Lipase Applications in Food Industry

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Lipase applications in food industry
Article in Indian Journal of Biotechnology · April 2007
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Indian Journal of Biotechnology
Vol 6, April 2007, pp 141-158
Lipase applications in food industry

R Aravindan*, P Anbumathi and T Viruthagiri
Biochemical Engineering Laboratory, Department of Chemical Engineering
Annamalai University, Annamalainagar 608 002, India
Received 13 September 2005; revised 16 January 2006; accepted 17 March 2006
Lipases are the most pliable biocatalyst and bring about a wide range of bioconversion reactions, such as hydrolysis,
interesterification, esterification, alcoholysis, acidolysis and aminolysis. Lipases can act on a variety of substrates including
natural oils, synthetic triglycerides and esters of fatty acids. They are resistant to solvents and are exploited in a broad
spectrum of biotechnological applications. Lipase catalyzed transesterification, hydrolysis and esterification are the
important class of reactions for food technology applications in fats and oil industry, dairy industry, pharmaceuticals and
bakery industry. Lipases are very peculiar as they hydrolyse fats into fatty acids and glycerol at the water-lipid interface and
can reverse the reaction in non-aqueous media. Novel biotechnological applications, like biopolymer synthesis, biodiesel
production, treatment of fat-containing waste effluents, enantiopure synthesis of pharmaceuticals and nutraceutical agents,
have been established successfully. The present article extends the frontier of lipase technology towards food processing
applications and discusses the important characteristics of lipases and its sources. Various methods of lipase immobilization
for food technology applications, various assay methods for lipase, production of lipase by submerged and solid state
fermentation strategies, and various purification methods available have been discussed in detail.
Keywords: ester, fatty acids, food applications, lipases, lipase sources, triglycerides
IPC Code: Int. Cl.8 C12N9/20
Introduction Lipases posses the unique feature of acting at the

Much of the early interest in enzymology interface between an aqueous and a non-aqueous
was developed by scientists like Pasteur, Payen and phase. They synthesize esters from glycerol and long
Persoz, who were associated with food, wine and chain fatty acids when the water activity is low. A
beer industries1. The presence of lipase in bacteria “true lipase” will split emulsified esters of glycerine
had been observed as early as 1901 AD for and long chain fatty acids, such as triolein and
Bacillus prodigiosus (now Serratia marcescens), tripalmitin. The growth of application of commercial
B. pyocyaneus (now Pseudomonas aeruginosa), enzymes is very significant and promising,
B. fluorescens (now P. fluorescens) and particularly in food industry during the past 35 years5.
Staphylococcus pyogenesaucreus (now S. aureus) by The panorama of lipase utilization encompasses
the microbiologist Eijkmann2. Lipases (triacylglycerol many other industries. It has wide range of applica-
acylhydrolases EC: 3.1.1.3) are ubiquitous enzymes, tions in oleochemical, detergent, organic industries,
which are found in animals, plants, fungi and bacteria, leather industry, environmental management, cosme-
and are of considerable physiological significance and tics and perfume industry, biomedical applications
industrial potential. Lipases are serine hydrolases3 and and biosensors3,6. At present, lipases are used for the
contain the consensus sequence G – X1 – S – X2 – G generation of enantiomerically enriched primary and
as the catalytic moiety, where G – glycine, S – serine, secondary alcohols and to a lesser extent for chiral
X1 – histidine and X2 – glutamic or aspartic acid4. The carboxylic acids and secondary amines7.
biological function of lipase is to catalyze the
Esters of short and medium chain carboxylic acids
hydrolysis of triacylglycerols to give free fatty acids,
and alcohol moieties synthesized by lipase play a
diacylglycerols, monoacylglycerols and glycerol.
relevant role in the food industry as flavour and aroma
__________ constituents. Esterification by lipases appears to be an
*Author for correspondence:
attractive alternative to bulk chemical routes. In fact,
Tel: 91-4144-238150
E-mail: [email protected] ester synthesis using lipase can be performed at room
142 INDIAN J BIOTECHNOL, APRIL 2007
temperature, pressure and at neutral pH in reaction Table 1Commercial lipases produced by Nova Nordisk10
vessels operated either batch wise or continuously.
Products obtained by these methods are more pure Brand name Mechanism Application
compared to the products obtained by alternative
Lipopan ® Hydrolysis and Baking industry
chemical means, because chemical catalysis may oxygen uptake
sometimes leads to non-specific and unwanted by- Lipozyme ® Interesterification Oils and fats industry
products and also requires high temperature, pressure Novazym ® 27007 Hydrolysis Pasta/noodles
and frequent regeneration of catalyst. Use of lipases Palatase Hydrolysis Dairy industry
Novozyme ® 871 Emulsification Pet food industry
alleviates complex downstream processes and thus,
leads to reduction in overall operation costs.
flavouring. Several traditional chemical markets are
However, the major drawback with this system is the
increasing with products derived from bioprocesses or
low conversion when compared to complex chemical
hybrid chemical/biocatalytic processes. In this review
processes.
the various microbial sources, substrates and
The industrial enzyme market for non-therapeutic
properties of lipase are discussed, with emphasis on
use, such as food, detergents, textiles, leather, pulp
recent developments of lipases in food processing
and paper industry, reached US $ 1 billion in 20008.
industries.
Lipases are produced by several microorganisms,
namely bacteria, fungi, archea, eucarya as well as by
animals and plants. Commercially useful lipases are Microbial Sources for Lipase
usually obtained from microorganisms that produce a Lipases occur widely in nature, but microbial
wide variety of extracellular lipases3. Microbial lipases are commercially significant because of low
lipases are of special interest because of their stability production cost, greater stability and wider
in organic solvents and their lack of requirement for availability than plant and animal lipases. They may
cofactors, their broad substrate specification and their originate from fungi, molds or bacteria and most of
high enantioselectivity8. In 1994, Nova Nordisk them are formed extracellularly. This ready
introduced the first recombinant commercial lipase, availability has created an enormous spin-off with
Lipolase®, which originated from the fungus respect to the enantioselective hydrolysis and
Thermomyces lanuginosus (formerly Humicola formation of carboxyl esters7.
lanuginosa) and was expressed in Aspergillus oryzae9. The enormous biotechnological potential of micro-
In 1995, Genencor International produced two bial lipases are related to their exquisite chemoselec-
bacterial lipases, Lumafast® from P. mendocina and tivity, regioselectivity and stereoselectivity12,13. They
Lipomax® from P. alcaligenes. Nova Nordisk markets are readily available in large quantities because many
a range of enzymes for various industrial purposes of them can be produced in high yields from
among which most of the enzymes are utilized mainly microorganisms6. The crystal structures of many
in food processing industries. Table 1 comprises some lipases have been solved, considerably facilitating the
of the commercial lipases produced by Nova Nordisk design of rational engineering strategies. Finally,
and their applications in food processing industries10. lipases do not usually require cofactors nor do they
Lipases are indispensable for the bioconversion of catalyze side reactions.
lipids (triacylglycerols) from one organism to another Most of the industrial microbial lipase is derived
and within the organisms, and they possess the unique from fungi and bacteria14. Fungi are preferable
feature of acting at an interface between the aqueous because fungal enzymes are usually extracellular,
and non-aqueous (i.e. organic) phase; this feature facilitating extraction from fermentation media15. In
distinguishes them from esterases6. This property of modern processes fungal genes are expressed in
lipases implies that the kinetics cannot be described bacterial systems. Some lipase producing microorga-
by the Michaelis-Menten equation, which is valid nisms and their applications in various food processes
only if the catalytic reactions take place in a were listed in Table 216-35. Though several microbial
homogenous phase. Some important reactions lipases are produced commercially, the high cost of
catalyzed by lipases are listed below11. lipase seems to be a major factor in its successful
The applications of enzymes in the food industry application. This may be overcome by immobilization
are many and diverse, ranging from texturing to for reuse of the enzyme13.
ARAVINDAN et al: LIPASE APPLICATIONS IN FOOD INDUSTRY 143
Table 2—Microbial sources and their applications carbon sources), nitrogen and phosphorous sources
in food processing and mineral salts. Compounds such as olive oil, oleic
Microorganisms Food applications
Refer- acid and span 80 seem to play an essential role in
ences lipase synthesis. Lipases production by B. sphaericus
Aspergillus oryzae Synthesis of saturated 16 has been investigated and maximum lipase activity,
fatty acids i.e. about 5.2 µmoles/mL.min, was obtained by using
Aspergillus sp. Flavour synthesis 17
Bacillus subtilis Bread making 18 an optimized medium with sesame oil as an inducer38.
Candida rugosa Flavour synthesis 19 The yeast Candida rugosa, which is an industrial
C. antarctica, Synthesis of lipophilic 20 producer of lipase, has been shown to secrete
C. cylindracea AY30, antioxidants extracellular lipase upon induction by fatty acids.
Humicola lanuginosa,
Pseudomonas sp. &
Lipase production may also be induced by adding
Geotrichum candidum oleic acid as the carbon source. This lipase is
C. parapsilosis Hydroxamic acids (food 22 composed of several isoforms with slightly differing
additive) catalytic properties. In the same yeast, the production
C. rugosa Cheese production 23,24 of a constitutive lipase was induced by using glucose
Chromobacterium Synthesis of aroma and 25
viscosum flavour compounds as carbon source14. Reychang et al36 revealed that the
Geotrichum sp. Bread making 26 presence of tween 80 and tween 20 in the culture
Mucor miehei Geraniol ester synthesis 27 medium not only promoted lipase production but also
M. miehei & Synthesis of short chain 28 changed the production of multiple forms in cultured
C. antarctica flavour thio ester in
solvent free medium
C. rugosa. Various non-conventional carbon sources,
M. miehei & Rhizopus Production of flavour 29 like beef tallow, wool scour effluent, whey and n-
arrhizus esters hexadecane, are also used to produce lipase. Other
Penicillium camembertii Production of glycerol- 30 significant factors influencing lipase production
glycolipids include nitrogen sources, initial broth pH, growth
P. chrysogenum Food industry waste 31
treatment temperature and dissolved oxygen concentration14.
P. roquefortii, A. niger Fragrance development 32 Lipase production by a thermophilic Bacillus
& R. arrhizus in dairy production species was increased several fold when magnesium,
Pseudomonas sp. Production of PUFA 33
Pseudomonas sp. Food processing and oil 34
iron, and calcium ions were added to the production
manufacture medium. Lipase production by Bacillus species A
Rhizomucor meihei Surfactants for baking 35 30-1 (ATCC 1841) required a complex medium39 that
industry, Dairy products, contained Ca2+, Mg2+, Na+, Co2+, Fe2+, K+, Mn2+, Mo2+
Noodles and Zn2+. Tween 80 and tween 20 increased lipase
Staphylococcus warneri Production of flavour 33 productivity by 2.9 and 5.7 fold, respectively with
& S. xylosus esters p-nitrophenyl butyrate as substrate. Lipase produced
Production of Lipases in the presence of tween 20 had much greater thermal
Microbial lipases are produced mostly by stability than that of lipase produced either in the
submerged culture but solid state fermentation can absence or in the presence of tween 8036. Generally
also be used. Generally the lipase production is bacterial lipases are glycoproteins but some
organism specific and it is released during the late extracellular bacterial lipases are lipoproteins. Lipases
logarithmic or stationary phase14,37. The cultivation from S. aureus and S. hyicus are stimulated by
period also varies with the microorganism and fast divalent ions, such as Ca2+, and the chelator EDTA
growing bacteria were found to secrete lipase within acts as an inhibitor. The pH optima of these enzymes
24 h. The production of lipase is mostly inducer vary between 7.5 and 9.0.
dependent, and in many cases oils act as good Fungal lipases have benefits over bacterial lipases
inducers of the enzyme14. Certain other inducers also for the fact that present day technology favours the
have a profound effect on the stimulation of lipase use of batch fermentation and low cost extractive
production. They include triglycerides, free fatty methods. Lipase producers are widespread in the
acids, hydrolysable esters, bile salts and glycerol37. fungal kingdom and are of much biotechnological
The organisms are normally grown in a complex interest in both research and applications. The main
nutrient medium containing carbon (oil, sugars, mixed fungal producers of commercial lipases are A. niger,
A. terreus, A. carneus, C. cylindracea, H. lanuginosa when compared to the submerged fermentation as the
(T. lanuginosus), Mucor miehei, Rhizophus arrhizus, medium employed can be obtained at low cost and the
R. delemar, R. japonicus, R. niveus and R. oryzae. down stream processing is also easier.
Media supplemented with glucose stimulated
Purification of Lipases
maximum lipase production in case of all these fungi.
Novel purification technologies are available to
Polysaccharides, such as glycogen, hyaluronate,
obtain homogeneity of lipase from a large number of
laminarin, gum arabic and pectin, stimulated the
bacteria and fungi, and from a few plant and animal
production of lipase in Serratia marcescens and
sources. The purification of lipases normally involves
Saccharomycopsis lipolytica. This might be due to the
several steps depending upon the purity desired for
detachment of lipase from the oil surface. A similar
food application.
effect was found with lecithin on R. japonicus37.
Since most of the microbial lipases are
Aeration has a variable effect on lipase production extracellular, the fermentation process is usually
by different organisms. The degree of aeration followed by the removal of cells from the culture
appears to be critical in some cases since shallow broth, either by centrifugation or by filtration. The
layer cultures (moderate aeration) produced much cell-free culture broth is then concentrated by
more lipase than shake cultures (high aeration). ultrafiltration, ammonium sulphate precipitation or
Lipase production using P. fragi, P. aeruginosa and extraction with organic solvents. About 80% of the
R. oligosporus was reduced by vigorous aeration. purification schemes attempted have used a
Increased growth and increased lipase production, precipitation step, 60% of which use ammonimum
followed by a rapid decrease of lipase activity was sulphate and 35% use ethanol, acetone or an acid,
observed in P. fragi with continuous shaking37. followed by a combination of several chromato-
Bacterial lipases from various Bacillus species graphic methods, such as gel filtration and affinity
were over expressed in E. coli using conventional chromatography. The final step of gel filtration
overexpression systems; however, many enzymes normally yields a homogenous product. Purification
(e.g. from Pseudomonas sp. and Burkholderia sp.) methods for lipases from various sources are listed in
were not amenable to these systems2,40. A study by Table 317,31,43-52.
Tang et al41 revealed that the fusion with Trx, After these pre-purification steps, novel
transformation into AD A9A (DE3) and recombinant purification technologies like (i) membrane separation
expression at low temperature are reliable and simple processes, (ii) immunopurification, (iii) hydrophobic
techniques for the production of recombinant lipase. interaction chromatography using epoxy-activated
Moreover, this strategy avoids contamination of the spacer arm as a ligand and polyethylene glycol
active enzyme with inactivated protein from the immobilized on sepharose, (iv) polyvinyl alcohol
inclusion bodies. polymers as column chromatography stationary
Rivera et al42 studied the lipase production by phases, and (v) aqueous two phase systems are
several filamentous fungi in solid state fermentation commonly employed53. The use of hydrophobic
(SSF). Penicillium candidum, M. miehei and interaction chromatography was generally found to
P. camembertii were selected to be the high yielding result in satisfactory enzyme recovery and fold
lipase producers with low protease levels and high purification.
dairy flavour generation. The kinetics of lipase An acid resistant lipase from A. niger has been
production by P. candidium by SSF was compared purified from crude commercial preparations by size
with submerged fermentation. Lipase production exclusion on Bio-gel-p-100 and ion exchange on
started earlier in SSF, generated a two fold higher Mono-Q. The lipase produced by R. delemar was
maximum concentration and was quite stable on the purified with a recovery of 3-4%. The lipase from
5th d of incubation. On the other hand, lipase R. japonicus NR400 was purified to homogeneity by
production by submerged fermentation reached its chromatography on hydroxyapatite, octylspharose and
maximum after 2 d of fermentation and the maximum sephacryl S-20037.
activity decreased rapidly after the 5th d of incubation.
These results make SSF an interesting alternative for Methods for Lipase Assay
microbial production of lipase. The solid state A number of assay protocols are employed for
fermentation offers lower production cost of lipases lipase assay due to the wide substrate specificity of
Table 3—Purification methods for lipase

Microorganism Purification method Reference
Pseudomonas spp.
P. fluorescens HU380 Phenyl-toyopearl fractionation (batch wise), DEAE – sepharose column 43
chromatography, Superdex – 200 HR FPLC
Pseudomonas sp. Biogel P-10 chromatography, Superose 12B chromatography 44
P. fluorescens strain 2D Ammonium sulphate precipitation, Hydrophobic interaction chromatography 45
P. aeruginosa Ammonium sulfate precipitation, Hydroxyapatite column chromatography 46
P. aeruginosa EF2 Ultrafiltration, Anion exchange chromatography (MonoQ), Gel filtration (superose) 47
FPLC
Bacillus spp.
Bacillus sp. Ammonium sulfate, Acrinol treatment, DEAE-sephadex A-50, Toyopearl HW-55F, 47
Butyl toyoperal 650M
Bacillus sp. Acetone fractionation, Two acetone precipitations, Octyl-sepharose CL-4B, Q- 48
Sepharose, Sepharose-12
B. thermoatenulatus Calcium soap, Hexane extraction, Methanol precipitation, Q-sepharose (ion exchange) 17
Rhizopus spp.
R. japonicus NR400 Hydroxy apatite, Octyl-sepharose and Sephacryl S-200 49
R. arrhizus Ammonium sulfate fractionation and Sephadex G-100 gel filtration 50
R. oryzae Acetone precipitation (80%), Sephadex G-100 51
Penicillium spp.
P. chrysogenum Ultrafiltration, Phenyl-sepharose, Mono Q HR5/5 and PD-10 column on sephadex G-25 31
P. citrinum Extraction and back-extraction using AOT reversed micelles in isooctane and 52
phenyl – superose column
lipases. Determination of lipase activity at the lipid- assay using olive oil as a substrate because of its
water interface is also indicative of free lipase. As simplicity, accuracy and reproducibility. A few
with all reactions catalyzed by enzymes, activity spectrophotometric assays are based on methods
measurements can be carried out using various which render colour to fatty acids released after
physico-chemical methods by monitoring the disap- hydrolysis of triacylglycerols. A unit lipase activity13
pearance of the substrate or by the release of the relates to the release of 1 µmole of free fatty acid
product. Numerous methods are available for from emulsified olive oil or triolein or tributyrin per
measuring the hydrolytic activity as well as the min at specified temperature and pH values. Specific
detection of lipase. The methods may be classified activity of lipases is expressed as units of lipolytic
as9: (i) Titrimetry, (ii) Spectroscopy (Photometry, activity per microgram of extra cellular protein.
Fluorimetry and Infrared), (iii) Chromatography, (iv)
Radio activity, (v) Interfacial tensiometry, (vi) Turbi- Properties and Characteristics of Lipases
dimetry, (vii) Conductimetry, (viii) Immunochem- Lipases are reported to be monomeric proteins,
istry, and (ix) Microscopy. The general triacylglycerol having molecular weight in the range of 19-60 kDa.
hydrolysis reaction catalyzed by lipases can be The physical properties of the lipases mainly depend
written as: on factors such as the position of the fatty acid in the
glycerol backbone, the chain length of the fatty acid,
Triacylgly- → Diacylgly- → Monoacylgly- → Glycerol and its degree of unsaturation. These features also
cerols cerols cerols affect the nutritional and sensory value of a given
+ Free + Free + Free triglyceride40. Many lipases are active in organic
fatty acids fatty acids fatty acids
solvents where they catalyze a number of useful
This reaction shows that the activity of lipases can reactions including esterification14.
thus be assayed by monitoring the release of either Lipase activity generally depends on the
free fatty acids or glycerol from triacylglycerols or availability of large surface area and requires extreme
fatty acid ester. The extensive reviews by Beisson mild conditions. Several structural studies on lipases
et al12 and Gupta et al9 describe in detail the various have provided clues to the understanding of
methods available for lipase assay. Today the most hydrolytic activity, interfacial activation and
widely used lipase assay protocol is the titrimetery stereoselectivity6. A survey by Linko and Wu19 on
major commercial lipases revealed that Aspergillus triacylglycerols has been observed in A. arrhizus,
lipases were highly selective for short-chain acids and R. delemar, C. cylindracea and P. aeruginosa. Owing
alcohols; C. rugosa lipases for propionic acid, butyric to this property these enzymes may be used to isolate
acid, butanol, pentanol and hexanol; and M. miehei optically pure esters and alcohols37,58.
and R. arrhizus lipases for long-chain acids and The use of organic media at low water activity
acetates21. This information on enzyme catalyses has offers a unique possibility to tune stereoselectivity
been valuable for the production of flavour esters54. through variation of the solvent. Since enzymes
Increase in lipase activity depends on the possess delicate and soft structures, any solvent may
concentration of ammonium sulphate solution used exert a significant influence on the catalytic properties
during the purification process53. The kinetics of the of an enzyme. Thus, an enzyme’s specificity may be
lipolysis reactions have been discussed by Brokerhoff altered by varying the properties of the solvents. The
and Jensen55. stereoselectivity of an enzyme catalyzed reaction is
The activity of lipase is pH dependent. Some also true for lipases. It can be controlled by choosing
lipases are stable over a wide range of pH values. the appropriate organic solvent7. Rooney and
Lipases are generally stable at or near a neutral pH; Weatherely39 studied the lipase catalyzed hydrolysis
some are stable up to pH 4.0 and 8.037. Extracellular of high oleate sunflower oil in a stirred liquid-liquid
lipases produced by A. niger, Chromobacterium reactor and observed maximum enzyme activity at the
viscosum and Rhizophus species are particularly oil-water interphase, in turn maximum overall rate of
active at acidic pH. An alkaline lipase active at pH reaction.
11.0 has been isolated from P. nitroaeducens37.
Lipases are capable of reversing the reaction that Substrates for Lipase
leads to esterification and interesterification under Glycerides are the natural substrate for lipases; they
certain experimental conditions, such as in the possess a chiral alcohol moiety. It was understood that
absence of water37. Cofactors are not essential for the lipases were particularly useful for the resolution or
expression of lipase activity but divalent cations, such asymmetrization of esters bearing a chiral alcohol
as calcium, stimulate the activity. The lipase activity moiety.
is inhibited drastically by Co2+, Ni2+, Hg2+ and Sn2+ General guidelines for the design of substrates for
and is slightly inhibited by Zn2+, Mg2+, EDTA and lipase were formulated by the IUPAC7:
SDS37, 56. Spontaneous and cyclic AMP induced lipase
formation is greatly enhanced in S. marcescens SM-6 i. The center of chirality should be located as close
exposure to glycogen, hyaluronate, pectin B and gum as possible to the site of the reaction (i.e. the ester
arabic37. carboxyl group) to ensure optimal chiral
The temperature stability profiles of lipases, recognition. Thus, esters of secondary alcohols
determined by half-life values, show maximum are usually more selectively transformed than
stability at lower temperatures. The maximum lipase those of primary alcohols.
activity of Epipactis gigantea and many other lipases ii. There is wide tolerance for the nature of both
were found in the range of 30-35°C. Thermophilic substituants R1 and R2 but they should differ in
bacterial lipases obtained from organisms from size and/or polarity to aid the chiral recognition
Icelandic hot springs had higher lipase activity at process. They may also be linked together to form
40-60°C37,57. cyclic structures. Polar groups, such as
Lipases may be divided into two groups according carboxylate, amide or amine—which would be
to the region-specificity exhibited with acyl glycerol heavily hydrated in an aqueous environment—are
substrate. Lipases from the first group show no not tolerated and, if they are required, they should
regiospecificity and release fatty acids from all three be protected with a lipophilic unit.
positions of glycerols. The second group lipases iii. The alkyl chain of the acid moiety (R3) should be
release fatty acids regio-specifically from the outer preferably of straight chain nature, possessing at
1 and 3 positions of acylglycerols. These lipases least three to four carbon atoms. Reaction rates
hydrolyse triacylglycerol to give free fatty acids may be improved by using ‘activated’ esters
1,2-(2,3)-diacylglycerols and 2-monoacylglycerol. bearing haloalkyl groups, e.g. Cl – CH2 – and Cl –
Partial stereo-specificity in the hydrolysis of (CH2)2 – for Type I and II, respectively (Fig. 1).
iv. The remaining hydrogen atom in both substrate

types must not be replaced by a substituent, since
esters of tertiary alcohols and tri substituted
carboxylates are usually not accepted by lipases.
v. The stereo-chemical preference of the most
commonly used lipases (e.g. from Pseudomonas
sp. and Candida sp.) for esters of secondary
alcohols follows an empirical model generally
referred to as “Kazlauskas rule”. These guidelines
may be followed to get fair accuracy to obtain the Fig. 1—Types of substrate for lipase
desired product.
The concept of lipase interfacial activation arises
from the fact that their catalytic activity generally
depends on the aggregation state of the substrates6.
The occurrence of lipase reaction at an interface
between the substrate and the aqueous phase is
because of the reversible nature of the enzyme
reaction. The enzymes acts on the substrate in a
specific or nonspecific manner, resulting in either the
complete hydrolysis of triacylglycerides into free fatty
acids and glycerol or, along with triglycerides,
monoacylglycerides, diacylglycerides, fatty acids and
glycerol are also formed. Strong interactions with
hydrophobic substrates at an interface are probably
caused by hydrophobic patches on the other lipase
surface. Such patches may also be responsible for self
association behaviour shown by the enzymes in
aqueous solutions37. Substrate specificity of lipases is
often crucial to their application for analytical and
industrial purposes. Specificity is shown both with
respect to either fatty acyl or alcohol parts of their Fig. 2—Diagrammatic representation of a lipase molecule
substrates58. Fig. 2 shows the diagrammatic showing its main features; the substrate can be any triglyceride37
representation of a lipase molecule with its substrate
(triglyceride). enhancement of lipase catalyzed reactions by
In general, lipases show little fatty acid specificity designing a suitable medium is still a largely
when incubated with most natural oils and fats, and empirical, despite the tremendous amount of data
high specificity when fish oil and milk fats are used as published to date7.
substrates. The presence of double bonds close to the
carboxyl groups in some fatty acids probably makes Molecular Modification
their esters resistant to attack by lipases. In contrast, Modern genetic engineering approaches with
M. miehei lipase preferentially releases butyric acid protein engineering may help in maximizing the
from milk fat, especially at low pH. Many microbes enzyme production for specific industrial need. The
produce two or more extracellular lipases with most suitable lipase for specific applications can be
differing fatty acid specificities, especially with obtained based on their substrate binding sites. Prior
respect to short-chain fatty acids. Geotrichum knowledge on structure function relationship is
candidum produces a lipase, which shows pronounced required. The review of Schmidt-Dannert59 on
specificity for the hydrolysis of esters of a particular recombinant microbes for lipase overexpression
type of long-chain fatty acid. Lipases show both provides a set of information to produce lipase for
regiospecificity and stereospecificity with respect to special applications. The most appropriate enzyme for
alcohol moiety of their substrates37,58. The selective particular reaction can be tailored via mutagenesis.
Protein engineering of lipases was studied based on isoforms. Lid swapping and DNA shuffling techni-
the sequence information since the mid of 1980s. ques could be used to improve the enantioselectivity,
P. mendocina lipase was the first enzyme to be thermostability and substrate specificity of CRL
engineered60. Thermostability of lipases can be isoform and increase their application in food
engineered by altering the amino acid sequence when processes67. A completely synthesized C. rugosa
designing for food application. A patent was lipase gene over expression in P. pastoris with high
registered on H. lanuginosa lipase with increased purity was reported by Brocca et al61.
temperature stability. Many lipases have been
engineered for thermostability, protease stability and Food Technology Applications
for oxidative stability60. A. niger was developed as an The majority of enzymes used in industry are for
important transformation host to overexpress food food processing, mainly for the modification and
enzymes since it has been considered GRAS breakdown of biomaterials68. A large number of fat-
(Generally Recognized As Safe) by the US Food and clearing enzymatic lipases are produced on an indust-
Drug Administration59. Among the isoforms rial scale. Most of the commercial lipases produced
lip 1 of C. rugosa was found to be most prominent. are utilized for flavour development in dairy products
The lip 1 gene was systematically modified by site- and processing of other foods, such as meat, vege-
directed mutagenesis to gain functional expression in tables, fruit, baked foods, milk product and beer69.
Saccharomyces cerevisiae61. Downstream from lip A Phospholipases have found industrial applications
gene of P. cepacia, an open reading frame lim A was in egg yolk treatment for the production of
identified. Only in the presence of this lim A, lip A mayonnaise and other emulsifiers, in lecithin
gene gets expressed in E. coli, B. subtilis and modification, and for the oil-degumming step in the
Streptomyces lividans62. Baker’s yeast strain transfor- refining of vegetable oils. Introduction of a microbial
med with plasmids containing LIP 1 and LIP 2 genes phospholipase (Lecitase Nova) has significantly
from Geotrichum sp. secreted high levels of lipase 1, improved the economy of enzymatic degumming of
but secretion of lipase 2 was comparatively higher. vegetable oils. In this process, the phospholipids are
Addition of this lipase 2 enriched the bread dough hydrolyzed and rendered more water soluble, hence
formulations26. facilitating their washout70. The function of
For the first time Sanchez et al18 expressed a phospholipase in egg yolk treatment is to hydrolyze
bacterial lipase gene (from B. subtilis) in baker’s egg lecithin, iso-lecithin, which improves the
yeast, S. cerevisiae. Lipase B that exhibited unique emulsifying capacity and heat stability. The egg yolk
substrate specificity for long-chain cis-9 unsatu- thus produced can be useful in the processing of
rated triacylglycerols were overexpressed in custard, mayonnaise, baby foods, dressings and in
Pichia pastoris63. Fickers et al64 developed a non- dough preparation. It can also be applied in the
genetically modified mutant strain Yarrowia lipolytica processing of sauces, like hallandise, bernaise and
CBS6303 with high productivity by chemical cafe de paris71.
mutagenesis. Utility of A. oryzae as a host for the Lipases have been successfully used as a catalyst
production of recombinant lipases was explained by for the synthesis of esters. The esters produced from
Huge-Jensen et al65. Schmidt-Dannert59 described the short-chain fatty acids are used as flavouring agents in
approaches for the production of recombinant lipase the food industry. Lipase immobilized on silica and
in different expression systems. Accumulation of microemulsion based organels were widely applied
active recombinant G. candidum lipase in P. pastoris for ester synthesis14,37. Accurate control of lipase
without any contaminating protein was reported by concentration, pH, temperature and emulsion content
Holmguist et al66. are required to maximize the production of flavour
The non universal codon of C. rugosa was and fragrance6. Table 4 describes some food
concerted to express universal serine triplets by site processing applications of microbial lipases14.
directed mutagenesis to gain expression of functional Uhling72 has explained the preparation of lipase
lipase in heterologous hosts67. Protein engineering of modified butter fat which found wide application in
purified C. rugosa lipase (CRL) isoforms allows the various food processes. Chocolates with coco butter
tailoring of enzyme function. This involves computer substitutes, bread, structured lipids like human milk
modeling based on available 3-D structures of lipase fat replacers, low calorie health oils, nutraceuticals,
Table 4—Lipase applications in the food industry14 Table 5—Examples of lipase in cheese production37
Food industry Action Product of application Cheese type Lipase source
Dairy foods Hydrolysis of milk Development of
Romano Kid/lamb pre-gastric
fat, cheese ripening, flavouring agents in
Domiati Mucor miehei
modification of milk, cheese and butter
Feta
butter fat.
Camembert Penicillium camemberti
Bakery foods Flavour Shelf-life propagation.
Mazarella Calf/kid pre-gastric
improvement
Parmesan
Beverages Improved aroma Alcoholic beverages,
Provolone
e.g. sake, wine
Fontina Mucor miehei
Food Quality improvement Mayonnaise, dressings
Ras
dressings and whippings
Romi
Health foods Transesterification Health foods
Roque fort Penicillium roqueforti
Meat and fish Flavour development Meat and fish product,
Cheddar Aspergillus oryzae/A. niger
fat removal
Manchego
Fats and oils Transesterification, Cocoa butter, margarine,
Blue
hydrolysis fatty acids, glycerol,
mono and diglycerides
cheeses of good quality was produced by using
EMC, etc. are few examples for lipase mediated food individual microbial lipases or mixtures of several
products. Since it is a new technology, more research preparations. Enzyme modified cheese (EMC) is
on usage of lipase to develop new commercial food produced when cheese is incubated in the presence of
products must be promoted. Oil from soybean is enzymes at elevated temperature in order to produce a
hydrolyzed by lipase in making Koji, a traditional concentrated flavour by lipase catalysis for use as an
Asian food73. Another soybean fermented food ingredient in other products, such as dips, sauces,
Tempeh utilized lipase from R. oligosporous74. This soups and snacks. The concentration of fat is 10 times
Tempeh forms a base material for many delicious, higher in EMC to that of normal cheese5,14,37,45,69.
easily digestible and nutritious food preparations, Table 5 describes the use of lipase in cheese making
providing a good number of human populations with and accelerated cheese ripening37. The free fatty acids
a valuable and affordable source of protein. In take part in simple chemical reactions that initiate the
addition to Tempeh, Kenkey and Mave (African food) synthesis of other flavour ingredients, such as aceto-
coupled with fermented vegetables and salads should acetate, β-keto acids, methyl ketones, flavour esters
be noted in this context6. and lactones37.
Dairy Industry Cocoa butter (CB) contains palmitic and stearic
Lipases are extensively used in the dairy industry acids and has a melting point of approximately 37°C,
for the hydrolysis of milk fat. The dairy industry uses leading to its melting in mouth which results in a
lipases to modify the fatty acid chain lengths, to cooling sensation. In 1976, Unilever filed a patent
enhance the flavours of various cheeses. Current describing a mixed hydrolysis and synthesis reaction
applications also include the acceleration of cheese to produce a cocoa butter substitute using an
ripening and the lipolysis of butter, fat and cream14, 37. immobilized lipase. This technology was
The free fatty acids generated by the action of commercialized by Quest-Lodrs Croklaan, based on
lipases on milk fat endow many dairy products, immobilized R. miehei lipase, which carries out a
particularly soft cheeses with their specific flavour transesterification reaction replacing plamitic acid
characteristics. The traditional sources of lipases for with stearic acid to give the desired stearic–oleic–
cheese flavour enhancement are animal tissues, stearic triglyceride14,40.
especially pancreatic glands (bovine and porcine) and Unduranga et al75 analyzed the production of a
pre-gastric tissues of young ruminants (kid, lamb and cocoa butter equivalent (CBE) through enzymic
calf). A whole range of microbial lipase preparations interesterification of palm oil midfraction (POMF)
have been developed for the cheese manufacturing with stearic acid in a solvent-free system using Nova
industry from M. miehei, A. niger, A. oryzae and lipase Lipozyme as a catalyst. Studies were carried
several others. In some cases microbial lipases have out both in batch and continuous packed bed reactors.
successfully replaced pre-gastric lipases. A range of POMF was found to be a suitable raw material for the
production of CBE, whose composition is close to
that of CB. The overall process is, however, action. The cultures and secondary flora, such as the
uneconomical due to the cost of the purification steps. P. roqueforti and P. camembertii in Blue-vein and
By using lipase catalyzed hydrolysis and Camembert cheeses respectively, are lipolytic and
alcoholysis of ester bonds in vitamin A and E esters, produce lipases, which are responsible for lipolysis. In
their level in different food formulae can be addition, lipases are usually added to Italian cheese,
determined. The supercritical fluid extraction (SFE) viz. paramesan, provolone, and romano, to intensify
method using immobilized C. antarctica preparation their flavour76. During ripening, there is a steady
is more beneficial to the oxidation prone vitamins A increase in the concentration of liberated fatty acids
and E. This extraction methodology should be and total soluble nitrogen. Lipases release the fatty
applicable for the determination of vitamins D2/D3, acids from triglycerides, thereby triggering the
K53 and β-carotene in milk powder and infant development of cheese flavour77.
formulae13,69.
The introduction of conjugated linoleic acid (CLA)
Gastric lipases have been used to accelerate
in dairy foods has been made possible through the
ripening and flavour development of many cheese
immobilization of lipases78. Lipases and proteases
types, including cheddar, provolone and ras cheeses.
have been used to accelerate ripening both
Lipase addition enhances the rate of fatty acid
individually and as a “cocktail”. The enzymes may be
liberation, which accelerates flavour development
added as such or they may be encapsulated. During
relative to control. These studies indicated that
cheese ripening, a series of enzymatic reactions
liberated fatty acid profiles of the accelerated process
proceed very gradually, modifying the fresh,
were identical to the control and the total quantities of
mechanically worked curd to the desired final ripe
short-chain liberated fatty acids (C4 to C6) were
cheese texture and flavour. The enzymes, lipases,
important for the development of typical cheddar
proteases and lactase hydrolyze lipids, proteins and
cheese flavour during ripening. The addition of calf
lactose, respectively in order to raise the level of
lipase and increasing the ripening temperature
flavour moieties and/or flavour processors76.
(from 7° to 53°C) result in a significant increase in the
liberation of fatty acids. The disadvantage with this is Fats and Oil Industry
that the lipase continues to be active after ripening Fats and oil modification is one of the prime areas
and can cause the development of strong rancid in food processing industry that demand novel
flavour. When a cock-tail of fungal protease and economic and green technologies79. Fats and oils are
lipase were used, cheddar cheese developed a highly important constituents of foods. Lipases allow us to
soluble proteins and free fatty acids and displayed modify the properties of lipids by altering the location
better flavour within three months of ripening. The of fatty acid chains in the glyceride and replacing one
level of enzyme added to accelerate cheese ripening is or more of these with new ones. In this way, a
also very important. High levels of enzyme during relatively inexpensive and less desirable lipid can be
ripening may result in excessive enzymatic reactions modified to a higher value fat14. Lipases catalyze the
that impart undesired characteristics and reduce the hydrolysis, esterification and interesterification of oils
yield. Adaptation of liposome technology for and fats. Among the lipolytic conversion of oils and
accelerated cheese ripening reduces bitterness and fats, esterification and interesterification are used to
losses in yield76. obtain value added products, such as specialty fats
Bacterial intracellular enzymes are released by cell and partial glycerides by using positional and fatty
lysis and contribute to flavour through lipolysis and acid specific lipases, and have greater industrial
other enzymatic actions. Microcapsules of cell free potential than fatty acid production in bulk through
extracts encapsulated in milk fat can be added to hydrolysis. Venkata Rao and Laxmanan13 constructed
carryout milk clotting. Cheeses made with intact an immobilized lipase membrane reactor for fat and
capsules contain substantially more enzymatic end oil hydrolysis, which yielded products that require
products than the one obtained by direct enzyme less downstream processing, thus reducing the overall
addition. The capsule stability can be improved by processing cost. The removal of phospholipids in
encapsulating in a high melting fraction of fat76. vegetable oils (de-gumming) using highly selective
Inherent milk lipase in cheese, made from microbial phospholipases is also a recently developed
unpasturized milk, affects considerable lipolytic environmental friendly process80.
Using granulation to immobilize lipases, it is ved by introduction of a microbial phospholipase

possible to produce a food grade, cost effective, (Lecitase Novo)70.
immobilized 1,3-regioselective (lipozyme TL 1M) Novozym 435 (C. antarctica lipase) catalyzed gly-
lipase targeted for the interesterification of cerolysis of commercial oils and fats to produce
commodity oils and fats for the production of frying monoacylglycerides (MGs) was investigated using a
fats, shortenings and margarine components70,81. tetra ammonium based ionic liquid as the reaction
Lipase catalyzed interesterificaiton of fats and oils to medium. A 90% yield of monoglycerides and nearly
produce modified acylglycerols cannot be obtained by 100% conversion of triglycerides in this ionic liquid
conventional chemical interesterification6. were achieved, which were markedly higher as
compared to yields in normal solvents. Excellent
Buisman et al20 used immobilized lipases
operational stability of the lipase and the reversibility
from C. antarctica (CAL-B), C. cylindracea Ay30,
of ionic liquid were also observed in consecutive
H. lanuginosa, Pseudomonas sp. and G. candidum for
reactions. This provides a new environmentally
the esterification of functionalized phenols for
benign ‘solution’ to the enzymatic modification of
synthesis of lipophilic antioxidants to be used in
fats and oils with industrial potentials86.
sunflower oil. There are many studies on the
hydrolysis of fats and oil by lipases used either in the Tailored vegetable oils with nutritionally important
pure form, in the immobilized form or in the cell structured triacylglycerols and altered physico-
bound form37. chemical properties have a big potential in future.
Microbial lipases may be exploited for retailoring of
The use of triacylglycerol lipase obtained from vegetable oils. Cheap oils may be upgraded to
genetically modified A. oryzae as a processing aid in nutritionally important structured triacylglycerols, like
the oils and fats industry for oil de-gumming, and in coco butter substitutes, low caloric triacylglycerols,
the food industry to improve emulsifying properties and PUFA and oleic oil- enriched oils. It is possible to
was technologically approved by Australia New change the physical properties of natural oils to
Zealand Food Authority (ANZFA) in 200268. A new convert them into margarines and hard butter with
process for immobilizing lipases based on the higher melting points, or into special low caloric
granulation of silica has dramatically simplified the spreads with short or medium chain fatty acids.
process and lowered the process cost. Such innovative Normally fat and oil modifications are carried out
methods are now widely implemented for the chemically by using directed interesterification. This
production of commodity fats and oils with no content process is energy intensive and non-specific. Lipase
of trans-fatty acids84. mediated modifications are likely to occupy a
A mathematical basis for the design and operation prominent place in oil industry for tailoring
of a continuous, packed bed rector was developed for structured-lipids since enzymatic modifications are
the interesterification of soybean oil, which contains specific and can be carried out at moderate reaction
22.7% oleoyl and 54.3% linoleoyl moieties as molar conditions79.
acyl moieties in hexane, with oleic acid using an Kajilal et al81 synthesized a structured lipid (SL)
immobilized Sn-1,3-specific lipase (Lipozyme IM) from natural vegetable oils so that it would contain
from M. miehei. The rate of change in oleoyl and EFAs and natural antioxidants. They incorporated
linoleoyl moiety compositions in soybean oil behenic acid into the Sn-1 and Sn-3 positions of
decreased due to the loss of catalytic activity of sunflower oil through a transesterification reaction
Lipozyme IM. In an organic solvent, Cossignani catalyzed by lipase. The synthesized product
et al82 studied the lipase catalyzed acidolysis of delivered 5.36 kCal/g and had an improved plastic
soybean oil with oleic acid to increase oleic acid nature, which increased their potential food
content. Lipase from the same source (R. miehei) was applications. Such foods are a special type of trans-
used to carryout acidolysis and a product having free solid fat. The SL was fed to rats and the results
minimal modification of Sn-2 position fatty acid compared to the control group, which was fed with
composition was obtained85. In the physical refining sunflower oil. No differences were observed in the
of vegetable oils, the degumming step can be carried amount of food consumed, which indicates the
out with a phospholipase. The economy of enzy- palatability and taste of the SL was quite similar to
matic degumming has significantly been impro- the native sunflower oil. Enzymatic interesterification
can also be used to produce oils and fats containing for organic acids with 8 or 10 carbon chain length,
nutritionally important polyunsaturated fatty acids whereas increase in carbon chain length from 4 to 8
(PUFAs), such as eicospentaenoic and docosa- for alcohol lowered the esterification yields.
hexaenoic acids87. Even though use of immobilized Esterification performance was also dependent on the
lipase in the interesterification of triglycerides was alcohol structure, with maximum activity occurring
first described in 1980s, the process was not for primary alcohol, whereas 40% reduced rate was
commercially viable then because of the high cost84. observed for secondary and tertiary alcohols92.
Lipases as Biosensors for Food Industry
Immobilized lipases are fast, efficient, accurate and Hexyl esters, green note flavour compounds, are
cost effective as sensors for the quantitative widely used for flavour and fragrance in food,
determination of triacylglycerol. This application is beverage and in pharmaceutical industries93. There is
important in the food industry, especially in fats and a growing demand for natural flavours containing
oils, beverages, soft drinks, pharmaceutical industries “green note” represented by hexanol (C6 alcohols)
and also in clinical diagnosis88. The basic concept of derivates. Hexyl butyrate is of special interest as it
using lipase as biosensors is to generate glycerol from represents a model of flavour ester. Owing to its
the triacylglycerol in the analytical sample and to ‘natural’ character, the biosynthesis of such esters by
quantify the released glycerol by a chemical or lipase catalyzed chemical reactions under mild
enzymatic method6. conditions has much current commercial interest. The
Wei et al89 developed a method for the ability of immobilized lipase (Lipozyme IM-77) from
determination of organophosphorous pesticides with a R. miehei to catalyze the transesterification of hexanol
surface acoustic wave impedance sensor by lipase and tributyrin was investigated by Chang et al94. They
hydrolysis. This method is also used to determine the concluded that reducing the amounts of co-substrate
dichlorvous residues in the root, stem and blade of (tributyrin) would reduce the production cost of
Chinese cabbage. Lipases may be immobilized onto synthesize hexyl butyrate. While, the transes-
pH/oxygen electrodes in combination with glucose terification of hexanol with triacetin, catalyzed by
oxidase, and these function as lipid biosensors and immobilized lipase from M. miehei (Lipozyme
may be used in triglycerides and blood cholesterol IM-77), was studied by Shieh and Chang91. The
determinations90. results showed that reaction temperature and substrate
molar ratio were the most important parameters and
Flavour Enhancement by Lipases
the content of added water had less effect on
The production of low molecular weight esters as
conversion. This optimization study has commercial
flavour compounds by biotechnological processes has
potential. Methyl ketones are major group of
potential interest for the food industry. The use of
compounds which contribute to cheese flavour and
naturally available substrates and enzymes is an
are derived from the fatty acids liberated by lipolysis.
essential part of the process design. Laboret and
These fatty acids have been shown to be
Perrand91 performed direct esterification of citronellol
enzymatically oxidized by secondary flora into
and geraniol with short-chain fatty acids, catalyzed by
methylketones through the β-oxidation pathway. In
free lipase from M. miehei, which gave high yields in
addition, the oxy fatty acids found in milk fat may
n-hexane. The initiative to scale-up applications was
also be the source of methylketones76.
also attempted, using a reactor that allowed the
production of ester in quantities up to 100 cm3. Bakery Industry
M. miehei lipase was immobilized on magnetic In baking industry, there is an increasing focus on
polysiloxane-polyvinyl alcohol particles by covalent lipolytic enzymes. Recent findings suggest that
binding with extended high activity. The performance (phospho) lipases can be used to substitute or
of the resulting immobilized biocatalyst was supplement traditional emulsifiers since the enzymes
evaluated in the synthesis of flavour esters using degrade polar wheat lipids to produce emulsifying
heptanes as solvent, in which ester synthesis was lipids in situ10,83,95. Lipase was primarily used to
maximized for substrates containing excess acyl enhance the flavour content of bakery products by
donor. The biocatalyst selectivity for the carbon chain liberating short-chain fatty acids through esterifi-
length was found to be different between organic cation. Along with flavour enhancement, it also
acids and alcohols. High reaction rates were achieved prolonged the shelf-life of most of the bakery
products. Texture and softness could be improved by fatty acids and stearic acid. Commercially immobili-
lipase catalyzation92. zed Sn-1, 3-specific lipase, lipozyme RM IM,
An artificially expressed lipase in A. oryzae was obtained from R. miehei was used as the biocatalyst
used as processing aid in the baking industry96. All for the acidolysis. For both oleic and stearic acids, the
hydrolytic enzymes, including lipase, were found to incorporation level increased with reaction time. The
be effective in reducing the initial firmness and SLs produced in this study by Sahin et al99 have
increasing the specific volume of breads97. potential use in infant formulae. They also state that
Recombinant yeast with Geotricum LIP 2 gene greater collaboration between industry and academia
expressed a protein, which exhibited similar will hasten and increase successful commercialization
biochemical properties. Fermented dough prepared of enzymatic processes101.
with this recombinant yeast rendered the bread with
Other Food Processing Industries
higher loaf volume and more uniform crumb
In recent times, lipases have been commonly used
structure26. Yeast with bacterial lipase gene LIP A
in the production of a variety of products, ranging
resulted in higher productivity of enzyme and found
from fruit juices to vegetable fermentation6. Lipases
use in bread making as a technological additive18.
facilitate the removal of fat from meat and fish
Increased butter falvour for baked goods was
products14. Cao et al reported a lipase-catalyzed solid
generated by hydrolysis of butterfat with suitable
phase synthesis of sugar fatty acid esters102. Pandey
lipase72.
et al6 reviewed the direct conversion of alkane diols
Dietetics into their monoesters and diesters, which has strong
With increasing consumer awareness of the risks implications for the food and pharmaceutical
associated with high fat intake, there is growing industries, because the products have non-ionic
demand for low caloric fats and fat replacers, but surfactant activity and can be used as monomeric
these cannot be exposed to high temperatures. The units in cross-linking in polymerization. The
majority of reduced caloric fats and fat substitutes processing of sausages with microbial lipases is an
available today contain fatty acids that are not emerging are of meet technology14.
naturally present in edible oils and fats but may match An interesting finding is the addition of lipase to
the chemistry and function of natural fats. The noodles, resulting in significantly softer textural
drawback is that such products lack nutritionally characteristics in noodles despite having the relatively
important essential fatty acids (EFA). low levels of the substrate acylglycerols present in the
A positional analysis of the structured triglycerols formulations101.
formed showed an increase in preference of the lipase In confectionary, 1,3-regioselectivity of lipases was
action for the primary positions compared to the exploited in the process development of a fat
secondary positions. The targeted structured production containing high concentration of
triglycerols with palmitoyl moieties in the Sn-2 1,3-disteraroyl-2-monoloein87. This fat could be used
position and medium chain acyl moieties in the Sn-1,3 as a substitute for shea stearine in the formulation of
positions should be useful in food formulation for cocoa butter equivalents. Fats designed to inhibit
infant nutrient and clinical as well as parental bloom formation in chocolate products have also been
nutrition applications. The use of a good grade papain produced by these types of enzyme esterification
containing lipase ensures ready acceptance of such reactions87.
products54.
[
C. rugosa lipases have many applications in the
Osborn and Akoh98 has discussed the modification food and flavour industry, in the production of ice
of vegetable oil for the production of infant formula cream and single cell protein, biocatalytic resolution
with highly absorbing TAGs. It is composed of of life saving pharmaceuticals, carbohydrate esters
MCFA (Medium Chain Fatty Acids) and PUFAs in and amino acid derivatives not obtainable by
the same positions and amounts as those found in conventional chemical synthesis103.
human milk. Structured lipids (SLs) containing Immobilized lipase from C. antarctica (Novozym
palmitic, oleic, stearic and linoleic acids, resembling 435) has been applied to perform the enzymatic
human milk fat (HMF), were synthesized by esterification of bioactive compounds with fatty acids.
enzymatic acidolysis between tripalmitin, hazelnut oil Various bioactive compounds, like vitamins,
secondary metabolites such as kojic acid from plants suprofen and ketoprox), the potential anti viral agent
and microorganisms, can be acylated to generate lamivudine, and for the enantiospecific synthesis of
products useful in the cosmetic, pharmaceutical, fine alkaloids, antibiotics, vitamins, and anti-
chemical, food and feed industries. A convenient arteriosclerotic, anti tumour and antiallergic
scaleable procedure for the downstream processing of compounds6.
the ester product comprises hexane-solid extraction of Nutraceuticals are food components that have
the unreacted lauric acid and water ethyl acetate health benefits beyond traditional nutritional value.
extraction of the unreacted pyridoxine, yielding lauric Novel biotechnology tools, like immobilization, have
acid-pyridoxine monoester as a white powder with also been applied for the isolation and incorporation
more than 90% purity, which is soluble in vegetable of such food components in ordinary foods.
oil100. Regioselective modification of polyfunctional Successful synthesis of nutraceuticals has been
organic compounds is yet another rapidly expanding reported by employing immobilized lipases, such as
area of lipase application. The enzyme has also been those from C. antarctica and Lactobacillus ruteri19.
used in conjugation with a microbial cocktail for the Lipases are also used in the synthesis of the artificial
treatment of fat rich effluents from ice cream plants. sweetener sucralose by regioselective hydrolysis of
This could also be utilized in waste processing of octaacetylsucrose2.
many food industries37.
Some limiting problems for such processes are: (1)
Lipases for Pharmaceutical Application insufficient enantioselectivity, (2) limited enzyme
Microbial lipases are used to enrich PUFAs from activity, (3) difficulties in recycling the lipase, and (4)
animal and plant lipids, and their mono and inherent practical limitations of the kinetic resolution
diacylglycerides are used to produce a variety of arising from the fact that 50% conversion is the
pharmaceuticals44. PUFAs are increasingly used as maximum possible19.
food additives, pharmaceuticals and nutraceuticals
because of their metabolic benefits. Many PUFAs are Safety Evaluation
essential for normal synthesis of lipid membranes and It is important that the lipases produced by
prostaglandins. Microbial lipases are used to obtain microbial sources do not exhibit any toxicity when
PUFAs from animal and plant lipids, such as used in food applications. The evaluation of safety
menhaden oil, tuna oil and borage oil. Free PUFAs involves mainly testing for acute, subacute and
and their mono and diacylglycerides are subsequently subchronic oral toxicity and mutagenic potential6.
used to produce a variety of pharmaceuticals14. Lipase G from P. camembertii, which is used as a
Liposomes are used in the medical field to optimize processing aid in the food industry, was subjected to
the action of drugs by transporting them to target various safety evaluations. As a result, it was
areas, thus circumventing drug waste inactivation and classified as a nonpathogenic and as safe for the
anatomical barriers19. enzyme production worker and the consumer106. No
safety concern was identified with lipase derived from
Considerable effort is being made to obtain R. oryzae under controlled fermentation conditions
optically, pure compounds, which are pharmacologi- and the toxicological test results concluded that it
cally more active than its antipode. Profens, a class of could be used as a food additive107. A. niger enzymes
nonsteroidal anti-inflammatory drugs, are active in the are considered GRAS with a restriction that the new
(s)-enantiomer form. Lee et al104 and Xie et al105 and unknown isolates should be checked for
synthesized pure (s)-ibuprofen using lipase-catalyzed ochratoxin A production before they are developed as
kinetic resolution via hydrolysis and esterification, production organisms108. At high levels R. miehei
respectively. lipase expressed in A. oryzae showed consequential
In addition to racemization in situ, lipases are also effects upon body weight and energy metabolism109.
capable of catalyzing synthetic reactions, which has Lipase D from R. oryzae, which is used for selective
led to the production of life saving drugs. Efficient hydrolysis of triglycerides and for interesterification
kinetic resolution processes are available for the of edible fats and oils, exhibits no adverse effect when
preparation of optically active homochiral used as described in the processing of dietary fatty
intermediates for the synthesis of nikkomycin-B, non acids and glycerides of fatty acid110. A lipase
steroid anti-inflammatory drugs (naproxen, ibuprofen, artificially expressed in A. oryzae, which is used as a
processing aid in the baking industry, appears safe for years and indications are that this growth will be
consumers and no occupational health precaution in sustained for many years.
manufacture was recommended due to its low
environmental impact96. Enzyme preparation from C. References
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Biotechnol, 81 (1999) 131-142.
develop novel cost-effective technologies for
4 Svendsen A, Sequence comparisons within the lipase family,
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some areas in food industry with commercial 7 IUPAC, Pure Appl Chem, 69 (1997) 1613-1632.
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Lipase applications in food industry

Article in Indian Journal of Biotechnology · April 2007

Aravindan Rajendran Anbumathi Palanisamy

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Lipase applications in food industry

Received 13 September 2005; revised 16 January 2006; accepted 17 March 2006

Introduction Lipases posses the unique feature of acting at the

Table 3—Purification methods for lipase

iv. The remaining hydrogen atom in both substrate

Using granulation to immobilize lipases, it is ved by introduction of a microbial phospholipase

20 Buisman G J H, Van-Heltersen C T W, Kramer G F H, 37 Ghosh P K, Saxena R K, Gupta R, Yadav R P & Davidson S,

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