevis, Lb. fermentum, Lb. frumenti, Lb. paralimentarius, [142]
starter Lb. plantarum, Leuc. argentinum, W. cibaria

Molecular
Teff Lb. fermentum, Lb. gallinarum, Lb. pontis, Lb. vaginalis, Leuc. holzapfelii, P. [47]
Molecular pentosaceus
(continued)
125
126

Cereal flour type
Method of
Teff – use of a starter Lb. amylovorus, Lb. brevis, Lb. fermentum, Lb. frumenti, Lb. paralimentarius, [142]
Molecular Lb. plantarum, Lb. pontis, Lb. reuteri, Lb. sanfranciscensis, P. acidilactici
Mexico Maize (pozol) Lb. alimentarius, Lb. casei, Lb. delbrueckii, Lb. lactis, Lb. plantarum, S. suis [143]
Molecular
Morocco Sourdough ferments, Lb. brevis, Lb. buchneri, Lb. casei, Lb. plantarum, Leuc. mesenteroides, [16]
traditional starter Pediococcus spp.
sponges
Phenotypical
Soft wheat flour Lb. buchneri, Lb. casei, Lb.delbrueckii, Lb. plantarum, Lb. sanfranciscensis, [144]
Phenotypical Leuc. mesenteroides, P. pentosaceus
Nigeria Maize Lb. brevis, Lb. casei, Lb. fermentum, Lb. plantarum, Leuc. mesenteroides, [145]
P. acidilactici
Portugal Maize (broa, Northern E. casseiliflavus, E. durans, E. faecium, Lb. brevis, Lb. curvatus, Lc. lactis [21]
Portugal) subsp. lactis, Leuconostoc spp., S. constellantus, S. equines
Phenotypical
Russia Rye Lb. brevis, Lb. fermentum, Lb. plantarum [146]
Phenotypical
Spain Wheat Lb. brevis, Lb. plantarum [10]
Phenotypical
Wheat Lb. brevis, Lb. cellobiosus, Lb. plantarum, Leuc. mesenteroides [12, 147]
Phenotypical
Sudan Sorghum (kisra) Lb. amylovorus, Lb. fermentum, Lb. reuteri [14]
Phenotypical
Kisra E. faecalis, Lb. fermentum, Lb. helveticus, Lb. reuteri, Lb. vaginalis, Lc. lactis [148]
Molecular
G. Huys et al.
Cereal flour type
Method of
5
Sweden Rye-wheat Lb. acidophilus, Lb. brevis, Lb. delbrueckii, Lb. farciminis, Lb. fermentum, Lb. [149]
Phenotypical plantarum, Lb. rhamnosus, Lb. sanfranciscensis, W. viridescens
Rye Lactobacillus sp., P. pentosaceus [150]
Phenotypical
Thailand Wheat Lb. casei, Lb. plantarum [42]
Turkey Wheat C. divergens, Lb. acetotolerans, Lb. amylophilus, Lb. brevis, Lb. plantarum, Lb. [151]
Phenotypic sakei, P. acidilactici, P. pentosaceus, T. halophilus
USA Wheat Lb. sanfranciscensis [75]
Phenotypic
Wheat Lb. paralimentarius, Lb. plantarum, Lb. sanfranciscensis [121]
Molecular
C. Carnobacterium, E. Enterococcus, Lb. Lactobacillus, Lc. Lactococcus, Le. Leuconostoc, P. Pediococcus, S. Streptococcus, W. Weissella
127
128 G. Huys et al.
Single isolations of yeast and LAB species have caused former misinterpretations of
their association with certain sourdough-producing regions, not only because of the
random isolation itself but also regarding the single habitat (sourdough) explored. Thus,
the association of, for instance, Lb. spicheri with German rice sourdoughs [80] might
represent accidental discoveries [57, 68, 69]. Instead, the dedicated use of basic raw
materials as well as the technological procedures applied rather determines the stability
and persistence of the yeast and LAB communities involved in the sourdough fermen-
tation process. Indeed, the presence of Lb. sanfranciscensis in wheat sourdoughs, which
was for a long time the sole habitat wherein this LAB species could be found [now, it
has been detected in rye sourdoughs (Table 5.4) and the insect gut as well [159]], can
be ascribed to its selection by the type of technology applied, i.e., backslopping
practices, temperature of incubation of the dough, pH of the dough, and/or microbial
interactions [113, 121, 135, 160, 161]. However, the use of certain raw materials,
encompassing cereal types and other ingredients such as adjunct carbohydrates, salt,
yoghurt, herbs, etc., and operational practices, such as dough yield and refreshment
times, may be linked to local traditions, may favor particular microorganisms as a result
of trophic and metabolic relationships and interactions (both cooperation and antibio-
sis), and hence may associate specific LAB and/or yeast species with specific geo-
graphical regions. For instance, the use of rye may select for amylase-positive
homofermentative Lactobacillus amylovorus, although higher temperatures cause a
shift toward the predominance of heterofermentative lactobacilli [120, 162]. The domi-
nance of mainly heterofermentative LAB species in traditional sourdoughs is caused by
a highly adapted carbohydrate metabolism, a dedicated amino acid assimilation, and
environmental stress responses [57, 58, 68, 163–166]. In particular, maltose, as the
most abundant fermentable energy source in dough, is metabolized via the maltose
phosphorylase pathway and the pentose phosphate shunt by strictly heterofermentative
LAB species such as Lb. sanfranciscensis, Lactobacillus reuteri, and Lb. fermentum.
This efficient maltose metabolism coupled to the use of external alternative electron
acceptors such as fructose, together with specific pathways such as the arginine deimi-
nase (ADI) pathway, and various environmental stress responses such as response to
acidic conditions, increases its competitiveness in the harsh sourdough environment
[167–172]. Moreover, maltose-positive LAB species such as Lb. sanfranciscensis often
form a stable association with maltose-negative yeast species such as C. humilis,
thereby preventing competition for the same carbohydrate sources [58, 164, 165]. Also,
the production of specific inhibitory compounds, maintained through backslopping,
such as the antibiotic reutericyclin produced by Lb. reuteri, may favor the dominance
of this LAB species, as is the case for certain German type II sourdoughs [173].
5.2.2 Influence of Cereals and Other Raw Materials
Cereal flours are not sterile. Their microbiological stability is related to their low
water activity. As yeasts and LAB naturally occur on plant materials, cereal flours
carry both groups of microorganisms and competitive yeast and LAB species reach
numbers above those of the adventitious microbiota upon fermentation of a

flour-water mixture [57]. However, whether the type of flour mainly directs the
growth of sourdough LAB species remains controversial [25, 26, 47, 89, 105, 107,
121, 123, 135]. For instance, whereas it was assumed that rice sourdough fermenta-
tion selects for Lb. spicheri [80], this LAB species cannot always be found in rice
sourdoughs [123]. This has been ascribed to competitiveness of the microorganisms
that are present in the sourdough ecosystem. Indeed, microbial interactions between
the spontaneous microbiota and an added sourdough starter culture may lead to the
dominance of autochthonous LAB species and/or strains. Among other mecha-
nisms, competitiveness may explain the apparent prevalence of LAB species in
specific sourdough preparations, such as evidenced by single reports on Lb. amylo-
vorus in rye sourdoughs [120], Lactobacillus sakei in amaranth sourdoughs [122],
and Lb. pontis in teff sourdoughs [47]. Yet, spontaneous sourdough fermentations
carried out in the laboratory with flour as the sole nonsterile ingredient indicate that
the type and quality (microbiological and nutritional) of the cereal flour used is
indeed an important source of autochthonous LAB and yeasts occurring in the ripe
sourdoughs [40, 107]. Hence, the flour plays a key role in establishing stable micro-
bial consortia within a short time. In this context, it has been shown that laboratory
sourdoughs based on wheat, rye, or spelt, backslopped daily for 10 days at 30 °C,
whether or not initiated with a Lb. sanfranciscensis starter culture [as tested in the
case of wheat sourdough fermentations in the study of Siragusa et al. [135]], reach
an equilibrium of LAB species through a three-step fermentation process: (1) preva-
lence of sourdough-atypical LAB species (e.g., Enterococcus spp. and Lc. lactis
subsp. lactis); (2) prevalence of sourdough-typical LAB species (e.g., species of
Lactobacillus, Leuconostoc, Pediococcus, and Weissella); and (3) prevalence of
highly adapted sourdough-typical LAB species (e.g., Lb. fermentum and Lb. plan-
tarum) [107–109, 135]. Indeed, it has been shown that the LAB species Lb. fermen-
tum (strictly heterofermentative) and Lb. plantarum (facultatively heterofermentative)
dominate several sourdough fermentation processes, irrespective of the type of flour
or the addition of starter cultures that are not robust enough [47, 108, 123, 136–138,
142, 172, 174, 175]. Concerning yeasts, C. glabrata and W. anomalus prevail dur-
ing laboratory sourdough fermentations [40]. Further, it has been shown that previ-
ous introduction of flour into the bakery environment helps to build up a so-called
house microbiota that serves as an important inoculum for subsequent bakery sour-
dough fermentations [155]. Indeed, LAB strains adapted to the sourdough and bak-
ery environment (apparatus, air, etc.), which have been shown to be genetically
indistinguishable, may be repetitively introduced in consecutive sourdough batches
during backslopping. The widespread use of bakers’ yeast may be responsible for
the prevalence of S. cerevisiae in bakery sourdoughs [26, 29, 31, 34–36, 39, 40].
However, there are also indications through reliable molecular data of a large strain
diversity of S. cerevisiae in single sourdoughs, that suggest an autochthonous wheat
flour origin of this yeast species in sourdough too [27, 30, 34]. Supportive of an
autochthonous origin of S. cerevisiae is also the presence of this species in rye flour [29].
Yet, during laboratory fermentations with flour as the sole nonsterile ingredient and
without added bakers’ yeast, other species such as C. glabrata and W. anomalus
130 G. Huys et al.
emerge [40]. Anyway, the direct environment is another important source of

(accidental) contamination of the flour by LAB and yeasts. Consequently, hygienic
conditions in the sourdough and bakery environments will play a role as well.
Finally, microorganisms occurring on cereals and subsequently in sourdoughs may
be of intestinal origin, due to fertilization practices on the grain fields, mouse feces
or insects in the flour mills, or fecal contamination of the sourdough production
environment [70, 175–178]. It may explain the opportunistic presence of
Lactobacillus acidophilus, Lactobacillus johnsonii, Lb. reuteri, and Lb. rossiae,
which are common gastrointestinal inhabitants.
5.2.3 Influence of Technology
Besides the cereals and other dough ingredients, which are mainly responsible as the
source of metabolic activity in the form of flour enzymes and endogenous microor-
ganisms, specific technological process parameters determine the species diversity,
number, and metabolic activity of the microorganisms (whether or not added) present
in the stable, ripe sourdough. These process parameters include chemical composi-
tion and coarseness of the flour, leavening and storage temperature, fermentation
time, pH, redox potential, dough yield, refreshment time and number of propagation
steps, and interactions between the microorganisms [57, 102, 164, 165, 179].
Different types of sourdough exist, on the basis of the processing conditions and/
or technology used for production, with a specific microbiota occurring in each type
[57, 180]. Type I or traditional sourdoughs are manufactured by continuous, (daily)
backslopping, at ambient temperature (<30 °C), to keep the microorganisms in an
active state. Therefore, mother doughs are used as an inoculum for subsequent
doughs by addition of the desired amount of dough to a fresh flour-water batch
according to defined cycles of preparation. These small-scale sourdough produc-
tions are used in traditional (home-made) sourdough bread making. Natural sour-
doughs frequently harbor Lb. sanfranciscensis and C. humilis/K. exigua as prevalent
LAB and yeast species, respectively. Type II or industrial sourdoughs are produced
through one-step propagation processes of long duration (typically 2–5 days) at a
fermentation temperature above 30 °C and with high water content. These large-
scale sourdough productions result in semifluid preparations, which are used as
dough acidifiers or flavor ingredients. Lb. amylovorus, Lb. fermentum, Lb. pontis,
and Lb. reuteri are commonly found in type II wheat and rye sourdoughs. Type III
sourdoughs are prepared in dried form to be used as nonliving acidifier supplement
and flavor carriers for (sourdough) bread production. In contrast to type I doughs,
doughs of types II and III require the addition of baker’s yeast for leavening.
Commercially available bulk starter cultures to prepare type II and III sour-
doughs aim at standardizing the end products through acidification of and flavor
formation in the dough [69, 181, 182]. New trends tend to develop starter cultures
that lead to improved functional properties other than acidification and flavor for-
mation, such as texture improvement, antibacterial and antifungal activities, and
health-promoting effects [69, 166]. In this context, strain robustness and fitness
towards microbial competitors and environmental conditions should be the driving
force in the selection of useful starters for sourdough fermentation processes, as it
has been shown that autochthonous strains often emerge [69, 135, 138, 142].
However, studies on the industrial exploitation of sourdough starter cultures are
scarce [183, 184]. Recently, the use of starter cultures in type I propagated sour-
doughs has been investigated [89, 123, 135, 185]. It is of course well known that the
fermentation temperature affects the ratio of lactic acid to acetic acid [185, 187]. In
general, homofermentative LAB starter cultures are used at high temperature and
for short fermentation times (e.g., 37 °C for 36 h) and heterofermentative LAB
starter cultures are used at low temperature and for long fermentation times (e.g.,
25 °C for 48 h), resulting in sourdoughs with mainly lactic acid and acetic acid,
respectively. However, it would be of great value to know the circumstances for the
expression of other functional properties that are of added value to sourdoughs
[182].
The fermentation temperature, one of the criteria to distinguish type I and II sour-
doughs, is essential for the community dynamics and stability of a sourdough micro-
biota [29, 160, 175, 179, 185, 188, 189]. For instance, spontaneous wheat sourdough
backslopping fermentations (type I) carried out at 23 °C for 10 days select for Le.
citreum instead of Lb. fermentum that prevails at 30 °C and 37 °C [175]. Similarly,
rye fermentations initiated with commercial sourdough starter cultures maintain the
presence of Lb. mindensis and Lb. sanfranciscensis at 25 °C (type I), but select for
Lactobacillus crispatus and Lb. pontis at 30 °C and for Lb. crispatus, Lb. frumenti,
and Lb. panis at 40 °C (both type II) [184]. Whereas Lb. sanfranciscensis prefers
long fermentation times at relatively low temperature, conditions that often prevail
during type I sourdough preparations, this species grows optimally at 32 °C [159,
189]. However, whereas C. humilis grows optimally at 27–28 °C but does not grow
above 35 °C [160, 189], the association of Lb. sanfranciscensis-C. humilis grows
optimally at 25 °C and 30 °C and may explain its stability between 20 °C and 30 °C
[160, 190]. The abundance of Lb. sanfranciscensis in wheat sourdoughs made at
ambient temperature indicates a low competitiveness of other LAB species such as
Lb. fermentum that prefers higher temperatures for optimal growth. Similarly, tem-
perature may be responsible for a selection toward Lb. helveticus during Sudanese
sorghum sourdough fermentations, which are carried out at 37 °C [148].
For the growth of sourdough LAB, also the pH plays an important role [160, 179,
188, 189]. For instance, Lb. sanfranciscensis cannot grow below pH 3.8–4.0 [160,
189], whereas C. humilis is not influenced by the pH [190]. An optimal pH for
growth of around 5.0 has been found for Lb. sanfranciscensis. This pH value cor-
responds approximately to that observed during the first stage of dough fermenta-
tion. However, the growth of lactobacilli is favored over yeast growth at pH values
above 4.5 [160]. Hence, the rate of acidification of the dough may determine the
level of Lb. sanfranciscensis in the dough. Natural sourdough fermentations dis-
playing higher pH values are often dominated by a different microbiota, encom-
passing Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and
Weissella, which are commonly present in the cereal flour [57, 133] or during the
132 G. Huys et al.
early fermentation process but die off when a significant pH decrease occurs upon
fermentation [89, 108].
Although sourdough fermentation proceeds anaerobically, the presence of oxy-
gen in the beginning of the fermentation and when small amounts of dough (high
ratio of surface to volume) are used may favor certain LAB and yeast species [179,
190, 192]. For instance, mild aeration has a positive influence on the competitive-
ness of Lb. amylovorus DCE 471 [179]. Similarly, P. kudriavzevii can only grow
when enough oxygen is available during fermentation [190]. Further, the ionic
strength and salt concentration of the dough affects microbial growth [160, 179,
193, 194]. Similarly, the presence of organic acids in and the buffering capacity of
the flour influence growth of both yeasts and LAB [160, 179]. In general, sourdough
LAB are acid-tolerant and their growth is favored in the presence of salt, as is the
case for, for instance, Lb. amylovorus DCE 471 [188, 193]. Alternatively, the growth
of C. humilis and S. exiguus is completely inhibited by 4% NaCl; also, the growth
of these yeasts is strongly inhibited in the presence of acetic acid and to a much
lesser extent by lactic acid [160, 179].
Whereas backslopping practices select for mainly heterofermentative LAB, the
amounts of dough used for backslopping and the frequency of the refreshments
determine the community dynamics and stability of the sourdough microbiota as
well. The amount of backslopping dough defines the initial pH and in this way
influences the growth and acidification rates of the LAB species involved [107, 189,
190]. Also, the amount of backslopping dough determines the dough yield and
hence the availability of water (water activity of the dough). Short refreshment
times may select for rapidly growing LAB species, which in turn depends on the
fermentation temperature and influences the acidification rate. In this regard, Lb.
fermentum is most competitive at 30 °C and 37 °C with backsloppings every 24 h,
while a mixture of Lb. fermentum and Lb. plantarum prevails at 30 °C with back-
sloppings every 48 h [175]. This may explain why Lb. sanfranciscensis is some-
times missed during laboratory-scale fermentation processes [136, 175]. Also, a
short refreshment time seems to favor C. humilis during sourdough fermentation
compared to S. cerevisiae [190].
Finally, interactions between LAB and yeasts are an important aspect for the
community dynamics and stability of the sourdough microbiota [50, 57, 165, 189,
195, 196]. Interactions encompass both cooperative and antagonistic ones. During
some sourdough fermentation processes yeasts cannot develop at all, perhaps
because of inhibition of yeast growth by nutritional competition or the presence of
inhibitory compounds [47, 123, 175]. In other processes, mutualistic interactions
lead to stable associations, not only between LAB species and yeasts (besides Lb.
sanfranciscensis/C. humilis, also Lb. sanfranciscensis/K. barnettii, Lb. plantarum/S.
cerevisiae, and Lb. brevis/Candida spp.) but also among LAB species (e.g., between
Lb. sanfranciscensis and Lb. plantarum or Lb. paralimentarius) [40, 41, 58, 69,
142]. Nevertheless, the competitiveness of LAB and yeasts in sourdough seems to
be strain-specific and not species-specific, as has been shown for Lb. sanfranciscen-
sis [135] and Lb. plantarum strains [138] in wheat sourdoughs recently.
5.3 Isolation of Sourdough Yeasts and Lactic Acid Bacteria
5.3.1 Isolation of Sourdough Yeasts
Yeast isolation from mature sourdoughs is relatively uncomplicated as a stable

sourdough usually harbors a homogeneous yeast population as part of the resident
microbiota. However, the follow-up of different developmental stages of a sour-
dough or the search for minor components requires a strategy that is optimized
towards the detection of subdominant components. Detailed information on food
yeast isolation was provided by Deak [197]. Here, only a brief discussion of the
currently practiced yeast isolation methods from sourdough is given, including
some references to more complete methodological resources. After sample homog-
enization and dilution, yeast growth is suitably effected on solid media. This
sequence of manipulations should be performed with minimal delay to avoid the
settling of yeast cells and cell death. Diluents, usually distilled water, peptone water,
saline, or Ringer solution, may influence the resulting cell counts, with peptone
water having resulted in the highest cell recovery [198]. Overviews of classical
growth media and isolation techniques for yeasts and foodborne yeasts are given by
Yarrow [199] and Beuchat [200], respectively. The cultivation media used in sour-
dough analyses are rich media containing complex compounds such as peptone
(e.g., Sabouraud agar), tryptone (e.g., Wallerstein Laboratory nutrient agar), yeast
extract (e.g., yeast extract peptone dextrose agar), malt (e.g., yeast and malt extract
agar, wort agar), and potato infusion (e.g., potato dextrose agar), together with an
additional component to inhibit bacterial growth. Most often chloramphenicol is
used as an antibiotic that can be added to the medium before sterilization without
losing its activity. Acidification of the medium is sometimes used to restrict bacte-
rial growth, while this is known to affect growth of some yeasts (namely of the
genus Schizosaccharomyces). However, the acidity of the sourdough lets it appear
unlikely for acid-sensitive strains to be present in a ripe sourdough. In general, most
yeasts show good vegetative growth at room temperature, although some may grow
at subzero temperatures and others up to 45 °C [201]. Cardinal growth temperatures
of yeasts are species- and strain-specific. The most suitable incubation temperature
for sourdough yeasts would be the temperature at which the sourdough is in its most
active state. The most frequently applied temperatures are 25–30 °C. First yeast
growth can under such conditions usually be observed after 2–3 days, while daily
inspection of the plates for 5 up to 10 days is recommended to allow full differentia-
tion of colony morphology and detection of more slowly growing components.
Other than the consideration of growth conditions, the selection of yeast colonies
for further characterization and identification is the most important factor influencing
the completeness of a diversity survey. Even though sourdough samples often pres-
ent a homogeneous yeast population, one needs to bear in mind that the colony
morphology of different yeast species is often very similar. Each observed morpho-
type should therefore be sampled more than once. A logical strategy would be to
recover a number of colonies of each type that represents a reasonable percentage
134 G. Huys et al.
of the total number of colonies of that particular morphotype. Each morphoptype’s

percentage provides important species abundance data in the sourdough if more
than one yeast species is isolated. The generation of pure cultures is crucial before
characterization and identification of the isolates. The purity should be tested micro-
scopically and on antibiotic-free medium to exclude any carry-over of the accompa-
nying bacterial microbiota.
5.3.2 Isolation of Sourdough LAB
Isolation of LAB from sourdough environments is challenging for three main rea-
sons. First, sourdoughs are complex ecosystems not only in terms of their microbial
composition but also in terms of the interactive effects among types of breadmaking
processes and ingredients. The utilization of soluble carbohydrates by LAB and,
thus, their energy yield are greatly influenced by the associated yeasts and vary
according to the type of carbohydrates [195]. However, as many media for selective
isolation of LAB incorporate yeast-inhibiting agents such as cycloheximide, pima-
ricin, and amphotericin B, the trophic interaction between LAB and yeasts is in
these cases disturbed, which may affect the recovery potential of LAB strains that
strongly rely on this association. Secondly, sourdough fermentation is a dynamic
process in which fast-acidifying LAB initially dominate the ecosystem and are then
gradually replaced by typical sourdough LAB that largely contribute to the organo-
leptic and textural properties of the end product. Depending on whether the early
subdominant LAB and/or the final dominant LAB are the target of the isolation
approach, it may thus be necessary to include multiple samples taken at different
time points. Finally, the LAB communities in sourdoughs may consist of metaboli-
cally very diverse groups, including obligately homofermentative and facultatively
or obligately heterofermentative species. As some of these species have specific
growth requirements in terms of the incubation medium and conditions (e.g., tem-
perature, pH, atmosphere, etc.), it seems inevitable that different medium formula-
tions and/or sets of incubation parameters are required to cover the entire metabolic
LAB spectrum present in a sourdough sample.
Initially, sourdough LAB were mostly isolated on de Man-Rogosa-Sharpe (MRS)
medium [202], which is the general medium used for the isolation and enumeration
of lactobacilli from fermented food products. The MRS medium contains glucose
as the main carbohydrate source. Triggered by growing insights in the species diver-
sity of sourdough-associated LAB, a number of more specialized media have been
developed for the selective isolation of typical sourdough species. For the specific
detection of Lb. sanfranciscensis, Kline and Sugihara [75] proposed the SourDough
Bacteria medium which contains maltose as the carbohydrate source in addition to
freshly prepared yeast extract (FYE) to further enhance growth. The Sanfrancisco
medium was developed for the isolation and description of Lb. pontis and Lb. min-
densis [79, 203]. This medium contains three carbohydrates (maltose, fructose, and
glucose), FYE, cysteine and rye or wheat bran. In parallel to the design of new
media, several authors also described variations of the original MRS medium
formulation for isolation of sourdough LAB. Vogel and co-workers [203] proposed
a modified MRS medium, referred to as MRS “Vogel”, with higher pH value (6.3),
whereas the MRS5 medium [185] contains the three major carbohydrates present in
the sourdough ecosystem (i.e., maltose, fructose, and glucose) in addition to cystein
and a vitamin mixture. In subsequent studies, the MRS5 medium has been success-
fully used for the isolation of several novel Lactobacillus species from sourdough
such as Lb. spicheri [80], Lb. namurensis [87], and Lb. crustorum [86]. From these
recent descriptions, it thus appears that the use of a modified MRS formulation with
a lowered pH (<6.0) and supplemented with an additional carbon source such as
maltose and/or fructose as well as with amino acids and vitamins under anaerobic
conditions is one of the most successful strategies for the isolation of (new) sour-
dough LAB species. In a recent study, the qualitative and quantitative performance
of 11 elective and selective culture media was compared for isolation of lactobacilli
from type I sourdoughs [204]. On the basis of the identification results obtained
with protein profiling, the largest species diversity was recovered on maltose-con-
taining MRS medium. However, the fact that MRS5 medium allowed the isolation
of a specific (but unidentified) subpopulation only found on this medium indicates
that there is no single efficient medium for the recovery of all lactobacilli from type
I sourdoughs.
5.4 Identification of Sourdough Yeasts and Lactic Acid

Bacteria
Traditionally, identification of sourdough microorganisms relied on (selective) cul-

turing, selection and purification of a limited number of isolates, and identification
of purified isolates with phenotypic and/or genotypic methods. Although this
approach has significantly contributed to our current knowledge of the sourdough-
associated yeast and LAB species diversity, the use of culture media holds a number
of intrinsic limitations. In a culture-based approach, species with very specific nutri-
ent and growth conditions may only sporadically or even not be recovered which
leads to an underestimation of the actual microbial species diversity present in the
complex sourdough ecosystem [68]. In contrast, culture-independent techniques
that are based on phylogenetic dissection of the metagenomic DNA extracted
directly from the sample allow one to unravel the species diversity and dynamics of
sourdough yeasts and LAB without the need to isolate and culture its single compo-
nents. However, also these DNA-based methods have a number of limitations,
including poor detection capacity of subdominant species and inadequate taxonomic
resolution between phylogenetically closely related species. Depending on the aim
of the study, conventional culturing and molecular methods are therefore often com-
bined to obtain a more complete picture of the microbial species diversity of sour-
dough ecosystems [41, 106].
136 G. Huys et al.
5.4.1 Culture-Dependent Approaches
5.4.1.1 Yeasts
Identification is the localization of individuals in a classification scheme by means

of diagnostic characteristics resulting in the assignment of names. The diagnostic
characteristics to be used are provided in the species descriptions and, in the case of
yeasts, are collected in the monograph “The yeasts: a taxonomic study,” currently in
its fourth edition [1], with the fifth edition about to be released [204]. While the
fourth edition still included instructions for the phenotypic identification of yeasts
[199], the fifth edition reformulates them as phenotypic “characterization” instead
[205]. This is a consequence of the need to use DNA-based methods to recognize
the since 1998 twofold increased number of yeast species. Nevertheless, the accu-
rate description of fermentation and assimilation abilities as well as other pheno-
typic characters of yeasts continues to be of interest in technological, ecological and
taxonomic frameworks.
Among the DNA-based methods currently applied to yeast identification the
partial sequencing of the large subunit (LSU) ribosomal ribonucleic acid genes
occupies a key position [206]. The DNA sequences of the variable regions D1 and
D2 located at the 5’ end of the LSU of virtually all known yeast species are docu-
mented in the public databases of the International Nucleotide Sequence Database
Collaboration (INSDC, including GenBank, the European Molecular Biology
Laboratory, and the DNA Data Bank of Japan). The entries of the three submis-
sion hubs are bundled, daily updated, and made available for searches by the
NCBI (www.ncbi.nlm.nih.gov/nucleotide/). Few distinct yeast species show no
or low sequence divergence in the D1/D2 LSU rDNA, can therefore not reliably
be distinguished, and require complementary analyses such as Saccharomyces
bayanus and S. pastorianus [206], Hanseniaspora meyeri and Hanseniaspora
clermontia; Hanseniaspora guilliermondii and Hanseniaspora opuntiae [207],
Meyerozyma guilliermondii and Meyerozyma caribbica; Trichomonascus ciferrii
and Candida mucifera; K. marxianus and Kluyveromyces lactis [55],
Debaryomyces hansenii, Debaryomyces fabryi and Debaryomyces subglobosus
[208]. Genetic regions that show in most cases larger divergence than the D1/D2
LSU rDNA and for which substantial sequence records have been accumulated
include the ITS region of the ribosomal gene cluster. This region is favored by
mycologists as the barcoding locus of fungi, although no common threshold
value to distinguish intraspecific from interspecific variation can be defined
[209, 210]. No systematic evaluation of ITS sequence variation to answer this
question for ascomycetous yeast species exists to date. In comparison to sequenc-
ing, RFLP analysis of ITS sequences offers simplified access to partial DNA
sequence information. The accessed information is determined by the recogni-
tion sites of the applied restriction enzymes and typically includes only a few
nucleotides, necessitating a range of restriction enzymes to reliably distinguish
the species in question.
Single-copy protein-coding gene sequences also accumulate in the public

databases and may be used to complement those of D1/D2 LSU and ITS sequences
in cases where the ribosomal genes do not allow a conclusive species identification.
Such protein-coding genes include the actin gene (ACT1) [55], translation elonga-
tion factor 1 alpha (TEF1), the mitochondrial cytochrome oxidase 2 gene (COX2)
[211], and the largest and second largest subunits of the RNA polymerase II gene
(RPB1, RPB2) [212]. In contrast to the multicopy ribosomal DNA (e.g., D1/D2
LSU, ITS), single-copy nuclear genes may be more difficult to amplify, as the design
of universal primers effective in phylogenetically distant species is not always pos-
sible and as only single primer binding sites are available in a haploid genome in
contrast to hundreds of binding sites for ribosomal genes. Highly variable mito-
chondrial genes (e.g., COX2) bear the possibility of having been subject to a differ-
ent evolutionary path than the nuclear genome, in other words, are prone to potential
horizontal gene transfers across species borders. As databases for complementary
gene sequences are far more restricted than for the D1/D2 LSU, one needs to assure
the existence of reference sequences. Databases that allow searching the available
sequences for specific strains, such as the yeast database of the Centraalbureau voor
Schimmelcultures, The Netherlands (www.cbs.knaw.nl/yeast/BioloMICS.aspx) and
the StrainInfo portal (www.straininfo.net/), Ghent University, Belgium, may be
used for the selection of complementary sequencing targets. The comparison of a
query sequence with reliable type strain sequences is essential, as type strains are
the only valid taxonomic reference for a species. Sequence alignments outside the
commonly consulted BLAST (Basic Local Alignment and Search Tool) tabulated
results are helpful, because type strain sequences are often not recognizable from
the sequence entry title line. It is recommended to include type strain sequences of
the phylogenetically most closely related species in these alignments to confirm the
differentiation of the species in question by the given DNA region.
While the so far discussed methods use genetic information of few or single loci,
the techniques commonly known as DNA fingerprinting exploit genetic information
that is distributed throughout the genome. A large variety of protocols exists and the
more reproducible among them are based on the specific binding of PCR primers to
mini- or microsatellite sequences in contrast to arbitrary binding realised in RAPD-
PCR. The primer most frequently applied to sourdough yeasts was derived from a
ubiquitous minisatellite sequence found in the protein II gene of the bacteriophage
M13 [213]. The primer referred to as M13 results from a consensus sequence of 12
partially incomplete repeats. After its use in a PCR assay as the single primer and
visualization of the PCR reaction products by agarose gel electrophoresis, usually
species-specific banding profiles based on the different lengths of amplifiable
sequences enclosed or flanked by M13 minisatellites are observed. The important
influence of experimental factors such as the DNA extraction method and PCR and
electrophoresis parameters on the resulting profiles implies the need to include type
strains for ideally side-by-side-comparisons if a complete identification is to be per-
formed. However, PCR-fingerprinting without type strains may be used to group
larger numbers of isolates and to select those that are representative of each group
for identification by DNA sequencing [40].
138 G. Huys et al.
The species S. cerevisiae has been observed to show extensive pheno- and
genotypic intraspecies diversity (reviewed in [214]). Part of such strain diversity has
been traced by molecular analyses to genomic variability associated with Ty-element
insertion sites [215]. Ty elements belong to a group of eukaryotic transposable ele-
ments that are also called retrotransposons because of some similarities with retrovi-
ruses. Ty-elements are flanked by long terminal repeat (LTR) sequences, in turn
formed by repeated delta elements. Delta elements are also found in larger numbers
independently from LTR sequences and have been used to design and optimize
specific PCR primers to amplify the sequences between two delta elements in S.
cerevisiae, resulting in an effective strain-typing method for this species [216, 217].
5.4.1.2 LAB
Although largely abandoned and replaced by molecular tools, characterization and

identification of sourdough LAB species by phenotypic methods is in some cases
still useful, or even mandatory when it concerns new species descriptions. The con-
ventional phenotypic approaches for identification of sourdough LAB species may
include physiological and chemotaxonomic tests and determination of major fer-
mentation pathways, carbohydrate utilization patterns, lactic acid configuration,
and peptidoglycan types. To determine carbohydrate patterns and enzymatic prop-
erties in a faster and more reproducible way, miniaturized biochemical test systems
such as the API system (Biomérieux, France) can be used for phenotypic character-
ization of sourdough LAB species [23]. However, it should be stressed that the
identifications obtained by comparison with commercial databases such as those
linked to API are only tentative and need verification with other taxonomic meth-
ods. A more advanced phenotypic identification approach is offered by chemotaxo-
nomic methods, which are based on the use of analytical methods to detect and
characterize one or several chemical cell components. Protein profiling by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been used for
identification of LAB isolates recovered from Italian [127] and Greek [72] sour-
doughs. SDS-PAGE of cellular proteins generally offers sufficient discrimination of
LAB isolates at species level but may fail to discriminate between species in the Lb.
acidophilus group [218] and the Lb. plantarum group [71], both of which are promi-
nent members of sourdough ecosystems. Although not yet evaluated for sourdough
LAB, the use of mass spectrometry (MS) methods is probably the most powerful
phenotypic approach currently available for classification and identification of bac-
teria [219]. One of these methods, matrix-assisted laser desorption/ionization-time-
of-flight (MALDI-TOF) MS, allows one to measure peptides and other compounds
in the presence of salts and to analyze complex peptide mixtures, which makes it an
ideal method for measuring nonpurified extracts and intact bacterial cells. The
resulting MALDI-TOF MS spectra can be used to generate identification libraries
for simple and high-throughput identification of unknown bacterial isolates. De
Bruyne and co-workers [220] constructed such identification libraries for the LAB
genera Leuconostoc, Fructobacillus, and Lactococcus, and reported that 84% of the
leuconostocs and fructobacilli and 94% of the lactococci were correctly identified at
species or subspecies level. Identification accuracies for the former two groups fur-
ther increased to 94–98% when machine learning was applied, which indicates the
important role played by advanced techniques for the analysis of complex MALDI-
TOF MS profiles.
Essentially, molecular approaches for identification of sourdough LAB are either
DNA fingerprint- or sequence-based. DNA fingerprinting methods rely on the use
of restriction enzyme analysis, the use of specific or random PCR primers, or a
combination thereof. Ribotyping, one of the first DNA fingerprinting techniques
used in bacterial taxonomy [221], relies on a combination of restriction analysis of
total genomic DNA and Southern hybridization to visualize a subset of restriction
fragments with labeled rDNA probes targeting conserved domains of ribosomal 16S
and 23S rRNA encoding genes. Although ribotyping generally provides high dis-
criminatory power at species to subspecies level, it has been used only sporadically
as an identification technique for LAB species. In specific cases, ribotyping has
proven particularly useful in the classification and identification of sourdough LAB,
for example for discrimination between the genomically and phenotypically highly
similar sourdough LAB W. cibaria and W. confusa [63] and for intraspecific differ-
entiation of Lb. sanfranciscensis strains from different sourdoughs [121]. Despite
its high resolution at species as well as at strain level, amplified fragment length
polymorphism (AFLP) fingerprinting was also only used sporadically for LAB
identification purposes. Essentially, AFLP combines the power of restriction frag-
ment length polymorphism with the flexibility of PCR-based methods by ligating
primer-recognition sequences (adaptors) to the digested DNA [222]. This whole-
genome fingerprinting technique has proved useful to support the description of the
sourdough species Lb. hammesii [82], Lb. crustorum [86] and Lb. namurensis [87]
and for molecular source tracking of Lb. spicheri, Lb. plantarum and Lb. sanfranci-
scensis in the production environment of artisan sourdough bakeries [155]. In con-
trast to the aforementioned methods, RAPD-PCR and repetitive DNA element
(rep)-PCR are technically less demanding DNA fingerprinting techniques based on
a single PCR step. Both methods are fast, relatively inexpensive, and exhibit a high
discriminatory power ranging from genus to intraspecific level, which explains their
wide application range for the identification and classification of LAB. RAPD-PCR
has been used in multiple studies on sourdough LAB for species identification
[129], strain differentiation [80, 89, 120, 126, 127, 131, 148, 158] and strain moni-
toring purposes [70, 122, 135, 138, 223]. The reproducibility of RAPD-PCR is
highly influenced by various factors, such as DNA purity and concentration and
minimal differences in the PCR temperature programme [224], for which reason
this method is less suitable for interlaboratory comparisons. Because of the use of
longer PCR primers complementary to bacterial interspersed repetitive DNA ele-
ments such as ERIC, BOX, REP or (GTG)5 and higher annealing temperatures,
rep-PCR protocols are more robust and display a higher level of reproducibility
[225]. rep-PCR using the (GTG)5 primer, i.e., (GTG)5-PCR, has been found particu-
larly useful for differentiation of sourdough LAB at the (sub)species up to the strain
level [74, 105, 106, 155, 226, 227]. For high-resolution differentiation of individual
140 G. Huys et al.
sourdough LAB strains by DNA fingerprinting, however, AFLP fingerprinting and

pulsed-field gel electrophoresis (PFGE) are the most powerful. Essentially, PFGE
involves the electrophoretic separation of genomic macrorestriction fragments
obtained by digestion with rare-cutting enzymes in an alternating electric field. In
sourdough studies, PFGE has been used as a typing method to differentiate among
strains within Lb. plantarum and Lb. sanfranciscensis [126, 135, 174].
Sequence-based analysis approaches for identification of sourdough LAB have
long relied on the use of 16S rRNA genes, and this has become a standard approach
to obtain a first preliminary view of the taxonomic diversity among a set of unknown
isolates recovered from a sourdough ecosystem [72, 80, 148]. In many of these
studies, only partial 16S rRNA gene sequences are determined and used in compari-
sons with public sequence databases. In many cases, the use of partial sequences
will only allow a tentative identification, of which the reliability is likely to improve
when the entire 16S rRNA gene is sequenced [228]. Despite its established use as a
standard method for identification of LAB species, 16S rRNA gene sequencing
does not allow differentiation of phylogenetically closely related species [63–66,
71]. The growing availability of whole-genome sequences has triggered the search
for alternative genes that offer a higher taxonomic resolution than the 16S rRNA
gene. The use of protein-encoding genes or so-called housekeeping genes essen-
tially combines the technological advantages of 16S rRNA gene sequencing and the
taxonomic resolution offered by a number of fingerprinting methods. Sequencing of
one or preferably multiple of these genes as taxonomic markers is a crucial step
forward in the development of standardized and globally accessible methods for the
identification of LAB. Housekeeping genes such as pheS (encoding the phenylala-
nyl-tRNA synthase) and rpoA (encoding the DNA-dependent RNA polymerase
alpha-subunit) display higher divergence rates than the 16S rRNA gene, and allow
discrimination between closely related LAB species with almost identical 16S
rRNA gene sequences [67, 229]. Several studies on sourdough LAB species diver-
sity have used such protein-encoding genes as phylogenetic markers in a single-
locus sequence approach. In conjunction with (GTG)5-PCR fingerprinting, the pheS
gene has been successfully used for the identification of LAB species from sour-
dough fermentations at laboratory scale [107] and from Belgian artisan bakery sour-
doughs and their environment [105, 106, 155], as well as for unraveling the
intraspecific diversity in the sourdough species Lb. rossiae [74]. Settanni and co-
workers [156] used the recA gene, encoding a protein essential for repair and main-
tenance of DNA, in a multiplex PCR assay to discriminate between the
phylogenetically highly related Lb. plantarum, Lb. pentosus and Lb. paraplantarum
in sourdough ecosystems. The recA gene has also been used in combination with the
16S rRNA gene to unravel the identity of LAB isolates recovered during wheat flour
sourdough type I propagation [135]. Sequences derived from the tuf gene, which
encodes the elongation factor Tu, have revealed a higher discriminatory power com-
pared to 16S rRNA gene sequences and have been used to support the delineation
of the new sourdough species Lb. secaliphilus [85]. Although single-locus sequence
analysis approaches are now commonly used within specific LAB groups, it has
been argued that the phylogenetic information obtained from only a single gene
may be influenced by lateral gene transfer (LGT) and may lead to incorrect
identifications. To compensate for possible LGT events, it has been suggested that
multilocus sequence analysis (MLSA) of at least five housekeeping genes from
diverse chromosomal loci and with wide distribution among taxa is required to reli-
ably distinguish a species from related taxa [230]. After a more thorough evalua-
tion, however, Konstantinidis and co-workers [231] concluded that three genes are
sufficient to anticipate the possible effects of LGT in MLSA-based identification
schemes. For LAB, MLSA based on the combined sequence analysis of the genes
atpA, pheS, and rpoA has been successfully explored for species identification of
enterococci [229], lactobacilli [67], leuconostocs [232], and pediococci [233]. For
sequence-based differentiation of LAB at strain level, multilocus schemes typically
include six or seven housekeeping genes. The resulting multilocus sequence typing
(MLST) approach has so far mainly been applied to study community structure,
evolution and phylogeography of bacterial pathogens [234]. A few MLST schemes
have been specifically developed for Lactobacillus species, including Lb. casei
[235, 236], Lb. plantarum [237] and Lb. salivarius [238].
5.4.2 Culture-Independent Approaches
The first approaches used to identify sourdough microorganisms independent of

culturing relied on the use of oligonucleotide probes targeting ribosomal gene
sequences specific for individual species or groups of species. The majority of these
probe-based methods made use of partial 16S rRNA gene sequences that were
identified as molecular signatures unique to specific LAB species [203, 239].
Gradually, the relatively laborious probe hybridizations were replaced by faster
community PCR assays using species-specific oligonucleotide primers. The success
of both approaches strongly depended on rigorous in silico probe or primer design
and required in vitro and in vivo validation using taxonomically well-characterized
type and reference strains and spiked sourdough samples, respectively. Species-
specific PCR primers complementary to signature sequences in the 16S or 23S
rRNA gene or in the 16S–23S rRNA intergenic spacer region have been applied for
the culture-independent identification of sourdough LAB species [26, 156, 161,
240–242]. By combining multiple sets of primers, several typical sourdough LAB
species can be simultaneously detected. In this way, Settanni and co-workers [156]
developed a two-step multiplex community PCR assay that enabled rapid
identification of up to 16 Lactobacillus species in sourdough samples. The introduc-
tion of real-time PCR technology has allowed one to further increase the sensitivity
of PCR-based identification assays and enables the simultaneous detection and
quantification of food microorganisms [243]. For this purpose, SYBR Green-based
real-time PCR assays based on the detection of the pheS gene have been used for
source tracking of Lb. plantarum and Lb. sanfranciscensis in traditional sourdoughs
and their production environments [155].
142 G. Huys et al.
Whereas probe- and primer-based identification approaches offer the specificity

and selectivity required to detect and monitor specific LAB in sourdough samples,
they were not designed to offer a complete picture of the predominant LAB species
diversity or to reveal new or unknown LAB species diversity in sourdough ecosys-
tems. In contrast, community fingerprinting methods such as denaturing gradient
gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis
(TTGE) do not require prior knowledge of the ecosystem’s diversity and are univer-
sally applicable to study the species diversity and dynamics of complex bacterial
communities in food environments [244]. The universal use of DGGE fingerprinting
is based on the sequence-dependent separation of a mixture of equally sized PCR
amplicons generated from a common taxonomic marker such as the 16S rRNA
gene. For the design of PCR primers, the V1, V3, and V6-V8 hypervariable regions
of the 16S rRNA gene are most commonly used. Taxonomic information on indi-
vidual members of the sample community can be obtained by band position analysis
provided that an identification database is available, clone library analysis, sequenc-
ing of excised and purified DGGE bands or hybridization using species-specific
probes. Major drawbacks of DGGE fingerprinting include its inability to detect sub-
dominant (i.e., <1%) community members and the fact that a single strain or species
may be represented by multiple bands in the DGGE profile due to heterogeneous
rRNA operons and/or heteroduplex molecules. Either using universal or group-
specific 16S rRNA gene primers, DGGE has been widely applied to inventorize
LAB communities in sourdoughs [41, 47, 106, 128, 245, 246] and to investigate the
dynamics, adaptation, and source of predominant sourdough LAB communities [34,
39, 80, 107, 123, 142, 155, 185]. Likewise, primers targeting the 26S LSU rDNA
have been used for DGGE fingerprinting analysis of sourdough yeast communities
[29, 46]. To maximally cover the microbial species diversity present in a sourdough
ecosystem, a number of DGGE studies have combined the use of 16S and 23S
rRNA gene primers to determine in parallel the predominant LAB and yeast compo-
sition of sourdough samples [33, 39, 41, 47, 123, 142, 245]. Compared to DGGE,
TTGE has been used to a much lesser extent for culture-independent analysis of the
sourdough microbiota [112]. In many of the cited studies, the sequence heterogene-
ity of the multicopy 16S rRNA gene is mentioned as an important limitation in
DGGE, as this may lead to an overestimation of the LAB species diversity. The
degree of overestimation can be estimated by scoring individual bands by position
analysis with a reference database and/or by band sequencing. Alternatively, single-
copy genes that do not exhibit this heterogeneity such as rpoB have been evaluated
for DGGE fingerprinting of LAB species during food fermentations [247].
Microarray technology represents one of the most recent culture-independent
approaches to study the diversity and identify individual members of the sourdough
microbiota. Phylogenetic microarrays, containing partial 16S rRNA gene sequences
as targets, are ideally suited for this purpose but are currently not available for sour-
dough microbiota. Alternatively, a functional gene microarray can be used when the
original annotation information allows one to link the responding oligonucleotides to
the original species. Weckx and co-workers [108, 109] used a LAB functional gene
microarray to analyze time-related RNAsamples that represented the metatranscriptome

of sourdough fermentations maintained by daily backslopping. The resulting set of
hybridization data allowed one to monitor the LAB community dynamics in the sour-
dough ecosystem and to identify the major LAB species that contributed to the estab-
lishment of a stable ecosystem through its three successive phases.
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Chapter 6
Physiology and Biochemistry
of Sourdough Yeasts
M. Elisabetta Guerzoni, Diana I. Serrazanetti, Pamela Vernocchi,

and Andrea Gianotti
6.1 Introduction
Although baker’s yeast is used worldwide as a leavening agent for the manufacture
of baked products, an overwhelming multitude of baked goods are also produced
with the aid of sourdough. These include above all breads from wheat, rye and mix-
tures thereof as well as the well-known Italian products such as Panettone, Colomba,
Pandoro and different types of brioches.
Traditional cultivation methods in combination with phenotypic and genotypic
identification adopted to characterise the yeasts of ripe dough revealed the presence
of some species belonging, especially, to the genera Saccharomyces and Candida
[1–6]. Their taxonomic features and diversity are described in Chap. 5. Based on
studies focused on the yeast population of sourdough, the most frequent species
were Saccharomyces cerevisiae and Candida milleri [7, 8]. In some cases, Candida
milleri accounted for 65.8% of the isolates in artisanal sourdough produced without
the addition of baker’s yeast [9].
This chapter is mainly focused on the description of: (1) yeast responses to phys-
ico-chemical conditions and their fluctuation during sourdough fermentation; (2)
implications of such responses for fermentation products and flavour compounds,
including quorum-sensing molecules; and (3) the production of baker’s yeast and its
use in the bread-making industry.
M.E. Guerzoni (*) • D.I. Serrazanetti • P. Vernocchi • A. Gianotti

Food Science Department (DISA), University of Bologna,
Via Fanin, 46, Bologna, Italy
e-mail: [email protected]; [email protected];
[email protected]; [email protected]

156 M.E. Guerzoni et al.
6.2 Effects of Physico-chemical Conditions on Yeast Growth

and Physiology
Cereal dough is a dynamic system that is characterised by continuous changes in

nutrient availability during processing. This system is subjected to simultaneous
release, by endogenous or added amylases, of fermentable carbohydrates such as
glucose, maltose and fructose, and their uptake and consumption by yeasts and lactic
acid bacteria.
The availability of specific nutrients maintains yeast fermentation and, in industrial
fermentations, nutrient-related stresses have several practical implications [10]. In
particular, the concentration of fermentable carbohydrates, as well as the dynamics
of their release by amylases depends on many factors including cereal type,
environmental and cultural factors, grain storage and milling conditions. In turn, the
activity of amylases is conditioned by temperature and, especially, water activity
(Aw) [11, 12]. During the first phase of bread making, the readily fermentable sugar
concentration is probably limiting. Figure 6.1 describes the effect of commercial
amylase supplementation on the rate of volume increase of doughs with different
concentrations of NaCl (Fig. 6.1).
Depending on the type of flour and bread-making technology, starvation condi-
tions can also be envisaged. The imbalance between yeast consumption and starch
hydrolysis might lead to rapid depletion of soluble carbohydrates. Gene expression
response to nutrient starvation involves changes in gene expression that persist until
the starvation is alleviated [13, 14]. Saldanha et al. [15] studied the physiological
response to limitation by diverse nutrients in batch and steady-state (chemostat)
cultures of S. cerevisiae. The yeast does not respond to the changing environment
with a small adjustment in key control points, but with the coherent transcriptional
regulation of a large set of genes. Cells adjust their growth rate to nutrient availabil-
ity and maintain homeostasis in the same way either in batch or in steady state
conditions (continuous culture). Therefore, the patterns of gene expression in the
steady state closely resemble those of the corresponding batch cultures just before
the yeasts exhaust the nutrients.
Yeasts can use a diverse array of compounds as nitrogen sources and, upon
demand, are capable of expressing catabolic enzymes of many different pathways.
Extensive studies on nitrogen metabolism and its regulation have been carried out
with S. cerevisiae. Nitrogen compounds such as ammonia, glutamine and glutamate
are used by yeasts, but asparagine is the preferred nitrogen source. When these pri-
mary nitrogen sources are not available or are present only in low concentrations
that limit growth, many other nitrogen sources are used, for example purines, amides
and most of the amino acids [16]. Yeasts may also grow on ammonium ions as the
sole nitrogen source, since they possess the entire repertoire of genes that encode for
the enzymes responsible for biosynthesis of all amino acids. Ammonium is directly
assimilated into amino acids, notably glutamate and glutamine, which serve as
donors of the amino group for the other amino acids. The main route for assimilation
of ammonium is the reaction of the NADPH-dependent glutamate dehydrogenase
6 Physiology and Biochemistry of Sourdough Yeasts 157
Fig. 6.1 Combined effect of NaCl and a-amylase supplementation on the rate of volume increase
of doughs inoculated with S. cerevisiae and incubated at 30°C for 3 h (Gianotti et al.
unpublished)
(GDH), which forms glutamate from a-ketoglutarate and ammonium. Whenever

the concentration of ammonium ions is low, glutamine synthase is activated. This
forms glutamine from a-ketoglutarate and ammonium in an ATP-dependent reaction.
Glutamine is absolutely required as the prominent precursor of several biosynthetic
pathways for asparagine, tryptophane, histidine, arginine, carbamoyl-phosphate,
CTP, AMP, GMP, glucosamine and NAD. Saccharomyces cerevisiae does not use
nitrate as a nitrogen source but very few species of yeasts have that capacity. Nitrate
assimilation occurs via the activity of the NADPH-dependent nitrate reductase,
which forms nitrite. Further, nitrite is reduced to ammonium by the NADPH-dependent
nitrite reductase. The type of nutrients available defines the internal metabolic flow,
while their abundance often limits the rate of biomass production and the energy
available for growth [17].
The use of the various nitrogen sources is highly regulated. It requires the
synthesis of specific catabolic enzymes and permeases, which are subject to nitrogen
catabolite repression. The de novo synthesis of permeases and catabolic enzymes is
controlled at the level of transcription, and often requires two distinct positive signals.
A global signal indicates nitrogen derepression, and a second, pathway-specific

signal indicates the presence of a substrate or of an intermediate of the pathway.
This control permits the selective expression of enzymes for a specific catabolic
pathway from many potential candidates within the nitrogen regulatory circuit.
However, some systems are controlled only by nitrogen metabolite repression and
not by induction [16].
The cell yield of yeasts seemed to be unaffected by the presence of lactic acid
bacteria in sourdough. Saccharomyces cerevisiae grown as a monoculture in dough
attained 8 log CFU/g [18]. Almost the same value of cell count was found under
co-culture with lactic acid bacteria. Conversely, the maximum specific growth rate
was negatively affected. According to Meroth et al. [6], cell counts of endogenous
yeasts of ca. 7.5 log CFU/g were reached during the first fermentation period at
30°C, with a dough yield of 200, and remained stable until the end of fermentation.
During fermentation of traditional sourdough products (e.g. Panettone or Colomba),
the total yeast counts reached the level of 8–8.5 log CFU/g, after addition of baker’s
yeast [19]. Yeast cells may encounter different environmental states. Maintaining
optimal functionality in the presence of such external variability is a central evolu-
tionary constraint. Feedback mechanisms directly link gene expression with internal
need. Physiological variables such as the rate of biomass production, the cellular
pools of nutrients or the energy feedbacks, to properly tune gene expression with the
corresponding functional needs, are part of these strategies [17].
Yeast cells starved for particular amino acids or treated with an inhibitor of protein
synthesis (cycloheximide) may exhibit a phenomenon referred to as the stringent
response. In S. cerevisiae, the stringent response results in a rapid inhibition of the
synthesis of rRNA (but not of tRNA or mRNA) and it is thought to be signalled
when cells sense a rapid decrease of the overall rate of protein biosynthesis. Nitrogen
compounds and, especially, amino acids are not the limiting factors for yeasts.
Saccharomyces cerevisiae and the other species, which naturally occur in sour-
doughs, are prototrophic for amino acids. Figure 6.2 shows the concentration of
various free amino acids during fermentation of kamut flour with baker’s yeast and
sourdough. Compared to the dough leavened with baker’s yeast, the fermentation by
sourdough lactic acid bacteria caused an increase of some amino acids. Fermentation
with baker’s yeast resulted in the decrease of free amino acids due to yeast metabo-
lism (see below). Because of the high value of pH during fermentation with yeasts
alone, the activation of flour endogenous proteinases is poor, and the concentration
of free amino acid increases only after the yeasts have reached the stationary phase
of growth [20].
Co-fermentation with lactic acid bacteria and yeasts determines environmen-
tal fluctuations in terms of availability of nutrients, synthesis of organic acids,
pH decrease and changes of the rheological properties of sourdough. Brandt
et al. [21] evaluated the effect of process parameters on the growth and metabo-
lism of L. sanfranciscensis and C. milleri during rye sourdough fermentation.
pH did not affect the growth of the yeast in the range 3.5–5.5, whereas the growth
of L. sanfranciscensis was inhibited at pH 4.0. The concentration of NaCl of 4%
(wt/wt of flour) inhibited the growth of L. sanfranciscensis but not that of C. milleri.
Fig. 6.2 Difference in the content (g/100 g) of various amino acids in ripe dough (K1) and
sourdough (SK1) of kamut flour
The effect of such fluctuations on the synthesis of lactate, acetate, ethanol and
CO2 agreed with that on growth. Gänzle et al. [7] used a predictive model to
describe the effect of temperature, pH, organic acids and NaCl on the growth of
L. sanfranciscensis and C. milleri. A basic metabolic activity was also found
under conditions where growth was inhibited.
6.2.1 The Yeast Carbohydrate Metabolism
Yeasts are classified as facultative anaerobes, i.e. they are capable of both fermentation
and respiration. When oxygen is unavailable yeasts carry out fermentation. Under
respiration, the hydrogen (electrons) from NADH + H+ is passed to oxygen in the
electron transport chain yielding approximately 2.5 ATPs per NADH + H+. During,
fermentation the hydrogen (electrons) is passed on acetaldehyde to form ethanol
yielding no ATPs per NADH + H+.
Yeasts accumulate energy in the form of ATP from different organic compounds
by using different metabolic pathways. Most yeasts use carbohydrates as their main
carbon and energy sources, but there are also yeasts that may use non-conventional
carbon sources. The preferred energy source for yeasts is glucose, and glycolysis is
the general pathway for conversion of glucose to pyruvate, whereby the synthesis
ATP is coupled to the generation of intermediates and reducing power in the form
of NADH for the biosynthetic pathways. Yeasts respire when oxygen is present and
the repression absent; pyruvate enters into mitochondria where it is oxidatively
Fructose
Fructose
Fru-6-P
Fru-1,6-P
Fba1
Dihydroxyacetonephosphate Glyceraldheyde-3-phosphate
NADH
Gut2 Gdp1, Gdp2
NAD Dak1, Dak2
Glycerol-3-phospate
Fps1 Gut1 Gpp1, Gpp2
+ NAD(P)H
NAD(P)
Glycerol Dihydroxyacetone
Ypr1, Gcy1
Glycerol
Gup1, Gup2
Fig. 6.3 Part of the glycolytic pathway and pathways for production of glycerol. Expression of the
genes encoding the proteins that are underlined is stimulated after osmotic shock
decarboxylated to acetyl CoA by means the pyruvate dehydrogenase multi-enzyme

complex. This reaction links glycolysis to the citric acid cycle, where acetyl CoA is
completely oxidised to give two moles of CO2 and reductive equivalents in the form
of NADH and FADH2. The citric acid cycle is an amphibolic pathway, as it combines
both catabolic and anabolic functions. This latter function is the consequence of the
production of intermediates for the synthesis of amino acids and nucleotides.
During alcoholic fermentation, yeasts re-oxidise NADH to NAD in a two-step
reaction starting from pyruvate. The first reaction is via decarboxylation through
pyruvate decarboxylase, followed by the reduction of the acetaldehyde to ethanol,
catalysed by the alcohol dehydrogenase (ADH). Simultaneously, glycerol is generated
from dihydroxyacetone phosphate to ensure, with an alternative pathway, the regen-
eration of NAD according to the mechanism described in Fig. 6.3.
Generally, the level of glycerol produced is ca. 0.03 mmol/g of sourdough.

Paramithios et al. [18] reported that the synthesis of glycerol is positively affected
by the co-cultivation of S. cerevisiae with L. sanfranciscenis and L. brevis. Exposure
of baker’s yeast to salt stress also increases glycerol production [22].
The glycolytic pathway and related enzymes were conserved during evolution,
even though the mechanisms of controlling the carbon and energy metabolism have
been adapted to the needs of each species. Saccharomyces cerevisiae switches to a
mixed respiro-fermentative metabolism, resulting in ethanol production, as soon as the
external glucose concentration exceeds 0.8 mmol/kg [23]. Hence, S. cerevisiae controls
fermentation versus respiration primarily in response to the concentration of glucose.
The aerobic synthesis of ethanol by S. cerevisiae depends on the relative capacities of
the fermentative and respiratory pathways. High glucose levels result in the glycolytic
rate exceeding that of the pyruvate dehydrogenase (Pdh) reaction, thereby generating
an overflow towards pyruvate decarboxylase (Pdc) and, hence, ethanol production. At
low external glucose levels and in the presence of oxygen, S. cerevisiae does not
synthesise ethanol [24]. The uptake of glucose into S. cerevisiae is controlled by multiple
hexose transporters (Hxts) [25], which have different substrate specificity and affinity,
and are expressed under different, overlapping conditions [26]. When S. cerevisiae is
exponentially growing under aerobic conditions with glucose or fructose as carbon
sources, glucose degradation proceeds mainly via aerobic fermentation. When yeast is
growing under aerobic conditions on mannose or galactose, degradation proceeds
simultaneously via respiration and fermentation.
Control of carbohydrate metabolism in yeasts is of both fundamental and practical
significance. It is regulated by different mechanisms depending on the genus and
species as well as on environmental conditions. These mechanisms have been called
Pasteur, Kluyver, Custers and Crabtree effects, glucose or catabolite repression, and
genera or catabolite inactivation [27]. Table 6.1 describes these regulatory phenomena
in S. cerevisiae and other genera, including also the species occurring in sourdough.
The term Crabtree effect defines the inhibition of the synthesis of respiratory
enzymes in the presence of oxygen and high concentration of glucose. Candida and
Pichia, which are frequently associated with sourdough, do not show the Crabtree
effect. Depending on environmental conditions, especially, the type of carbon
sources, yeasts adjust the energy metabolism according to a process referred to as
carbon catabolite repression. Two extreme cases are exponential growth on glucose
or ethanol, which lead to the almost exclusive fermentation of the former with
extensive secretion of ethanol or to exclusive respiration to carbon dioxide, respec-
tively. Both, fully respiratory and fermentative metabolism are mediated by differ-
entially active transcription factors. During fermentation, the transcription factor
complex of Tup1p, Ssn6p and Mig1p mainly represses the expression of respiratory,
gluconeogenic and alternative carbon source utilisation genes [28]. The minimal
activity of the citric acid cycle for biosynthetic purposes is ensured mainly by the
Rtg transcriptional activators [29]. During respiratory growth on non-fermentable
carbon sources, the respiratory genes of the citric acid cycle and the respiratory
chain are highly induced. This is triggered by the Hap transcription factors, a global
activator complex of respiratory genes [29]. Activation of genes for gluconeogenesis
during growth on non-fermentable carbon sources is achieved by the transcriptional
Table 6.1 Regulatory phenomena (Adapted from Barnett and Entian [27])
Yeast species and
Name What happens genera Underlying factors
Pasteur effect Sugar used faster S. cerevisiae Oxidised cytochrome
anaerobically than inactivates
aerobically 6-phosphofructokinase
Custers effect D-glucose is fermented Brettanomyces and Much acetic acid
to ethanol and CO2 Dekkera spp. is produced via an
faster in aerobic than NAD+-aldehyde
in anaerobic conditions dehydrogenase.
Consequently,
anaerobically, the high
NADH:NAD+ ratio
inhibits glycolysis
Kluyver effect Ability to use Candida spp., Pichia Probably caused mainly
oligosaccharide or spp. by slower uptake of
galactose sugar anaerobically
aerobically, but not
anaerobically, although
glucose is fermented
Crabtree effect Adding glucose to tumour Decrease of ADP
cells lowers the concentration in
respiration rate mitochondria
activators Cat8p and Sip4p [28–31]. These are the key elements of the yeast
transcriptional regulatory network for the extreme cases of fermentative and fully
respiratory growth [32].
Saccharomyces cerevisiae carries out respiration and fermentation simultane-
ously at high growth rates, even under fully aerobic conditions. This phenomenon
occurs under chemostat culture conditions by the shift from biomass formation
towards fermentative products at increased dilution rate. The Crabtree effect has
important consequences for industrial processes aiming at producing yeast biomass,
where the formation of fermentative by-products is undesired. Goddard [33]
emphasised the importance of the Crabtree effect on the competitive advantage of
S. cerevisiae during growth in ecosystems characterised by elevated concentrations
of carbohydrates. The competitive advantage is lacking, when fermentable carbohy-
drates are limiting as occurs in the sourdough ecosystem.
An alternative route for glucose oxidation is the hexose phosphate pathway, also
known as the pentose phosphate cycle, which provides the cell with the pentose
sugars and cytosolic NADPH necessary for biosynthetic reactions. The second step
of this pathway is the dehydrogenation of glucose-6-phosphate to 6-phosphoglu-
conolactone and the generation of one mole of NADPH (by glucose-6-phosphate
dehydrogenase). Subsequently, 6-phosphogluconate is decarboxylated via the activity
of phosphogluconate dehydrogenase to ribulose-5-phosphate and a second mole of
NADPH is generated. Besides generating NADPH, the other major function of this
pathway is the production of ribose sugars, which serve for the biosynthesis of
nucleic acid precursors and nucleotide coenzymes. The reduced carriers, NADH
and FADH2, are reoxidised in the respiratory chain located in the inner mitochon-
drial membrane. The energy released during the transfer of electrons is coupled to
the process of oxidative phosphorylation via ATP synthase, an enzyme complex
also located in the inner mitochondrial membrane and designed to synthesise ATP
from ADP and inorganic phosphate.
For the majority of industrial fermentations with bakers’ yeast, the elevated capac-
ity to ferment available carbohydrates is an important characteristic, especially when
the biomass is exposed to high sugar concentrations and/or absence of oxygen.
6.2.2 Stress Response in Sourdough Yeasts
Yeasts are exposed to constant fluctuations of their growth conditions. Consequently,

they have to develop sophisticated responses to adapt to and survive under a variety
of conditions. Yeasts, as well as other organisms, employ a concerted response to
external stress [34]. One mechanism that yeast cells use to protect the cell from the
effects of environmental variation is to initiate a common gene expression program.
This program includes about 900 genes, whose expression is stereotypically altered
when yeast cells are shifted to stressful environments. The genes that participate in
this response amount to almost 14% of the currently predicted genes in the yeast
genome [35].
The “general stress” transcription factor could be identified in the zinc-finger
transcription factor (Msn2) [14]. Normally, Msn2 is exported from the nucleus, and
a cyclin-dependent kinase (Srb10) is concomitantly repressed. Under stress, Msn2
re-localises to the nucleus and, with the relief of Srb10 repression, activates tran-
scription. The stress response is rapid, but quickly attenuated. Bose et al. [36]
showed that this attenuation is caused by a nuclear-dependent degradation of Msn2.
Msn2 rapidly disappeared from cells after heat or osmotic shock.
Process parameters, including temperature, dough yield, oxygen, pH, as well as
the composition of starter cultures, determine the quality and handling properties of
sourdough [37] and the metabolic response of microorganisms responsible for the
fermentation process [38]. The exposure of microbial cells to stressful and fluctuating
conditions during fermentation involves a broad transcriptional response with many
induced or repressed genes. The selective pressure exerted by environmental condi-
tions encountered by yeast cells during sourdough fermentation, accounts for the
consolidated dominance of selected yeast species. Nutrient availability likely modu-
lates the microbial ecology of sourdough. However, within the sourdough ecosys-
tem there are numerous mechanisms whereby one species may influence the growth
of another [38]. Although autochthonous bacteria and yeasts are adapted and com-
petitive in their respective environment, the dough environment can be described as
a stressful environment for microorganisms [39].
The conditions of the sourdough microenvironment that principally affect yeast
responses and growth rate are: nutrient availability (starvation), pH (acid stress),
dough yield and presence of sugars, salts and polysaccharides (osmotic stress),
oxygen (oxidative stress), temperature fluctuations (heat shock and cold stress) and
interactions between lactic acid bacteria and yeasts (e.g. S. cerevisiae, C. milleri and
L. sanfranciscensis), and between yeasts (e.g. S. cerevisiae and C. milleri).
6.2.2.1 Low Temperature
In bakery practice, the temperature of the sourdough is an important parameter to

control the growth of lactobacilli and yeasts [21]. Häggman and Salovaara [40]
studied the effect of process parameters, including low temperature, on the leaven-
ing of rye dough. Under the experimental conditions, the endogenous C. milleri was
responsible for leavening, especially when the temperature was set at 22°C. The low
temperature of fermentation also slowed the acidification rate, thus favouring an
extended production of CO2 by C. milleri. An appropriate modulation of the tem-
perature and the use of temperatures ranging from 4°C to 8°C is a practical approach
to control the fermentation rate and to program the working times.
The effects of the low temperature and other environmental parameters on yeast
physiology are conditioned by the exposure dynamics [41]. Transcriptional
responses during adaptation to suboptimal temperatures permitting growth (10–
20°C) [42, 43] differed from those found after exposure to temperatures below
10°C, where growth ceases [44, 45]. Two different mechanisms of response are
distinguished: (1) the early cold response (ECR), occurring within 12 h; and (2) the
late cold response (LCR), occurring later than 12 h [43].
Tai et al. [41] used chemostat cultivation to compare different culture conditions
and/or microbial strains at fixed specific growth rate. They observed 15% of the
genes that showed a consistent transcriptional response in previous batch-culture
studies on cold adaptation [42, 43, 45] were also identified under chemostat condi-
tions at 30°C [46]. In batch cultures, the exposure of S. cerevisiae to low tempera-
tures invariably induces an increased synthesis of storage carbohydrates (especially
trehalose) and transcriptional up-regulation of genes involved in storage carbohy-
drate metabolism [47]. Transcriptional induction of the trehalose-biosynthesis genes
TPS1 and TPS2 is consistently observed in cold-shock studies, and after exposure
to near-freezing conditions. Several other genes such as HSP12, HSP26, HSP42,
HSP104, YRO2 and SSE2, and those encoding the three cell-wall mannoproteins
(Tip1p, Tir1p, and Tir2p), fatty-acid desaturase (Ole1p), which influences the mem-
brane fluidity, and Nsr1p, a nucleolar protein required for pre-rRNA processing and
ribosome biogenesis, were consistently associated with cold shock [41, 43, 45]. The
stress response element (STRE) binding factors Msn/Msn4 are implicated in the
coordinate regulation of low-temperature-responsive genes [43, 47]. Consistently,
many genes induced upon a temperature downshift were also induced under a vari-
ety of other stress conditions [43]. Contrarily to batch cultures, where the low tem-
perature adaptation is accompanied by marked ESR (Environmental Stress
Response) (29% of the differentially expressed genes in batch cultures at low tem-
perature respond to ESR as well), only three ESR-induced genes (YCP1, VPS73,
Fig. 6.4 Yeast recovery ability in terms of volume increase of a dough inoculated with the selected
strain (P6). The dough was initially stored for 7 days at 4°C (yellow square), and then incubated at
35°C
and EMI1) showed higher transcription at 12°C under chemostat cultures.

Conversely, 88 ESR-induced genes showed a consistently lower transcript level at
12°C [41]. Studies on cold adaptation in batch cultures of S. cerevisiae revealed a
clear transcriptional up-regulation at low temperature of chaperone-encoding genes
such as HSP26 and HSP42 [43, 44]. The proteins encoded by these genes prevent
the aggregation of cytosolic proteins during heat shock [41].
In bakery practice, refrigeration of dough or sourdough is used to control
fermentation. Under the refrigerated conditions, yeasts have to maintain and then to
recover their fermentative capacity in a very short period of time (15–30 min). At
4–8°C, many yeast strains continue to ferment at a slow rate and induce a slight
increase of the dough volume. The fermentation stops at 4°C. Only selected strains
recover their leavening capacity when the dough temperature is raised to 28–35°C
(Gottardi et al. unpublished data) (Fig. 6.4). As shown in Fig. 6.4, these strains
increased the dough volume within 15 min also when the dough had been kept for
7 days at 4°C.
6.2.2.2 Acidity
The acidity of the sourdough depends on lactic and acetic acid production by lactic
acid bacteria [40]. Usually, low temperatures of sourdough fermentation delay the
lactic acidification and decrease the time of yeast exposure to high acidity. A low
fermentation temperature was suggested as a means to improving the synthesis of
CO2 by yeasts [40].
Because only the non-dissociated acids diffuse into the cell, the type of acid
more than the pH determines yeast inhibition [49]. Mainly the level of dissociation
of acetic acids affects the leavening capacity [40]. The non-dissociated form of the
acetic acid inhibits yeasts by acidifying the cytoplasm, which causes physiological
stress or suppresses metabolic activity [48]. This especially happens when the pH of
the sourdough is below the pKa of acetic acid, 4.76. Non-dissociated acetic acid
decreased the leavening capacity of C. milleri when grown together with the hetero-
fermentative L. brevis compared to homofermentative species. Candida milleri
adapts to a wide range of pH (3.5–6.0) and has a good inherent acid tolerance, the
leavening activity is obviously affected by the fermentation process [7].
The leavening power of baker’s yeast is strongly influenced by the environmen-
tal conditions of storage [49]. Pre-treatment at 30°C with organic acids (malic,
succinic and citric acids), under a wide range of pH values, was assayed before
use [49]. The treatment with organic acids variously increased the fermentative
activity. When the pH of baker’s yeast, containing citric acid, was raised from 3.5
to 7.5, both the fermentative and maltase activities increased. Glycerol-3-phosphate
dehydrogenase activity and the levels of internal glycerol also increased in the
presence of citrate. On the contrary, baker’s yeast containing succinic acid at pH
7.5, showed a decreased viability during storage, despite the maintenance of high
fermentative activity.
6.2.2.3 Osmotic Stress
Aw is defined as the chemical potential of the free water in solution. Low and interme-
diate values of Aw limit the growth of yeasts. The Aw of the cytosol of yeast cells is
lower than that of the surrounding medium, corresponding to a higher osmotic pres-
sure (turgor pressure). This turgor pressure drives water into the cell based on the
concentration gradient. Turgor pressure is counteracted by the limited ability for
expansion of the cell wall and thus determines the shape of the cells [50]. The ability
to survive under rapid changes of Aw is an intrinsic characteristic of the microbial cell.
Survival mechanisms in response to osmotic downshift or upshift allow passive water
loss or uptake. In response to altered osmolarity, yeasts cells develop mechanisms to
adjust to high external osmolarity and to maintain or to recover an inside-directed
driving force for water adaptation. These mechanisms are based on sensing the
osmotic changes [50] and accumulating chemically inert osmolytes, for example
glycerol. The high osmolarity glycerol (HOG) signalling system plays a central role
in the osmotic adaptation of yeasts. Saccharomyces cerevisiae monitors osmotic
changes through the sensor histidine kinase (Slu1) localised at the plasma membrane.
Under optimal environmental conditions, Slu1 is active and inhibits signalling. Upon
loss of turgor pressure, Slu1 is inactivated, and this results in the activation of the
nitrogen-activated protein (NAO) kinase cascade and phosphorylation of the NAP
kinase (Hog1). Active Hog1 accumulates in the nucleus, where it affects gene
expression. Two HOG target genes encode for enzymes involved in the synthesis of
glycerol. Because of the presence of three hydroxylic functions that attract water
clouds, glycerol serves as an osmolyte to increase the intra-cellular osmotic pressure
[50]. Since glycerol is more reduced than the substrate glucose, its s ynthesis also
affects the redox metabolism. Therefore, the redox metabolism needs to be adjusted
accordingly. The pathway for the synthesis of glycerol is shown in Fig. 6.3. The
NAD-dependent glycerol-3-phosphate dehydrogenase (Gpd) and the glycerol-3-
phosphatase (Gpp) catalyse the two step reactions. Saccharomyces cerevisiae also
possesses genes that might encode the enzyme glycerol dehydrogenase (GCY1 and
YPR1) and the dihydroxyacetone kinase (DAK1 and DAK2). These two enzymes are
involved in the pathway for glycerol degradation [50]. The pathway for glycerol via
Gpd and Gpp converts NADH to NAD, while the conversion of glycerol to dihy-
droxyacetone phosphate via Gcy1p and Dak1p reduces NADP to NADPH. Hence, the
glycerol-dihydroxyacetone phosphate cycle mainly acts as transhydrogenase for the
interconversion of NADH to NADPH. Since stress conditions require high levels of
NADPH to manage reactive oxygen species, this pathway assumes an important role.
The capacity for other NADPH-generating reactions, such as those of the pentose
phosphate pathway, is also increased under stress conditions. When salt-stressed yeast
is inoculated in dough with high sugar content, the fermentation time was significantly
reduced and bread-specific volume increased due to glycerol accumulation and a less
dense gluten network. Moreover, two-step industrial fermentation, including a pre-
adaptation to osmotic stress, in order to enhance flavour, has been proposed [51].
6.2.2.4 Membrane Lipids as Modulators of Stress Tolerance in Yeasts
Biological membranes are the barrier that separates cells from their environment
and are the primary target for damage during environmental stress. Sudden changes
of environmental conditions cause alteration in the organisation and structure of
membrane lipids, and alter the function of many cellular activities. Homeoviscous
adaptation to low temperature maintains the molecular order of membrane lipids as
well as the activity of membrane-associated enzymes and transporters. To date,
most of the research in this field has focused on the connection between the physical
state of the membrane and cold and ethanol tolerance. In organisms producing etha-
nol, including yeasts, intra-cellular ethanol freely diffuses into an external medium.
At high concentration, the ethanol present in the external medium acts as a chemical
stress. A long-standing conundrum is the mechanism by which cells growing in the
presence of a high concentration of ethanol modify their membrane composition.
The plasma membrane lipid is the main site of impediment and interaction with
ethanol. The altered lipid composition, following the exposure to ethanol, combats
the deleterious effect. The ethanol-dependent modification of phospholipids’ fatty
acid composition was also shown in S. cerevisiae [52]. The addition of ethanol
(0.5–1.5 M) to S. cerevisiae leads to a progressive decrease of the proportion of
saturated fatty acids (SFAs) (mainly 16:0) and to a corresponding increase of the
mono-unsaturated fatty acid residues, especially 18:1. This increased unsaturation
favours an increase of the fluidity of the membrane. On the basis of similar observa-
tions in Escherichia coli, it was suggested that increased unsaturation was not due
to the additional synthesis of unsaturated fatty acids (UFAs), but was the result of
the decrease of levels of SFAs. An exception to this hypothesis was the increase of
18:0 in the presence of ethanol [52–54]. It is assumed that the presence of ethanol
Table 6.2 Total cell fatty acid composition in 48 h S. cerevisiae cells in relation to different
incubation temperatures (15, 20, 30, and 40°C) [55]
Strain
S. cerevisiae BG7FI S. cerevisiae 635
Temperature (°C) Temperature (°C)
Fatty acida 15 20 30 40 15 20 30 40
C6:0 0.00 0.38 0.02 0.00 0.00 0.00 0.00 0.00
C8:0 0.00 0.46 0.21 0.06 0.00 0.16 0.00 0.33
C10:0 1.44 6.47 2.05 0.49 1.06 2.55 6.06 0.76
C10:1 0.00 0.54 0.00 0.00 0.00 0.27 0.00 0.00
C12:0 3.72 7.86 8.11 3.72 3.43 8.59 18.21 3.09
C12:1 0.11 0.61 0.93 0.16 0.24 0.57 0.76 0.37
C14:0 4.02 2.08 1.52 1.63 5.28 4.45 3.56 1.90
C14:1 0.81 0.76 0.62 0.23 1.78 1.57 1.25 0.20
C16:0 33.19 37.05 41.28 38.34 25.84 32.57 33.77 33.73
C16:1 33.62 26.13 25.06 30.46 37.26 28.85 19.47 30.67
C18:0 8.13 8.73 11.19 12.83 6.00 7.17 8.55 11.31
C18:1n9 12.41 8.29 7.82 9.67 17.17 12.50 7.03 15.34
C18:1n11 0.44 0.24 0.51 2.01 0.48 0.35 0.25 1.64
C18:2 2.12 0.39 0.68 0.42 1.47 0.41 1.09 0.64
Unsaturation 0.51 0.37 0.36 0.43 0.59 0.45 0.31 0.49
degreeb
C16:1/C16:0 1.01 0.70 0.61 0.79 1.44 0.88 0.58 0.91
C18:1/C18:0 1.58 0.98 0.74 0.91 2.94 1.79 0.85 1.50
Chain lengthc 16.16 15.46 15.86 16.27 16.15 15.74 15.12 16.33
a
Expressed as a percentage of total fatty acids
b
D/mol = [1(mol%monoenes)/100 + 2(mol%dienes)/100 + 3(mol%trienes)]/100
c
S(PC)/100, where P is percentage of a fatty acid and C is its carbon atom number
on both sides of the plasma membrane causes the separation of the two monolayers
and the concurrent increased synthesis of C18 fatty acid residues, which helps in the
maintenance of membrane integrity [52].
The decrease of the degree of fatty acid unsaturation along with the temperature
increase is widely documented. The incorporation of exogenous oleic acid has been
reported to increase the tolerance of yeasts to the lethal effect of temperatures
between 41°C and 47°C [55]. Table 6.2 shows the modulation of the membrane
fatty acid composition of two strains of S. cerevisiae in a temperature range from 15
to 40°C [55]. For both strains, the relationship between unsaturation level and tem-
perature showed two maximum values at 15 and 40°C. The latter corresponds to the
maximum fermentation temperature for these strains. These results suggest that the
role of the unsaturation level has to be considered not only in terms of the contribu-
tion to membrane fluidity when temperature decreases but also as a mechanism to
protect the cell membrane from damage generated by oxidative and thermal stresses
[55, 56]. It was postulated that an oxygen-consuming desaturase prevents the accu-
mulation of oxygen and reactive oxygen species such as H2O2 in yeasts. During
sourdough fermentation, yeasts are exposed to H2O2 [57]. It has been observed that
the co-inoculation of S. cerevisiae and L. sanfranciscensis into dough gives rise to
the release of medium-chain fatty acids and of 2(5H)-furanones presumably

originating from peroxidation of membrane-associated UFAs, followed by a
sequence of b-oxidation reactions [58, 59]. The biosynthesis or integration of UFAs
in the yeast membrane is reported as a mechanism to detoxify H2O2 and protect
cells from oxidative stress [59] and this has already been observed in L. helveticus
[60]. ROS with consequent peroxidation of membrane fatty acids has been observed
in S. cerevisiae after a cycle of freezing/thawing [61].
Sterols are membrane-associated lipids that play an important role in the tolerance
to physico-chemical stresses including ethanol exposure. The ability of sterols to
increase stress tolerance is well documented [62]. Oxygen is required for the cycliza-
tion of squalene to sterols. Thus, the presence of oxygen during the early phase of
yeast fermentation is considered a limiting factor for growth and fermenting due to
the membrane-stabilising effect of sterols. Also the synthesis of UFAs is prevented by
anaerobic conditions. When sterol and UFA biosynthesis is prevented by anaerobic
conditions, yeasts integrate sterols and UFAs from the fermentation media in their
membranes. Cereals contain a large variety of nutrients including phytosterols, which
serve as a source of membrane sterols for yeasts also in anaerobic conditions.
6.3 Minor Yeast Metabolites in Sourdough
In addition to ethanol and CIO2, yeasts generate a large spectrum of metabolites

during sourdough fermentation. Yeasts are responsible for characterising the aroma
profile, or so-called “fermented taste” in the production of bread and alcoholic
beverages. This profile consists of a complex mixture of flavour compounds but the
characteristic impact on flavour is determined mainly by fusel alcohols and their
derivatives. Commonly known fusel alcohols are 3-methyl-1-butanol, 2-methyl-1-
butanol and 2-methyl-1-propanol. When ammonia is used as the nitrogen source,
fusel alcohols can be synthetized via the isoleucine, valine and leucine (ILV) pathway
[63]. However, their concentration drastically increases when branched chain amino
acids (BCAAs) are present in the media. Yeasts convert free amino acids mainly by
the Ehrlich pathway [64] (Table 6.3). The Ehrlich pathway assumes the conversion
of the BCAAs to fusel alcohols by three enzymatic steps. The first step is a transam-
inase, in which the amino group of BCAAs is transferred to 2-oxoglutarate, resulting
in branched chain oxoacids and glutamate. The second step is the decarboxylation
step that converts the branched chain oxoacid to the branched chain aldehydes. The
last step is the reduction step in which branched chain aldehydes are reduced to
branched chain alcohols or so-called fusel alcohols ([65] Fig. 6.5). For instance,
S. cerevisiae and other sourdough yeasts convert leucine and phenylalanine to
3-methylbutanol and 2-phenylethanol, respectively [64]. The Strecker reaction during
baking also generates a-dicarbonyl compounds such as methylglyoxal
(2-oxopropanal), which leads to the corresponding aldehydes and acids [66].
The presence of yeasts (Saccharomyces and Hansenula genera) during sourdough
fermentation favoured an increased synthesis of alcohols, esters and some carbonyl
compounds compared to sourdough fermentation without the addition of yeasts [67].
170
Table 6.3 Higher alcohols and their precursor amino acids

Amino acids Ketoacids Aldehydes Organic acids Alcohols Esters
Ile a-Keto-3-methyl- 2-Methylbutanal 2-Methyl butyric acid 2-Methylbutanol
pentanoic acid
Leu a-Ketoisocaproic acid 3-Methylbutanal; 3-Methylbutyric acid 3-Methylbutanol Ethyl-3-methylbutanoate
2-Methylpropanal 2-Methyl propanoic 2-Methylpropanol
acid
Val a-Ketoisovaleric acid 2-Methylpropanal 2-Methyl propanoic acid 2-Methylpropanol Ethyl isobutanoate
Phe Phenyl pyruvate Benzaldehyde Benzoic acid Phenylmethanol Ethyl benzoate
Phenylacetaldehyde Phenylacetic acid Phenylethanol Phenylethyl-acetate
Trp Indole-3-pyruvate Indole-3-acetaldehyde Indole-3-acetic acid
Met a-Keto methylthio Methional Methylthiobutyric acid Methionol Ethyl-3-methylthio
butyrate propionate
Methylthio-acetaldehyde Methanethiol Methylthioacetate
M.E. Guerzoni et al.
Fig. 6.5 Schematic representation of metabolic routes of L-leucine in yeasts, leading to 4-methyl-2-
oxopentanoate, 3-methyl-1-butanol and 3-methylbutyrate (Adapted from [65])
Especially, the levels of ethanol, methylpropanol, 2- and 3-methylbutanol as well as

of ethyl acetate and diacetyl were considerably increased in sourdoughs with the
addition of yeasts. Diacetyl is synthesised either by lactic acid bacteria or yeasts
[68]. Although it is difficult to distinguish between the contribution of the two
groups of microorganisms, processing parameters such as high water content and
temperature may favour the growth of yeasts [69–71] and, consequently, their con-
tribution to the synthesis of volatile compounds during fermentation. There was a
clear difference between the volatile compound profiles of wheat dough fermented
with Dekkera bruxellensis compared to S. cerevisiae. Contrarily to S. cerevisiae,
Dekkera sp. did not generate the same levels of 3-methylbutanol but formed high
concentrations of esters such as the odour-intense ethyl 2-methylbutanoate [72].
Table 6.4 shows the metabolites synthesised by L. sanfranciscensis and S. cerevisiae
in doughs with different dough yields [51]. When the dough was inoculated with
L. sanfranciscensis, isobutanol, acetoin, 1-hexanol, ethyl octanoate and butyric acid
were found. When the dough was inoculated with S. cerevisiae at a dough yield
value of 146, the highest synthesis of volatile compounds was found, which included
alcohols and short- and medium-chain fatty acids.
6.4 Quorum Sensing on Sourdough Yeasts
Several reports have shown quorum sensing-like phenomena in fungal species. The
morphological transition, from the filamentous and mycelial form to the yeast form,
or vice versa, was always found. The regulation of the switch between the filamentous
Table 6.4 Selected metabolites produced by L. sanfranciscensis and S. cerevisiae inoculated in

dough with various dough yields (data expressed as peak chromatographic area) [51]
L. sanfranciscensis S. cerevisiae
DY 220 DY 146 DY 220 DY 146
Compound
Ethanol 7.72 7.80 8.10 8.34
Isobutanol 6.19 7.46 7.15 7.31
Isoamyl alcohol 7.60 7.47 7.77 7.97
Acetoin 6.42 6.69 5.63 6.19
2,3-Butanediol n.d. n.d. 5.55 5.35
Butyric acid 6.42 6.48 6.59 6.57
Ethyl decanoate n.d. n.d. 6.12 5.44
Ethyl-9-decenoate 5.78 6.29 n.d. n.d.
Hexanoic acid 7.64 7.34 6.79 6.95
Phenylethanol 6.23 6.28 7.87 7.93
Ethyl hexadecanoate n.d. n.d. 6.17 6.41
Standard deviation values of the three repetitions in this experiment were less than 10%
n.d. Below detection limit
and yeast forms of the parasitic fungus Histoplasma capsulatum provided the first
example of an apparent quorum-sensing mechanism in eukaryotes [73].
Saccharomyces cerevisiae may show the morphological transition from the yeast
to the filamentous forms in response to environmental cues. Growth under nitrogen-
limiting conditions is an example, which activates several signal transduction
pathways. One of these routes is the Ras-cAMP-dependent protein kinase (PKA)
pathway. Chen and Fink [74] provided some intriguing molecular clues to PKA sig-
nalling. The conditioned medium of S. cerevisiae stationary-phase cultures markedly
induces the filamentous growth. Induction was partially related to the fivefold stimu-
lation of the transcription of FLO11, a gene essential for filamentous growth. The
active molecules were purified and were found to be phenylethanol and tryptophol, two
aromatic alcohols derived from phenylalanine and trytophan, respectively, which are
always present in yeast-fermented foods. The addition of both alcohols causes the
more vigorous filamentous growth and the induction of FLO11. On the basis of the
bacterial paradigm, these compounds have the features of quorum-sensing molecules.
The synthesis of these molecules is the highest during the stationary phase of growth,
the addition of tryptophol induces the expression of genes (ARO9 and ARO10), which
are required to convert tryptophan to tryptophol, and mutants unable to synthesise these
alcohols show a markedly decreased filamentous growth and FLO11 expression.
Preliminary results on inter-species communication during sourdough fermenta-
tion have been reported [51]. As shown in Fig. 6.6, the exposure of C. milleri to the
cell-free wheat flour hydrolyzed (WFH) medium, previously inoculated with
L. sanfranciscensis, induced morphological changes and early autolysis of the yeast.
Figure 6.7 shows another possible effect of the inter-species signalling mecha-
nism, which is mediated by lactic acid bacteria metabolites. After 2 h of exposure to
Fig. 6.6 Cells of S. cerevisiae exposed for 4 h to cell-free liquid Wheat Flour Hydrolyzed (WFH)
previously inoculated with L. sanfranciscensis
Fig. 6.7 GC-SPME profile of volatile compounds produced by C. milleri when exposed for 4 h to
cell-free CM in which L. sanfranciscensis (LSCE1) had been incubated for 4 h in WFH with
(a) and without starch (b). 1 Ethanol, 2 isoamyl alcohol, 3 acetoin, 4 ethyl octanoate, 5 acetic acid,
6 1-octanol, 7 isobutyric acid, 8 butyric acid, 9 isovaleric acid, 10 ethyl-9-decenoate, 11 hexanoic
acid; 12 phenylethanol, 13 octanoic acid, 14 c-octalactone, 15 c decalactone, 16 decanoic acid, 17
ethyl-9-hexadecenoate, and 18 dodecanoic acid [51]
the cell-free wheat flour hydrosylate fermented L. sanfranciscensis, C. milleri

released metabolites belonging to the family of lactones. Glutamic acid was sug-
gested as the presumptive precursor of certain g-lactones in yeasts [75]. D-Lactones
were obtained from 11-hydroxy fatty acids during fermentation with S. cerevisiae,
Sporobolomyces, Geotrichuum and Candida spp.
6.5 Baker’s Yeast in the Bread-Making Industry
The production of baker’s yeast biomass represents a highly competitive multi-billion

dollar global industry. The large variety of bread-making processes and recipes used
around the world place considerable demands on baker’s yeasts. These demands
translate into technological and economic challenges for baker’s yeast industries.
During production and dough fermentation, cells of baker’s yeasts are exposed to
environmental stresses such as freeze-thaw, dehydration and oxidation. Damage to
cell macromolecules (e.g. proteins, nucleic acids and membranes) is avoided
through microbial adaptation and tolerance to multiple stress conditions [76].
6.5.1 Baker’s Yeast Production
The first stage in the production of baker’s yeast consists of growing the yeast from
a pure culture in a series of fermentation vessels. Baker’s yeast is recovered from the
last step of fermentation through centrifugation to concentrate the biomass.
Subsequently, filtration further concentrates the yeast cells. The collected pellet is
blended in mixers with small amounts of water, emulsifiers, and oil. Finally, the
mixed press cake is extruded, cut and either wrapped for shipment or dried to form
dry yeast.
6.5.1.1 Raw Materials
The species S. cerevisiae is used for producing compressed yeast. Different strains
of S. cerevisiae are required to produce each of the two dry yeast products, Active
Dry Yeast (ADY) and Instant Dry Yeast (IDY). Instant dry yeast is produced from a
faster-reacting yeast strain than that used for ADY. Cane molasses and beet molas-
ses are the principal substrates for yeast growth. Molasses contains 45–55% (wt/wt) of
fermentable carbohydrates (sucrose, glucose and fructose). The amount and type
of cane and beet molasses used depend on the types and costs, and on the presence
of inhibitors and toxins. Usually, a blend consisting of both cane and beet molasses
is used. Once the molasses mixture is blended, the pH is adjusted to 4.5–5.0 to prevent
bacterial growth. The substrate is clarified to remove sludge and is then sterilised
with high-pressure steam. After sterilisation, it is diluted with water and held in
tanks until use. Additional nutrients, including nitrogen, potassium, phosphate,
magnesium and calcium, with traces of iron, zinc, copper, manganese and molybde-
num, are added. Usually, nitrogen is supplied by adding ammonium salts, aqueous
ammonia or anhydrous ammonia. Phosphates and magnesium are added in the form
of phosphoric acid or phosphate salts and magnesium salts. Vitamins such as biotin,
inositol, pantothenic acid and thiamine are also required. The latter is added to the
feedstock, while the others are already present in the molasses malt.
6.5.1.2 Fermentation
Yeast cells are grown in a series of fermentations to decrease the synthesis of ethanol
and CO2. The fermentation is operated under aerobic conditions. The initial stage of
propagation takes place in the laboratory. A portion of the pure yeast culture is
mixed with molasses malt in a sterilised flask, and the yeast is allowed to grow for
2–4 days. The entire content of the flask is used to inoculate the first fermentation
vessel. Batch fermentations are carried out, where the yeast is allowed to grow for
13–24 h. Usually, one or two vessels are used for this stage of the process. The batch
fermentations are essentially a propagation of the flask fermentation, except for the
use of sterile aeration and aseptic transfer for the subsequent stage. Following pure
culture fermentations, the yeast mixture is transferred into an intermediate vessel
under batch or fed-batch conditions. The following fermentation is a stock fermen-
tation stage (Fig. 6.8). The content from the intermediate vessel is pumped into the
stock vessel, which is equipped for increasing feeding under aeration. This stage is
termed stock fermentation, since after the fermentation is completed, the yeast biomass
is separated from the bulk of the vessel through centrifugation, which produces a
stock of yeast for the next stage. The next stage, pitch fermentation, also produces a
stock of yeast. Aeration is vigorous, and molasses and other nutrients are increas-
ingly fed into the vessel. The liquor from this vessel is usually shared into several
parts for pitching the final trade fermentation. Alternatively, the yeast is separated
by centrifugation and stored for several days before use. The final trade fermenta-
tion has the highest degree of aeration. A large air supply is required and the vessels
are often started in a staggered fashion to reduce the size of the air compressors. The
initial fermentation lasts 11–15 h. After which the molasses have been fed into the
vessel, the liquid is aerated for 0.5–1.5 h to allow a further maturation of the yeast.
This step has a stabilising effect for the subsequent refrigerated storage. The content
of the yeast biomass increases stage by stage: ca. 120 kg in the intermediate vessel,
420 kg in the stock vessel, 2,500 kg in the pitch vessel, and 15,000–100,000 kg in
the trade vessel. Once the optimum quantity of yeast is grown, the biomass is recov-
ered from the final trade vessel by centrifugation. The centrifuged yeast biomass is
further concentrated by a filter press or rotary vacuum filter. The filter press forms a
filter cake containing 27–32% of solids. The rotary vacuum filter forms a cake with
ca. 33% of solids. The filter cake is then blended in mixers with small amounts of
water, emulsifiers, and cutting oils to form the end-product. The final packaging
steps vary depending on the type of yeast product.
Fig. 6.8 Process layout of the different commercial baker’s yeast formulas
For the compressed yeast formula, emulsifiers are added giving the yeast a white,
creamy appearance and inhibiting water spotting of the yeast cake. A small amount
of oil, usually soybean or cottonseed oil, is added to help the extrusion of the yeast
through nozzles to form continuous ribbons of yeast cake. The ribbons are cut, and
the yeast cake is wrapped and cooled to less than 8°C, being ready for the market
under refrigerated conditions. For dry yeast preparation, after filtration the biomass
is sent to an extruder, where emulsifiers and oils (different from those used for com-
pressed yeast) are added to texturise it and to help extrusion. After extrusion in thin
ribbons, the yeast is cut and dried under batch or continuous drying systems.
Following drying, the yeast is vacuum-packed or packed under nitrogen gas before
heat sealing. The shelf-life at room temperature is 1–2 years.
6.5.2 General Characteristics of Fresh and Dry Baker’s Yeast
Baker’s yeast consists of living cells of S. cerevisiae. To describe the characteristics

of baker’s yeast, two types of formula are considered: fresh and dry yeast.
6.5.2.1 Fresh Baker’s Yeast
Fresh baker’s yeast is commercialised as block or compressed yeast, granulated

yeast and liquid (cream) yeast. Compressed yeast is available in blocks with variable
consistency. The consistency varies from high plasticity (kneadable) to friable
(crumbly texture). Granulated yeast is in the form of small granules. Liquid yeast is
a suspension of yeast cells in water with a cream-like viscosity. The dry matter content
of fresh baker’s yeast varies depending on the formula, the fermentation performance,
and the consistency/friability. Liquid and compressed yeast with high plasticity and
friability have a dry matter content of 15–21, 27–31 and 30–35%, respectively. The
dry matter of granulated yeast is 31–37%. The nitrogen content of the dry matter has
a value of ca. 8.0%. The ash content of the dry matter is ca. 6%, and the value of pH
is usually ca. 5.0. Fresh baker’s yeast is suitable to be used directly in dough. As it
is a perishable product, it should be stored under refrigerated conditions.
6.5.2.2 Dry Baker’s Yeast
Dry baker’s yeast is sold as active dry yeast (ADY) and instant dry yeast (IDY).
ADY is characterised by spherical particles with a diameter of 0.2–3 mm. The yeast
is reactivated under warm water at ca. 38°C (not exceeding 45°C) and then used as
fresh yeast. IDY consists of porous cylindrical particles with a diameter of 0.5 mm
and length up to a few millimetres. Rehydration in water is not necessary and it may
be added directly to the flour. Depending on the activity and recipe, IDY is added to
the flour at a level that corresponds to one-third of that of fresh yeast. Direct contact
of the yeast with salt, fat or sugar has to be avoided to prevent osmotic stress and
dispersion during rehydration of the flour. Careful rehydration of the flour with
water at room temperature is needed. The dry matter ranges from 92–96 to 93–97%
for ADI and IDY, respectively. The density is 75–95 and 55–80% for ADI and IDY,
respectively. The nitrogen content of the dry matter has a value of ca. 8.0% and the
ash content of the dry matter is ca. 6%,with a pH of usually ca. 6.0.
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Chapter 7
Physiology and Biochemistry
of Lactic Acid Bacteria
7.1 Introduction
In the past decades, studies on the physiology and biochemistry of sourdough

lactic acid bacteria provided insight into the microbial ecology of sourdough as
well as the effect of the metabolic activity of lactic acid bacteria on flavor, texture,
shelf-life, and nutritional properties of leavened baked goods. Lactic acid bacteria
are the dominant microorganisms of sourdough. Their metabolic versatility favors
adaptation to the various processing conditions and the metabolic interactions with
autochthonous yeasts determine mechanisms of proto-cooperation during sour-
dough fermentation [1–3]. Lactobacillus species are most frequently found in
sourdough fermentations although species belonging to the genera Pediococcus,
Enterococcus, Lactococcus, Weissella and Leuconostoc were also identified ([4–6],
see Chap. 5). A large number of Lactobacillus species were first identified from
sourdoughs or fermentation processes of cereals [5]. This chapter gives an over-
view of the general growth and stress parameters, carbohydrate and amino acid
metabolism, synthesis of exopolysaccharides and antimicrobial compounds, and
the conversion of phenolic compounds and lipids of lactic acid bacteria during
sourdough fermentation.
M. Gänzle (*)
Department of Agricultural, Food and Nutritional Science, University of Alberta
Edmonton, Canada
M. Gobbetti

184 M. Gänzle and M. Gobbetti
7.2 General Growth and Stress Parameters
The large diversity of lactic acid bacteria associated with sourdough fermentation
(Chap. 5), is matched by a comparable diversity of general growth and stress parame-
ters. Different processes of sourdough fermentation select for organisms with different
growth parameters. Generally, type I sourdoughs, which are characterized by fre-
quent back-slopping to achieve leavening without addition of baker’s yeast, select
for organisms growing rapidly in cereal substrates. Under these conditions,
Lactobacillus sanfranciscensis is often found as the predominant organism. Type II
sourdoughs, which are characterized by long fermentation times and high fermenta-
tion temperatures, select for acid-tolerant organisms and L. pontis, L. fermentum,
L. reuteri and related organisms are frequently found [6–8].
This effect of fermentation parameters on sourdough microbiota is reflected by
the response of the growth of sourdough lactic acid bacteria to pH, temperature, and
NaCl concentration. Mathematical models for the growth of sourdough lactic acid
bacteria were developed for L. sanfranciscensis [9], L. pontis [10], L. amylovorus
[10, 11] and L. plantarum [12]. The optimum temperature for growth is species
specific; mesophilic organisms grow optimally between 30 and 35°C, while thermo-
philic organisms do not grow at ambient temperature (25°C) and grow optimally
between 40 and 45°C [9–11, 13]. Traditional sourdough fermentations in Europe
are typically carried out in the temperature range of 25–35°C (Chap. 4) and are thus
dominated by mesophilic lactic acid bacteria. Many industrial processes and cereal
fermentations in tropical climates are conducted at higher temperatures and accord-
ingly select for thermophilic lactic acid bacteria [7, 8].
The optimum pH of sourdough lactic acid bacteria is typically between 5.0 and
6.0 [9–11], matching the pH of sourdough after inoculation with 5–20% of a
previous batch of sourdough. Remarkably, the pH of wheat flour, about 6.2–6.5, is
close to the maximum pH permitting growth of L. sanfranciscensis, pH 6.7 [9]. The
minimum pH of growth of L. sanfranciscensis and L. pontis was determined as pH
3.9 and pH 3.5, respectively. Growth and lactic acid production by L. plantarum
continues until a pH of 3.1 is reached [12]. The higher pH tolerance of L. pontis
corresponds to its competitiveness in type II sourdoughs, which select for acid-tol-
erant lactic acid bacteria. The acid tolerance of L. pontis and related organisms
adapted to type II sourdoughs is dependent on conversion of arginine and glutamate,
which consume intracellular protons, and on the formation of exopolysaccharides.
These metabolic pathways are discussed in more detail below.
Lactic acid bacteria tolerate concentrations of undissociated acetic and lactic
acids that exceed by far those concentrations that are typically encountered in sour-
dough [9–11]. Growth of L. sanfranciscensis and L. pontis is observed at lactate
concentrations of up to 300 and 500 mmol/L, respectively [9, 10]; both organisms
also tolerate high concentrations of acetic acid. This high tolerance for organic acids
contrasts with the response of yeasts, which are inhibited by undissociated organic
acids but not by low pH. Moreover, because the pH but not the organic acid concen-
tration limits the growth of lactic acid bacteria in sourdough, the selection of cereal
7 Physiology and Biochemistry of Lactic Acid Bacteria 185
substrates with a high buffering capacity, for example whole wheat flour or bran,
allows the production of sourdough or sourdough products with a high concentration
of organic acids and a corresponding high total titrable acidity.
The response of sourdough lactic acid bacteria to high salt concentrations is also
species specific. Generally, obligate heterofermentative lactobacilli are more sensi-
tive to NaCl in comparison to other lactobacilli. For example, the heterofermentative
L. sanfranciscensis and L. pontis are inhibited by 4% NaCl whereas L. plantarum
and L. amylovorus tolerate up to 6% NaCl [9, 10, 12].
7.3 Metabolism of Carbohydrates
Lactic acid bacteria belong to three metabolic categories: (1) obligately homofer-
mentative organisms, which ferment hexoses through the EMP (Embden-Meyerhof-
Parnas) pathway to lactate as the major end product of carbohydrate metabolism
(Fig. 7.1). Pentoses are not fermented. Examples of obligately homofermentative
lactobacilli occurring in sourdough include L. delbrueckii, L. acidophilus,
L. farciminis, L. amylovorus, and L. mindensis. (2) Obligately heterofermentative
organisms, which ferment hexoses and pentoses through the 6-PG/PK
(6-phosphogluconate/phosphoketolase) pathway and synthesize equimolecular
amounts of lactate and ethanol or acetate. CO2 is additionally produced from hexo-
ses (Fig. 7.2). Key examples of obligately heterofermentative lactobacilli in sour-
dough are L. sanfranciscensis, L. rossiae, L. brevis, L. pontis, and L. fermentum.
(3) Facultatively heterofermentative organisms, which ferment hexoses through the
EMP pathway, and pentoses and gluconate through the 6-PG/PK pathway. Examples
of facultatively homofermentative lactobacilli occurring in sourdough include L. plan-
tarum, L. alimentarius, L. paralimentarius, and L. curvatus [13].
Homofermentative metabolism of hexoses under anaerobic conditions yields
2 mol ATP per mol hexose. In contrast, heterofermentative metabolism of hexoses
under anaerobic conditions yields only 1 mol ATP per mol hexose unless co-substrates
are present (Fig. 7.1). Contrarily to other fermented foods where obligately homo-
fermentative species have the major role, obligately heterofermentative lactic acid
bacteria are dominant in sourdough fermentations [6]. Several factors determine the
dominance of heterofermentative strains: (1) the metabolism of maltose via maltose
phosphorylase activity, simultaneous fermentation of hexoses and pentoses through
the 6-PG/PK pathway, and the use of fructose and other substrates as an external
acceptor of electrons; (2) the optimal pH and temperature which often coincides
with the values of sourdough fermentation; (3) the capacity of showing alternative
phenotype responses and to markedly adapt under various environmental stresses;
and (4) the synthesis of a large spectrum of antimicrobial compounds [6]. The vari-
able sources of fermentable carbohydrates in sourdough determine phenotypic
responses that include the use of external acceptors of electrons, the preferential
and/or simultaneous use of nonconventional energy sources, and the interaction with
exogenous or endogenous enzymes from the flour.
Glucose
Out
1 ATP
ADP
Glucose-6-P
2
In
Fructose-6-P
ATP
3
ADP
Fructose-1,6-DP
4
5
Glyceraldehyde-3-P Dihydroxyacetone-P
(2) Pi (2) NAD+
6
(2) NADH+2H+
(2) 1,3-Phosphoglycerate
ADP
7
ATP
(2) 3-Phosphoglycerate
8
(2) 2-phosphoglycerate
9 H2O
(2) Phosphoenolpyruvate
ADP
10
ATP
(2) Pyruvate
NADH+H+
11
NAD+
(2) Lactate
Fig. 7.1 Embden-Meyerhof-Parnas (EMP) pathway; homolactic fermentation. The final products
of glucose metabolism are in bold. (2) indicates the formation of two moles of each compound. 1
Glucokinase, 2 glucose-6-phosphate isomerase, 3 phosphofructokinase, 4 fructose 1,6-bisphos-
phate aldolase, 5 triosephosphate isomerase, 6 glyceraldehyde 3-phosphate dehydrogenase, 7
3-phosphoglycerate kinase, 8 phosphoglycerate mutase, 9 enolase, 10 pyruvate kinase, 11 lactate
dehydrogenase
7.3.1 Use of External Acceptors of Electrons
Because the energy yield of heterofermentative metabolism of hexoses is low,

the competitiveness of obligate heterofermentative lactobacilli depends on the
use of external electron acceptors. The practical relevance of the use of external
acceptors of electrons is represented by the substantial modification of the
Sucrose Maltose
Glucose
18 H+
Fructose Out
H+
Maltose
Pi
Glucose 1 Glucose-1-P
ATP
ADP
2 3 In
Glucose-6-P
NAD+
4
NADH + H+
6-P Gluconate
5 NAD+
CO2 NADH + H+
Ribulose-5-P
Xylulose-5-P
Pi
8 Acety l-P 7
Glyceraldehyde -3-P
Acetate CoA
ADP 9 Pi NAD+
ATP Pi 12
NADH+H+
Acetyl-CoA 1,3-Phosphoglycerate
ADP
NADH+H+ 10 13
CoA ATP
NAD+
3-Phosphoglycerate
Acetaldeide
NADH+H+ 14
11
NAD+ 2-Phosphoglycerate
Ethanol
15 H2O
Phosphoenolpyruvate
ADP
16
ATP
Pyruvate
NADH+H+
17
NAD
Lactate
Fig. 7.2 6-Phosphogluconate/phosphoketolase pathway (6-PG/PK); heterolactic fermentation.

The final products of glucose metabolism are in bold (Adapted from [3]). 1 Maltose phosphorylase,
2 hexokinase, 3 phosphoglucomutase, 4 glucose-6-phosphate dehydrogenase, 5 6-phosphogluconate
decarboxylase, 6 epimerase, 7 phosphoketolase, 8 acetate kinase, 9 phosphotransacetylase, 10
aldehyde dehydrogenase, 11 alcohol dehydrogenase, 12 glyceraldehyde 3-phosphate
dehydrogenase, 13 3-phosphoglycerate kinase, 14 phosphoglycerate mutase, 15 enolase, 16 pyru-
vate kinase, 17 lactate dehydrogenase, 18 levansucrase
Fructose R=O GSSG O2, H2O2

NADH+H+ NADH+H+ NADH+H+ NADH+H+
1 2 3 4
NAD+ NAD+ NAD+ NAD+
Mannitol R-OH 2 GSH H2O2, H2O
Fig. 7.3 Examples of some reactions that allow NADH + H+ co-factor reoxidation (Adapted from
[3]). 1 Mannitol dehydrogenase, 2 alcohol dehydrogenase, 3 glutathione dehydrogenase, 4 NADH
oxidase. GSSG, oxidized glutathione; GSH reduced glutathione; R=O, aldehyde (e.g., hexanal);
R-OH, corresponding alcohol (e.g., hexanol)
fermentation quotient (see Chap. 4) which positively influences the sensory and
shelf-life characteristics of sourdough baked goods [2]. Additional ATP is syn-
thesized when acetyl-phosphate is employed for acetate synthesis through ace-
tate kinase (Fig. 7.2). The synthesis of acetate and ATP as alternative metabolites
from acetyl-phosphate requires the availability of co-substrates to oxidize
NADH that were generated in the upper branch of the 6-PG/PK pathway. When
external acceptors of electrons are available, the recycling of NADH is achieved
without the need to synthesize ethanol from acetyl-phosphate. Most heterofer-
mentative lactic acid bacteria are capable of fructose reduction to mannitol to
achieve co-factor regeneration (Fig. 7.3). Fructose is quantitatively converted to
mannitol by most heterofermentative lactic acid bacteria under acidic condi-
tions [3, 14]. When maltose-negative and maltose-positive sourdough lactic
acid bacteria are associated, fructose conversion may have a further role [2].
Most strains of Weissella spp. differ from other heterofermentative lactic acid
bacteria because they do not convert fructose to mannitol with concomitant
acetate formation [15]. The activity of the mannitol dehydrogenase of L. san-
franciscensis LTH2581 is optimal at 35°C and pH 5.8–8.0 [16]. Once synthe-
sized, mannitol could be further used as an energy source by strains of
L. plantarum. This occurs under anaerobiosis and in the presence of ketoacids
(e.g., pyruvate) as electron acceptors [17].
Oxygen is also used as an external electron acceptor and is reduced to H2O with
H2O2 as an intermediate ([2, 18]; Fig. 7.3). Aerobiosis also induced the expression
of a 12.5-kDa superoxide dismutase (SOD), probably Mn2+ dependent [18]. Overall,
lactic acid bacteria possess various enzymes that are involved in the detoxification
of oxygen radicals. NADH-peroxidases and the system involved in the transport of
L-cysteine are specifically used by sourdough lactobacilli to detoxify H2O2 [19].
The latency phase of growth and cell yield of L. sanfranciscensis CB1 are positively
influenced by traces of oxygen and Mn2+ [18]. When aldehydes are available in the
environment, L. sanfranciscensis showed R-specific activity by NADH-dependent
alcohol dehydrogenase [20, 21] (Fig. 7.3). For instance, the reduction of hexanal to
hexanol activates the acetate kinase pathway and the synthesis of acetic acid. Also
the reduction of oxidized glutathione (GSSG) into reduced glutathione (GSH), via
glutathione dehydrogenase, protects against oxidative stress, and allows the
synthesis of acetic acid [22] (Fig. 7.3).
Acetate H2O
Oxaloacetate 3
Citrate Malate Fumarate
1 6
NADH+H+
NADH+H+ NAD+ 5 7
2
CO2 NAD+
CO2
4
Pyruvate Lactate Succinate
NADH+H+ NAD+
Fig. 7.4 Citric acid metabolism (Adapted from [3]). 1 Citrate lyase, 2 oxaloacetate decarboxylase,
3 malate dehydrogenase, 4 lactate dehydrogenase, 5 malolactic enzyme, 6 fumarase. 7 succinate
dehydrogenase
7.3.2 Metabolism of Organic Acids
Sourdough lactic acid bacteria exhibit strain-dependent metabolism of citrate, malate,

and fumarate; a few species are also capable of anaerobic lactate metabolism. Citrate,
malate, and lactate conversion consumes intracellular protons and thus increases the
acid tolerance of lactic acid bacteria. The initial steps of citrate metabolism by lactic
acid bacteria are transport by citrate permease and the reaction catalyzed via citrate
lyase (Fig. 7.4). Two alternative destinations are possible for oxaloacetate: the first
allows the synthesis of succinic acid; the second proceeds via decarboxylation into
pyruvate [23]. Lactococcus lactis converts part of the pyruvate into a-acetolactate.
This reaction takes place when external acceptors of electrons (e.g., citrate) are avail-
able, resulting in a surplus of pyruvate with respect to the amount needed to regenerate
NADH through lactate dehydrogenase. a-Acetolactate is reduced to acetoin or nonen-
zymatically converted to diacetyl, an important flavor compound of the bread crumb
[24]. Diacetyl formation is not observed in obligate heterofermentative lactobacilli and
conversion of citrate to succinate is the most common route for other sourdough lactic
acid bacteria. Nevertheless, the synthesis of diacetyl was found in sourdoughs fer-
mented with L. plantarum, L. farciminis, L. alimentarius and L. acidophilus [25].
Lactobacillus sanfranciscensis uses the route of the pyruvate to convert citrate into
lactate and acetate, also including the co-fermentation with maltose [26, 27]. The
regeneration of the co-factor during the reaction catalyzed by the lactate dehydroge-
nase allows the additional synthesis of acetate. Lactate formation from citrate does not
cause a decrease of the value of pH, and, therefore, the citrate metabolism of L. san-
franciscensis during sourdough fermentation is not limited by the low pH [3]. The use
of citrate during sourdough fermentation thus favors an increase of lactate and acetate
concentrations. Malate and fumarate are also converted to lactate by L. sanfranciscen-
sis [3, 26] (Fig. 7.4). In sourdough, the synthesis of pyruvate and lactate may also result
from the catabolism of nonconventional substrates (e.g., amino acids). For instance,
serine could be deaminated into ammonium and pyruvate. This latter may be, in turn,
reduced to lactate. Pyruvate may be synthesized directly (e.g., alanine) or indirectly
(e.g., aspartic acid) through the reaction of transamination [17].
Lactate conversion has been described for L. parabuchneri, an isolate from ting, a
lactic-fermented sorghum sourdough [28]. Lactate conversion to propanediol proceeds
through NADH-dependent reduction of lactate to lactaldehyde and 1,2 propanediol.
The reduction of two NADH to NAD+ allows the concomitant formation of acetate
from lactate [29]. Lactate conversion to 1,2-propanediol occurs mainly in the
stationary phase of growth and the consumption of lactate improves the stationary
phase survival of L. parabuchneri [30].
The practical relevance of the use of nonconventional energy sources is more
complex and less diffuse within the population of sourdough lactic acid bacteria. In
some cases, the addition (e.g., citrate) is needed for conditioning of the microbial
metabolism. In other cases (e.g., deamination or transamination of amino acids), it
is possible to note a large phenotype variability depending on the biotypes.
7.3.3 Preferential and/or Simultaneous Use of Energy Sources
Lactic acid bacteria use various sources of energy according to hierarchical features
that are determined through mechanisms of global control, mainly regulated at the
transcription level [31]. When bacteria are subjected to a mixture of energy sources,
they preferentially use the substrate that ensures the highest cell yield. When
growing on glucose-containing mixtures, L. amylovorus DCE 471 always consumed
glucose most rapidly, which seemed to steer growth during the early phase. Maltose
consumption started only when low levels of glucose were reached [32]. The pres-
ence of glucose repressed the fermentation of fructose, maltose, and sucrose of
L. paralimentarius and Weissella cibaria [33]. However, maltose, sucrose and fruc-
tose metabolism is not repressed by glucose in the sourdough-adapted species
L. reuteri and L. sanfranciscensis. Levansucrase, the only enzyme responsible for
sucrose metabolism in L. sanfranciscensis and a major contributor to sucrose
metabolism in L. reuteri is constitutively expressed or regulated independent of the
carbohydrate supply [35, 39]. In L. reuteri, sucrose phosphorylase is induced by
sucrose or raffinose but not repressed by glucose [40]. The efficient and preferential
metabolism of maltose and sucrose by several obligate heterofermentative lactoba-
cilli from sourdough likely reflects adaptation to cereal substrates where sucrose
and raffinose are the major carbon sources in the resting grain while maltose is the
major carbon source that is liberated by cereal enzymes during fermentation.
Metabolism via maltose phosphorylase or sucrose phosphorylase activity allows
a higher energy yield because the chemical energy of the glycosidic bond is employed
for ATP synthesis [34, 35]. Both enzymes are frequently found in heterofermentative
lactic acid bacteria from sourdough. Expression of maltose phosphorylase in L. san-
franciscensis and L. reuteri is constitutive and not repressed by glucose, in contrast,
hexokinase activity is observed only if glucose is available. During growth on malt-
ose, L. sanfranciscensis phosphorylyses maltose and accumulates glucose in the
environment, according to the molar ratio of ca. 1:1 (Fig. 7.2) [34, 36]. Glucose
accumulated by L. sanfranciscensis is available for maltose-negative yeasts and lac-
tic acid bacteria. Nevertheless, the release of glucose from the cell takes place only
when the activity of the enzyme hexokinase is not induced [37]. The accumulation of
glucose was not found for some strains of L. sanfranciscensis that were cultivated in
the presence of maltose and fructose. Therefore, it was hypothesized that once liber-
ated the glucose is used through the activity of the enzyme hexokinase, which is in
turn induced by the presence of fructose [38].
Some strains of L. sanfranciscensis have the capacity to express b-glucosidase
activity, but this activity is repressed by glucose [41]. Lactobacilli from sourdough
also show simultaneous rather than consecutive fermentation of pentoses and hexo-
ses. Compared to growth on maltose as the only energy source, L. alimentarius
15 F, L. brevis 10A, L. fermentum 1 F e L. plantarum 20B showed the highest
growth and acidification rates, and the highest cell yield when cultivated in the pres-
ence of xylose, ribose, and arabinose [42]. Other sourdough lactic acid bacteria
showed the highest performance in terms of growth and synthesis of acetic acid
when cultivated in the presence of a mixture of pentose carbohydrates [43]. The
presence of pentoses induces the synthesis of the phosphoketolase enzyme in facul-
tatively heterofermentative lactic acid bacteria (e.g., L. plantarum) and allows the
second half of the 6-PG/PK pathway to proceed (Fig. 7.2). The selection of lactoba-
cilli with the capacity to ferment pentose carbohydrates is thus a suitable alternative
to sucrose addition to increase acetate formation.
7.4 Proteolysis and Catabolism of Free Amino Acids
7.4.1 Proteolysis
Lactic acid bacteria are characterized by multiple amino acid auxotrophies. Lactic
acid bacteria depend on substrate-derived proteases or on the activity of their
proteolytic system to satisfy the nitrogen metabolism [44]. The proteolytic sys-
tems of lactic acid bacteria includes serine cell-envelope-associated proteinase
(CEP – PrtP) which is associated to the cell wall, oligopeptide and amino acid
transporters, and a large number of intracellular peptidases [45] (Fig. 7.5).
Contrary to lactic acid bacteria in dairy fermentations, L. sanfranciscensis ATCC
27651T and most other sourdough lactobacilli do not possess a cell-envelope-
associated proteinase and depend on cereal-associated proteases [46, 47]. The
comparison between sourdoughs and chemically acidified doughs showed that the
degradation of the native proteins is mainly due to the activity of cereal endoge-
nous proteinases [46, 48, 49]. The acidification by sourdough lactic acid bacteria
favors the activation of aspartate-proteinases from cereals, which have an optimal
pH of activity that ranged between 3.0 and 4.5 [50]. Nevertheless, selected strains
of sourdough lactobacilli showed the capacity to hydrolyze albumins, globulins,
and gliadins during fermentation [51–53]. Oligopeptides (4–40 amino acids) are
transported inside the bacterial cell and hydrolyzed through a complex system of
peptidases. An overview of the intracellular peptidases of L. sanfranciscensis is
shown in Fig. 7.6. Several intracellular peptidases of L. sanfranciscensis CB1
were biochemically characterized: a 65-kDa metal-dipeptidase (PepV), with
protein
acid
PrtP +
alkaline
biosynthesis -
oligopeptides ATP
Amino acids
H+
Fig. 7.5 Proteolytic system of lactic acid bacteria (Adapted from [44]). PrtP, Cell-envelope-
associated proteinase (CEP)
PepO PepE
Phe Ser
Endopeptidase
PepF
PepN (general aminopeptidase)

PepC (general aminopeptidase)
Glu/Asp PepA (narrow specificity aminopeptidase)
Pro Prolyl-oligopeptidase
Pro PepP (aminopeptidase P)
Leu PepL (leucyl aminopeptidase)
Pro PepI (proline iminopeptidase) Exopeptidase
PepV (general dipeptidase)
Carboxypeptidase
Pro
Carboxypeptidase P / Prolyl-carboxypeptidase
Pro
PepX (X- prolyl dipeptidyl aminopeptidase)
Gly Pro Gly Ile
Dipeptidyl-peptidase IV
Leu Pro Gly
Dipeptidyl-peptidase II
Pro
PepQ (prolidase)
Pro
PepR (prolinase)
PepT (tripeptidase)
Fig. 7.6 Peptidase system of lactic acid bacteria (e.g. Lactobacillus sanfranciscensis) and substrate
specificity (Adapted from [50])
Gliadins ( ),
Glutenins disulfide bonds:
, glutenins LMW (Low Molecular Weight),
, glutenins y-HMW (High Molecular Weight), Primary proteolysis by cereal proteinases
, glutenins x-HMW (High Molecular Weight), Microbial reduction of disulfide bonds
, hydrolysis products,
, peptides
Secondary proteolysis by
microbial enzymes, amino acids
catabolism Addition of enzymes
(fungal or malt-derived)
- Peptides hydrolised to free amino acids

- Production of volatile compounds
responsable for the aroma
- Comple degradation of proteins to small
peptides and free amino acids
Fig. 7.7 Proteolytic scheme that occurs during sourdough fermentation. Figure shows substrates,
enzymes and the activities of primary and secondary proteolysis (Adapted from [50])
elevated specificity towards dipeptides containing hydrophobic amino acids; a

75-kDa general aminopeptidase (PepN); and an X-prolyl-dipeptidyl aminopepti-
dase (PepX), which exclusively hydrolyzes substrates with the N-terminal
sequence X-Pro [51, 54]. The capacity to hydrolyze oligopeptides adjacent to
proline residues was shown in selected sourdough lactobacilli. However, the pro-
teolytic system of lactic acid bacteria does not cleave peptides with the sequence
motif XPP [55]. Overall, sourdough fermentation increases amino acid concentra-
tions compared to doughs that are chemically acidified [48, 52]. Proteolysis dur-
ing sourdough fermentation proceeds in two stages (Fig. 7.7): (1) primary
proteolysis that releases oligopeptides, and is mainly operated by cereal endoge-
nous proteinases that are activated at low pH and by accumulation of thiols; and
(2) secondary proteolysis that releases free amino acids and small-sized peptides,
and is exclusively operated through peptidase activity of lactic acid bacteria.
The degradation of the cereal proteins has a fundamental importance for the
rheology and sensory features of leavened baked goods. High molecular weight
glutenins (HMWG) and gliadins, following the decreasing order of solubility of
fractions g, a, and w, are hydrolyzed into alcohol-soluble oligopeptides [47]. The
partial hydrolysis of glutenins during sourdough fermentation results in the depo-
lymerization and solubilization of the glutenin macropolymer (GMP), thus affect-
ing the viscoelastic properties of the dough. The degree of polymerization of GMP
is also influenced by reducing agents. The reduced glutathione (GSH) is the most
important reducing agent in wheat dough. GSH may be subjected to an exchange
reaction with the thiol groups of the gluten proteins, thus reducing the intermolecu-
lar disulfide bond and favoring the decrease of the molecular mass of the GMP [50].
Sourdough heterofermentative lactic acid bacteria possess glutathione reductase
activity (see Sect. 3.1), which reduces the extracellular oxidized GSSG to GSH. The
continuous recycling of GSSG into GSH keeps the level of SH groups in the dough
elevated and increases the number of SH groups in the gluten proteins [56].
The proteolytic system of sourdough lactic acid bacteria contributes to the accu-
mulation of bioactive peptides in sourdough. Fermentation of rye malt sourdoughs
with L. reuteri resulted in the accumulation of angiotensin-converting-enzyme inhib-
itory peptides [55]; the formation of peptides with antioxidant activity was also dem-
onstrated [57]. Sourdough fermentation in combination with fungal enzymes was
also demonstrated to decrease the concentration of gluten to levels of less 10 ppm,
which are tolerated by celiac patients [52, 58]. This hydrolyzed wheat flour was used
for the manufacture of baked goods and administered to celiac patients for 60 days.
As shown by hematology, immunology and histological analyses, the consumption
of 10 g of equivalent gluten per day was absolutely safe for celiac patients [59, 60].
Nine peptidases were partially purified from the pooled cytoplasm extract of the
above-selected sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the
most common immunogenic peptide generated during digestion of Triticum species.
At least three peptidases, general aminopeptidase type N (PepN), X-prolyl dipeptidyl
aminopeptidase (PepX), and endopeptidase (PepO) were necessary to detoxify the
33-mer without generation of related immunogenic peptides. After 14 h of incuba-
tion, the combination of at least six different peptidases totally hydrolyzed the 33-mer
into free amino [61]. Peptidase activities of sourdough lactobacilli show consider-
able diversity at the strain level [55, 62] and the expression of genes coding for
peptidases and peptide transport is under control of the concentration of peptides that
are present during sourdough fermentation [46].
7.4.2 Amino Acid Metabolism
Peptides and free amino acids represent the substrates for microbial conversion, or
are transformed during baking to volatile flavor compounds (Table 7.1). Catabolism
of free amino acids by sourdough lactic acid bacteria not only has sensory
implications but also increases acid resistance and the microbial energy yield under
Table 7.1 Examples of amino acid precursors and derived carbonylic compounds, which are gen-
erated during sourdough fermentation and/or during baking
Amino acid precursor Derived carbonylic compound
Leucine 3-Methylbutanol
Isoleucine 2-Methylbutanol
Valine 2-Methylpropional
Alanine Acetaldehyde
Methionine Methional
Phenylalanine Phenylacetaldehyde
Threonine 2-Hydroxypropional
starvation conditions [63, 64]. Major pathways for amino acid conversion by lactic
acid bacteria include decarboxylation reactions, transamination, and metabolism by
lyases (for review, see [65]). These pathways lead to the synthesis of ketoacids,
aldehydes, acids and alcohols, which are important flavor compounds of baked
goods (Table 7.1) [66]. Major pathways that are relevant in sourdough fermentation
are outlined in more detail below.
The catabolism of arginine (Arginine Deiminase, ADI) was studied in depth on
sourdough lactic acid bacteria (Fig. 7.8). Three enzymes are involved, arginine
deaminase (ADI), ornithine carbamoyl transferase (OTC), and carbamate kinase
(CK). A fourth protein, located at the cell membrane which acts as transporter,
allows the antiporter exchange between arginine and ornithine [67]. The activity of
ADI, OTC, and CK enzymes is a species-specific property of several obligately
heterofermentative species, including L. rossiae, L. reuteri, L. brevis, L. hilgardii,
L. fermentum, L. pontis, and L. fructivorans [68]. Generally, arginine is quantita-
tively converted to ornithine by ADI-positive lactic acid bacteria during sourdough
fermentation [48]. The activity of ADI, OTC, and CK of L. rossiae CB1 is well
adapted to the acidity (pH 3.5–4.5) and temperature (30–37°C) conditions of sour-
dough fermentation [69]. The ADI pathway in L. fermentum IMDO 130101 is up-
regulated in response to temperature and salt stress conditions [70]. During
sourdough fermentation, the expression of the ADI pathway favors: (1) microbial
growth and survival, which determines a constant composition of the microbial
population in the sourdough; (2) the enhanced tolerance of lactic acid bacteria to
acidity by contributing to homeostasis of the intracellular pH; and (3) an increased
synthesis of ornithine which is converted, during baking, into 2-acetyl-1-pyrroline,
the compound responsible for the typical flavor of the bread crust.
Glutamine is the most abundant amino acid of wheat proteins; L. sanfranciscen-
sis and other sourdough lactobacilli convert glutamine into glutamate (Fig. 7.9).
Glutamine conversion to glutamate improves the adaptation of lactobacilli to sour-
dough acidity due to consumption of protons and liberation of ammonia which
increases the extracellular pH. The synthesis of glutamate also has a positive effect
on the sensory properties of leavened baked goods [71]. Glutamate is alternatively
decarboxylated to g-aminobutyrate (GABA), or converted to a-ketoglutarate (aKG)
by glutamate dehydrogenase (EC 1.4.1.3). Heterofermentative lactobacilli preferably
Fig. 7.8 Arginine deiminase 2-acetyl-pyrroline

catabolism (ADI) in
sourdough lactic acid baking
bacteria (Adapted from [3]).
1 Arginine-ornithine ornithine arginine
1
antiporter, 2 arginine Out
deaminase, 3 ornithine
transcarbamylase, 4
carbamate kinase In
arginine
H+
2
NH4+
citrulline
3
Carbamoyl-phosphate
ornithine
H+ + ADP
4
ATP
CO2+ NH4+
Out In
pH < 4.4 pH = 7
glutamine glutamine
H2O
1
NH3
NH3
glutamate glutamate α-ketoglutarate
α
3
H+ NADH + H+
2 4
NAD+
CO2
γ-aminobutyrate γ-aminobutyrate α-hydroxyglutarate
Fig. 7.9 Glutamine and glutamate metabolism of sourdough lactic acid bacteria. 1 Glutaminase,
2 glutamate decarboxylase, 3 glutamate dehydrogenase, 4 alcohol dehydrogenase
employ aKG as an electron acceptor to regenerate NADH [20]. aKG also acts as
the preferred amino acceptor in transamination reactions with leucine, phenylala-
nine, and other amino acids [72]. After peptide transport, the transamination reac-
tions are the second limiting factor for the conversion of amino acids by lactic acid
bacteria [73]. Consequently, the addition of a-ketoglutarate into the food matrix or
the selection of glutamate dehydrogenase positive strains considerably increases the
benzaldehyde
3
1 2
phenylalanine phenylpyruvate phenyllactate
4
5
phenylacetate 6 phenylacetaldehyde 7 phenylethanol
Fig. 7.10 Phenylalanine catabolism in Lactobacillus plantarum and Lactobacillus sanfranciscen-

sis. The most abundant metabolic products are underlined, those responsible for the aroma com-
pounds are in bold (Adapted from [3]). 1 Transaminase, 2 dehydrogenase, 3 chemical oxidation,
4 multienzyme complex, 5 decarboxylase, 6 and 7, dehydrogenase
conversion of amino acids. Glutamate dehydrogenase activity is variable depending

on the biotype of lactic acid bacteria [73]. This enzyme is also dependent on NADH
+ H+, which links the catabolism of glutamate of L. sanfranciscensis DSM20451 to
the regeneration of NADH during the central metabolism of carbohydrates (Fig. 7.9)
[74]. Glutamate decarboxylase (GAD) activity was also found in sourdough lactic
acid bacteria [75, 76]. GAD converts L-glutamate to GABA, through a single-step
a-decarboxylation. GABA, a four-carbon nonprotein amino acid, has several
functional activities. Sourdough fermentations with L. plantarum, and Lc. lactis or
L. reuteri allowed the synthesis of GABA in concentrations of up to 9 g/kg, compa-
rable to those found in functional preparations [75, 76]. Glutamate decarboxylase-
mediated acid resistance was shown to contribute to the persistence of L. reuteri in
type II sourdough fermentations [64].
The catabolism of phenylalanine by L. sanfranciscensis and L. plantarum is shown
in Fig. 7.10. Leucine, isoleucine, and valine undergo a similar mechanism. Alternative
pathways for leucine metabolism were proposed but remain to be validated by enzy-
matic and genetic analyses [77]. Phenyl-lactate or phenyl-acetate are the main end
products of the catabolism of phenylalanine. For L. plantarum TMW1.468, a gluta-
mate-dehydrogenase negative strain, the synthesis of phenyl-lactate is increased
through addition of aKG but not with glutamate and citrate added. Conversely, for
L. sanfranciscensis DSM20451, a glutamate-dehydrogenase positive strain, the
synthesis of phenyl-lactate is increased through addition of aKG, glutamate and pep-
tides containing glutamate. However, aKG and glutamate favor the conversion of
phenylalanine only when fructose or citrate are also present, probably owing to the
co-factor dependence of glutamate dehydrogenase [74]. During sourdough fermenta-
tion, L. plantarum TMW1.468 and L. sanfranciscensis DSM20451 synthesize ca.
0.35–1.1 mM of phenyl-lactate; the concentration of 1.5 mM inhibits the growth of
L. sanfranciscensis DSM20451 during growth in mMRS at pH 5.3 [3].
Cystathionine lyase activities are also diffuse within sourdough lactic acid bac-
teria (Fig. 7.11) [78]. A homotetrameric 160-kDa cystathionine g-lyase was purified
and characterized from L. reuteri DSM 20016 [79]. The enzyme is active towards a
large number of amino acids and amino acid derivatives, including methionine, and
allows the synthesis of ammonia, a-ketobutyrate, and low-molecular-weight volatile
sulfur compounds.
1 2
NH3 + α-ketobutyrate + cysteine cystathionine homocysteine + pyruvate + NH3
3
Fig. 7.11 Cystathionine metabolism (Adapted from [169]). 1 Cystathionine-g-lyase, 2 cystathio-

nine-b-lyase, 3 cystathionine-b-synthetases
7.5 Synthesis of Exopolysaccharides
The formation of exopolysaccharide by lactic acid bacteria during sourdough

fermentation improves culture survival and stress resistance, impacts dough rheology
and bread texture, and allows production of bread with specific nutritional functionality.
EPS from lactic acid bacteria can be categorized on the basis of their composition or
their biosynthesis. EPS composed of only one type of constituting monosaccharide are
classified as homopolysaccharides (HoPS). Examples include levan, which is composed
of fructose, or dextran, which is composed of glucose. Heteropolysaccharides (HePS)
are composed of two or more constituting monosaccharides. Lactic acid bacteria employ
two alternative routes for EPS synthesis, intracellular synthesis from sugar nucleotides
by glycosyltransferases, and extracellular synthesis from sucrose by glucansucrases or
fructansucrases. EPS produced by glucansucrase or fructansucrase activity are invari-
ably HoPS composed of glucose or fructose, respectively [80, 81]. EPS produced by
intracellular glycosyltransferases predominantly are HePS with glucose, galactose,
N-acetylglucosamine, N-acetylgalactosamine, rhamnose, or fucose as constituting
monosaccharides [82–84]. Noticeable exceptions of HoPS produced by intracellular
glycosyltransferases include a galactan from Lc. lactis [85] and b-glucan produced by
Pediococcus parvulus and Oenococcus oeni [86].
Large-scale screening of strain collections of sourdough lactic acid bacteria
revealed that very few isolates are capable of HePS formation [39, 87–89]; only one
report documents formation of HePS in sourdough [90]. In contrast, HoPS forma-
tion is a frequent trait of sourdough lactic acid bacteria and any sourdough is likely
to contain at least one HoPS-forming strain [39, 88]. Moreover, the in situ formation
of HoPS during sourdough fermentation as well as the effect of HoPS on bread
quality is well documented ([91, 92]), for a review see [93] and Chap. 8). Subsequent
paragraphs will hence only briefly discuss HePS formation and focus on HoPS
formation and applications.
7.5.1 EPS Biosynthesis and HoPS Structure
HePS formation by lactic acid bacteria is mediated by large gene clusters that are
encoded on plasmids or the chromosome (for reviews, see [82–84, 94]). EPS gene
clusters generally code for proteins regulating EPS biosynthesis (EpsA in
Streptococcus thermophilus Sfi6), polymerization, export, and chain length
determination (EpsB, EpsC, and EpsD in S. thermophilus Sfi6), and one or several
Enzyme properties: - sucrose

- pH and temperature optimum - fructose, glucose
- polymer length, linkage type, - maltose
Strain properties: and degree of branching - other acceptor sugars
- general growth parameters - hydrolysis to transferase ratio (pentoses)
- glucansucrase /
fructansucrase expression Substrate:
- formation of lactate and - buffering capacity (pH profile of
acetate (utilization of fermentation)
fructose as electron - concentration of acceptor
acceptor) III Hydrolysis carbohydrates and release during
- other metabolic properties fermentation.
relevant for bread quality - presence of other structure forming
polymers (e.g. pentosans, gluten)
II Oligosaccharide Process conditions:

lactate, formation
acetate, - time, temperature of fermentation
ethanol, CO2 - Level and type of sucrose addition
(mannitol) (batch / fed-batch)
I Exopolysaccharide formation
Influence on bread quality: texture, shelf life, and content of dietary fibre
Fig. 7.12 Factors influencing yield of exopolysaccharides and the influence of exopolysaccha-
rides on bread quality. Glucansucrases catalyze three alternative reactions: (I) polymerization of
glucose or fructose to exopolysaccharides, (II) glycosyl transfer to acceptor carbohydrates to form
oligosaccharides, (III) sucrose hydrolysis to glucose and fructose. EPS properties and yield are
additionally dependent on the concentration of substrate-derived acceptor carbohydrates, and the
ambient pH and temperature
phosphor-glycosyltransferases or glycosyltransferases (EpsE, EpsF, EpsG, and EpsI

in S. thermophilus Sfi6). EPS is synthesized by glycosyltransferases with sugar
nucleotides derived from glucose-6-phosphate as substrates. The repeating unit of
the polysaccharide, consisting of two to eight monosaccharides, is assembled by
sequential addition of monosaccharides to the membrane-bound lipid carrier undec-
adeprenyl pyrophosphate. The repeating unit is exported and polymerized extracel-
lularly. HePS biosynthesis requires energy-rich sugar nucleotides as precursors, and
the assembly and export of the repeating units competes with the peptidoglycan
biosynthesis; therefore, HePS yields are relatively low, typically well below 1 g/L.
HoPS synthesis is mediated by extracellular cell-wall associated or soluble glu-
cansucrases or fructansucrases [80, 81]. Both groups of enzymes are classified as
retaining glycosyl hydrolases and use sucrose as substrate. Fructansucrases but not
glucansucrases also employ raffinose, stachyose, or verbascose as substrate [40].
Catalysis by glucansucrases and fructansucrases proceeds through a covalently
linked glucosyl-enzyme or fructosyl-enzyme intermediate, respectively, with subse-
quent transfer of the glycosyl moiety to a growing polymer chain, a suitable acceptor
carbohydrate, or water (Fig. 7.12). Sucrose hydrolysis or oligosaccharide formation
are thus alternative reactions to polysaccharide synthesis [80, 95–97]. The ratio of
sucrose hydrolysis to oligo- or polysaccharide synthesis depends on the enzyme
structure as well as the substrate concentrations [98, 99]. Because the catalytic mech-
anism retains the chemical energy of the glycosidic bond of the substrate, HoPS
synthesis does not require energy-rich substrates or co-factors. Several recent reviews
provide an excellent overview on structure-function relationships of glucansucrases

and fructansucrase [80, 81, 100].
HoPS produced from sucrose are composed only of fructose or glucose but never-
theless have a large diversity with respect to linkage type, or degree of branching, and
molecular weight. Dextran is mainly composed of a−(1 → 6) linked glucose molecules
with a varying degree of a−(1 → 3) or a−(1→ 2) linkages and a−(1 → 3,6) branching
points. Dextran is produced by many strains of Leuconostoc spp. and Weissella spp.
but dextran-forming Lactobacillus spp. were also described [80, 81, 88]. Mutan is
mainly composed of a−(1 → 3) linked glucose moieties and is produced by
Streptococcus spp. and L. reuteri [80, 81]. Glucans with alternating a−(1 → 6) and
a−(1 → 3) or alternating a−(1 → 4) and a−(1 → 6) linkages are referred to as alternan
and reuteran, respectively. Reuteran formation has to date exclusively been observed
in strains of L. reuteri [81]. The current knowledge on structure-function relationships
of glucansucrases allows the manipulation of the linkage type [101] as well as the
molecular weight and degree of branching [102]. Information on structure-function
relationships of glucansucrases has also been used for identification of wild-type glu-
cansucrases producing polymers with desired properties [103]. The linkage type in the
oligosaccharides formed by glucansucrases generally matches the linkage types found
in the corresponding polysaccharide [81, 97]. For example, L. reuteri 121 produces
a−(1 → 4) and a−(1 → 6) linked reuteran with a relative molecular weight of 8 × 107;
in the presence of glucose or maltose as acceptor carbohydrates, maltose and isomalt-
ose or isomaltotriose and panose are produced. Fructans produced by lactic acid bac-
teria include the predominantly b−(2 → 1) linked inulin and the predominantly
a−(2 → 6) linked levan; both polymers contain b−(2 → 1 → 6) branching points [80,
81, 98, 104]. A majority of fructan-producing sourdough isolates form levan.
Remarkably, levansucrases form predominantly inulin-type fructo-oligosaccharides
and hetero-oligosaccharides from sucrose [95, 98].
7.5.2 Ecological Function of HoPS Production and HoPS

Formation in Dough
The ecological function of glucansucrases and fructansucrases for cereal-associated

lactic acid bacteria was related to biofilm formation, sucrose and raffinose utilization,
and stress resistance. In oral streptococci, glucansucrases and fructansucrases are
primarily responsible for sucrose-dependent biofilm formation on the tooth enamel,
and are considered major virulence factors of these organisms [105]. Analogous to
the biofilm formation by oral streptococci, colonization and biofilm formation by
L. reuteri in nonsecretory epithelia of the proximal intestinal tract of animals was
found to be dependent on HoPS formation. The reuteransucrase of L. reuteri
TMW1.106 was a major contributor to formation of the extracellular biofilm matrix,
the levansucrase in the same organism functions as a glucan-binding protein. Both
enzymes are required for colonization of reconstituted lactobacilli-free mice by
L. reuteri TMW 1.106 [106].
Glucansucrases and fructansucrases use one of the two constituting monosaccharides

of sucrose for poly- or oligosaccharide synthesis, the other monosaccharide, fructose
and glucose, respectively, accumulates in the fermentation substrate. Levansucrases also
release the a-galactosides melibiose, manninotriose, and manninotetraose from
raffinose, stachyose, and verbascose, respectively [40]. In L. sanfranciscensis LTH2590,
levansucrase is the only enzyme capable of sucrose and raffinose hydrolysis [40, 98]. In
L. reuteri and Lc. mesenteroides, levansucrase, glucansucrase and sucrose phosphory-
lase constitute alternative pathways for sucrose (and raffinose) metabolism. Metabolism
by extracellular levansucrases is the preferred metabolic route for raffinose, stachyose,
and verbascose, presumably because the resulting a-galactosides have a smaller degree
of polymerization, which facilitates transport across the cytoplasmic membrane [40].
Glucansucrase activity invariably accumulates fructose and thus supports acetate forma-
tion by heterofermentative lactic acid bacteria [98]. Because high quantities of acetate
have detrimental effects on the structure of wheat bread [107], dextran-producing
Weissella spp. that do not convert fructose to mannitol with concomitant acetate forma-
tion exhibited superior performance in baking applications when compared to
L. reuteri or L. sanfranciscensis [91, 92, 108].
A contribution of HoPS formation to stress resistance of lactic acid bacteria has
particularly been demonstrated for L. reuteri. Here, HoPS or fructo-oligosaccharides
increase survival at acid conditions during the stationary phase [103], and improves
survival of freeze-dried L. reuteri during storage [109]. The protective effects of
levan and fructo-oligosaccharides are partially attributable to their specific interac-
tion with phospholipids of biological membranes [109, 110]. However, protective
effects are not limited to levan and fructo-oligosaccharides; dextran and b-glucan
also improved acid resistance and stationary phase survival [86, 111].
Lactic acid bacteria producing HoPS from sucrose in laboratory medium
generally also produce HoPS during growth in sourdough [39]. HoPS concentrations
typically range from 1 to 8 g/kg dough [39, 91, 92, 98, 112]; more than 10 g/kg
were reported in optimized, pH-controlled fermentations [113]. Glucansucrases
and fructansucrases are extracellular enzymes, thus, their activity is governed by
ambient conditions rather than intracellular pH and substrate concentrations
(Fig. 7.12). The optimum pH of levansucrases and glucansucrases of sourdough
lactobacilli ranges from 4.5 to 5.5, matching the pH profile of sourdough fermenta-
tions [98, 99]. HoPS yields in sourdough were maximized by pH-controlled fer-
mentations at a pH of 4.7 [113]. Because sucrose acts both as glycosyl-donor and
glycosyl-acceptor in HoPS formation, the sucrose concentration has a decisive
influence on the ratio of sucrose hydrolysis, oligosaccharide formation, and poly-
saccharide formation. Below 10 g/kg sucrose, sucrose hydrolysis is the predominant
reaction [98, 114]. Increasing sucrose concentrations initially increase the forma-
tion of levan over hydrolysis. After a further increase of the sucrose concentration,
sucrose increasingly acts as a glycosylacceptor and oligosaccharide formation is the
predominant reaction catalyzed by glucansucrases or fructansucrases [98, 114, 115].
In sourdough fermentation with 100 g/kg sucrose, L. sanfranciscensis LTH2590
produced 11 g/kg fructo-oligosaccharides but only 5 g/kg levan [98]. High reuteran
yields from L. reuteri TMW1.106 were achieved in pH static fed-batch fermentations
that maintained sucrose concentrations at 10% throughout fermentation [113]. The

addition of acceptor carbohydrates other than sucrose also influences HoPS yields
in sourdough fermentations. Maltose is a stronger acceptor for glucansucrases
compared to glucose [97, 115]. Correspondingly, dextran yields in wheat sourdoughs,
characterized by high maltose concentrations throughout fermentation, were
substantially lower compared to yields by the same strains in sorghum fermentations,
which are characterized by low maltose and high glucose concentrations [15].
Maltose, xylose, and arabinose also act as alternative acceptor carbohydrates for
levansucrase activity [95, 96].
Exopolysaccharide-producing sourdough starter cultures have found industrial
application to improve the textural properties of bread [116]. In addition to the func-
tion of HoPS as hydrocolloids in baking applications, specific HoPS were found to
prevent pathogen adhesion to eukaryotic cells [117] and to exhibit prebiotic activity
[92, 118]. HoPS-producing lactic acid bacteria thus enable the formulation of baked
goods with specific health properties.
7.6 Antimicrobial Compounds from Sourdough

Lactic Acid Bacteria
Leavened baked goods can become contaminated by spores of the genus Bacillus, which
survive baking, yeasts, mainly belonging to the genera Pichia and Zygosaccharomyces,
which colonize the surface and negatively affect the sensory properties, and, especially,
by moulds, mainly belonging to the genera Penicillium, Aspergillus and Cladosporium
which alter the color and the sensory properties, and, in some cases, synthesize myco-
toxins. More than 40 species of fungi were described as contaminants of baked goods.
Although chemical preservatives (e.g., sorbate and propionate, ethanol) are routinely
used for preventing the contamination of leavened baked goods, sourdough lactic acid
bacteria show a number of natural bio-preservative features that are complementary to
chemical preservatives, or can even substitute their use. Antimicrobial compounds from
sourdough lactic acid bacteria were additionally shown to influence sourdough micro-
biota, and to contribute to the stability of individual strains.
The inhibitory activity of sourdough lactic acid bacteria is generally attributable
to rapid consumption of oxygen and fermentable carbohydrates, and the formation
of lactate with concomitant reduction of the pH. Additional metabolites with specific
antimicrobial activity include diacetyl, hydrogen peroxide, acetate and other short-
chain fatty acids, and reuterin. Acetate formation by heterofermentative lactobacilli
in sourdough is readily adjusted by addition of sucrose or pentoses [39], and con-
tributes to shelf-life extension of bread (see below). The odor threshold of diacetyl,
butyrate, and caproate is substantially lower than the concentrations required for
antimicrobial activity; these compounds can thus not be accumulated in bread with-
out adverse effects on the sensory bread quality. Although individual sourdough
lactic acid bacteria are capable of reuterin synthesis, reuterin formation has not been
achieved in cereal fermentations [119].
7.6.1 Antifungal Compounds from Sourdough

Antifungal compounds synthesized by sourdough lactic acid bacteria include

diacetyl, hydrogen peroxide, acetate, propionate, caproate, 3-hydroxy fatty acids,
phenyllactate, cyclic dipeptides, reuterin, and fungicidal peptides [120]. Numerous
reports describe a substantial increase of the mould-free shelf life of bread owing to
antifungal activities of sourdough lactic acid bacteria [121, 122]. However, metab-
olites of lactic acid bacteria responsible for the antifungal effect remain in most
cases unknown. Acetate inhibits fungal growth only at concentrations that
significantly impair the sensory and textural quality of bread [30, 107, 123].
Phenyllactate and 4-hydroxyphenyllactate were initially characterized as antifun-
gal metabolites from L. plantarum ITM21B [124]. Lactobacilli capable of produc-
ing phenyllactate and hydroxyphenyllactate delayed the growth of Aspergillus
niger and Penicillium roqueforti for up to seven days of bread storage [125]. The
concentration of phenyllactate accumulated by lactobacilli in sourdough fermenta-
tion, however, is about 100 fold lower than its minimal inhibitory concentration
[74, 126] and the antifungal effect of the sourdough is thus not attributable to phe-
nyllactate accumulation. Likewise, the levels of antifungal cyclic dipeptides in
bread are 1,000 fold lower than their MIC, but above the threshold imparting a
metallic and bitter taste [127]. Accordingly, the antifungal effect of sourdough lac-
tic acid bacteria is attributed to a synergistic activity of several compounds. The
antifungal effect of L. reuteri, L. plantarum, and L. brevis in the presence of Ca
propionate (0.2%, w/w) [128] delayed fungal growth by 8 days compared to the
bread started with baker’s yeast alone. It was hypothesized that a synergistic activ-
ity between acetic acid and phenyllactate with chemical preservatives was respon-
sible for this effect. The antifungal effect of L. buchneri and L. diolivorans against
growth of four moulds on bread was attributed to a combination of acetate and
propionate [30]. The preservative effect of L. amylovorus [121, 122] was attributed
to the synergistic activity of more than ten antifungal compounds, including phe-
nyllactate, phenolic acids, fatty acids, and cyclic dipeptides [129].
In addition to the antifungal metabolites of lactic acid bacteria, inhibitory pep-
tides derived from the substrate may also contribute to the preservative effect.
A water-extract from beans in combination with sourdough fermented with L. brevis
AM7 contained three natural inhibitory compounds, two phaseolins (NCBI n. gi
130169, gi 403594) and one lectin (NCBI n. gi 130007) [130]. Antifungal peptides
were also identified from the water-extract of sourdough. The combined activity of
the above compounds determined a delay in fungal growth of up to 21 days, lead-
ing to a shelf life for the bread that was comparable to that found when using Ca
propionate (0.3% w/w). A very extensive bread shelf life was also achieved by
using sourdough lactic acid bacteria and amaranth flour or wheat germ [131, 132].
Sourdough fermented with the nonconventional yeast Wickeramomices anomalus
LCF1695 and L. plantarum was shown to exhibit strong antifungal activity based
on synergistic activities between the inhibitory peptides liberated by L. plantarum
and ethyl-acetate produced by W. anomalus [133]. Fungal contamination was

delayed by 28 days under pilot plant bakery conditions. Although indubitable
progress has been achieved in the making and storage of baked goods, fungal con-
tamination remains one of the main problems in terms of long-term shelf life of
bakery products. The search for novel methods of bio-conservation appears to be
one of the most promising tools in this regard, even though a combination of vari-
ous inhibitory compounds appears to be inevitable to markedly extend the storage
of leavened baked goods.
7.6.2 Antibacterial Compounds from Sourdough

Antibacterial metabolites from lactic acid bacteria include reuterin and the organic
acids described above. However, the emphasis of research related to antibacterial
activities was placed on bacteriocins, ribosomally synthesized peptides with anti-
bacterial activity against closely related organisms, and reutericyclin. The pre-
vention of bread spoilage by rope-forming bacilli does not require the selection of
specific protective cultures. Growth of endospores of Bacillus spp. is readily inhib-
ited by modest acidification as is characteristic for sourdough bread [134–136].
Acetate and propionate are more effective against rope-forming bacilli than lactic
acid [134]. Remarkably, the bacteriocins nisin and pedicoin, either included as
additives, or generated in situ by bacteriocin-producing lactic acid bacteria, were
ineffective [134].
Bacteriocin formation by sourdough lactic acid bacteria was described particu-
larly for strains of L. sakei, L. plantarum and L. amylovorus (for a review, see [137,
138]). Bacteriocins appear not to be suitable for extending the shelf life of bread but
formation in sourdough fermentation was shown to enhance the stability of sour-
dough microbiota. Lactococcus lactis M30, a lacticin 3147-producing strain [139],
produced a bacteriocin during fermentation. The inhibitory activity persisted under
the low values of pH of sourdough and after thermal treatments that corresponded
to baking temperatures. Similarly, the bacteriocin-producing strain L. amylovorus
DCE471 persisted for a long time during sourdough propagation [140]. The com-
petitiveness of L. pentosus 2MF8, which synthesized a bacteriocin-like inhibitory
substance [141] and Lc. lactis subsp. lactis M30, which synthesized lacticin 3147
[139] were studied during sourdough propagation. Lactococcus lactis subsp. lactis
M30 showed a larger spectrum of inhibition compared to L. pentosus 2MF8, and did
not inhibit the growth of L. sanfranciscensis. After 20 days of back-slopping, the
persistence of Lc. lactis subsp. lactis M30 inhibited the indicator strain L. plan-
tarum 20, without interference in the growth of L. sanfranciscensis CB1. The above-
described features of bacteriocins, together with the demonstration of the in situ
inhibitory activity, encourage the use of antimicrobial compounds to facilitate the
persistence of the starter cultures and the conditioning of the microbial interactions
that occur during sourdough fermentation.
Reutericyclin is a tetramic acid derivative with a broad spectrum of activity

against Gram-positive bacteria, including rope-forming bacilli [142, 143]. To date,
all reutericyclin-producing strains were isolated from an industrial rye sourdough.
Reutericyclin is produced to active concentrations in sourdough and the persistence
of L. reuteri strains during long-term propagation of a rye sourdough was attributed
to the synthesis of reutericyclin (for a review, see [144]). The use of reutericyclin-
producing L. reuteri as a sourdough starter also caused a delay in the growth of
Bacillus sp. during bread storage [144].
7.7 Metabolism of Phenolic Compounds and Lipids
The major phenolic compound in wheat and rye is ferulic acid bound to cell wall
polysaccharides [145]. Changes in the ferulic acid content during sourdough
fermentation are predominantly the result of oxidation reactions and cereal enzymes
[146, 147]. In contrast, cereal grains used in African cereal fermentations, particularly
sorghum and millet, are rich in phenolic compounds, including phenolic acids, phe-
nolic acid esters, desoxyanthocyanidins, and tannins [148, 149]. In these cereal
grains, metabolism of phenolic compounds influences product properties, and the
content of antimicrobial phenolic compounds influences the microbial ecology of
sorghum sourdough fermentations [150]. A review on the metabolism of food phe-
nolics by lactic acid bacteria, based predominantly on isolates from wine and veg-
etable fermentations, is provided by Rodriguez et al. [151].
Conversion of phenolic compounds is based on glycosyl hydrolases, for example
b-glucosidase and a-rhamnosidase, which convert flavonoid glycosides to the cor-
responding aglycones [152, 153], esterase degrading methyl gallate, tannins, or
phenolic acid esters [154], and decarboxylases and reductases with activity on phe-
nolic acids [151]. The strain-specific metabolism of phenolic compounds is particu-
larly well described for L. plantarum [151, 155]. Decarboxylation of hydroxyl
cinnamic acids generates the corresponding vinyl derivatives [156]. Reductases
hydrogenate the double bond of hydroxyl cinnamic acids or their decarboxylated
vinyl derivatives [151, 156]. The spectrum of activities is strain specific; organisms
capable of conversion of hydroxy cinnamic acids harbor reductase activity, decar-
boxylase activity, or both [150, 151, 156]. Analysis of phenolic compounds during
sorghum sourdough fermentation revealed that strain-specific glycosyl hydrolase,
decarboxylase, and phenolic acid reductase contributed to the conversion of pheno-
lic compounds [151].
Phenolic compounds, including phenolic compounds from sorghum and mil-
let, exhibit strong antimicrobial activity [148, 150]. Particularly the activity of
hydroxy benzoic and hydroxy cinnamic acids is well characterized [157]. The
resistance of lactic acid bacteria towards phenolic acids is highly strain specific
and the strain-specific capability for phenolic acid conversion corresponds to
resistance [157, 158]. Because the conversion of hydroxy cinnamic acids by
reductase- or decarboxylase activities reduced the antimicrobial activity of caffeic
acid two to fivefold, metabolism was recently identified as a mechanism of

detoxification [157]. To date, lactic acid bacteria capable of conversion of pheno-
lic compounds were predominantly isolated from fermented beverages (wine,
whisky, beer), or African sorghum fermentations. These organisms are likely a
suitable source for starter cultures of phenolic-rich cereals and pseudocereals
used in gluten-free baking [159].
Lipid metabolism in sourdoughs is poorly described and particularly lipase activ-
ity of sourdough lactic acid bacteria appears not to be relevant. However, sourdough
fermentation influences lipid oxidation and the influence of lipid oxidation products
on bread flavor. During flour storage and during dough mixing, chemical oxidation
and cereal-derived lipoxygenase, respectively, convert free linoleic acid to lipid per-
oxides. Peroxides are chemically converted to lipid aldehydes, i.e., (E)-2-nonenal
and (E,E)-2,4-decadienal, with a strong influence on the flavor of the bread crumb
[21]. Heterofermentative lactobacilli reduce these aldehydes to the corresponding
alcohols with a much lower flavor threshold through alcohol reductase activity.
Homofermentative lactobacilli may increase lipid oxidation through formation of
hydrogen peroxide [21]. Baker’s yeast also reduced flavor-active aldehydes result-
ing from lipid oxidation, but slower and through different metabolic pathways [21].
Thiol accumulation by sourdough lactic acid bacteria [56] may additionally influence
lipid oxidation. Cysteine and related thiol compounds reduce linoleic acid perox-
ides to the corresponding hydroxy fatty acids [160], and thus interrupts the reaction
cascade leading to flavor-active aldehydes.
7.8 Cell-to-Cell Communication
Bacteria synthesize, release, sense, and respond to small signaling molecules that
are defined as auto-inducers. The signaling molecules accumulate in the environ-
ment and lead to a series of physiological and biochemical responses when the
quorum (threshold concentration) is reached. The term quorum sensing is derived
from this consideration, and is mostly used to describe cell-to-cell communication
[161]. Overall, the mechanisms of intraspecies communication include the use of
acyl-homoserine lactones (AHL) and auto-inducing peptides (AIP) for Gram-
negative and -positive bacteria, respectively [162, 163]. The interspecies communi-
cation is mainly based on signaling molecules such as furanone derivatives. The
mechanisms involve the LuxS protein or AIP molecules and the three-component
regulatory system (3CRS) [161]. Several studies considered the microbial dynamics
during sourdough fermentation [164]. Within this complex food ecosystem, an
understanding of the mechanisms of interspecies communication should give new
insights into the physiological response of sourdough lactic acid bacteria and the
interactions in an heterogeneous microbial community that govern growth and
metabolism. The mechanism of cell communication was studied in L. sanfrancis-
censis CB1 co-cultivated with other sourdough lactic acid bacteria [165]. The high-
est number of dead and/or damaged cells of L. sanfranciscensis CB1 was found
when co-cultured with L. plantarum DC400 or L. brevis CR13. The co-cultivation

with L. rossiae A7 did not interfere with survival compared to the mono-culture.
The analysis of the proteome revealed the induction of several cytoplasm proteins
of L. sanfranciscensis CB1, especially when associated with L. plantarum DC400.
The majority of the induced proteins had a key role in the mechanisms of response
to environmental stresses, leading to the hypothesis that co-cultivation with some
species of lactic acid bacteria could be considered like an environmental stress
which promoted cell communication. A central role for the LuxS protein was estab-
lished, while also furanone derivatives were identified as presumptive signaling
molecules. The synthesis of volatile compounds and the activity of peptidases also
decreased when L. sanfranciscensis CB1 was cultivated together with L. plantarum
DC400. The mechanisms of cell communication were also studied in L. plantarum
DC400 [166]. The co-cultivation of this strain with other sourdough lactic acid bac-
teria did not interfere with survival compared to the mono-culture. Nevertheless, the
analysis of the proteome revealed the induction of proteins involved in the response
to environmental stresses and cell communication. Lactobacillus plantarum DC400
synthesized the pheromone plantaricin A (PlnA) at variable concentrations that
depended on the associated microbial species [167]. The co-cultivation with L. pen-
tosus, L. brevis and other sourdough lactic acid bacteria species did not modify the
synthesis of PlnA compared to the mono-culture conditions and did not show effects
on the viability and survival of the other species. On the contrary, the co-cultivation
with L. sanfranciscensis increased the synthesis of PlnA and caused a decrease of
the viability of this species. The same effect was found during growth of L. sanfran-
ciscensis in the presence of the chemically synthesized PlnA. Under these environ-
mental conditions, the analysis of the proteome revealed the over-expression of
proteins that had a central role in the energy metabolism, catabolism and biosynthesis
of proteins and amino acids, stress responses, homeostasis of the redox potential, and
programmed cell death. Notwithstanding antimicrobial activities due to substrate
competition and synthesis of inhibitory compounds (e.g., phenyl lactic acid), and the
elevated capacity to adapt to changing environments, it could be hypothesized that
the dominance of L. plantarum during sourdough fermentation [168] may relate to
the synthesis of the pheromone/bacteriocin PlnA as stimulated by mechanisms of
quorum sensing.
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Chapter 8
Sourdough: A Tool to Improve Bread Structure
Sandra Galle
8.1 Introduction
The quality of bread is characterized by its flavor, nutritional value, texture, and shelf
life [1]. In the baking industry, these characteristics are improved by addition of bread
improvers or enzymes. Alternatively, the addition of sourdough influences all aspects
of bread quality and thus meets the consumer demands for a reduced use of additives.
As sourdough is an intermediate but not an end product the microbiological activity
has to be determined on the bases of their impact on bread quality. Biochemical
changes during sourdough fermentation occur in protein and carbohydrate compo-
nents of the flour. The rate and extent of these changes greatly influence the properties
of the sourdough and consequently the quality of the bread dough and bread structure.
The effects are associated with the metabolites produced by lactic acid bacteria (LAB)
and yeast during fermentation, including organic acids, exopolysaccharides (EPS),
enzymes, and CO2. The following chapter presents the impact of sourdough fermenta-
tion on structure-forming components of bread, and bread texture.
8.2 Components Determining Dough and Bread Structure
Gluten proteins of wheat create unique viscoelastic properties of dough, which

allow dough to expand in response to formation of carbon dioxide and to retain most
of this gas in the dough. Polymeric glutenins give strength and elasticity to wheat
dough, whereas the monomeric gliadins are responsible for the viscous properties
of the dough [2, 3]. Embedded in the protein network are starch granules that form
S. Galle (*)
Department of Agricultural, Food and Nutritional Science, Edmonton, University of Alberta,
Alberta, Canada

218 S. Galle
a continuous structure with gluten during kneading. In freshly baked bread, the
main networks are the continuous gluten network which forms a matrix between the
swollen, gelatinized starch granules and the transient starch network, consisting of
entangled, gelatinized starch polymers [4]. During cooling the main changes occur
in the starch fraction, where amylose forms a partially crystalline amylose network,
with crystalline amylose and amylose-lipid complex. The amylose network together
with the gluten network formed during baking determines the resilience of the fresh
bread. Additionally pentosans (arabinoxylans) and transient gelatinized amylopec-
tin networks also contribute to the structure formation of freshly baked wheat bread.
During storage bread texture becomes harder largely because of physical changes
that occur in the starch-protein matrix of the bread crumb [5]. Retrogradation is the
process by which starch reverts to a more crystalline form after gelatinization.
Because amylose is already completely retrograded in fresh bread, the amylopectin
retrograding over time is primarily responsible for bread staling [6]. In addition,
water migrates within the crumb and from crumb to crust. This water loss is another
important factor contributing to the bread staling process [4].
Compared to wheat flour, rye flour differs with regard to gluten composition,
starch gelatinization, and a-amylases activity [7]. Consequently, factors determin-
ing structure formation in rye dough and rye bread structure differ from wheat
dough and bread. Whereas sourdough was an essential ingredient for ensuring bak-
ing properties of dough containing more than 20% of rye flour, its addition to wheat
dough remains optional. The use of sourdough has gained increased interest in the
last decade as a means to improve the quality and flavor of wheat bread [1]. However,
there is much disparity in the results concerning the effect of sourdough on the final
wheat product and the use of sourdough has been shown to either decrease [8–10]
or increase final bread volume [10–15].
8.3 Organic Acids
The production of organic acids and consequently the pH drop in sourdough have a
major effect on structure-forming components like starch, gluten, and arabinoxy-
lans (Table 8.1). The primary effect of acids on the protein fraction is the increased
swelling and solubility of gluten proteins [16, 17]. This effect is explained by a posi-
tive net charge of the proteins in an acidic environment. Increased intramolecular
electrostatic repulsion causes gluten proteins to unfold and expose more hydropho-
bic groups. The presence of strong intermolecular electrostatic repulsive forces pre-
vents the formation of new bonds. This results in softer dough with less stability and
shorter mixing time [18, 19]. Furthermore, softness of the gluten promotes swelling
and increased water uptake [20]. Partial acid hydrolysis of starch also exerts posi-
tive effects on the starch granules leading to an increased water binding capacity [8,
21]. In addition to the impact of low pH on dough components, secondary effects of
acidification and fermentation time include changes in the activity of cereal enzymes.
Flour proteases have their pH optima in the acidic range and increased proteolysis
8 Sourdough: A Tool to Improve Bread Structure 219
Table 8.1 Effects of sourdough metabolites on bread structure

Metabolites Effect on flour components Effect on dough/bread structure
Organic acids Increased swelling and solubility of the Shorter mixing time, less
gluten stability of the dough
Increased water uptake of gluten and Increase of elasticity and
starch softness of the dough
Increased proteolysis of gluten proteins Both increase and reduction
through flour endogenous proteinases of bread volume
Organic acids Increased solubility of pentosans Improved volume and crumb
through acid hydrolysis and endog- structure of rye and wheat
enous pentosanases bread
Organic acids Inhibition of endogenous a-amylases Bakeability of rye bread
Enzymes Proteolysis Weakening of the gluten
Glutathione-reductase structure, dough softening
Exopolysaccharides Increased water absorption Increased softness of dough and
bread texture, increased volume
Interaction with gluten-starch network Increased shelf life (anti-
Inhibiting retrogradation of starch staling)
CO2 Expansion of gas cells Leavening of dough and bread
occurs in doughs at pH 4 in comparison to nonacidified systems [22]. In comparison

to straight dough processes, the increased activity of cereal proteases is also attrib-
utable to longer fermentation times. The rheological consequence of gluten degra-
dation is a major reduction of elasticity and firmness of the sourdough and subsequent
bread dough [10, 23, 24]. Whether this has positive or negative effects on bread
volume and staling depends on the acidity profile and gluten network.
Physicochemical changes in the protein network resulting from sourdough fermen-
tation enhance gas retention and allow greater expansion due to softer and more
extensible doughs [11, 13, 25]. Confocal laser-scanning microscopy visualized the
effect of incorporation of sourdough on the dough microstructure: Relative to the
fine well-oriented network of the control, the gluten in the dough with added sour-
dough had a more amorphous nature and there were greater areas of aggregated
material composed of thicker protein strands [23]. The presence of thicker strands
could also account for a greater increase in loaf volume [26]. Increased volume cor-
relates with the increased softness of the crumb, and is associated with a reduced rate
of staling [13, 27]. However, if the acidity of sourdough is further increased, bread
volume decreases [8–10]. Quantitative analysis of gluten in sourdough and chemi-
cally acidified doughs showed that gliadins and glutenins are hydrolyzed [28, 29].
Especially high molecular glutenins are completely degraded, which leads to a
strong gluten softening. Weaker gluten increases the expansion of dough, but also
decreases gas retention. Accordingly, the acidity level of sourdough and subsequent
bread dough must be carefully controlled to attain increased volume [30].
The drop in pH during fermentation not only affects cereal proteases but modulates
amylase activity. Acid conditions partially inactivate amylases. This is an important
aspect in rye baking since excessive a-amylase activity results in a sticky crumb, a
very open grain, and a reduction in loaf volume [7, 21]. In wheat flour, a-amylases are
220 S. Galle
virtually absent, while b-amylases are abundantly present, but the latter have little if any
activity on starch granules and are inactivated before starch gelatinization [4].
In rye flour proteins are not capable of forming a network similar to wheat gluten,
here water-binding pentosans take over the function in the structure-forming process
[21]. Pentosans mainly consist of arabinoxylans (AX), which can be subdivided into
water extractable (WEAX) and water unextractable arabinoxylans (WUAX). WUAX
have a deleterious influence on bread quality, in contrast WEAX increase bread vol-
ume due to the high water-binding capacity and the ability to undergo oxidative
gelation [31] (see Chap. 2). Acidification has a pronounced effect on the solubility of
AX due to acid hydrolyzation of WUAX, therefore increasing the proportion of
WEAX [32]. Furthermore, during sourdough fermentation pH optima for endoge-
nous rye enzymes like L-arabinofuanosidase, endo-xylanase, and xylosidase are
reached [33]. Also in wheat endoxylanases cause a reduction of WUAX and increase
the level of WEAX [6]. Thus, beneficial WEAX can increase during sourdough fer-
mentation. In rye doughs, this improves physical properties by increasing the elastic-
ity of the dough and the subsequent bread crumb. The bread crumb is characterized
by an improved mouthfeel, improved crumb structure, and volume [7, 31, 33]. In
wheat baking the increased solubility of pentosans in sourdough also contributes to
enhanced bread volume and improved softness [6, 13, 34].
8.4 Enzymes from LAB Contributing to Dough

and Bread Structure
Proteolysis in sourdoughs is mainly based on the pH-mediated activation of endogenous

flour proteases [28, 29, 35] (Table 8.1). Lactobacilli used in sourdough fermentation
may also exhibit strain-specific proteolytic activity [36, 37], but appear to play a minor
role in the overall proteolysis. Although LAB do not influence overall proteolysis
when compared to aseptically acidified doughs, they affect the pattern of hydro-
lyzed products, increasing the amount of dipeptides and amino acids. In fact, the pro-
teolysis by LAB induced softening of the dough in comparison to chemically acidified
doughs [37]. Furthermore, the choice of a highly proteolytic starter culture, such as
Enterococcus faecalis, substantially contributed to the gluten proteolysis [38].
The significant role of enzymes produced by LAB has been proposed to explain
observed differences in the staling of sourdough breads [12, 13]. Sourdough breads
with comparable acidity levels had varying staling rates in terms of firmness and
starch retrogradation. LAB strains possessing proteolytic and amylolytic activities
were most effective in delaying staling [12].
In addition to the pH-dependent cereal proteases and LAB-liberated proteases,
glutathione reductase expressed by heterofermentative lactobacilli contributes to
depolymerization of gluten protein [39] (Table 8.1). Glutathione reductase reduces
extracellular oxidized glutathione (GSSG) to the reducing agent glutathione (GSH)
leading to increased SH groups in the gluten proteins. GSH undergoes thiol-
exchange reactions with gluten proteins and decreases intermolecular disulfide
cross-linking, resulting in a decreased molecular weight of the glutenin macropoly-

mer (GMP). Only small amounts of reducing agents are necessary to strongly affect
the disulfide network of glutenins [40].
8.5 Exopolysaccharides
Some strains of LAB synthesize EPS from sucrose. EPS exhibit a positive effect on
the texture, mouthfeel, taste perception, and stability of fermented food. Moreover,
prebiotic effects have been described for specific EPS [41–43].
EPS can be classified in homopolysaccharides (HoPS) and heteropolysaccharides
(HePS) on the basis of their biosynthetic pathways. HoPS are synthesized from
sucrose by extracellular glucansucrases or fructansucrases and contain only one
type of monosaccharide, glucose or fructose, respectively. HePS are synthesized by
intracellular glycosyltransferases and consist of one or several monosaccharides
(see Chap. 7). HePS formation by LAB is used in dairy fermentation to improve the
texture of yoghurt and other fermented dairy products, but was recently also
employed in sorghum sourdough fermentation [44]. Glucan and fructan synthesis
was observed for Leuconostoc spp. and Weissella spp., which synthesize dextrans
[45]. Lactobacillus reuteri, L. panis, L. pontis, L. frumenti, and L. sanfranciscensis
produce fructans (levan or inulin) and glucans (dextran, reuteran, or mutan) [43]. It
was reported that any sourdough is likely to contain at least one EPS-producing
strain [43, 46]. In addition to EPS, glucan and fructansucrases can synthesize gluco-
and fructooligosaccharides, respectively. In sourdough L. reuteri, L. sanfranciscen-
sis and Weissella cibaria were shown to synthesize FOS, 1-kestose, and
isomaltooligosacchrides, respectively [47–49]. Together with EPS, in particular
levan from L. sanfranciscensis, oligosaccharides have been described for their
beneficial health effects. The production of EPS by sourdough LAB exerts two dif-
ferent functions: it improves the nutritional value (Chap. 9) of the bread and improves
bread texture.
Bacterial HoPS in sourdough act as hydrocolloids. The effect of bacterial EPS and
hydrocolloids on dough and bread is based on two functions: (1) water binding at the
dough stage and (2) the interaction with other dough components such as proteins
and starch. This interaction influences structural networks during fermentation and
the baking process [50]. During sourdough fermentations, LAB can produce EPS in
amounts that are sufficient for improving the dough structure [49]. Dextran from
Leuconostoc mesenteroides was reported to contribute to the long storage stability of
Panettone [51]. The application of in situ formed EPS can therefore reduce the
requirement for hydrocolloids to meet the consumer demands for clean labels and
reduced costs. Furthermore, levan produced in situ was more effective compared to
externally added levan [52]. The technological benefits of in situ formed EPS depend
on EPS properties and polymer yield. These are in turn dependent on the carbohydrate
supply of the fermentation substrate. Depending on the strain, flour, dough yield,
and fermentation conditions can be selected to obtain optimal EPS production [53].
222 S. Galle
Fig. 8.1 Wheat bread prepared without EPS containing sourdough (a) compared to bread prepared
with sourdough containing EPS (b) (Data from Galle [10])
EPS formation in dough reduced elasticity and strength, demonstrating the

interaction of EPS with the structure-forming components [10]. The addition of
sourdough fermented with HoPS-producing strains in wheat and rye dough induced
increased softening and freshness of the crumb and improved specific volume of the
bread (Fig. 8.1) [10, 14, 51]. The baking industry currently employs in situ formed
EPS and their potential as bread improvers is of growing importance. A process was
developed to adapt sourdough microbiota to high sucrose, and to reach a content of
25% dextran [54]. Dextran interacts with the gluten network and builds structure
resulting in improved dough stability and gas retention. Its high water-binding capac-
ity accounts for the freshness of the baked product. Linear, high molecular weight
dextran has a greater effect on bread volume compared to more branched, high
molecular weight dextran [51, 54, 55]. When the same amounts of reuteran, levan, or
dextran were added to wheat dough, dextran exhibited the best effect on viscoelastic
properties of dough and the volume of the final bread [43].
Sucrose metabolism of LAB not only yields EPS but also glucose and fructose.
Glucose supports gas production by yeast and contributes to dough leavening [10,
25, 56]. Fructose stimulates mannitol and acetate formation by heterofermentative
LAB. Acetate has antimicrobial properties, but high acetate concentrations nega-
tively influence flavor and bread structure. Beneficial effects on bread volume of
levan and reuteran from L. sanfranciscensis and L. reuteri, respectively, were miti-
gated by the detrimental effects of acetate (Fig. 8.2) [9, 10]. Different from hetero-
fermentative lactobacilli, Weissella spp. form only minor amounts of acetate and at
the same time significant amounts of dextran [15, 47, 48]. The application of
dextran-containing Weissella-sourdoughs provided mildly acidic bread with
improved structure and shelf life. Incorporation of higher amounts of Weissella-
sourdough leads to a higher concentration of dextran in the final bread dough with-
out negative impacts on the flavor and texture of the bread [10, 15].
4
Specific Volume [mL g-1]
0
Control 20% WSD 10% WSD 20%RSD 10%RSD
Fig. 8.2 Specific volume of bread prepared with 10 and 20% EPS-containing Weissella-sourdough
(WSD) compared to bread made with 10 and 20% EPS- and acetate-containing L. reuteri-sourdough
(RSD) (Data from Galle [10])
The findings of the influence of EPS in wheat and rye baking can be transferred
into gluten-free baking (Table 8.2) and are discussed in detail in Chap. 10. In sum-
mary, EPS formed during sourdough fermentation from sucrose can be successfully
applied to improve bread-making performance, however, it is necessary to select an
EPS-producing starter culture not solely based on its polymer properties and poly-
mer yield but also according to its by-products and fermentation performance.
8.6 CO2 Formation
Sourdough typically contains yeasts and lactic acid bacteria. Interactions of yeasts
and lactobacilli are important for microbial activity and CO2 formation in sour-
dough [57]. Gas formation by microorganisms is necessary in order to leaven bread.
In sourdough, carbon dioxide is produced by both heterofermentative LAB and
yeasts. The contribution of each group to the overall gas volume depends on the
starter culture and the dough technology applied [21, 25]. An increase in loaf vol-
ume was observed when sourdough with increased fermentation time was added to
the dough, but when baker’s yeast was applied the gas production by yeasts
superseded the gas production by LAB [11, 21, 30]. Unless sourdough is used as the
sole leavening agent, it is generally assumed that sourdough improves gas retention
but not gas production in bread dough [11, 21].
224
Table 8.2 In situ formed EPS and their effect on bread quality
EPS Strains Amounts [g kg−1 SDa] Effects on bread structure References
Dextran Ln. mesenteroides 0.6–16 g/kg Increases volume (wheat) [10, 15, 48, 51, 56]
W. cibaria Decreases firmness (wheat and sorghum)
W. confusa Improves freshness (wheat, rye, sorghum)
Glucan W. cibaria 2.5 g/kg Increases dough viscosity (wheat) [14]
L. plantarum Increases volume (wheat)
Decreased firmness (wheat)
Levan L. reuteri 1.5–5.2 g kg−1 No improvement (wheat, sorghum) [9, 48]
L. sanfranciscensis
Fructan L. reuteri 3.3 g kg−1 Decreases firmness (sorghum) [56]
Decreases freshness (sorghum)
Reuteran L. reuteri 0.6–5.1 g kg−1 Decreases firmness (sorghum, wheat) [10, 56]
Improves freshness (sorghum, wheat)
Decrease in dough strength and elasticity (wheat,
sorghum)
HePS L. buchneri n.d. Decreases dough strength and elasticity (sorghum) [44]
a
SD … sourdough; n.d. … HePS amount not detectable, HePS formation in sourdough was confirmed by gene expression
S. Galle
8.7 Alternative Fermentations and Synergistic Affects

of Sourdough and Dough Additives
The nutritional importance of dietary fiber has been demonstrated in many studies.
Acceptable loaf volume with high-fiber, whole-grain bread is difficult to obtain. The
fermentation of bran allows enhanced water absorption and textural modification of
bran particles. The use of fermented bran in combination with an enzyme mixture
(a-amylases, xylanase and lipase) improved the volume, texture, and shelf life of
high-fiber wheat bread [58]. Combined use of exogenous enzymes and sourdough
in wheat baking also enhanced the rate of acidification, improved bread volume,
and retarded staling [13, 59, 60]. Recently, sourdough in combination with cryopro-
tectants and/or conventional additives (e.g., honey, hydrocolloids) in frozen dough
technology were used to overcome problems such as prolonged final leavening
time, lower loaf volume, and poor bread characteristics [61].
8.8 Conclusion
The addition of sourdough improves the quality of bread and has a long tradition in
production of wheat and rye bread. Organic acid, EPS, enzymes, and CO2 synthesized
during sourdough fermentation by LAB and yeast are responsible for the positive
changes in dough and bread quality. Organic acids positively influence structure-
forming compounds of wheat and rye dough, particularly gluten, starch, and arabi-
noxylans. Furthermore, the production of organic acids and consequently the drop in
pH induces/inhibits endogenous enzymes like proteinases, pentosanases, and amy-
lases, also interfering with structural components. EPS formed from sucrose during
fermentation can replace hydrocolloids, currently used as dough and bread improvers.
However, it has to be considered that the metabolism of the sourdough microflora can
also have negative impacts on the bread quality, i.e., increased acidification compro-
mises crumb structure and masks beneficial effects of EPS. An improved knowledge of
metabolites formed during sourdough fermentation and their interaction with the dough
components enables a more direct optimization of the resulting bread quality. The stud-
ies presented in this review demonstrate that the use of an optimized sourdough process
in the production of bread provides a feasible technology for producing breads with
improved texture, volume, and shelf life. Sourdough technology can be useful to reduce
or eliminate the level of additives often used in baked products, and furthermore helps
to meet consumer demands for clean labels, natural products, and reduced costs.
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Chapter 9
Nutritional Aspects of Cereal Fermentation
with Lactic Acid Bacteria and Yeast
Kati Katina and Kaisa Poutanen
9.1 Introduction
Sourdough fermentation is best known and most studied for its effects on the sensory
quality and shelf life of baked goods. Acidification, activation of enzymes and their
effects on the cereal matrix as well as production of microbial metabolites all pro-
duce changes in the dough and bread matrix that also influence the nutritional qual-
ity of the products. The nutritional quality is formed through the chemical
composition and structure of the fermented foods, i.e. content and bioavailability of
nutrients and non-nutrients. Sourdough fermentation can change all of these, as
previously reviewed by Poutanen et al. [1] and Katina et al. [2].
Sourdough fermentation has been traditionally applied to whole grain foods, and
it is a good means of making whole grain bread more palatable. Rye bread is an
extreme example of this, as most of the whole-grain rye bread is made through
sourdough fermentation [3]. Sourdough fermentation, also in the form of pre-treat-
ing raw materials, is again gaining interest also in mixed flour and dietary-fibre-
enriched baking [4], where it also can change the properties of the dietary fibre
complex. Fermentation has been studied for reducing the glycaemic response of
bread [5, 6], and for increasing the uptake of minerals [7]. Microbial metabolism
during sourdough fermentation may also produce new nutritionally active com-
pounds, such as vitamins [8] and potentially prebiotic exopolysaccharides [9].
This chapter will deal with nutritionally relevant changes in cereal starch, protein,
dietary fibre, vitamins, minerals and some phytochemicals, and discuss the potential
of microorganisms to produce new compounds.
K. Katina (*) • K. Poutanen

VTT Technical Research Centre of Finland,
PB 1000, 02044 VTT Espoo, Finland
e-mail: kati.katina@vtt.fl

230 K. Katina and K. Poutanen
9.2 Effects on Cereal Biopolymers
9.2.1 Starch
Dietary carbohydrate is the major source of plasma glucose. An increase in the

amount of rapidly digestible carbohydrate in the diet causes a rapid increase in
blood glucose levels and a large demand for insulin in the postprandial period. The
major carbohydrate sources in the Western diet contain rapidly digestible starch,
and many common starchy foods like bakery goods, breakfast cereals, potato prod-
ucts and snacks produce high glycaemic responses. There are strong indications that
the large amounts of rapidly available glucose derived from starch and free sugars
in the modern diet [foods with high glycaemic index (GI) and high insulin index
(II)] lead to periodic elevated plasma glucose and insulin concentrations that may be
a risk factor to health [10].
Most processed starchy foods have low to medium moisture contents, thus
their digestion is basically a solid–liquid two phase reaction, and the enzyme
(particularly a-amylase) needs first to diffuse into the hydrated solid food matrix,
bind to the substrate, and then cleave the glycosidic linkages of the starch
molecules [11]. Factors affecting the binding of a-amylase to substrates [e.g.
inhibition by the hydrolysis products (maltose and maltotriose)] will slow down
the enzymatic reaction and thus digestion of starch. Other physiological factors
affecting starch digestibility include gastric emptying, enzyme inhibitors and
viscosity in the digestive tract [12].
Macro- and microstructure of cereal foods has a profound influence on the digest-
ibility of starch, as reviewed by Singh et al. [13]. Especially, the characteristics of
starch per se are of crucial importance for glucose response. Amylose-rich starches
are more resistant to amylolysis than waxy or normal starches. The major intrinsic
factors affecting raw starch digestibility include the supramolecular structure (pack-
ing of crystallites inside the starch granule), the ratio of amylase and amylopectin,
the amylopectin fine structure, and the surface characteristics of starch granules
[14]. In vitro, native starches are hydrolysed very slowly, and to a limited extent, by
amylases [15–17]. When starch is used in food processing, starch gelatinisation, i.e.
the process of disrupting starch crystalline structure with heat and moisture, usually
results in a decrease or loss of the slow digestion property of native cereal starches
[18]. Gelatinised starch will exist for example in bakery products in a partially or
completely amorphous state. Thus, the more gelatinised starch is, the more rapidly
it will be digested [19]. In many common starchy foods, such as in regular white
wheat bread, the starch is highly gelatinised and product structure very porous,
resulting in rapid degradation of starch in the small intestine and a very rapid rise of
blood glucose level (high GI).
There are several mechanisms leading to slow digestion of gelatinised starch
[20]. The first group of important factors is related to the state of starch in the food
matrix. Starch retrogradation, which is the reassociation of amylose and amylopec-
tin to form double helices and possible crystalline structures, promotes slow
9 Nutritional Aspects of Cereal Fermentation... 231
digestibility. The molecular structure of amylopectin is also an important factor, as

high branch density has been shown to be linked to slow digestibility. Lowering the
degree of starch gelatinisation and partially retaining the A-type crystalline struc-
ture of related starches is one effective way to increase the content of slowly digest-
ible starch in food products. The second group of factors include impact of chewing
on food structure, gastric emptying rate, transit time in the small intestine and the
properties of digestive enzymes [16].
The means to slower starch digestibility in wheat flour-based products such as
bread, biscuits and breakfast cereals are rare, if the addition of a high amount of
intact kernels is excluded due to the resulting inferior product quality and consumer
preferences. For wheat bread, the use of pre-fermentation technology (sourdough)
or the addition of soluble fibres were identified in a recent review as the only sug-
gested means to reduce GI [21].
The fermentation of the wheat and rye flour matrix with lactic acid bacteria
(sourdough process) has been shown to lower GI of wholemeal barley bread [19,
22] and wheat bread [5, 23–25], and insulin index (II) of rye breads with varying
dietary fibre (DF) content [26]. Several mechanisms have been proposed to be
involved in sourdough processing contributing to reduced starch digestibility.
Formation of organic acids, especially lactic acid, during fermentation has been
suggested to be a main reason. The physiological mechanisms for the acute effects
of acids appear to vary. Whereas lactic acid lowers the rate of starch digestion in
bread [22], acetic and propionic acids appear instead to prolong the gastric empty-
ing rate [27]. Chemical changes taking place during sourdough fermentation have
been postulated to diminish the degree of starch gelatinisation [19], which would
partly explain the lower digestibility of sourdough-fermented cereal foods.
Sourdough fermentation has been also shown to promote the formation of resistant
starch, which has slower digestibility [28].
At the product level, tissue integrity, porosity and structure of starch are impor-
tant characteristics influencing glycaemic response. Rye breads baked from whole-
meal or white rye flour with very different fibre contents produced lower insulin
responses than white wheat bread, when the food portion size was standardised to
provide 50 g of starch [26]. The breads were baked with a sourdough process and
with 40% of a total amount of rye flour being pre-fermented before incorporation
into the dough. The results suggested that with all rye breads, regardless of bran
content, less insulin was needed to regulate blood sugar from the same amount of
starch in comparison to normal wheat bread. The influence is probably due to the
more rigid and less porous structure of rye bread, and because of the presence of
organic acid formed during sourdough fermentation [29].
There also may be other mechanisms for the sourdough to regulate GI/II of the
products. For example, pH-dependent proteolysis generally occurs during sour-
dough fermentation [30] producing significant amounts of peptides and amino acids
in the sourdough. These may have a role in regulating glucose metabolism [31].
Furthermore, the results of Katina et al. [32] demonstrate that sourdough fermenta-
tion increases the amount of free phenolic compounds, which may also have an
impact on lowering the GI/II [6, 33].
However, not all the sourdough breads automatically have low GI/II [34]. In
general, a rather low pH of sourdough and subsequent bread is required to obtain
lowered GI or II; typical values being 3.5–4 for sourdoughs and 3.8–5.1 for sour-
dough breads [5, 24, 25, 35, 36]. The efficacy of individual acids reducing GI is
not completely clarified [6], and may vary between different bread types. In addi-
tion, such a low pH will in many cases reduce bread volume and increase density,
which have been shown to promote low GI per se also in regular wheat breads
[37]. Furthermore, the sensory quality of highly acidic breads may be a limiting
factor for consumer acceptability of such breads, and means for enhancing the
efficacy of fermentation while maintaining higher pH levels would be desirable.
Further studies will be needed to clarify the direct influence of sourdough metabo-
lites (acids, peptides, and exopolysaccharides) on starch digestibility, and the
indirect impact of sourdough fermentation on cereal matrix properties (density,
liberation of phenolic compounds, state of protein, and formation of resistant
starch), which all influence digestibility.
9.2.2 Protein
Protein degradation that occurs during sourdough fermentation is among the key
phenomena that affect the overall quality of sourdough bread as reviewed by Gänzle
et al. [30]. Proteolysis by sourdough fermentation has been found to be higher than
in just yeasted doughs. During dough fermentation, the proteolysis by LAB releases
small peptides and free amino acids, which are important for rapid microbial growth
and acidification and as precursors for the flavour development of leavened baked
products [38]. Furthermore, this proteolytic activity might be used as a tool to
reduce certain allergen compounds. Cereal proteins are one of the most frequent
causes of food allergies. Wheat proteins may induce a classical allergy affecting the
skin, gut or respiratory tract, exercise-induced anaphylaxis, occupational rhinitis or
asthma [36, 39], and protein modification with fermentation offers possibilities to
reduce their allergy-causing properties. For example, De Angelis et al. [36] demon-
strated the capacity of probiotic VSL#3 to hydrolyse wheat flour allergens. Albumins,
globulins, and gliadins extracted from wheat flour, a chemically acidified and started
doughs, and total proteins extracted from breads were analysed by immunoblotting
with pooled sera from patients with an allergy to wheat. Several IgE-binding pro-
teins persisted after treatment of baker’s yeast bread with pepsin and pancreatin.
The signal of all these IgE-binding proteins disappeared after further treatment by
VSL#3. Utilisation of the VSL#3 strain as a starter for bread making, caused a
marked degradation of wheat proteins, including some IgE-binding proteins. De
Angelis et al. [36] showed that the IgE-binding profile of the bread manufactured by
VSL#3 was largely different from that of baker’s yeast bread. The IgE-binding pro-
teins that persisted in the bread made with VSL#3 were completely degraded by
pepsin and pancreatin.
Intensive degradation of prolamin of wheat and rye has also opened new possibilities
to use these cereals even as part of gluten-free diets [23, 40, 41]. Controlled proteolysis
in wheat and rye doughs was suggested to reduce gluten levels to such an extent that the
products were tolerated by celiac patients [42]. While such sourdoughs with extended
fermentation time are not suitable for bread production as such, they can be incorporated
as baking improvers into gluten-free recipes. It was shown in a 60-day clinical trial that
biscuits and cakes produced using a hydrolysed wheat product made using sourdough
lactobacilli and fungal proteases were not toxic to patients with celiac disease [43].
The quality of gluten-free bread is often inferior when compared to conventional
(wheat) products [2]. However, by degrading prolamins of wheat or rye with a pro-
teolysis-intensive sourdough process, it is possible to produce good quality gluten-
free bread with sourdough technology [40, 42]. The concept of complete elimination
of gluten, however, is controversial. Gluten is considered essential for wheat baking
and the complete elimination of gluten from wheat and rye, albeit possible, is tech-
nically challenging in industrial baking operations. The use of germinated rye in
sourdoughs may avoid, in part, such controversy because the water binding as well
as gas retention in rye doughs are mediated by pentosans which remain unaffected
by proteolysis [30]. De Angelis et al. [23] demonstrated that fermentation by selected
sourdough lactic acid bacteria to decrease celiac intolerance to rye flour [44, 45]
used flour from germinated wheat and rye grains to enhance the proteolysis and
efficient degradation of wheat and rye prolamins.
Recently, it has been demonstrated that sourdough fermentation can promote
the formation of bioactive peptides [46–48]. Bioactive peptides are defined as
specific protein fragments that have positive effects on body functions or condi-
tions and that may influence human health. Usually, bioactive peptides correspond
to specific sequences from native proteins, which are released through hydrolysis
by digestive, microbial, and plant proteolytic enzymes, and their levels generally
increase during food fermentation. Coda et al. [46] summarised that bioactive
peptides, on the basis of in vitro and in vivo studies, have demonstrated a large
spectrum of biological functions, such as opioid-like, mineral-binding, immuno-
modulatory, antimicrobial, antioxidative, antithrombotic, hypocholesterolemic,
and antihypertensive activities. The ability of selected lactic acid bacteria to
produce antioxidant peptides during sourdough fermentation by using various
cereal flours as substrates was demonstrated [46]. The radical-scavenging activity
of water/salt-soluble extracts (WSE) from sourdoughs was shown to be significantly
(P < 0.05) higher than that of chemically acidified doughs. Twenty-five peptides of
8–57 amino acid residues were identified in their study and nearly all sequences
shared compositional features that are typical of antioxidant peptides. All of the
purified fractions showed ex vivo antioxidant activity on mouse fibroblasts
artificially subjected to oxidative stress. Recently, interest in antioxidant peptides
derived from food proteins has increased, and evidence that bioactive peptides pre-
vent oxidative stresses associated with numerous degenerative aging diseases (e.g.
cancer and arteriosclerosis) is accumulating [49].
Rizzello et al. [47] exploited the potential of sourdough lactic acid bacteria to
release lunasin, an anticarcinogenic peptide, during fermentation of cereal and
non-conventional flours. They used selected lactic acid bacteria as sourdough

starters to ferment wholemeal wheat, soybean, barley, amaranth, and rye flours.
Sourdough-originated lunasin was identified in their study and the concentration of
lunasin was shown to increase up to two to four times during fermentation.
From a practical standpoint, baked cereal goods are currently manufactured by
highly accelerated processes. Long-term fermentations by sourdough, characterised
by a cocktail of acidifying and proteolytic LAB and yeasts, have been almost totally
replaced by the indiscriminate use of chemical and/or baker’s yeast leavening
agents. In these technological circumstances, cereal components (e.g. proteins) are
subjected to very mild or no degradation during manufacture, resulting in less easily
digestible foods compared to traditional and ancient sourdough baked goods [41].
9.2.3 Dietary Fibre
Dietary fibre consists of the plant polysaccharides and lignin that are resistant to
hydrolysis by the digestive enzymes of man. A high consumption of dietary fibre
may lower the risk of cardiovascular disease, diabetes, hypertension, obesity, and
gastrointestinal disorders [50, 51]. Cereal foods are an important source of dietary
fibre, and because of their role as a staple food provide an important food group to
increase the currently too low intake of dietary fibre. Sourdough fermentation pro-
vides two main options for enhancing utilisation of fibre-enriched products: (1) It is
important technology in the manufacture of whole grain bread, especially rye bread,
and (2) it may be used to modify fibre-rich cereal ingredients such as bran and germ
for improved technological functionality.
Wholemeal rye and wheat are very good sources of dietary fibre. However, a
high content of fibre poses technological challenges for baking. For whole-grain rye
baking, sourdough fermentation is an essential part of the process [2]. Without sour-
dough wholemeal rye or wheat-rye flour mixes are very difficult to process, and
sourdough improves the overall quality and shelf life of whole-grain rye breads.
The rye sourdough process not only improves flavour and texture of rye bread but
enables consumption of wholemeal rye, which is well known for its high nutritional
quality and health-promoting properties.
Bran sourdough (or bran pre-ferment) is a potential means to improve the quality
of high fibre bread [4, 52–54]. The use of bran sourdough improves loaf volume and
crumb softness of high-fibre wheat breads [4, 52, 55] and bread with 10-% fermented
bran has been reported to provide the best sensory properties of bread [53]. The
impact of fermentation is assumed to be related to control of endogenous microbiota
of bran, endogenous xylanase activity and subsequent solubilisation of arabinoxy-
lans in bran fermentation [4]. Enzyme activity and gluten characteristics of dough
containing fermented bran will be modified by the acidity produced during fermen-
tation, and subsequently decreased pH. The fibre content of the bran does not change
significantly in a short fermentation time but can decrease slightly during prolonged
fermentation due to hydrolysis of cell wall structures (Katina, unpublished data).
Use of enzymes (a-amylase, xylanase, lipase) in combination with yeast fermentation

of bran has been shown to increase the volume of the subsequent bread, and soften
its texture significantly [55]. Use of fermented bran improves carbon dioxide reten-
tion of the dough, and the use of enzymes strengthens that effect. Also, addition of
insoluble arabinoxylans and xylanase enzymes has been shown to increase the vol-
ume of the sour dough bread [56]. Arabinoxylans function as the source material of
xylose and arabinose, which accelerate the acidification rate and positively interfere
with the metabolism of sourdough microflora.
Sourdough fermentation improves the technological functionality of bran as a baking
ingredient, but it most probably also changes the quality of dietary fibre. The physiologi-
cal effects of dietary fibre depend on the chemical but also physical characteristics,
including degree of polymerisation of the polysaccharides, presence of side chains and
degree of cross-linking, particle size and cell wall integrity [51]. Because of solubilisa-
tion of arabinoxylan, sourdough fermentation may influence its fermentation pattern and
also produce prebiotic oligosaccharides [57]. It may also influence the bioaccessibility
of phytochemicals associated with the dietary fibre complex, as shown below.
Wheat germ, in addition to vitamins and lipids, contains a significant amount of
dietary fibre. Fermentation of wheat germ has recently been noticed to enhance the
volume of the bread and decrease the rate of firmness [58]. The use of wheat germ
as a source of dietary fibre for bread is still moderate because of its poor shelf-life
stability. The high lipase and lipoxygenase activities cause sensitivity to oxidation
which leads to the release of free fatty acids and, consequently, to the appearance of
rancidity in baked goods. Sourdough fermentation stabilised and enhanced some
nutritional and chemical properties of the wheat germ. Because of lactic acidification,
the lipase activity of the sourdough fermented wheat germ has been shown to be
lower than that found in the raw wheat germ [58].
9.3 Micronutrients
9.3.1 Vitamins
Whole-grain cereal foods are an important source of vitamins, such as thiamine,

vitamin E and folates. Yeast fermentation increases the folate content during the
pre-fermentation process of both wheat flour and bran [32, 59] and rye [32, 59, 60],
causing over a doubling of folate in rye fermentation [60]. The presence of yeast has
been shown to be a crucial factor for increased folate production in rye sourdough
as sourdough bacteria had only slight effects on the synthesis of folates [61]. Yeast
strains have been shown to be different in their capability to produce folate, and thus
a high folate-producing strain could be used as an alternative to folate fortification
[62, 63]. The folate content in fermented cereal foods can be further increased by
the use of malted or germinated grains, as reviewed by Jagerstad et al. [64].
Conversely, 25–38% reduction of folate content in yeast and LAB fermented breads
have been reported by Gujska et al. [65].
Thiamine content has been reported to increase especially in elongated yeast

fermentation [8, 66], but also to decrease in the actual baking process [67]. Prolonged
yeast or sourdough fermentation maintained the original content of vitamin B1 in
whole wheat baking in contradiction to a short process, which reduced its amount.
Whole wheat breadmaking with yeast (from kneading to final bread), with long fer-
mentations time resulted in a 30% enrichment in riboflavin. The fermentation step
can thus improve the retention of vitamins in the baking process. The use of both
yeast and sourdough did not have a synergistic effect on B-vitamin levels [8].
Production of the B2 vitamin with strain selection for enrichment of pasta and bread
has also been recently demonstrated by Capozzi et al. [68]. The applied approaches
resulted in a considerable increase of vitamin B2 content (about two- and threefold
increases in pasta and bread, respectively), thus representing a convenient and
efficient food-grade biotechnological application for the production of vitamin B2-
enriched bread and pasta. This methodology may be extended to a wide range of
cereal-based foods, feed, and beverages. However, sourdough or yeast fermentation
do not automatically increase the levels of all vitamins; decreased levels have been
observed for vitamin E during sourdough preparation and dough making [69], and
for levels of tocopherol and tocotrienol in rye sourdough baking [60].
9.3.2 Minerals
Whole grains are a good source of minerals, including calcium, potassium, magne-
sium, iron, zinc and phosphorus. As the bran fraction of the grain also contains
phytate (myo-inositol hexaphosphate), the bioavailability of minerals may be lim-
ited. This has a large impact especially in developing countries, where iron deficiency
is a common nutritional disorder, especially among children and women. Grains
contain 3–22-mg phytic acid per gram [70], concentrated in the aleurone layers.
Phytate has strong chelating capacity and forms insoluble complexes with dietary
cations, thus impairing mineral absorption. Phytases are able to dephosphorylate
phytate, forming free inorganic phosphate and inositol phosphate esters, which have
less capacity to influence mineral solubility and bioavailability. It has been shown
that iron was more bioavailable in mice when fed in sourdough bread vs. straight
dough bread [71], and absorption of zinc, magnesium, and iron was higher in rats
when bread was baked using sourdough [72].
Grain endogenous phytase activity is accelerated in the acidic environment pro-
duced in sourdough fermentation. Lactic acid bacteria and yeasts may also possess
some phytase activity. The pH optimum of wheat phytase is pH 5.0, and that of
yeast is somewhat lower, i.e. pH 3.5 [73]. A moderate decrease of pH to 5.5 in fer-
mentation reduces phytate content of whole wheat flour by 70% due to enhanced
action of endogenous phytase present in the flour [74]. It was suggested that the
endogenous flour phytase activity was much more influential than the microbial
phytase of the sourdough. No major phytase activity was found in screening of 50
lactic acid bacteria strains isolated from sourdoughs [75], even though in studies
with phytic acid as the only carbon source sourdough-originated lactic acid bacteria
have been reported to utilise it [76, 77]. Phytase activity has been detected in com-
mercial baker’s yeasts [78], and variable activities were detected in traditional sour-
dough starters containing both yeast and lactic acid bacteria [79, 80]. Yeast strains
high in phytase activity have also been suggested to be potential phytase carriers in
the gastrointestinal tract [81].
Phytase action is dependent on the fermentation conditions: flour particle size,
acidity, temperature, time and water content [82, 83]. Sourdough fermentation has
been shown to be more effective in solubilising minerals in whole-wheat flours than
its bran fraction. Bran particle size influenced calcium and iron solubilisation, which
only happened if the bran was finely milled [7]. Pre-fermentation of bran with lactic
acid bacteria increased phytate breakdown (up to 90%) and increased magnesium
and phosphorus solubility [84].
Selenium-enriched rye and wheat seeds have been used to produce fermented
sourdough bread, and studied in human volunteers for bioavailability of selenium
[85, 86]. The selenium enrichment was made by incubating the seeds in selenium
solution. The high content of selenium in raw material was reflected in high con-
tents in the sourdough bread and further in humans having consumed the bread.
9.3.3 Phytochemicals
Phytochemicals are biologically active compounds in the cereal grain and they
have been suggested to be among the factors contributing to the protective proper-
ties of whole grain foods [87]. The outer layers of grains, such as bran, contain
much higher levels of phytochemicals, such as phenolic acids, alkylresorcinols,
lignans, phytosterols, tocols and folate, than the inner parts [60, 88]. Processing
may decrease or increase the levels, and also modify the bioavailability of these
compounds as reviewed by Slavin et al. [89], and for the phenolic compounds of
rye as reviewed by Bondia-Pons et al. [90].
Wheat bread containing a sourdough-fermented wheat bran-flour mixture
was recently shown to provide higher antioxidant potential as compared to reg-
ular wheat bread [91]. Traditional rye sourdough has been shown to increase the
antioxidant activity (DPPH radical scavenging activity) in the methanol-
extracted fraction of rye sourdough, concurrently with increased levels of easily
extractable phenolic compounds [60]. Accordingly, the antioxidant capacity of
traditional rye breads baked with sourdough has been shown to be higher than
that of common white wheat bread, the highest values reported for breads made
with whole meal flour [67, 92].
Fermentation of rye or wheat bran with yeast and especially with added cell
wall-degrading enzymes was able to increase the level of free ferulic acid [4, 32,
93]. Ferulic acid is a structural component in cell walls, cross-linked to arabinoxy-
lan. Since most of the ferulic acid is covalently bound to the cell wall structures, its
bioaccessibility in physiological conditions is low, and bioprocessing can be used as
an effective means to increase the bioaccessibility of ferulic acid. Wheat bread

supplemented with bioprocessed bran increased the in vitro and in vivo
bioaccessibility of phenolic compounds as well as the colonic end metabolite 3-phe-
nylpropionic in breads, and exerted anti-inflammatory effects ex-vivo [93, 94].
9.4 Microbial Exopolysaccharides
Dietary non-digestible oligosaccharides (NDO) have been shown to modulate the

composition and activity of intestinal microbiota, and they may also exert health
benefits in humans by improving bowel function, prevention of overgrowth of
pathogenic bacteria through selective stimulation of non-pathogenic members of
intestinal microbiota and by increased production of short-chain fatty acids (SCFA)
[95]. Intestinal fermentation and health benefits of fructo-oligosaccharides, galacto-
oligosaccharides and xylo-oligosaccharides have been well documented in animal
and human studies [96, 97]. Recently, stimulation by isomalto-oligosaccharides
(IMO) of the growth of intestinal lactic acid bacteria in a rat model was also shown
by Ketabi et al. [95]. The relationship between diet, intestinal microbiota and host
nutrition is currently under active investigation, and the integration of the functional
analyses of gut microbiota and sourdough genomes and metagenomes may allow
for design of prebiotic molecules with specific functional properties [98].
Microbes are able to produce a variety of polysaccharides. Exopolysaccharides
(EPS) are sugar biopolymers that are secreted by bacteria, microalgae and by some
yeasts and filamentous fungi. They may protect cells from external stress factors
such as desiccation and antimicrobial substances, and mediate interactions of cells
with surfaces and other cells, thus playing an important role, for instance, in biofilm
formation. EPS can be divided into capsular polysaccharides that are more or less
tightly bound on cells, and extracellular slime which cells excrete to their surround-
ing medium. EPS production can usually be detected on solid and liquid medium,
respectively, from a slimy or ropy colony appearance and from an increase in
medium viscosity. Microbial EPS vary greatly in mass; from ~10 kDa to 1–2 mDa.
On the basis of their chemical composition, all microbial EPS can be broadly divided
into homopolysaccharides (= Hops), consisting of only one monosaccharide type,
and heteropolysaccharides (= Heps), made of two or more different monosaccharide
units. Additionally, various inorganic or organic constituents may be attached. The
possible complexities of polysaccharide structures are almost infinite as, for instance,
even a disaccharide may be linked in eight different ways [99, 100].
Lactobacilli from wheat and rye sourdoughs have been shown to produce EPS
[9, 101], and especially gluco-oligosaccharides [102] and fructo-oligosaccha-
rides, which have prebiotic properties [9]. For example, Lactobacillus sanfranci-
scensis LTH2590 produced 0.5–1% levan (flour basis) during 24-h fermentation
in wheat and rye doughs [101]. Tieking et al. [103] studied the ability of seven
fructan- or glucan-positive LAB (Lb. sanfranciscensis LTH 2581 and 2590, Lb.
frumenti TMW 1.103, 1.660, 1.669, Lb. pontis TMW 1.675, Lb. reuteri TMW
1.1.06) to produce these EPS during wheat dough fermentation in the presence of
12% sucrose (flour weight). For all the strains the production of the same EPS at
a level of 0.5–2 g kg−1 was shown. Levans from Lb. sanfranciscensis may also
exert probiotic effects as they are preferentially degraded by bifidobacteria in the
intestinal tract [101]. Formation of oligo- and polysaccharides with prebiotic
potential has also been shown by Lb. reuteri LTH5448 and Weissella cibaria
10 M in sorghum sourdoughs [104].
9.5 Future Prospects
Sourdough fermentation is a food processing method with a long history, tradition-

ally used mainly to improve product quality. During the past 15 years, the use of
microbial fermentation has also been proven to intensively modify the nutritional
quality of cereal foods. Because of complex microbial and food structure interac-
tions present in a sourdough system, fermentation can be tuned for multi-functional
nutritional modifications of both traditional and novel fermentable substrates.
In the future, sourdough technology can provide an effective means to utilise and
upgrade side streams from both food and non-food processing, provide novel pro-
tein functionalities and produce completely novel oligo- and polysaccharides for
new nutritional improvements such as fat or sugar replacement. They also show
potential in producing and influencing bioavailability of minor food constituents
with high biological activity. Next-generation fermentations with yeast and lactic
acid bacteria can thus be considered effective cell factories to modify cereal and
also other fermentable materials for nutritionally tailored food or feed.
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Chapter 10
Sourdough and Gluten-Free Products
Elke K. Arendt and Alice V. Moroni
10.1 Introduction: Gluten-Free Cereal Products
Celiac disease is one of the most common food intolerances, with an incidence of 1 in
every 100 people worldwide, a number that is set to rise [1]. A lifelong avoidance of
gluten-containing cereals and related products is the only effective treatment for
people who suffer from celiac disease. Foods that are not allowed in the gluten-free
(GF) diet are all the gluten-containing products prepared from barley, kamut, oat,
wheat and their derivates, in which the gluten content exceeds 20 mg/kg on a total
basis [2]. As the request for GF products is significantly rising, food technologists
and manufacturers are called upon to satisfy the increasing requirements of the GF
consumers [3]. In particular, people who suffer from celiac disease and those who
are allergic to gluten ask for high-quality GF products, with the same textural, sensorial
and nutritional properties as their gluten-containing counterparts [4, 5]. Nonetheless,
the replacement of gluten with other non-toxic ingredients in conventional products,
primarily bread and pasta, constitutes a major technological obstacle for the food
industry. In fact, gluten represents the structure-forming protein in flour and it is
responsible for the unique viscoelastic properties (extensibility, resistance to defor-
mation, mixing tolerance and gas-holding capacity) of the dough [6]. The proteins
present in GF flours do not possess these fundamental structural features, and, upon
E.K. Arendt (*)

School of Food Science, Food Technology and Nutrition,
National University of Ireland, Cork, Ireland
Department of Food and Nutritional Sciences,
University College Cork, Western Road, Cork, Ireland
A.V. Moroniv
School of Food Science, Food Technology and Nutrition,
National University of Ireland, Cork, Ireland
National Food Biotechnology Centre, National University of Ireland, Cork, Ireland

246 E.K. Arendt and A.V. Moroni
mixing, a weak batter, resembling a cake dough, is obtained [7]. Because of the
impaired rheological properties of the GF batters in comparison to conventional
doughs, most of the GF products available on the market are characterised by over-
all low quality, lacking flavour and showing poor textural characteristics and mouth
feel [4, 8]. Furthermore, as GF products are mainly made from starch and are gener-
ally not fortified [9], their contribution in terms of different nutrients, such as folate,
B vitamins, iron and dietary fibre, is poor [10, 11].
Over the last decade, most of the academic research in the GF field has been
focused on the improvement of the quality of GF breads, producing breads that would
meet the expectations of the GF consumers in terms of appearance, structure and
nutritional benefits. Recent advances have been made in the incorporation of nutrient-
dense whole grains in GF bread formulations [12–14]. In particular, increasing atten-
tion has been drawn to the utilization of pseudocereals, i.e. amaranth, buckwheat and
quinoa, for their excellent protein quality and high fibre, mineral and phytochemical
contents [15, 16]. Incorporation of pseudocereals in GF formulations was shown to
induce significant improvements in the baking quality of the GF dough and in the
nutritional benefits of the GF bread [12, 13, 17, 18]. However, these “healthy” flours
are not yet extensively used for the production of GF products. Additionally, incorpo-
ration of prebiotics was also shown to be a successful approach for improving the
dietary fibre content of GF breads produced from starch [19].
The attempts aimed at the improvement of the textural properties of GF bread are
primarily affected by the absence of standardised baking tests for GF flours. For
example, a recent study has shown how the physiochemical composition, i.e. starch
content, particle size and rate of damaged starch, of oat flour can dramatically
influence its bread-making performances [20]. However, no guidelines are currently
available for the evaluation of the baking quality of GF flours.
Various additives, such as starches, hydrocolloids, non-toxic proteins, enzymes
and combinations thereof have been investigated in an attempt to improve the
poor structural and gas-holding capacity of GF batters. Because of their structure-
forming properties [21], hydrocolloids have been extensively used to imitate the
viscoelastic properties of gluten [22]. In particular, hydroxyl-propril-methyl-cellulose
(HPMC), carbossi- or methyl-cellulose (CMC, MC), locust bean and guar gum,
xhantan and pectins have been efficiently incorporated in GF bread formulations
[9, 14, 23, 24, 25 ]). Overall, these investigations suggest that the obtained quality
improvements, i.e. improved gas retention, crumb texture, specific volume and
prolonged shelf life, are correlated to the amount of hydrocolloids used and the
interaction between the type of flour and type of hydrocolloid employed.
Non-toxic proteins can also be applied to promote structure formation in GF
breads. Incorporation of milk, legume and egg proteins can induce the formation of
a gluten-like matrix in the batter and, therefore, improve the volume and the crumb
texture of the final bread [14, 18, 26, 27]. As for hydrocolloids, the positive effects
of the added proteins strongly depend on the interaction between type of flour and
nature of the proteins [25]. However, the application of structuring proteins may
represent a matter of concern, as these ingredients can be potential allergens for
celiac patients [28].
10 Sourdough and Gluten-Free Products 247
Enzymatic processing of GF flours has also been extensively investigated. In

particular, both cross-linking promoting enzymes, such as transglutaminase
(TGase), cyclodextrin glycosyl transferase, glucose oxidase [23, 29, 30, 44] and
proteases [31] were shown to improve the viscoelastic properties of batter and the
final bread quality. TGase was proved to be particularly efficient when applied in
GF breads produced with rice and buckwheat flours, used individually or in com-
bination with other ingredients [32, 33]. In particular, Renzetti et al. [33] showed
that the mode of action and the effects that TGase exerts on the pseudoplastic
behaviour of the batter and, ultimately, on bread quality vary according to the raw
material used for baking. Glucose oxidase was also used as a bread improver in
rice and oat GF breads [23, 29]). Interestingly, protein hydrolysis was shown to
improve the baking quality of rice flour, by decreasing the resistance to deforma-
tion of the batter during the baking process [31]. Thus, the concept that not only
structure-promoting treatments can improve the bread-making performances of
GF flours brings about new opportunities for GF baking [30, 34].
Overall, even if over the last decade promising improvements in the quality of GF
breads have been obtained, the production of superior quality GF bread still repre-
sents a challenging task. Considering the lack of standardized baking tests, the high
costs of the investigated additives and the great variability of flour-additive interac-
tions, alternative ways to produce high-quality GF breads need to be investigated.
This chapter discusses the recent advances in the application of sourdough in GF
baking as a low-cost, efficient and natural tool to improve the quality of GF bread.
10.2 Sourdough Bread
Sourdough is a mixture of flour and water fermented with lactic acid bacteria (LAB)
and yeasts, which can be added as starter cultures or originate as contaminants in the
flour [35, 36]. During sourdough fermentation, the resident LAB are the main factor
responsible for the acidification of the dough and for the synthesis of aroma com-
pounds, exopolysaccharides, enzymes and anti-fungal compounds [37–39]. The
addition of sourdough can strongly influence the quality of bread, in terms of
enhanced texture, prolonged shelf life and improved organoleptic and nutritional
profile [5, 40]. A profound knowledge of the metabolic events and the microbiologi-
cal interactions occurring during sourdough fermentation is of crucial importance for
controlling the fermentation and ensuring constant quality of the sourdough bread.
However, while extensive research has been done on wheat and rye sourdoughs, for
example on their microbial composition and their functional and organoleptic prop-
erties [35], only little information is available for GF sourdoughs [41–51]. Despite
being limited in number, these studies indicate that the GF flours represent a unique
source of novel strains and that GF sourdough can be successfully applied for
modifying the rheological properties of the batters and for producing high-quality
GF breads (Tables 10.1 and 10.2). In the next paragraphs we will discuss recent
findings in the application of sourdough fermentation to GF flours.
248
Table 10.1 GF sourdough fermentations and their effects on the properties of GF batters and breads
Substrate/starter Sourdough properties Effects on GF batter Effects on GF bread
Sorghum flour – L. plantarum [48] Proteolysis of water soluble proteins Increased strength Improved bread volume and
|of the starch gel crumb structure
Sorghum flour – W. cibaria [52] Synthesis of dextran and GOS Softer crumb and presence
of undigested GOS
Sorghum flour – W. kimchii or Synthesis of dextran and GOS Improved viscoelastic Improved crumb structure, specific
W. cibaria MG1 [41] Production of low amounts of acetate properties (?) volume and delayed staling (?)
Composite formulation – Production of anti-fungal compounds Increased elasticity Increase in the shelf life
L. plantarum FST 1.7 [44] Activation of endogenous enzymes Delayed staling
Red sorghum – L. plantarum/ Hydrolysis of oligosaccharides Improved nutritional and sensorial

L. casei or L. reuteri/L. fermentum quality (?)
Pulse flour – L. reuteri [53] Hydrolysis of glycol esters
of phenolic compounds and flavonoid
glucosides
Amaranth – L. paralimentarius Improved viscoelastic
|AL28 or L. plantarum AL30 [42] properties
(?) predicted effects
E.K. Arendt and A.V. Moroni
Table 10.2 Microbiota of GF sourdoughs and traditional GF products

Starter (S)
Raw material Spontaneous
Product Dominant microbiotaa (Sp)b Reference
Sorghum
Sudanese kisra L. fermentum, L. reuteri, Sp [54–56]
L. vaginalis, L. helveticus,
L. pontis, P. pentosaceus, I. orientalis
Sudanese khamir P. pentosaceus, L. brevis, L. lactis, Sp [57]
L. cellobiosus, C. parapsilosis,
C. orvegnsis, R. glutinis
Botswana – L. plantarum, L. casei/paracasei, Sp Sekwati-Monang,
porridge L. buchneri, L. reuteri, L. perolens Gänzle,
unpublished
Rice
Industrial L. paracasei, L. paralimentarius, Sp [43]
sourdough L. perolens, L. spicheri, S. cerevisiae
L. fermentum, L. gallinarum, L. pontis, S
C. krusei, S. cerevisiae
Laboratory-scale L. gallinarum, L. plantarum, L. helveticus, S 50]
sourdough L. fermentum, L. kimchii, L. pontis,
I. orientalis, S. cerevisiae
Maize
Kenkey L. fermentum, L. reuteri, P. pentosaceus, Sp [58–60]
C. krusei, S. cerevisiae
Mexican pozol L. delbrueckii, L. casei, L. fermentum, Sp 61–63]
L. plantarum, Streptococcus spp.,
Leuconostoc spp., Weisella spp.
Laboratory-scale L. brevis, L. casei, L. fermentum, Sp [64, 65]
sourdough L. plantarum, Lc. mesenteroides,
L. dextranicum, P. acidilactici, C.
halbicans, S. cerevisiae, S. pombe
Laboratory-scale L. fermentum, L. paralimentarius, S [50]
sourdough L. helveticus, L. pontis, S. cerevisiae,
I. orientalis
Teff
Injera P. cerevisiae, L. brevis, L plantarum, Sp [66]
L. fermentum, Saccharomyces,
Torulopsis, Candida spp.
Laboratory-scale L. plantarum (S), L. paralimentarius (S), Sp and S [46, 47]
sourdough L. fermentum (S,Sp), L. sanfranciscensis
(S), L. frumenti (S), L. pontis (S,Sp),
L. reuteri (S), L. amylovorus (S),
L. brevis (S), L. vaginalis (Sp), L.
gallinarum (Sp), P. acidilactici (S),
P. pentosaceus (Sp), Lc. holzapfelii (Sp),
K. barnetti (S), S. cerevisiae (S,Sp),
C. glabrata (Sp)
(continued)

Starter (S)
Raw material Spontaneous
Product Dominant microbiotaa (Sp)b Reference
Buckwheat
Laboratory-scale L. plantarum (S,Sp), L. paralimentarius (S), Sp and S [46, 47, 50]
sourdoughs Lc. argentinum (S), L. sanfranciscensis
(S), W. cibaria (S,Sp), L. brevis (S), L.
fermentum (S, Sp), L. amylovorus (S), L.
helveticus(S), P. pentosaceus (Sp), Lc.
holzapfelii (Sp), L. graminis (Sp), L. sakei
(Sp), L. vaginalis (Sp),
L. crispatus (Sp), L. gallinarum (Sp), K.
barnetti (Sp)
Amaranth
Laboratory-scale L. plantarum (S,Sp), L. sakei (Sp), Sp and S [49, 50]
sourdoughs L. paralimentarius (S,Sp), L. fermetum
(S), L. helveticus (S), L. spicheri (S),
P. pentosaceus (Sp), Enterococcus spp.
(Sp), S. cerevisiae (S), C. glabrata (S)
Adapted from [5]
a
I. Issatchenkia, L. Lactobacillus, Lc. Leuconostoc, P. Pediococcus, R. Rhodotorula,
S. Saccharomyces, C. Candida
b
The fermentation was either started by addition of starter strains (S) or by the spontaneous biota
of the flour (Sp)
10.3 Ecology of GF Fermentations and Development of GF

Sourdough Starters
The species diversity of wheat and rye sourdoughs has been intensively investigated
by culture dependent and independent approaches. Heterofermentative species
belonging to the genus Lactobacillus are among the most frequently isolated, but
also species of the genera Leuconostoc, Pediococcus and Weissella were retrieved
in traditional sourdoughs [67]. It is widely accepted that the selection of the com-
petitive biota in wheat and rye sourdoughs is mainly driven by the fermentation
parameters [36, 68], whereas the role played by the flour and its autochthonous
microorganisms is under discussion [67].
Most ecological studies have been performed on sourdoughs produced from
wheat, rye or spelt, fermented at laboratory scale or previously processed in bakery
environments [69–73]. All together, these investigations indicate that the quality
and nature of the flour play only a marginal role in the selection of the competitive
species in conventional sourdoughs. In this regard, we must consider that wheat, rye
and spelt are three closely related cereals, and that the bakery environment has been
shown to define the predominant species in sourdoughs [73]. Therefore, a question
still remains unsolved: would this principle be applicable for alternative sour-
doughs? Recent studies indicate a different trend for GF flours [46, 50]. Vogelmann
et al. [50] investigated the adaptability of various LAB and yeast starter strains in
continuously propagated sourdoughs prepared from GF cereals and pseudocereals.
The authors concluded that some of the starter strains used could not persist over the
fermentation and their adaptability to the GF sourdoughs was strongly influenced
by the chosen flour. Unfortunately, because of the high number of different sub-
strates used, the authors could not identify the reasons for such variability. Moroni
et al. [46] recently investigated the development of buckwheat and teff sourdoughs
using commercial starters. The substrate used determined the persistence of specific
starter strains; whereas spontaneous species, originating from the flour, either out-
competed or co-dominated with the starter LAB and yeasts. The unique sugar com-
position of certain GF flours is a key factor in the selection of dominant species in
GF sourdoughs [41, 46, 47]. For example, when added as a starter strain,
Lactobacillus sanfranciscensis failed to grow in sorghum sourdough due to lack of
maltose at the beginning of the fermentation [41]. Instead, the high glucose level in
sorghum sourdough favoured the growth and metabolic activities of Weissella spp.
[41]. Similarly, Weissella cibaria was found to be highly competitive in buckwheat
sourdough, in which the initial content of the monosaccharide was higher than that
of maltose [46, 47]. Furthermore, the high ratio glucose/maltose favoured the
coexistence of maltose positive yeasts and LAB in teff sourdoughs [46]. All together,
these findings suggest that the nature of the GF flours used for sourdough fermentations
has a strong impact in the selection of the dominant LAB and yeast strains.
Furthermore, these studies clearly indicate that commercial starters as such cannot
be efficiently used for the production of GF sourdough. In fact, the main criteria for
selecting useful starters are that the starter’s strains must be highly adapted to the
GF substrate, dominate the fermentation and inhibit the growth of contaminant and/
or autochthonous strains [67].
Ecological studies on GF sourdoughs are essential for developing GF starters, but
to date only few data are available (Table 10.2). Most of these investigations have
been carried out on traditional products, mainly produced from maize, sorghum and
teff in tropical countries. These fermentation products were dominated by LAB spe-
cies that mainly overlapped with those frequently isolated in wheat and rye sour-
doughs [68]. In particular, L. fermentum, L. reuteri and L. plantarum were the ones
most frequently isolated (Table 10.1). However, an interpretation of the results
obtained in these studies is difficult due to the inappropriate techniques used for spe-
cies identification, and to the non-sterile conditions under which the fermentations
were carried out. More recently, the species diversity of laboratory-scale and indus-
trial sourdoughs produced from different GF cereals and pseudocereals has been
investigated through integrated approaches of culture dependent and independent
techniques (Table 10.1). These sourdoughs have either been developed using starter
cultures [43, 46, 50] or by spontaneous fermentation [47, 49, 64, 65]. As observed in
traditional products, L. fermentum, L. plantarum, and also L. paralimentarius were
present in virtually all the spontaneously and starter fermented GF sourdoughs from
rice, maize, buckwheat, teff and amaranth (Table 10.1). Instead, the dominance of
the most common sourdough species L. sanfranciscensis and L. pontis was substrate-
specific. Species such as L. gallinarum, L. graminis, L. sakei and Pediococcus
pentosaceus, which are not frequently isolated in conventional sourdoughs, were

present in various GF sourdoughs, in particular when produced by spontaneous fer-
mentation. The pseudocereals buckwheat and amaranth were found to be good sub-
strates for the growth of L. sakei, P. pentosaceus and L. paralimentarius, whereas
buckwheat sourdoughs induced inhibition of yeast growth [46, 47, 50, 51]. In con-
clusion, the ecological studies on GF fermentations suggest that GF flours represent
an important reservoir for novel, competitive LAB and yeast strains that can be
selected as starters for the production of stable GF sourdoughs. In a second stage,
these strains can be screened for their functional properties, such as production of
EPS, aroma and/or anti-fungal compounds and rate of acidification [5, 72, 74].
10.4 Proteolysis as a Tool to Improve the Baking

Performances of GF Flours
The proteolytic events occurring during sourdough fermentations of wheat and rye
flours have been exhaustively reviewed by Gänzle et al. [75]. Protein degradation
during sourdough fermentation is among the key phenomena that affect the overall
quality of sourdough bread, by inducing formation of precursors for flavour com-
pounds and by modifying the viscoelastic properties of the dough [75]. Because of
the gradual acidification of the dough by LAB, endogenous enzymes are activated
and exert primary proteolysis on flour proteins. In a second stage, the released pep-
tides are further hydrolysed into amino acids by intracellular peptidases of LAB, in
a strain-specific manner [76]. In general, most sourdough LAB do not possess extra-
cellular proteinase activity and prefer peptide uptake over amino acid transport [77].
LAB can affect the pattern of hydrolysed products by increasing the amount of
dipeptides and amino acids released in the sourdough ([76, 77]. As yeasts consume
amino acids during growth, amino acid accumulation in sourdough can occur only
after yeast growth has stopped [78–80]. Studies on the proteolytic events occurring
during sourdough fermentation of GF flours and their effects on GF bread quality
are still limited. Recently, sourdough fermentation was effectively applied for the
production of GF bread based on sorghum flour, potato starch and HPMC ([48];
Table 10.1). Superior quality bread could be produced only when the total amount of
sorghum flour was replaced by sorghum sourdough, fermented with the starter strain
L. plantarum. The authors ascribed this quality improvement mainly to the prote-
olytic events occurring on soluble sorghum proteins during sourdough fermentation.
The hydrolysed proteins did not interfere with the starch gel upon gelatinisation and
a stronger starch gel with superior structural properties was obtained. As shown by
confocal laser scanning microscopy (CLSM), the presence of small peptides in the
sourdough bread prevented the formation of protein aggregates in the crumb upon
cooking; whereas aggregates were formed in the chemically acidified control.
Elkhalifa et al. [81] investigated the molecular and structural changes occurring
during fermentation of sorghum flour for the preparation of the traditional Sudanese
food kisra. As shown by light and CLS microscopy, fermentation resulted in the
hydrolysis of the water-soluble proteins that constitute the outer shell of the starch
granules. As a result, smaller starch granules were released from the matrix and the
pasting properties of sorghum flour were modified. Proteolysis has also been inves-
tigated in the GF product towga, a traditional Tanzanian fermented food prepared
by fermentation of either sorghum, maize, cassava, millet or combinations thereof
[82]. Both spontaneous and starter-induced fermentations induced the activation of
proteinases and, depending on the fermentation conditions, an increase in the con-
tent of specific amino acids, such as glutamic acid, proline, ornithine, methionine
and lysine.
Overall, the proteolytic events occurring during sourdough fermentation can posi-
tively affect the baking quality of GF flours. Therefore, sourdough fermentation could
replace the use of proteolytic enzymes in GF bread formulations [30, 31], which would
allow a reduction in the cost of the bread and enable consumers’ acceptance issues to
be overcome. However, more studies are needed in order to understand which GF
flours can be positively treated by sourdough fermentation and what degree of prote-
olysis is required in order to enhance their baking performances.
10.5 Proteolysis for Reducing the Toxicity of Wheat Flour
During endoluminal digestion, a family of peptides rich in Pro and Gln are released
from prolamins of wheat and rye. These toxic peptides are responsible for the auto-
immune response in celiac patients [83, 84]. During sourdough fermentation of
wheat flour, gliadins are among the most affected proteins, where the extent of
hydrolysis of monomeric gliadins (a-, b-, g-, w-gliadins) is strain-specific [76, 85].
Di Cagno et al. [76] showed that selected LAB, possessing proteolytic activities,
could efficiently hydrolyse the 31–43 fragment of the toxic peptide A-gliadin in
wheat sourdough. The same authors further applied the proteolytic LAB for producing
non-toxic sourdough from a mixture of toxic and non-toxic flours, in which the
highly toxic 33-mer peptide was completely hydrolysed [86]. Breads produced with
this sourdough showed acceptable quality and when they were fed to celiac indi-
viduals, no alterations to the baseline values of the patients were observed. The
same pool of proteolytic LAB was proven to be efficient for detoxifying rye flour
[87] and, when used in association with L. sanfranciscensis, for producing non-
toxic wheat sourdough bread of acceptable quality [88]. Sourdough fermentation
can also be efficiently applied for eliminating the gluten that can eventually be present
as a contaminant in GF flours [89].
Prolonged sourdough fermentation of wheat and rye using specific LAB may
represent a novel technology for baking good-quality breads that can be consumed
by celiac individuals. Nonetheless, long-term in vivo tests are needed in order to
confirm the suitability of these products for celiac patients. Furthermore, manufac-
turers will have to face the obstacle of gaining the acceptance of consumers towards
GF products containing detoxified wheat and/or rye.
10.6 Exopolysaccharides: A Low-Cost Alternative

to Hydrocolloids in GF Breads
Exopolysaccharides (EPS) produced by LAB are alternative biothickeners that act

as viscosifying, stabilising, emulsifying or gelling agents in a wide range of food
products [90]. EPS are generally classified in two categories: homopolysaccharides
(HoPS), glucose or fructose polymers, and heteropolysaccharides, containing (ir)
regular repeating units [90]. To date, only HoPS have been shown to be useful in
bread making. Glucans and fructans are synthesised by extracellular glucan- or
fructansucrases, respectively, by various sourdough-associated LAB. Lactobacillus
reuteri, L. panis, L. pontis, L. frumenti and L. sanfranciscensis were shown to pro-
duce fructans (levan or inulin) and glucans (dextran, reuteran or mutan) [91]. In
particular, Leuconostoc spp. and Weisella spp. were proved to synthesise a large
variety of dextrans [92, 93]. The structure, molecular weight and the production
yield of HoPS varies among the producing LAB species and also depends on the
carbohydrate concentration and composition of the source [92, 94]. During sour-
dough fermentations, LAB can produce EPS in high amounts, sufficient for improving
the structural properties of the dough [95, 96]. In particular, in situ production of
EPS was shown to be more effective than external addition of the same polysac-
charide in the bread formulation [97]. Addition of sourdough fermented with HoPS-
producing strains in wheat dough had a dramatic effect on bread quality by inducing
softening of the crumb and increasing specific volume of the bread [92, 93, 95].
In addition to EPS, glucan- and fructansucrase can synthesise gluco- and fructo-
oligosaccharides (FOS), respectively [98]. FOS have been associated with prebiotic
effects [94, 99]. In particular, the levan produced by L. sanfranciscensis LHT2590,
was proved to stimulate bifidobacterial growth in vitro [100]. In sourdough, L. reu-
teri, L. acidophilus and L. sanfranciscensis LHT2590 showed the ability to produce
the prebiotic FOS 1-kestose [94, 94]. Therefore, EPS-producer strains can be applied
not only to improve the bread-making performance of the flour, but also to enhance
the nutritional value of the bread. These features render EPS the ideal replacement
for hydrocolloids in GF breads. However, to date, only few studies [41, 52] have
investigated EPS formation in GF sourdoughs and their feasibility for GF baking.
Schwab et al. [52] recently analysed the applicability of the EPS-producers
L. reuteri LHT5448 and W. cibaria 10M in GF sourdoughs (Table 10.1). Both strains
were shown to be suitable starters for sourdough fermentation of quinoa and sorghum,
and during fermentation they produced levan/FOS and dextran/GOS (galacto-
oligosaccharides), respectively. When applied in baking, sorghum sourdough fer-
mented with W. cibaria was shown to be the most effective. In fact, softer sorghum
breads were obtained upon addition of this sourdough, and the isomaltooligosaccha-
rides present in the dough were not digested by baker’s yeast during proofing [52].
The authors concluded that consumption of 300 g of sorghum GF bread prepared
with W. cibaria 10M would account for a significant intake of prebiotic GOS.
Galle et al. [41] have also screened EPS-forming Weissella strains for their potential
as starter strains in sorghum and wheat sourdoughs (Table 10.1). Independent from
the strain used, higher amounts of EPS were formed in sorghum sourdough than in
wheat, because of the higher concentration of glucose in the GF flour. In particular,
the amount of dextrans produced by W. kimchii and W. cibaria MG1 were high
enough for the sourdough to be applicable as a replacement for hydrocolloids in
bread. In both sourdoughs, Weissella strains also produced oligosaccharides, whose
structure was dependant on the chosen flour. Furthermore, both strains released fruc-
tose and formed only small amounts of acetate, a characteristic which is favourable
for bread production [101]. All together, these studies indicate that sourdough fer-
mented with EPS-forming Weissella strains can be applied in GF bread as a substi-
tute for hydrocolloids. In addition, different types of oligosaccharides can be formed
in the GF sourdough through manipulation of the carbon sources.
In conclusion, EPS-forming LAB can be successfully applied for improving the
baking performances of GF flours. However, screening must be performed in order to
identify EPS-producing strains that can be successfully applied as starter cultures in
GF sourdoughs. In addition, more investigations are needed to evaluate the applica-
bility of specific starters in GF sourdough breads, as the type of EPS and its interac-
tions with the matrix strongly influence the structural properties of the dough [93].
10.7 Starch Hydrolysis for Delaying the Staling of GF Bread
Retrogradation/recrystallisation of starch is one of the key events involved in bread

staling. During storage, the amylopectin network present in fresh bread gradually
turns into a semi-crystalline network, which is responsible for crumb firming [102].
Amylases, in particular maltogenic amylases, and malt are commonly applied in
bread baking as anti-staling agents [103]. Addition of sourdough to wheat bread can
retard staling and sourdoughs with high TTA (total titratable acidity) and low pH are
favourable for such purposes [104]. Even if most LAB species do not possess
amylolytic activities, amylolytic strains were isolated from cereal fermentations in
tropical climates [75, 105, 106]. In this regard, Corsetti et al. [107, 108] showed that
the biological acidification together with the proteolytic and amylolytic activities of
the selected starter strains delayed staling of sourdough wheat bread.
Staling represents one of the major issues in GF baking, since most GF breads
are mainly starch based. Research on the effects of sourdough fermentation in starch
hydrolysis and staling in GF bread is extremely limited. Malting and boiling com-
bined with fermentation of sorghum resulted in a decrease of crumb firmness and
dryness of sorghum-wheat composite bread [109]. Songré-Ouattara et al. [110] iso-
lated amylolytic strains of L. plantarum in pearl millet gruels and efficiently used
them as starter cultures for starch hydrolysis. The authors also suggested that more
amylolytic strains can be isolated from the traditional product [110]. Schober et al.
[48] showed that the combination of a-amylase and sourdough improved the qual-
ity of sorghum bread, but it did not have any positive effects on the staling rate.
Instead, chemical acidification had rather negative effects on the bread volume and
it dramatically increased the firmness over the whole storage period. Incorporation
of sourdough into a GF bread mixture was also shown to delay the firming process,
whereas chemical acidification increased the staling rate of the bread ([44];
Table 10.1).
Thus, depending on the flour and the starter strains, sourdough fermentation can
be used for controlling the firming process of GF bread. However, the mechanism of
bread staling is still under discussion and virtually no studies address this complex
event in GF formulations. Therefore, more investigations are needed in order to
characterise the activity of flour and bacterial amylases in GF sourdoughs. Through
in depth investigations, it will be possible to identify the best process conditions for
retarding staling of GF bread by application of sourdough, i.e. type and amount of
added sourdough and its combination with commercial amylases and/or malts.
10.8 Sourdough as a Natural Tool for Improving

the Shelf Life of GF Bread
The distribution of dust and mould spores in the bakery environment is the main
cause of bread spoilage [111]. Bread spoilage represents a main issue for the baking
enterprises, in so far that it induces economical losses and health risks for the
consumers. The most common bread spoilage moulds are Aspergillus, Cladiosporum,
Endomyces, Fusarium, Monilia, Mucor, Penicillium and Rhizopus [111]. An effec-
tive and natural way to improve the shelf life of bread consists in the application of
sourdough. Sourdough-associated LAB can produce various substances with anti-
fungal properties [112, 113]. Heterofermentative LAB release anti-fungal organic
acids, among which acetic and propionic acids are more effective than lactic acid
[113]. A mixture of organic acids, i.e. caproic, acetic, formic, propionic, butyric, and
n-valeric acids, produced by L. sanfranciscensis CB1 were shown to be the main
factors responsible for its anti-mould activity against Fusarium, Penicillium,
Aspergillus and Monilia [108]. Strains of L. plantarum were proven to have broad
anti-fungal activity, with 4-hydroxyphenlyllactic and phenyllactic acid being the
major inhibiting compounds produced by the sourdough-LAB [114–116]. Ryan
et al. [39] also showed that addition of sourdough fermented with the anti-fungal
strain of L. plantarum allowed a reduction of the calcium propionate level in wheat
bread by around 30%, without any negative effects on the shelf life. Reutericyclin is
a low molecular weight antibiotic active against Gram-positive LAB and yeasts
produced in active concentrations by L. reuteri [117]. In addition, L. reuteri strains
can produce reuterin, an anti-microbial substance active against bacteria, yeasts and
fungi (reviewed by [118]). Sourdough LAB are also effective against rope spoilage
induced by Bacillus spp., because of production of organic acids and other unknown
anti-bacterial compounds [119, 120].
Research on the application of sourdough for prolonging the shelf life of GF
breads is still in its infancy. Recently, Moore et al. [44] employed sourdough (20%)
fermented by the anti-fungal strain L. plantarum FST 1.7 in a composite GF bread
(Table 10.1). Lactobacillus plantarum FST 1.7 retained its inhibitory activity against
the test fungus Fusarium culmorum in the GF bread and it delayed the growth of the
mould for up to 3 days in respect to the non-acidified control. Thus, the potentiality
of sourdough as a shelf-life improver is retained in GF breads, but more investiga-
tions should evaluate this application. GF fermentations represent an important
source of novel LAB strains and their potentiality as anti-fungal starter strains
should be evaluated.
10.9 Sourdough Fermentation for Enhancing

the Health Benefits of GF Bread
Sourdough has the potential to enhance the nutritional benefits of bread by improv-
ing mineral bioavailability, lowering the glycaemic response and regulating the
accessibility of bioactive compounds in the flour (reviewed by [38]).
Phytic acid (PA) is the major storage form of phosphorous in grains and it is
considered an anti-nutritional factor. In fact, PA impairs mineral absorption by
strongly binding dietary cations to form insoluble complexes [121]. For celiac
patients, who suffer from micronutrient deficiencies, the presence of PA in GF bread
is particularly undesirable. Cereal grains contain phytases which can increase min-
eral bioavailability by dephosphorylating phytate to free inorganic phosphate and
inositol phosphate esters. Sourdough fermentation favours the activity of endoge-
nous phytases by creating optimal pH conditions for their activation [122, 123].
Lopez et al. [123] showed that sourdough fermentation enhanced phytate hydrolysis
twice as much in respect to conventional yeast fermentation in wheat and in whole
wheat bread. In addition, sourdough-associated LAB and yeasts were found to exert
phytase activity in traditional sourdoughs [124]. Accordingly, De Angelis and co-
workers [122] showed that fermentation by L. sanfranciscensis CB1 induced a
decrease of PA by over 50% in wheat dough.
To date no studies have investigated the fate of PA in GF sourdoughs destined for
bread production. Yet, phytase activity has been characterised during fermentation of
GF cereals. Fermentation induced a decrease in PA content of sorghum and pearl mil-
let [125]. Two phytase-positive strains, i.e. L. plantarum and L. fermentum, have been
recently isolated from the above fermentation products [126].
Sourdough fermentation can also increase the bioavailability of several nutri-
ents, such as folate, thiamine, vitamin B1 and antioxidants in wheat and rye bread
(reviewed by [38]). However, the excessive content of bioactive compounds in GF
substrates can hamper the nutritional attributes of the GF flour [53]. Gänzle et al.
[53] applied sourdough fermentation to red sorghum and pulse flour for reducing
their content of polyphenols and oligosaccharides, respectively (Table 10.1).
The a-galactosidase positive strain L. reuteri efficiently hydrolysed raffinose,
stachyose and verbascose in pulse flour. Additionally, sourdough fermentation of
red sorghum with binary combinations of LAB, i.e. L. plantarum / L. casei or L. reuteri
/ L. fermentum, resulted in quantitative hydrolysis of glycol esters of phenolic
compounds and flavonoid glucosides. Therefore, LAB-induced fermentation of GF

flours has the potential to improve their nutritional and sensorial characteristics
[53]. Furthermore, as shown by Galle et al. [41] and Schwab et al. [52] fermentation
of GF materials with selected EPS-producer strains can result in the release of
significant amounts of GOS with prebiotic activity.
The poor nutritional benefits of GF breads can be enhanced by sourdough fer-
mentation, which can efficiently decrease the content of anti-nutritional factors in
the GF flour and increase the content of prebiotic GOS. However, more studies are
needed in order to identify the best flour/starter combinations and process condi-
tions for producing GF bread of high nutritional quality.
10.10 Conclusions
Production of GF bread of superior structural and nutritional quality still represents

a major issue for food technologists. Sourdough fermentation of wheat and rye has
been used for improving the overall quality of bread since ancient times. Recent
studies have shown that its main positive effects on bread quality are retained when
sourdough fermentation is applied to GF materials. Proteolysis and EPS production
occurring during sourdough fermentation of GF substrates were shown to improve
the rheological properties of the bread dough and the final bread quality. Furthermore,
addition of sourdough has the potential of delaying the staling process through
starch hydrolysis. From a nutritional point of view, application of sourdough tech-
nology leads to both an increase in certain bioactive compounds and a decrease in
the content of anti-nutritional factors in GF flours. However, even if promising
results have been obtained, the use of sourdough in GF baking is only in its infancy
and more research should focus on this matter. For example, the potential of sour-
dough as a flavour-carrier has not been investigated in GF sourdoughs yet, even
though the lack of flavour is one of the main negative aspects of GF breads.
Following the rising interest in the use of sourdough in GF baking, investigations on
the ecology of GF fermentations are increasing. Through such studies, it will be
possible to select novel starter strains for producing GF sourdoughs with specific
properties for achieving the desired quality improvements in GF breads.
With more data available, the industrial production of GF sourdough starters and
GF sourdough breads is likely to become a reality in the market of GF products.
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Chapter 11
Sourdough and Cereal Beverages
Jussi Loponen and Juhani Sibakov
11.1 Introduction
Worldwide, a number of fermented solid, semi-liquid, and liquid products exist that
are produced by microbial incubations or fermentation utilising milk, meat, legumes,
tubers, and cereals as raw materials. However, only with cereal substrates is it possible
to produce fermented doughs, porridges, gruels as well as alcoholic and non-alcoholic
beverages, and also non-dairy yogurt alternatives. This chapter will focus on tradi-
tional cereal-based non-alcoholic fermented beverages and intends to introduce
products that differ in their manufacturing concepts. In principal, all porridges or
gruels may be turned into “beverage mode” by adding more water to the recipes
though sedimentation of solids may make this impractical. Nevertheless, processing
technology and specialty ingredients nowadays allow stabilisation of these into
homogenous and stable products. It is worth noting that most of the fermented cereal
beverages are traditionally consumed in certain geographical regions and, thus, the
majority can be considered as indigenous to specific groups of people.
11.2 Boza
Boza is a traditional cereal beverage made by fermentation of cooked, strained, and

sugared cereal slurry. The term boza refers to millet (Persian) but, in practise, flour
or semolina or cracked grains of wheat, maize, rice, or their mixtures are also used
J. Loponen (*)
Fazer Group, Helsinki, Finland
J. Sibakov
VTT Technical Research Centre of Finland, Espoo, Finland

266 J. Loponen and J. Sibakov
Fig. 11.1 Process chart for

boza preparation
as raw materials. The end product is a fairly thick liquid with pale yellow appearance,
and a mixed sweet and sour taste that is characteristic for boza. Starter cultures for
boza production may originate from a previous boza fermentation, sourdough, pure
cultures, or yoghurt. Arici and Daglioglu [1] review the characteristics of boza and
its history more in detail. Turkey and Bulgaria are the heartlands of boza.
The process of boza fermentation is illustrated in Fig. 11.1 (based on [1]).
Cooking of pre-processed (cleaned, milled) cereal raw materials into a slurry is fol-
lowed by a cooling and straining stage, sucrose addition (up to 20% w/w), inocula-
tion with a starter culture, overnight fermentation, and finally cooling and packaging
(Fig. 11.1). Water is added during processing in order to adjust the consistency of
the slurry. The fermented boza exhibits pseudoplastic rheological behaviour, i.e. its
apparent viscosity decreases with increased shear rate [2, 3].
Hancioğlu and Karapinar [4] were among the first to monitor boza fermenta-
tions. During a 24 h fermentation, acidification took place, the pH dropped from 6.1
to 3.5, and final cell counts of lactic acid bacteria and yeasts were 5 × 108 and 8 × 106,
respectively. Gotcheva et al. [5] observed that glucose accumulated and free amino
nitrogen levels decreased during boza fermentation, of which the latter observation
11 Sourdough and Cereal Beverages 267
Table 11.1 LAB and yeast strains isolated from boza fermentations. Literature data was collected
using criteria that the total cell counts for LAB and yeasts in boza were at minimum levels of 107
and 105, respectively; lower cell densities were considered irrelevant
Amylolytic Bacteriocin
LAB Weissella confusa [4]
Lactobacillus fermentum [4, 6, 7]
Lactobacillus paracasei × [6, 8]
Lactobacillus pentosus × [6, 8, 9]
Lactobacillus rhamnosus × [6, 8]
Pediococcus pentosaceus × [10]
Lactobacillus sanfranciscensis [4]
Lactobacillus coryniformis [4]
Lactobacillus plantarum × × [6–9]
Lactobacillus coprophilus [7]
Lactobacillus brevis [6, 7]
Lactobacillus acidophilus [7]
Lactobacillus raffinolactis [7]
Leuconostoc mesenteroides × [4, 7, 11]
Leuconostoc paramesenteroides [4]
Oenococcus oenos [4]
Yeasts Saccharomyces uvarum [4]
Saccharomyces cerevisiae [4, 7]
Geotrichum penicillatum [7]
Geotrichum candidum [7]
Candida tropicalis [7]
Candida glabrata [7]
indicates that extensive protein breakdown did not occur during the fermentation
process. Hayta et al. [2] showed that the soluble protein content increased during
boza fermentation, which was probably induced by acidification. These values and
tendencies are characteristic for boza fermentation. Indeed, the cooking stage prior
to starter inoculation is an important feature of boza, because the heating practically
inactivates the raw material associated enzymes and microbes, and consequently
results in a semi-sterile base free from enzymes and microbes. This feature allows
the usage and operation of starter cultures with specific activities. Of the lactic acid
bacteria, especially heterofermentative lactobacilli and leuconostocs dominate boza
fermentations (Table 11.1) whereas Saccharomyces species are the predominant
yeasts. In addition members of the Weissella, Lactococcus, Pediococcus, Geotrichum
and Candida families were identified (Table 11.1). Zorba et al. [12] used strains
previously isolated from boza as starters for new fermentation in order to develop
an optimal starter culture. A combination of Leuconostoc mesenteroides ssp. mes-
enteroides, Weissella confusa and Saccharomyces cerevisiae strains was suggested
as a feasible starter culture for the controlled production of boza as the trio elicited
the desired rheological and sensory properties of fully fermented boza [12].
In addition to improved sensory and rheological properties, the use of controlled

starter cultures in boza fermentation aimed at incorporating bacteriocin-producing
strains in boza fermentations; Todorov and Dicks [8, 10, 11] isolated a number of
bacteriocin-producing strains from boza and also partially characterised many bac-
teriocins (Table 11.1). Bacteriocins are small proteinaceous metabolites that are
excreted by the host strain during its stationary growth phase, and they exhibit lethal
or static effects against bacteria closely related to the host, which may prolong shelf
life and improve the safety of the products.
Boza, as a semi-sterile base, lacks the enzyme activity of cereal raw materials,
which in sourdough fermentation, for instance, play a significant role. This sets
requirements for starter strains to grow as well as offers some possibilities for food
technologists to take advantage of specific catalytic activities of starter strains of
even added exogenous catalysts. Petrova et al. [9] identified two amylolytic lacto-
bacilli strains from Bulgarian boza. The strains, Lactobacillus plantarum Bom 816
and L. pentosus N3, used starch as a sole carbohydrate source, and the amylolytic
activity of the cell-wall associated enzymes was optimal at pH 5.5 and 45 °C. No
research on the synthesis of extracellular polysaccharides (EPS) exists though boza
could serve as a potential matrix for this as it can be fermented with selected starter
cultures (e.g. EPS-producing starter cultures) and its carbohydrate composition
could be adjusted to meet the desired EPS-synthesis profiles. For instance, the syn-
thesis of homopolysaccharides by glucansucrases or fructansucrases of LAB is
boosted by high matrix sucrose content. In addition, future works could focus on
investigations related to other bioactivities such as vitamin B profile and their
changes during boza fermentation.
11.3 Togwa
Togwa is a Tanzanian beverage prepared from cereal porridge by liquefying the

starch paste with malt followed by fermentation. The flour used to make the por-
ridge is usually from maize, sorghum or finger millet, though cassava is a common
ingredient as well. The addition of germinated sorghum or finger millet, with high
amylolytic activity, induces saccharification of gelatinised starch and results in a
sweetened slurry with reduced viscosity. Ripe togwa from a previous fermentation
batch usually serves as a starter for the fermentation of the liquefied and saccharified
slurry. Togwa is described as having an opaque, brownish appearance and a slightly
floury mouthfeel [13]. A more detailed illustration of the manufacturing process for
togwa is provided in Fig. 11.2 (Modified after [13, 14]).
Lactic acid bacteria and yeasts are the dominant microorganisms in togwa.
Mugula et al. [14, 15] investigated some key fermentation parameters and identified
dominant microorganisms from Tanzanian togwa samples. The final pH-values for
togwa were between 3.1 and 3.5 and most of the LAB isolates were heterofermenta-
tive and roughly every third isolate produced dextran. Homofermentative L. plantarum,
however, dominated fermentations and three of its isolates showed amylolytic activity

togwa preparation
as they hydrolysed starch [14]. The dominance of L. plantarum was probably

because of its acid tolerance and its ability to effectively utilise different carbon
sources. Obligative heterofermentative Lactobacillus brevis, L. fermentum and
W. confusa, and homofermentative Pediococcus pentosaceus were also among
dominant LAB strains in togwa [14]. Issatchenkia orientalis was the predominant
yeast whereas S. cerevisiae, Candida pelliculosa and C. tropicalis were also among
the most common yeast isolates (Table 11.2).
Hellström et al. [16] isolated a number of yeast strains from togwa fermenta-
tions, identified them using API and molecular techniques, and determined their
phytase activity. Yeast strains with high phytase activity could serve as alternatives
for mineral fortification or the addition of phytases in order to improve the mineral
availability of fermented foods through elimination of the metal-chelating com-
pound phytate. The lactic acid bacteria may also promote phytate elimination as the
acidification increases the activity of endogenous cereal phytases [17].
In addition to the elimination of antinutritive compounds, the microflora of togwa
may produce beneficial compounds such as B vitamins in food fermentations.
Hjortmo et al. [18] investigated folate production in togwa fermented with previously
isolated togwa-adapted yeasts [16]. Tetrahydrofolate and 5-methyl-tetrahydrofolate
were the dominant forms. The concentration of the latter folate type substantially
Table 11.2 Dominant microbes isolated from togwa

Organism Relevant enzyme activities Reference
LAB Weissella confusa [14]
Lactobacillus fermentum [14]
Pediococcus pentosaceus [14]
Lactobacillus plantarum Amylolytic [14]
Lactobacillus brevis [14]
Yeasts Saccharomyces cerevisiae [14, 16]
Issatchenkia orientalis (Candida Phytase [16]
krusei)
Pichia guilliermondii [16]
Kluyveromyces marxianus [16]
Hanseniaspora guilliermondii Phytase [16]
Candida glabrata [16]
Pichia norvegensis [16]
Pichia burtonii [16]
Candida tropicalis [14, 16]
Candida pelliculosa (Pichia [14, 16]
anomala)
increased in a yeast-fermented maize base whereas the levels of tetrahydrofolate

remained unchanged. The increase of total folate was highest with a strain of
Candida glabrata, which resulted in a togwa with 23-fold folate content compared
to the control incubation without yeast; the final folate concentration in the
C. glabrata ferment was 700 ng per g dry matter [18].
Mugula et al. [19] investigated the proteolytic activity in togwa and verified that
the added malt was the main source of proteolytic activity. The amino acids leucine,
isoleucine, valine, ornithine, glutamic acid accumulated during togwa fermenta-
tions [19]. These amino acids are well known to influence flavour formation directly
or indirectly as, for instance, Leu, Val, Ile serve as precursors for Strecker aldehydes
that are typical amino-acid-derived flavour compounds in sourdoughs [20].
11.4 Mahewu
Mahewu is a traditional non-alcoholic spontaneously fermented beverage consumed

by the Bantu people of Southern Africa, for example in Zimbabwe. Gruel prepared
from maize or sorghum is cooled down and inoculated with sorghum malt (or wheat
flour) and left to stand for 1–2 days. During the incubation period, saccharification
and spontaneous fermentation take place. The dominant microflora of mahewu con-
sists of lactic acid bacteria, mainly Lactococcus lactis subsp. lactis [21] as well as
yeasts, although grain-associated fungi were also detected [22]. A major goal of
using malt combined with fermentation is to reduce the amount of tannins, which
are antinutritive polyphenols equipped with astringent sensory properties and therefore
generally regarded as highly unfavourable compounds. Bvochora et al. [22] showed
that the fermentation reduced the levels of tannins (specifically proanthocyanidins)
by more than 50% during 36 h mahewu preparation. According to Oyewole [23] the
pathogenic Campylobacter, Escherichia coli and Shigella bacteria could not sur-
vive in fermented mahewu products.
11.5 Bushera
Bushera is a fermented beverage traditionally consumed in Uganda; low-alcoholic

bushera is produced using a fermentation period of 1 day whereas longer fermenta-
tion results in an alcoholic beverage that is not suitable for children [24]. Bushera is
prepared from sorghum malt or millet malt. Its manufacture begins with the addi-
tion of malt flours of sorghum or millet to boiling water. During this boiling stage
the starch gelatinises, with saccharification also likely taking place. After the boil-
ing stage, the gruel is cooled down and fermentation is initiated by adding malt flour
to the system. After 1 day of fermentation a beverage with low alcohol content is
obtained.
11.6 Pozol
Pozol is a maize-based non-alcoholic beverage that originates from the Maya

regions of South-Eastern Mexico and Guatemala (citations in [25]). The beverage is
consumed by all family members, including infants, and it is a staple food of
Mayans. The pozol beverage is made by suspending spontaneously fermented maize
dough in water. Prior to fermentation, maize kernels are nixtamalised, i.e. cooked in
lime (1%) water and the hulls are removed. After this stage a second boiling stage
may be used or the nixtamalised dehulled kernels are directly washed and wet-
milled [26]. The milled mass (called masa or nixtamal) is moulded into oval-shaped
dough pieces, wrapped in banana leaves and left to stand at ambient temperature for
one to several days. During this period of time, spontaneous fermentation takes
place. At the start of incubation the pH of the dough is ~7 and during the first day it
declines to below pH 5, while after 6 days the pH is below 4 [25, 27].
Lactic acid bacteria (mainly from the genera Lactococcus, Lactobacillus,
Leuconostoc and Weissella) are numerically the dominant microbes (~109 cfu/g
FW) whereas yeasts and fungi are also present in substantial numbers [26–28]. As
in many other cereal fermentations amylolytic strains of LAB and yeasts as well as
dextran-producing strains apparently have adapted and, thus, are significantly pres-
ent in pozol [27]. Olivares-Illana et al. [29] characterised an inulosucrase active
strain of Leuconostoc citreum isolated from pozol that synthesised inulin-type poly-
mers. Of the fungus, all Geotrichum strains and nearly half of the yeast strains were
able to utilise lactate as a sole carbon source, which demonstrates a good adaptation
to sour conditions [27].
11.7 Chicha
Chicha is a fermented corn-based beverage consumed mainly in Southern Africa.

The manufacturing technique is quite unique, because human saliva is used as a
source of amylase enzyme, which converts the starch of corn into fermentable sug-
ars. The primary microorganisms are yeasts, especially S. cerevisiae, bacteria from
the species of Lactobacillus, Leuconostoc and Acetobacter, as well as moulds, such
as Aspergillus [21].
11.8 Kishk
Kishk is a traditional fermented product, which is still consumed in Egypt, Syria

and in many Arabic countries. It is made of dough, which contains salt and fer-
mented skim milk. In addition, oat groats, oat flour, pre-cooked wheat groats (also
called burghol) or wheat flour can be used as an ingredient of kishk. Most usually,
the dough consists of a mixture of fermented milk and burghol. The dough is
kneaded once a day for 6 days at 35 °C to hydrate and gelatinise the starch.
The fermentation of kishk is spontaneous and takes place during this period
mainly by L. plantarum, L. brevis, L. casei, Bacillus subtilis and certain yeasts
[21, 30].
After 6 days, the dough is moulded into small pellets, which are set into trays to
dry in the sunshine for a week. The dried pellets are ground either traditionally by
hand or in grain stores in industrial scale production. The beverage is produced by
dissolving the ground pellets into hot water. Fermented kishk products have ele-
vated levels of minerals and they are good sources of b-glucan and dietary fibre.
Fermentation and drying also affects the shelf life of the product positively [30].
11.9 Kvass
Kvass is a fermented cereal beverage consumed in Eastern Europe. It is produced

from rye malt, rye flour, stale rye bread and sucrose. In addition, barley malt or
flour, as well as other cereals can be used as ingredients. A special type of kvass malt
(fermented rye malt) is often used for the typical aroma characteristics of kvass.
Kvass resembles Turkish boza with respect to the composition of the final product
as well as the microflora [31, 32].
RYE, RYE MALT (SPECIAL KVASS MALT),

BARLEY, BARLEY MALT, OTHER CEREALS, STALE RYE BREAD
RICE, CORN AND STARCH.
MASHING CUTTING TO 2–3 cm CUBES
WORT CLARIFICATION DRYING
BOILING WORT CONCENTRATION

By vacuum evaporation MIXING WITH HOT WATER
ca. 1:10 solid to liquid ratio
COOLING / ADDITION OF WATER
INCUBATION
ADDITION OF STARTER (AND SUGAR) At room temperature for 12 h
FERMENTATION
At 12–30 ºC for 12–24 h
COOLING TO 4–6 ºC AND CLARIFICATION
ADJUSTMENT OF SUGAR CONTENT
SERVING
Fig. 11.3 Flow charts of two main kvass-making techniques (Modified according to [31, 32])
Baker’s yeast (S. cerevisiae) is the predominant microorganism in kvass

fermentations. In addition, lactic acid bacteria are added or already present as a
latent infection in the malt or in the bakery/brewery environment [31–33]. Dlusskaya
et al. [32] were among the first to characterise the microbes in kvass fermentations.
They concluded that L. casei, Lc. mesenteroides and Saccharomyces cerevisiae
were the dominating members of the kvass microflora.
Two main kvass-making techniques exist using either stale sourdough bread or
malt as raw materials (Fig. 11.3). When fermentation originates from stale sour-
dough bread, all sugars necessary for yeast growth are derived from the bread-making
process. In the second technique, gelatinised starch is cleaved by malt enzymes
during mashing. In both techniques, the liquefied sugars are fermented by yeast and
lactic acid bacteria at 12–30 °C for 12–24 h. During fermentation, no more than
about 1% of the sugars are converted into lactic acid, CO2 and ethanol [31, 32].
After fermentation, kvass is cooled down to 4–6 °C and clarified through
filtration or centrifugation. Traditionally intensive clarification was not used and,
thus, kvass contained high cell counts of viable yeasts and lactic acid bacteria, as
no heating was used after fermentation. The sugar content might be adjusted by
addition of another syrup after fermentation. As a rule of thumb, the quality of
kvass can be determined through the amount of sugar added. In general, cheaper
products contain a significantly higher amount of sugar than premium products,
which are made with a bigger proportion of high-quality wort extract. The alcohol
content of kvass varies and is influenced by the choice of ingredients, the mixture
of microorganisms, the duration and temperature of fermentation, as well as by
consumer preference. It can even be considered that kvass is spoiled if it contains
more than 1% of ethanol [31, 32].
Before the nineteenth century, kvass production was limited only to private
homes. Rye bread was soaked in water and spontaneously fermented with yeast and
lactic acid bacteria. Several days of fermentation resulted in an alcohol concentra-
tion lower than 1.5 vol-%. Berries, fruits, herbs or honey can be added either before
or after fermentation to adjust the flavour. Because of the high sugar and low alco-
hol concentration, and the absence of pasteurisation, kvass was only enjoyed for
short periods of time and could not be stored [31].
In the nineteenth century, most kvass producers specialised in particular raw
materials, leading to new flavours, such as apple, pear and peppermint kvass.
At the same time the growing urbanisation and industrialisation made homemade
kvass increasingly rare. In the 1960s, the success of the Coca-Cola Company
inspired the Russian government to assign chemists in Moscow the task of devel-
oping an economical method for kvass production. In the early years of industrial
production, the final product was filled and sold directly in large containers, which
were commonly situated on trailers on street corners and in market places. Now
the vast majority of kvass is sold in 1–3-l plastic bottles, which can be stored for
4–6 weeks [31].
11.10 Sourish Shchi
Sourish shchi is a Russian beverage with a low level of alcohol (below 2.5 vol-%).
It was popular in Russia during the eighteenth and nineteenth centuries, but it is still
in production. The origin of this beverage is most likely linked to the appearance of
champagne in Russia as it required champagne bottles and a second fermentation.
The six components usually involved in the preparation of sourish shchi (shchi is
old Russian and means “six”) were three kinds of malt: barley, rye, wheat; one kind
of flour: wheat or rye; buckwheat and honey [34].
In the beginning of the process, malted and unmalted grains are mashed sepa-
rately using hot water and then the two fractions are combined to produce a wort
(Fig. 11.4). Fermentation starts spontaneously through the naturally occurring
microbes found on grains, in the vessels and in the air. The wort is allowed to stand
in a warm place for 12–24 h. Once the wort begins to ferment, there follows one or
more inoculations with a “special sour” culture (from the sediment of the vessel
from the previous fermentation). For example, Dankovtsev et al. [34] used S. cerevisiae,
S. uvarum (carlsbergensis) and S. minor yeasts, as well as L. brevis as starter cul-
tures. The majority of the spontaneous sour inoculum in the experiments of
Dankovtsev et al. [34] consisted of wild and cultured yeast, such as the previously
mentioned starter strains, as well as Saccharomyces oviformis, and strains from the
genera Torula and Candida. The bacteria were identified as lactobacilli.

the preparation of sourish
shchi
After the final inoculation, it is important to monitor the “culmination” of the

fermentation, which is usually indicated by the thickness of the foam layer. When
fermentation is almost complete, the wort is cooled down and the beverage bottled
in champagne bottles. To ensure a sufficient level of carbon dioxide, sugar syrup or
honey is added just before bottling. The bottles are allowed to end ferment at very
low temperature. Sourish shchi differs from Russian kvass and other traditional
beverages in its sparkling nature due to a high CO2 concentration, the frothiness and
its resemblance in taste to champagne [34].
11.11 Hulu-Mur
Hulu-mur is a Sudanese non-alcoholic beverage made of sorghum (Sorghum

bicolor) and preferably using the cultivar Fetarita [35]. In the preparation of Hulu-
mur, both malted and unmalted sorghum grains are used (Fig. 11.5). First unmalted
sorghum dough is prepared and par-baked after which malted sorghum slurry and a
starter is added. Sorghum sourdough (used for kisra bread making) is used as a

hulu-mur preparation
starter. During the first fermentation stage, acidification takes place and the pH
drops from 5.9 to 3.7 [35]. After the first fermentation other ingredients (spices,
dates, and tamarinds) are added with some water and the mixture is allowed to fer-
ment a second time. Finally, the fully fermented dough is baked into small and thin
sheets, and the dry sheets are suspended in water to prepare the hulu-mur drink.
Relatively little research data on hulu-mur exist though Mahgoub et al. [36] inves-
tigated the micronutrient content and its changes in hulu-mur.
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bility of Boza, a traditional cereal-based fermented Turkish beverage. Eur Food Res Technol
213:335–337
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physical and sensory analysis. J Food Eng 52:95–98
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tion of the traditional Bulgarian beverage boza. Int J Food Sci Technol 36:129–134
6. Botes A, Todorov SD, von Mollendorff JW, Botha A, Dicks LMT (2007) Identification of
lactic acid bacteria and yeast from boza. Process Biochem 42:267–270
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of the Bulgarian cereal-based fermented beverage boza. Process Biochem 36:127–130
8. Todorov SD, Dicks LMT (2006) Screening for bacteriocin-producing lactic acid bacteria from
boza, a traditional cereal beverage from Bulgaria: comparison of the bacteriocins. Process
Biochem 41:11–19
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fermented beverage boza. Z Naturforsch 65c:218–224
10. Todorov SD, Dicks LMT (2005) Pediocin ST18, an anti-listerial bacteriocin produced by
Pediococcus pentosaceus ST18 isolated from boza, a traditional cereal beverage from Bulgaria.
Process Biochem 40:365–370
11. Todorov SD, Dicks LMT (2004) Characterization of mesentericin ST99, a bacteriocin pro-
duced by Leuconostoc mesenteroides subsp. dextranicum ST99 isolated from boza. J Ind
Microbiol Biotechnol 31:323–329
12. Zorba M, Hancioglu O, Genc M, Karapinar M, Ova G (2003) The use of starter cultures in the
fermentation of boza, a traditional Turkish beverage. Process Biochem 38:1405–1411
13. Kitabatake N, Gimbi DM, Oi Y (2003) Traditional non-alcoholic beverage, Togwa, in East
Africa, produced from maize flour and germinated finger millet. Int J Food Sci Nutr
54:447–455
14. Mugula JK, Nnko SA, Narvhus JA, Sørhaug T (2003) Microbiological and fermentation char-
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15. Mugula JK, Narvhus JA, Sørhaug T (2003) Use of starter cultures of lactic acid bacteria and
yeasts in the preparation of togwa, a Tanzanian fermented food. Int J Food Microbiol
83:307–318
16. Hellström AM, Vázques-Juárez R, Svanberg U, Andlid TA (2010) Biodiversity and phytase
capacity of yeasts isolated from Tanzanian togwa. Int J Food Microbiol 136:352–358
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Chapter 12
Perspectives
12.1 Microbial Ecology of Sourdough
The characterization of microbiota of a large number of industrial and artisanal

sourdoughs has greatly promoted our understanding of the microbial ecology of
sourdoughs, and resulted in the description of more than a dozen new species ([1–3],
Chap. 5). The origin of sourdough microbiota, however, remains unclear.
Environmental contamination appears to play a role for yeasts found in sour-
dough [4]. However, the lactic microbiota of back-slopped sourdoughs are mark-
edly different from the microbiota of spontaneously fermenting sourdoughs or the
raw materials, excluding the raw materials as a significant source of most lactoba-
cilli associated with sourdough. Moreover, back-slopped sourdoughs remain the
only known source of several Lactobacillus species. Significant changes in the
microbiota of sourdoughs were found during long-term propagation in artisanal
bakeries [4]. In other cases, sourdough microbiota remained stable at the strain level
over several decades of propagation. Genome sequencing of Lactobacillus sanfran-
ciscensis recently provided an unprecedented snapshot of microbial evolution in the
sourdough environment. Whole genome sequencing of two strains isolated from the
same industrial sourdough before and after 18 years of continuous back-slopping
revealed that the genome underwent only minimal changes during that time [5].
Human use of sourdough is thus unlikely to allow the evolution of specialized
microbial species, or sourdough-adapted lineages of Lactobacillus species.
M. Gänzle (*)
Department of Agricultural, Food and Nutritional Science, University of Alberta,
Edmonton, Canada
[email protected]
M. Gobbetti

Sourdough microbiota and the lactic microbiota of the intestine of humans and
animals show remarkable overlap, indicating that intestinal lactobacilli are a poten-
tial source of sourdough-adapted strains [1]. The substrate availability of the upper
intestine of mammals that consume cereal-based foods, the major site of coloniza-
tion by lactobacilli in swine, poultry and rodents [6], is remarkably similar to sour-
dough. Sucrose and maltose are the major carbon sources and carbohydrate
metabolism by sucrose phosphorylase or levansucrase and maltose phosphorylase
contributes to the ecological fitness of lactobacilli. The intestinal origin of sour-
dough strains was recently demonstrated for L. reuteri, which is both a gut symbiont
and a stable member of sourdough microbiota [1, 7]. Several L. reuteri lineages
have evolved to become specific for their respective hosts, i.e. humans, swine, poul-
try or rodents [7]. Multi-locus sequence analysis and analysis of host-specific physi-
ological and genetic traits assigned five L. reuteri isolates from back-slopped
sourdoughs to rodent- or human-specific lineages. Comparative genome hybridiza-
tion revealed that the sourdough isolate L. reuteri LTH2584 is genetically highly
related to the rodent isolate L. reuteri 100-23. Taken together, these results demon-
strate that sourdough isolates of L. reuteri that persisted in a back-slopped sour-
dough for over 50,000 generations initially originated from intestinal microbiota
[8]. Likewise, L. rossiae, L. pontis and L. fermentum were described as stable mem-
bers of intestinal and sourdough microbiota [9], and sourdough isolates of these
species may also originate from the intestine of animals.
The overlap of intestinal and sourdough microbiota allows the development of
probiotic cereal products employing probiotic lactobacilli as starter cultures. Vellie,
oat bran fermented with probiotic lactobacilli, provides a conceptual template for
such products [10]. Mesophilic lactobacilli are unlikely to originate from the intesti-
nal tract of mammals. However, L. sanfranciscensis was isolated from the intestinal
tract of fruit flies [11], which may provide a ubiquitous reservoir for the species.
Wheat and rye sourdoughs exhibit similar microbiota; their composition and
activity depends mainly on the process conditions (see Chap. 5). Durum wheat sour-
dough, commonly used for bread baking in Southern Italy, was suggested to select
for obligate heterofermentative lactic acid bacteria owing to the higher levels of
maltose, sucrose and amino acids [12]. Moreover, cereal fermentations in Africa
and South Asia predominantly use sorghum, millet, corn, rice, or teff as raw materi-
als [13]. The dominant species of lactic acid bacteria in fermentations with these
substrates only partially overlap with wheat and rye sourdoughs [14, 15]. The higher
ambient temperature in tropical climates selects for thermophilic microbiota.
Substrate-derived factors such as the carbohydrate availability and the presence of
antimicrobial phenolic compounds also contribute to the establishment of divergent
and substrate-specific microbiota. However, the specific effect of the raw materials
on the microbial ecology of sourdough is currently not fully understood. The
industrialization of food production in developing countries will result in a more
standardized fermentation process and the production of starter cultures (see e.g. [16]),
paralleling the development in the European baking industry in the past decades.
This development will also lead to an improved understanding of the microbial
ecology of cereal fermentations in tropical climates.
12 Perspectives 281
12.2 Sourdough and Product Quality
Current knowledge of the effect of carbohydrate metabolism, exopolysaccharide

production, and amino acid conversion allows the targeted selection of starter cul-
tures for improved bread quality (see Chap. 7). The availability of genome sequences
for sourdough-adapted lactobacilli [7, 17, 18] not only furthers the understanding of
the microbial ecology of sourdoughs, but also facilitates the elucidation of addi-
tional metabolic pathways converting amino acids, lipids, and phenolic compounds
and their effect on bread quality. The elucidation of the effect of individual meta-
bolic pathways on bread quality is supported by the development of tools related to
comparative genomics, proteomic and transcriptomic analyses, and the develop-
ment of novel tools for metabolic re-engineering of sourdough lactic acid bacteria
[7, 8, 18]. A more comprehensive analysis of the effect of strain-specific metabolic
traits on product quality in combination with a decrease of the expense associated
with genomic analyses for starter cultures will facilitate in silico strain selection as
a complement for the evaluation of cultures in application trials.
12.3 Sourdough and Nutrition
The comparison between baked goods made with baker’s yeast or chemical leaven-
ing and those made with sourdoughs clearly evidences that sourdough bread is more
digestible and has a higher bioavailability of minerals because substantial degrada-
tion of cereal components (e.g. proteins and phytate) occurs during fermenta-
tion [19]. This is already an undisputable reason for the preferred use of sourdough.
However, public policy and industrial product development in developed countries
are no longer predominantly related to the need to meet basic nutritional require-
ments by an increased bioavailability of macro- and micronutrients. In contrast,
nutritional intervention or the formulation of functional food aims to prevent or to
mitigate chronic diseases that are highly prevalent in developed countries. Chronic
diseases that are substantially influenced by diet include inflammatory bowel dis-
ease, diabetes, celiac disease as well as cardiovascular diseases, obesity and the
metabolic syndrome. Consequently, the formulation of functional food products has
become an important component in industrial product development and product
diversification. Examples include gluten-free products, products with reduced
sodium content or an increased level of dietary fibre, and food enriched with anti-
oxidative or bioactive compounds.
Wheat flour-based products such as bread, biscuits and breakfast cereals are
characterized by relatively high values of glycaemic index and insulin index.
Sourdough fermentation or the addition of soluble fibres represent the most promis-
ing tools to decrease starch digestibility and, consequently, the values of glycaemic
index and insulin index. Various mechanisms are responsible for this effect but these
remain to be elucidated in depth [20, 21]. Sourdough fermentation represents an
indispensable biotechnology for making wholegrain bread, especially rye bread,
and it may be used to modify fibre-rich cereal ingredients such as bran and germ,
which improves the technological functionality of these components.
During the last decade, a number of studies [22–26] showed that fungal proteases
or malt in combination with sourdough can result in complete hydrolysis of gluten
during long-term fermentation. As recently shown by two in vivo challenges, wheat
flour fermented with selected sourdough lactobacilli and fungal proteases is toler-
ated by celiac patients under remission. Sourdough fermentation also improves the
sensory acceptability of naturally gluten-free products and may prevent the moder-
ate cross-contamination by gluten.
Microbial metabolism during sourdough fermentation may also favour the syn-
thesis, release, and/or the bioavailability of a number of functional compounds such
as vitamins, phytochemicals, pre-biotic exopolysaccharides and bioactive peptides.
For example, antioxidant peptides (e.g. lunasin) are liberated from cereal proteins
during sourdough fermentation. These peptides may have promising preventive
activities towards oxidative stresses that are associated with degenerative aging dis-
eases (e.g. cancer and arteriosclerosis) [27].
In conclusion, the use of sourdough has evolved as an important tool in the devel-
opment of functional baked goods (see Chaps. 10 and 11). Our knowledge on the
intricate relationship between diet and chronic disease continues to increase. This
knowledge allows one to take advantage of the formation or modification of bioac-
tive compounds during sourdough fermentation to further expand the toolset for
development of sourdough products with specific nutritional functionality.
12.4 Industrial and Artisanal Use of Sourdough
In European countries, 30–50% of the bread production includes the use of sour-
dough or sourdough products. In North America, sourdough is a small but rapidly
growing segment of the market for baking improvers. The traditional use of sour-
dough as a leavening agent in artisanal bakeries retains its place in small or medium-
sized, specialized bakeries. Particularly the production of regional specialities, for
example Panettone, Pumpernickel or San Francisco Sourdough Bread, continues to
rely on the traditional use of sourdough as a leavening agent. However, for a major-
ity of industrial applications, the technological aim of sourdough use has shifted
from its traditional use as a leavening agent to use as a dough acidifier or baking
improver (see Chap. 1). This development is supported by the industry’s efforts to
develop “clean label” products. The use of sourdough allows bread production from
the ingredients flour and water with optional addition of yeast and salt, and thus can
replace numerous other ingredients to improve bread quality and shelf life. Moreover,
in-house sourdough fermentation can achieve substantial cost savings. Ingredients,
for example malt, emulsifiers, hydrocolloids, or enzymes, are replaced by the exper-
tise and equipment needed for sourdough fermentation. However, few industrial
bakeries master the automated and large-scale fermentation of sourdough. One of
the limiting factors is the availability of equipment allowing fermentation control
12 Perspectives 283
for use of sourdough as a leavening agent. Equipment for industrial sourdough

fermentation is typically designed to carry out (semi-)automated batch fermenta-
tions according to the fermentation scheme of traditional procedures, and is thus
incompatible with large-scale and continuous bread production [28]. Consequently,
sourdough fermentation is increasingly carried out by specialized suppliers to the
baking industry [29] . The use of sourdough in bakeries employs stabilized, usually
dried, preparations that are shelf stable. This second line of sourdough products in
addition to traditional fermentations allows for product innovation to match the spe-
cialized need of individual customers. Examples include ready-to-use, active sponge
doughs, dried sourdough products enriched with exopolysaccharides or flavour
compounds derived from the Maillard reaction, and starter cultures selected for
specific metabolic traits for improved bread quality.
It is noteworthy that lyophilized starter cultures for direct inoculation of bread
dough have not found widespread commercial use in baking applications, in con-
trast to the predominant use of starter cultures in meat and dairy fermentations.
Freeze-dried cultures fail to develop the required metabolic activity in straight
dough processes, and thus require revitalization in a pre-ferment or sponge dough
prior to use. In-house propagation of sourdough with occasional restoration of the
desired fermentation microbiota with cereal-based freeze-dried starter preparations
is thus a preferred option for many bakeries.
The increasing use of sourdough as a baking improver also allows the inclusion
of non-conventional organisms and raw materials. Continuous propagation of sour-
dough invariably selects for fermentation microbiota consisting of lactic acid bacte-
ria and yeasts. The industrial production of baking improvers, however, can be
started with other food-grade organisms that grow in cereal substrates and maintain
dominance over one or a few stages of fermentations. Bifidobacteria [30], propioni-
bacteria [31], fungi and acetic acid bacteria [32] all grow in cereal substrates and
have been employed in experimental cereal fermentations. Moreover, traditional
cereal fermentations employed in Africa, Asia, and Latin America for production of
steamed bread, beverages, porridges, vinegar, or condiments provide a source of
fermentation organisms that are highly adapted to cereal substrates. The metabolic
potential of these organisms vastly differs from sourdough lactic acid bacteria and
their use allows novel functionalities for baked products.
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Index
A B
Acidity, 165–166 Bacteriocin formation, 204
ADI catabolism. See Arginine deaminase Baked goods
(ADI) catabolism aroma and flavour compounds, 89
AFLP. See Amplified fragment length vs. baker’s yeast, 281
polymorphism (AFLP) and bread (see Bread)
AFM. See Atomic force microscopy (AFM) cereal, 15
Altamura bread, 90–91 classification, 47–48
American system, 87 degradation, cereal proteins, 194
Amino acid metabolism description, 47
ADI catabolism, 195, 196 flavor compounds, 195
cystathionine, 197–198 glutamate, 195
glutamine and glutamate, 195–197 leavening agents (see Leavening, agents)
peptides and free amino acids, 194–195 making process (see Baking process)
phenylalanine, 197 production, 94
Amplified fragment length polymorphism quality, 97
(AFLP), 139 quality assessment, dough, 73–79
Antibacterial compounds salt, sugar and fats, 56–57
bacteriocin formation, 204 sourdough, 1
prevention, bread spoilage, 204 water, 55–56
reutericyclin, 205 wheat flour, 194
Antifungal compounds, 203–204 Baker’s yeast
Antimicrobial compounds, production
sourdough LAB fermentation, 175–176
antibacterial, 204–205 raw materials, 174–175
antifungal, 203–204 use, molasses, 8
inhibitory activity, 202 Saccharomyces cerevisiae, 57
leavened baked goods, 202 Baking process
Arabinoxylans (AX) aim and modifications, 58, 59
technological properties, 17 artisanal production, 72
WEAX and WUAX, 219 colour intensity and vapour, 70
xylanase enzymes, 235 complex chemical reactions, 68, 70
Arginine deaminase (ADI) catabolism, continuous processes, 61
195, 196 description, 68
Atomic force microscopy (AFM), 78 discontinuous processes (see Straight-dough
AX. See Arabinoxylans (AX) and sponge-and-dough method)

DOI 10.1007/978-1-4614-5425-0, © Springer Science+Business Media New York 2013
288 Index
Baking process (cont.) large-scale, 9

dough makeup operations, 67–68 military, 7
GF flours, 252–253 milling process (see Milling)
heating causes and porous network, 68 oldest leavened and acidified, 2
industrial production, 73 pain viennois, 4
leavening, 66–67 quality, 29, 233
lipid effects, wheat flour, 36–38 recipe, 3
mixing, 61–66 rye and wheat sourdoughs, 30
ovens, 70–71 rye sourdough, 92
polar lipids, 38 San Francisco, 93–94
protein–sugar interactions, 70 sourdough, 247–250
and storage, bread area, 68, 69 starch gel, 15
temperature gradient and surface areas, 68 structure (see Bread structure)
vitamins, 236 white pan, 92–93
Basic local alignment and search tool Bread making
(BLAST), 137 baker’s yeast production, 174–176
Beverages biotechnology, novel types, sourdough, 99
boza, 265–268 brewer’s yeast, 4
bushera, 271 and cake production, 85
chicha and kishk, 272 continuous processes, 61
description, 265 discontinuous processes, 58–61
hulu-mur, 275–276 dough properties, 31
kvass, 272–274 dry baker’s yeast, 177
mahewu, 270–271 fresh baker’s yeast, 177
pozol, 271–272 glucans and fructans, 254
sourish shchi, 274–275 HMW-GS, 21
togwa, 268–270 industries, 100
Biodiversity, microbial species diversity. Parisian bakers, 3
See Microbial species diversity protocol, 88
BLAST. See Basic local alignment and search rye-flour, 92, 99
tool (BLAST) techniques and knowledge, 4
Boza WEAX, 17
characteristics, 266 wheat, 29
description, 265–266 yeast, 5
enzyme activity, 268 Bread structure
fermentation, process, 266 CO2 formation, 223
glucose and free amino nitrogen, 266–267 description, 217
LAB and yeast strains, 267 enzymes, 220
sensory and rheological properties, 268 EPS, 220–223
Bread fermentations and synergistic affects, 225
acidified and leavened, 7–8 gluten proteins, 217–218
Altamura, 90–91 organic acids, 218–219
baked goods, 13 rye flour, 218
and baked goods, 48 Bushera, 271
baker’s yeast, 86
black and white, 6
brewer’s yeast, 3 C
cereal, 28 Carbohydrate metabolism
dough strength and volume, 27 EMP pathway, 185, 186
and flour, 6 external acceptors, electrons
flour performance, 54–55 acetate and ATP synthesis, 188
French, 5, 91–92 description, 186
GF, 254–258 fructose, 188
gliadins and glutenins, 49 oxygen, 188
Greece, 2 heterofermentative strains, 185
Index 289
organic acids, 189–190 house microbiota and LAB species, 129

6-phosphogluconate/phosphoketolase microbiological stability, 128
(6-PG/PK) pathway, 185, 187 microorganisms, competitive yeast and
use, energy sources LAB species, 128–129
b-glucosidase activity, 191 phenolic compounds, 205
L. amylovorus DCE 471, 190 proteinases, 191
levansucrase, 190 proteins, degradation, 194
maltose phosphorylase, 190–191 raw, 2
Carbohydrates roasted/boiled, 6
NSP, 16–18 Chicha, 272
starch (see Starch) Chorleywood bread process (CBP), 60–61
CBP. See Chorleywood bread process (CBP) CLSM. See Confocal laser scanning
Cell-envelope-associated proteinase (CEP), microscopy (CLSM)
191, 192 Confocal laser scanning microscopy
Cell-to-cell communication, 206–207 (CLSM), 78
CEP. See Cell-envelope-associated proteinase Culture-dependent approaches
(CEP) LAB
Cereal fermentation, LAB and yeast AFLP and ribotyping, 139
biopolymers, 230–235 API system, 138
EPS, 238–239 DNA fingerprinting methods, 139
micronutrients, 235–238 housekeeping genes, 140
Cereal food LGT, 141
dietary fibre, 234 MALDI-TOF MS, 138–139
macro-and microstructure, 230 MLSA and MLST, 141
Cereal grains MS methods, 138
carbohydrates (see Carbohydrates) PFGE, 139–140
chemical composition, 12, 13 protein profiling and SDS-PAGE, 138
cool-season and rice, 11 RAPD-PCR, 139
description, 11 sequence-based analysis, 140
lipids (see Lipids) single-locus sequence analysis, 140–141
minerals, 38–39 yeasts
production, 11, 12 BLAST and PCR, 137
proteins, 18–36 DNA-based methods, 136
size and weight, 12 genetic regions and low sequence
spring, 12 divergence, 136
vitamins, 39 LTR sequences, 138
warm-season, 11 phenotypic “characterization”, 136
wheat, rye and barley, 11–12 S. cerevisiae and Ty elements, 138
Cereals. See also Cereal grains single-copy protein-coding gene
bakers’ yeast and fermentation sequences, 137
process, 129 Culture-independent approaches
beverages (see Beverages) community fingerprinting methods, 142
biopolymers microarray technology, 142–143
dietary fibre, 234–235 PCR assays and probe-based methods, 141
protein, 232–234 real-time PCR technology, 141
starch, 230–232 Cystathionine metabolism, 197–198
direct environment, 130
empirical techniques and fundamental
measurements, 73 D
fermentation, LAB and yeast (see Cereal Dietary fibre
fermentation, LAB and yeast)flour arabinoxylans function, 235
types, 129 bran sourdough, 234
foods, 1 cereal foods and sourdough fermentation,
GF products, 245–247 234
290 Index
Dietary fibre (cont.) oxidizing, 35

description, 234 phosphoketolase, 191
physiological effects and germ, 235 proteolytic, 253
rye and wheat, 234 EPS. See Exopolysaccharides (EPS)
Differential scanning calorimetry (DSC), 15 Exopolysaccharides (EPS)
Disulfide bonds biosynthesis and HoPS structure,
cysteines Ca and Cb, 27 198–200
description, 24 classification, 220
2D structures, C-terminal domain, 24, 26 description, 238
head-to-tail, 27 formation, 198
LMW-GS, 24, 27 FOS and GOS, 254
Dough rheology glucan and fructan synthesis, 220–221
fundamental measurements, 76–77 gluten-free baking, 223, 224
quality assessment HoPS, 221–222, 254
baking properties, 74, 75 IMO, 238
descriptive empirical measurements, lactobacilli, 238–239
73–74 NDO and SCFA, 238
Dough inflation system, 76 production and formation, HoPS,
Extensograph and Alveograph, 74 200–202
Farinograph and Mixograph, 74 strain collections, sourdough
Kieffer extensibility rig, 76 LAB, 198
Mixolab, 74 sucrose metabolism, 222–223
rheofermentometer, 74 Weissella strains, 255
textural properties, 76
Dry baker’s yeast, 177
DSC. See Differential scanning calorimetry F
(DSC) Fermentation
acidity, 165
bacteriocin, 204
E baker’s yeast production
Embden-Meyerhof-Parnas (EMP) pathway, emulsifiers, 176
185, 186 formulas, process, 175, 176
EMP pathway. See Embden-Meyerhof-Parnas stock fermentation, 175
(EMP) pathway yeast mixture, 175
Enzymes biogas/bioethanol, 11
ADI, OTC and CK, 195 boza, 265–268
breads, 30 bread
bread structure dough, 2
glutathione reductase, 220 structure (see Bread structure)
proteolysis, 220 bushera and pozol, 271–272
sourdough metabolites, 220, 221 and development, GF sourdough starters,
cross-linking promoting, 247 250–252
dihydroxyacetone kinase (DAK1 effect, parameters, 184
and DAK2), 167 EPS, 198
endogenous, 252 GF sourdough, 248
glycerol dehydrogenase (GCY1 grain, 4–5
and YPR1), 167 heterolactic, 187
hexokinase, 190–191 homofermentative metabolism, hexoses,
hydrolases, 49 185
hydrolytic, 86–87 homolactic, 186
hydrolyzing, 34–35 hulu-mur, 276
inhibitors, 35–36 industrial and artisanal use, 282–283
levansucrase, 190 kamut flour, amino acids, 158, 159
Index 291
kishk, 272 EPS, 254–255

kvass, 273–274 fermentations and development
LAB and yeasts, 158–159 dominant species, 251
leavened loaf, bread, 31 ecological studies, 251–252
leavening, 58–59 heterofermentative species, 250
and leavening, 2 Weissella spp., 251
lipid oxidation, 206 health benefits, 257–258
low temperature, 164, 165 HPMC, 246
mahewu, 270–271 non-toxic proteins, 246
microbial metabolism, 282 protein hydrolysis, 247
pentoses and hexoses, 191 proteolysis, 252–253
pH-controlled, HoPS, 201 shelf life improvement, 256–257
phenolic compounds, 205–206 starch hydrolysis, staling, 255–256
polish, 5 TGase, 247
and proofing rooms, 67 Glutenin macropolymers (GMP), 27–28
protein degradation, 30 GMP. See Glutenin macropolymers (GMP)
rye malt sourdoughs, 194
sourdough, 229, 257–258
and sourdough applications, 85–101 H
sourish shchi, 274–275 History and social aspects of sourdough
stages, proteolysis, 193 bread, 1
sterols, 169 Egyptians, 2
temperature, 131 fermentation and leavening, 2
togwa, 268–270 France
yeast metabolism, 159–163 back-slopping and bread-making
Flavor compounds, 189, 194–195 techniques, 4
French bread (pain au levain), 5, 91–92 bread making method, 3, 5
French system, 86–87 cervoise, 3
Fresh baker’s yeast, 177 dehydrate, 5
levain-chef, 4
pain mollet, 3–4
G poolish and pain viennois, 4–5
b-Glucans, 18 Saccharomyces cerevisiae, 5
Glutamine and glutamate metabolism, socio-cultural and socio-economic
195–197 factors., 3
Gluten travail sur 3 levains and levain tout
amylose network, 218 point, 4
enzyme activity, 234 Germany
free bread, 233 acidified and leavened, 7–8
free products (see Gluten-free (GF) baker’s yeast, 8
products) brewing and baking, 8
and gliadins, 49 chemical acidulants, 8–9
glutathione reductase, 220–221 commercialization, dried, 9
gluten-free baking, 223, 224 rye flour, 8
native, 27 Greece, 2
proteolysis, 220 Italy
quantitative analysis, 219 baker’s yeast, 7
swelling and water uptake, 218 black and white bread, 6
wheat (see Wheat gluten) Cato the Elder, 6
Gluten-free (GF) products characteristic shape, knotted and dark
batters, bread and flour, 246 red crust, 6
celiac disease, 245 “military bread”, 7
description, 245–246 PDO/PGI, 7
enzymatic process, 247 renaissance, 6–7
292 Index
Homopolysaccharides (HoPS) boza fermentations, 266–267

bread making, 254 bread structure, 220
glucansucrases and fructansucrases cereal fermentation, 229–239
levansucrases, 201 culture-dependent approaches, 136–141
maltose, 202 culture-independent approaches, 141–143
optimum pH, 201 definition, 8–9
oral streptococci, 200 diacetyl, 171
stress resistance, 201 fermentation, amino acids, 158
sucrose concentration, 201–202 inter-species signalling mechanism,
structure and EPS biosynthesis 172–174
dextran, mutan and glucans, 200 isolation, 134–135
fructans, 200 kvass, 273–274
gene clusters, 198–199 mahewu, 270
glucansucrases and fructansucrases, 199 phenotypic and genetic analyses, 96–97
sucrose hydrolysis, 199–200 physiology and biochemistry (see
HoPS. See Homopolysaccharides (HoPS) Physiology and biochemistry, LAB)
Housekeeping genes, 140 pozol, 271–272
HPMC. See Hydroxyl-propril-methyl- taxonomy
cellulose (HPMC) classification, 113–114
Hulu-mur, 275–276 dextran-producing species, 117
Hydroxyl-propril-methyl-cellulose fingerprinting methods, 117
(HPMC), 246 heterofermentative pediococci, 117
heterogeneous group and
homofermentative, 113
I Lactobacillus, 116–117
Identification molecular DNA, 116
culture-dependent approaches, 136–141 species diversity, sourdough
culture-independent approaches, 141–143 fermentation, 114–116
dominant and sub-dominant togwa, 268–269
microorganisms, 95 yeasts, 2
head-to-tail disulfide bond, 27 Lactobacillus
lactic acid bacteria and yeasts, 96–97 description, 116
IMO. See Isomalto-oligosaccharides (IMO) strain, 30
Isolation Lactobacillus plantarum
LAB, 134–135 anti-fungal strain, 256
sourdough yeasts, 133–134 fermentation processes, 129
Isomalto-oligosaccharides (IMO), 238 isolated amylolytic strains, 255
isolation, 116
phenylalanine catabolism, 197
source tracking, 141
K strains, 95
Kishk, 272 Lactobacillus sanfranciscensis
Kvass (L. sanfranciscensis)
baker’s yeast, 273 association, maltose-negative yeast
description, 272 species, 128
filtration, 273–274 description, 3
kvass-making techniques, 273 detection, 134
production, 274 fermentation, 131
genome sequencing, 279
growth and metabolism, 158–159
L laboratory-scale fermentation process, 132
LAB. See Lactic acid bacteria (LAB) metabolites, 171, 172
Lactic acid bacteria (LAB) peptidase system, LAB, 191–193
acidity, 165–166 phenylalanine catabolism, 197
Index 293
starter strain, 251 Metabolic proteins

wheat and/rye flour sourdoughs, 88 enzyme inhibitors, 35–36
wheat sourdoughs, 128 hydrolyzing enzymes
Lateral gene transfer (LGT), 141 carbohydrate-degrading, 34
Leavening proteolytic, 34–35
acidity, 165–166 oxidizing enzymes, 35
agent, 8 Microbial species diversity
agents cereals and raw materials, 128–130
Baker’s yeast, 57 origin
chemical, 57–58 Altamura and Pugliese bread, 118
extensographic analyses, 66 bread production and craftsmanship, 118
fermentation and proofing rooms, 67 LAB species and rye bread baking, 118
microalveoli and danish pastry, 66 leavening, 117
semi-solid mass and volume “sour”, 118
expansion, 66 strains, 119
viscoelastic properties and alveoli region-specific
retained, 66 maltose metabolism, 128
and fermentation, 2 maltose-positive LAB species, 128
microbial, 3 nonexhaustive overview, LAB, 119–127
strains, 165 single isolations, yeast and LAB
LGT. See Lateral gene transfer (LGT) species, 128
Lichenins, 18 technology
Lipids aeration and LAB, 132
composition backslopping practices, 132
nonstarch, 36, 37 chemical composition and coarseness,
phospho and glyco, 36, 37 130
starch, 36 fermentation temperature, 131
effects, baking performance, 36–38 growth, sourdough LAB, 131
membranes (see Membrane lipids) pH value, 131–132
metabolism, 206 starter cultures, 130–131
polar, 57 type II/ industrial sourdoughs, 130
polar, baking, 38 type III sourdoughs, 130
vitamins, 235 type I/traditional sourdoughs, 130
LMW. See Low-molecular-weight (LMW) Micronutrients
Lopez, H.W., 257 minerals, 236–237
Low-molecular-weight (LMW), 16 phytochemicals, 237–238
vitamins, 235–236
Milling
M breaking system, 51
Mahewu, 270–271 cereals, 39
MALDI-TOF. See Matrix-assisted laser chemical composition, wheat regions, 50
desorption/ionization-time-of-flight cleaning, 53–54
(MALDI-TOF) composition, flour extraction rate, 52–53
Matrix-assisted laser desorption/ionization- debranning and pearling, 54
time-of-flight (MALDI-TOF), description, 49
138–139 durum wheat, 52
Membrane lipids external layers and germ, caryopsis, 52
homeoviscous adaptation, 167 “flour extraction yield”, 49–50, 52
SFAs and UFAs, 167 grains and sieving, 18
sterols, 169 kernel and wheat operations, 51
temperature, fatty acid composition, sieving/grading section, 51–52
168–169 sizing/purification system, 51
294 Index
Milling (cont.) O
stages and flour classification, 50 Organic acids
starchy endosperm, 49 amylase activity, 219
Minerals citrate metabolism, 189
constituents, 38–39 L. parabuchneri, 190
micronutrients, 236–237 malate and fumarate, 189
and phytochemical, 246 optimum pH, 218–219
Mixing, baking process quantitative analysis, 219
different times and speeds, 62 swelling and solubility, gluten proteins, 218
dough development, 62–63 WEAX and WUAX, 219
“level of absorption”/“hydration”, 61 Osborne, T.B., 18
microscopic level, 62 Osmotic stress, 166–167
mixers Ovens
advantages, plant, 65–66 Ancient, 70
Artofex, 63 artisanal bakeries, 71, 72
carousel system, 65 characteristics, 70
Chorleywood method, 63 cyclotherm, 70
classification, 63 “direct and indirect firing system”, 70
high-speed and auxiliary equipment, 63 heat, 70–71
planetary and mixing tools, 64–65 industrial bakeries and shutters control, 71
physical characteristics, flour, dough and microwave and modern rack, 71
bread, 62 Oxidative gelation, 17
stiff and soft/slack doughs, 61
MLSA. See Multilocus sequence analysis
(MLSA) P
MLST. See Multilocus sequence typing Panettone cake, 93
(MLST) Pentosans, 17
Molecular weight distribution (MWD) PFGE. See Pulsed-field gel electrophoresis
GMP, 27–28 (PFGE)
native gluten proteins, 27 Phenolic compounds, 205–206
native wheat storage (gluten) proteins, Phenotypic and genetic analyses,
27, 28 LAB and yeasts, 96–97
rye storage proteins, 28 Phenotypic and genotypic analyses, 135
Multilocus sequence analysis (MLSA), 141 Phenylalanine metabolism, 197
Multilocus sequence typing (MLST), 141 Physico-chemical parameters
MWD. See Molecular weight distribution acetic acid plays, 98
(MWD) dough yield (DY), 97–98
endogenous and exogenous factors,
sourdough characteristics and
N performances, 97, 98
NDO. See Non-digestible oligosaccharides fermentation quotient (FQ), 99
(NDO) pH, 98
Near-infrared spectroscopy (NIR), 78–79 TTA, 98–99
NIR. See Near-infrared spectroscopy (NIR) Physiology and biochemistry, LAB
NMR. See Nuclear magnetic resonance amino acid metabolism, 194–198
(NMR) antimicrobial compounds
Non-digestible oligosaccharides (NDO), 238 (see Antimicrobial compounds,
Nonstarch polysaccharides (NSP) sourdough LAB)
arabinoxylans (AX), 17 carbohydrate metabolism, 185–191
b-glucans, 18 cell-to-cell communication, 206–207
nutritional point, 16 description, 183
NSP. See Nonstarch polysaccharides (NSP) EPS, 198–202
Nuclear magnetic resonance (NMR), 79 general growth and stress parameters,
Nutrition, 281–282 184–185
Index 295
lipid metabolism, 206 Q

phenolic compounds, 205–206 Quality assessment, dough
proteolysis, 191–194 image analysis, 77–78
Physiology and biochemistry, sourdough microscopy, 78
yeasts rheology (see Dough rheology)
bread-making, 174–177 spectroscopy, 78–79
carbohydrate metabolism (see Yeast Quorum sensing
metabolism) cell-free wheat flour hydrolyzed (WFH),
catabolic enzymes and permeases, 172, 173
157–158 cell-to-cell communication, 206
cereal dough, 156 Histoplasma capsulatum, 171–172
cultivation methods, 155 inter-species signalling mechanism,
description, 155 172–174
effect, process parameters, 158–159 morphological transition, S. cerevisiae, 172
fermentation, kamut flour, 158, 159
gene expression, 156
growth, Saccharomyces cerevisiae, 158 R
metabolites (see Yeast metabolites) Reutericyclin, 205
NaCl and a-amylase supplementation, Rye sourdough bread, 92
156, 157
nitrogen metabolism and regulation,
156–157 S
quorum sensing, 171–174 San Francisco bread, 93–94
stress response (see Stress response) Saturated fatty acids (SFAs), 167
yeast cells, 158 SCFA. See Short-chain fatty acids (SCFA)
Phytochemicals, micronutrients, 237–238 SFAs. See Saturated fatty acids (SFAs)
Pliny the Elder, G., 6 Short-chain fatty acids (SCFA), 238
Pozol, 271–272 Sourdough
Proteins vs. active sourdoughs, 99–100
amino acid composition, flour, 18, 19 Altamura bread, 90–91
bioactive peptides, 233 biological starter, 94
cereal and wheat, 232 bran, 234
degradation, prolamin, 233 bread
gluten and WSE, 233 GF fermentations and properties,
LAB and baked cereal goods, 234 batters, 247, 248
metabolic (see Metabolic proteins) LAB and yeasts, 247
Osborne fractions microbiota, GF products, 247, 249–250
albumins and globulins, 19 bread and baked good characteristics, 94
cereal flour, 18–19 bread structure (see Bread structure)
geno type, 20 and cereal beverages(see Beverages)
prolamins, 19 definition, 85–86
quality, gluten-free bread, 233 fermentation, 66–67, 229, 257–258
storage (see Storage proteins) French bread (pain au levain), 91–92
VSL#3 and proteolysis, 232 history and social aspects of sourdough,
Proteolysis 1–9
baking performances, GF flours, 252–253 industrial and artisanal use, 282–283
bioactive peptides, 194 leavening, 60
CEP, 191, 192 microbial ecology
degradation, cereal proteins, 194 back-slopped sourdoughs, 279
peptidases, L. sanfranciscensis, 191–193 genome sequencing, 279
reducing toxicity, 253 sucrose and maltose, 280
stages, fermentation, 193 wheat and rye, 280
Pulsed-field gel electrophoresis (PFGE), nutrition, 281–282
139–140 panettone cake, 93
296 Index
Sourdough (cont.) degradation prevention, 8

performance evaluation description, 13
media cycloheximide, 96 dietary carbohydrate and solid–liquid two
microbiological and physico-chemical phase reaction, 230
parameters, 95 endosperm, 49
phenotypic and genetic analyses, 96–97 fermentation, wheat and rye flour matrix,
physico-chemical parameters, 97–99 231
physiology and biochemistry, LAB, gelatinised and amylose-rich, 230
183–207 gelatinization, 218
physiology and biochemistry, sourdough GI and II, 230, 231, 232
yeasts, 155–177 granules, 14
preparation and storage (see Sourdough hydrolysis, 255–256
preparation and storage) interaction, lipids, 16
product quality, 281 macro-and microstructure, cereal foods, 230
read vs. straight dough bread, 236 retrogradation, 218, 230–231
rye, 17, 30 rye breads baked and wheat flour-based
rye sourdough bread, 92 products, 231
San Francisco bread, 93–94 structure, processing
shelf life improvement, 256–257 amylose molecules and staling
sponge dough, 90 endotherm, 16
vs. stabilized sourdoughs, pure cultures, DSC and mixing starch absorbs, 15
100–101 LMW and crystallization, 16
starters, 250–252 viscosity measurements and
strains, Lactobacillus plantarum, 95 retrogradation processes, 15
type I, 88–89 water-protein, 79
type II and III, 89 Starter cultures
wheat, rye/spelt, 94 boza
white pan bread, 92–93 fermentation, 268
yeasts (see Sourdough yeasts) production, 266, 267
Sourdough and civilization. See History and CO2 formation, 223
social aspects of sourdough dried grapes, 6
Sourdough preparation and storage food production, 280
American system, 87 Lb. sanfranciscensis, 129
DY 225–250, 88 meat and dairy fermentations, 283
French system, 86–87 sourdoughs, 251–252
liquid, 88 starch hydrolysis, 255
Sourdough yeasts type II and III sourdoughs, 129–130
identification, 135–143 Storage proteins
isolation, 133–134 classification, 48
taxonomy classification and primary structures
analyzed, yeast diversity, 107–111 amino acid sequences, 22
basidiomycetous and genera, 106 HMW group, 21–22
C. humilis/C. milleri, 112–113 interchain disulfide bonds, 22
classification, 106 LMW group, 23–25
DNA-DNA, 112 MMW group, 22
HaeIII, 113 PAGE, 21
Hansenula and Pichia, 106 partial amino acid sequences, domain B
Saccharomyces and saccharomycetales, of HMW-GS, 22, 23
106 quantitative composition, 23, 26
species, ecosystem, 107, 112 wheat, rye, barley and oats, 20–21
vegetative growth, 105 corn, millet, sorghum and rice, 33
Sourish shchi, 274–275 disulfide bonds, 24–27
Starch enzymes, 30
amylose and amylopectin, 13–14 fertilization, 28–29
characteristics, 230 germination and infections, 29
Index 297
heat and high pressure, 30 U

MWD, 27–28 UFAs. See Unsaturated fatty acids (UFAs)
oxidation, 29–30 Unsaturated fatty acids (UFAs), 167, 169
wheat gluten, 30–33
Straight-dough and sponge-and-dough
method V
baker’s yeast, 60 Vitamins
CBP, 60–61 constituents, 39
characteristics, 58, 59 micronutrients, 235–236
fermentation, 58 and soluble sugars, 52
ingredients, 60
physical characteristics, flour, dough
and bread, 60, 62 W
polish, poolish/viennese methods, 60 Water extractable (WEAX), 219
Stress response Water unextractable arabinoxylans
acidity, 165–166 (WUAX), 219
environmental, 128 WEAX. See Water extractable (WEAX)
gene expression program, 163 Wheat flour
low temperature, 164–165 barley and honey, 6
membrane lipids (see Membrane lipids) Egyptians, 2
osmotic stress, 166–167 glycolipids, 37
process parameters, 163 nonstarch lipids, 36
transcription factor, Msn2, 163 pH, 184
yeast responses and growth rate, 163–164 polar lipids, 37–38
“specific baking activity”, 37
toxicity reducing, 253
T Wheat gluten
Taxonomy cereals, 30
LAB, 113–117 chemical bonds disulfide linkages, 31
sourdough yeasts, 105–113 covalent and noncovalent bonds, 32
TEM. See Transmission electron microscopy cysteine and sodium metabisulfite, 31
(TEM) dityrosine and isopeptide bonds, 31
Togwa dough retains, 30–31
dominant microbes, 269, 270 hydrated monomeric and oligomeric
folate production, 269–270 proteins, 31
germinated sorghum, 268 hydrophobic bonds, 32
LAB and yeasts, 268–269 interchain disulfide structure, 2D model,
manufacturing process, 268, 269 32, 33
proteolytic activity, 270 oxygen and S-S linkages, 31
yeast strains, 269 “plasticizer”/“solvent”, 31
Tradition SH-SS interchange reactions, 31
biotechnology, 3 WUAX. See Water unextractable
breads represent temporary, 7–8 arabinoxylans (WUAX)
cereal flour proteins, 18
culture, 1
GF sourdoughs and products, 249–250 Y
Italian sourdough bread, 90–91 Yeast
mixing, 62–63 baker’s, 5, 7, 8, 57
San Francisco bread, 93–94 brewer’s, 3–5
sourdoughs, 88–89, 130 cereal fermentation, 229–239
values, 7 CO2 formation, 223
Traditional use of sourdough, 282–283 glucose, 222
Transmission electron microscopy (TEM), 78 lactic acid bacteria, 96–97
TTA, 98–99 microscopic observations, 2
298 Index
Yeast (cont.) Pasteur, Kluyver, Custers and Crabtree

physiology and biochemistry effects, 161–162
(see Physiology and biochemistry, respiration and fermentation,
sourdough yeasts) S. cerevisiae, 162
Yeast metabolism Yeast metabolites
alcoholic fermentation, 160 amino acids, Ehrlich pathway, 169, 170
carbon and energy sources, ethanol, methylpropanol, 2-and
159–160 3-methylbutanol, 171
glycolytic pathway, glycerol production, “fermented taste”, 169
160–161 fusel alcohols, 169, 171
hexose phosphate pathway, 162–163 L. sanfranciscensis and S. cerevisiae,
hydrogen (electrons), 159 171, 172

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Handbook on Sourdough Biotechnology

Marco Gobbetti • Michael Gänzle

ISBN 978-1-4614-5424-3 ISBN 978-1-4614-5425-0 (eBook)

Library of Congress Control Number: 2012951618

© Springer Science+Business Media New York 2013

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

1 History and Social Aspects of Sourdough ........................................... 1

11 Sourdough and Cereal Beverages ........................................................ 265

Stefan Cappelle, Lacaze Guylaine, M. Gänzle, and M. Gobbetti

1.1 Sourdough: The Ferment of Life

S. Cappelle (*) • L. Guylaine

M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, 1

1.2 History and Social Aspects of Sourdough in France

Fould-Springer facilitated the development of bread making based on yeast to the

1.3 History and Social Aspects of Sourdough in Italy

1.4 History and Social Aspects of Sourdough in Germany

and widespread production of leavened and acidiﬁed bread; however, these

bread as bread where acidity is exclusively derived from biological acidiﬁcation.

Peter Koehler and Herbert Wieser

2.1 Introductory Remarks

P. Koehler (*) • H. Wieser

M. Gobbetti and M. Gänzle (eds.), Handbook on Sourdough Biotechnology, 11

Table 2.1 Cereal production in 2010 [1]

Table 2.2 Chemical composition of cereal grains (average values) [2, 3]

2.2.1.1 Amylose and Amylopectin

2.2.1.2 Starch Granules

In the endosperm starch is present as intracellular granules of different sizes and

2.2.1.3 Changes in Starch Structure During Processing

On cooling granule remnants that are enriched in amorphous amylopectin

2.2.1.4 Interaction with Lipids

2.2.2 Nonstarch Polysaccharides (NSP)

2.3.1 Osborne Fractions

2.3.2 Storage Proteins of Wheat Rye, Barley, and Oats

2.3.2.1 Classification and Primary Structures

The nomenclature of types is rather confusing and inconsequential. On the one

Fig. 2.1 Schematic structure

2.3.2.2 Disulfide Bonds

B-hordein 112–184 YVHPSILQQLNPCKVFLQQQCSPVPVPQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQFRHEAIRAIVYSIFL

Gellrich et al. [65]

Because HMW-GS do not occur as monomers, it is generally assumed that they

2.3.2.3 Molecular Weight Distribution

Most information on the quantitative MW distribution (MWD) of native storage

Fig. 2.3 Molecular weight

comparison with total glutenins; the HMW-GS combination 1Dx5 + 1Dy10

2.3.2.4 Influence of External Parameters

of albumins/globulins are scarcely inﬂuenced, whereas those of gluten proteins

The baking process involves a drastic heat-treatment of proteins, with temperatures

2.3.2.5 Wheat Gluten

The covalent structure of gluten proteins is complemented by noncovalent bonds

2.3.3 Storage Proteins of Corn, Millet, Sorghum, and Rice

2.3.4 Metabolic Proteins

2.3.4.1 Hydrolyzing Enzymes

The many carbohydrate-degrading enzymes include a-amylases, b-amylases,

Enzymes that hydrolyze proteins are called proteinases, proteases, or peptidases.

Other Hydrolyzing Enzymes

2.3.4.2 Oxidizing Enzymes

Lipoxygenase is present in high levels in the germ. It catalyzes the peroxidation of

2.3.4.3 Enzyme Inhibitors

animals. Some inhibitors appear to be bifunctional inhibiting amylases as well as

M.A. Pagani () • G. Bottega • M. Mariotti ()