Mol Biol Rep (2014) 41:1035–1048

DOI 10.1007/s11033-013-2949-9

Major proteins in goat milk: an updated overview on genetic

variability
Maria Selvaggi • Vito Laudadio • Cataldo Dario •

Vincenzo Tufarelli

Received: 7 February 2013 / Accepted: 20 December 2013 / Published online: 1 January 2014
Ó Springer Science+Business Media Dordrecht 2013

Abstract Milk and dairy products are very important in located in Asia and Africa. The domestication of goat is
Mediterranean diet because of their health promoting and well known since ancient times, as it is thought to have
organoleptic properties. In many developing countries, arisen about 10,000 years ago in the mountains of Iran [1].
goat rearing has a key role in livestock production. What In the past goats were considered as marginal species for
makes goats so popular is their ability to provide high subsistence farming of poor populations underestimating
quality food under diverse climatic conditions and resil- their economic role and potentialities. To date, goats are no
ience to extreme and capricious environments. In the last more only synonymous of underdevelopment and poverty
years, the interest concerning caprine milk has been in fact goat milk plays a vital role in human nutrition in the
increasing also to find a new exploitation for local breeds. area acknowledged as the cradle of modern civilization.
To promote the goat dairy products there is a clear need to What makes goats so popular is their ability to provide high
know the quality and the technological aspects of milk quality food under diverse climatic conditions, as well as
produced. That being so, the purpose of this study was to their resilience to extreme and capricious environments [2].
review the available literature on the major goat milk Although it may not be important in certain parts of the
proteins with a particular attention to recent findings on world, the contribution of goat milk production to the
their genetic variability. Moreover, the main effects of economic and nutritional wellbeing of humanity is unde-
different protein variants on milk yield and composition niable in many developing countries, especially in the
were also discussed. Mediterranean, Middle East, Eastern Europe and South
American countries. In the last years, interest concerning
Keywords Milk  Casein  Whey protein  Dairy goats  caprine milk has been increasing also to find a new
Polymorphism  Genetic variants exploitation for local breeds. In developed countries
belonging to Europe, Oceania and North and South
America, goat milk production is assuming an increasingly
Introduction economic relevance, especially due to the production of
goat cheeses which are suitably selected as a gourmet food,
Milk and dairy products are very important in Mediterra- and receive the highest prices among cheeses on the market
nean diet because of their health promoting and organo- [3]. Consumption of goat milk and dairy products is also
leptic properties. In many developing countries, goat associated with beneficial health effects. New potential
rearing plays a key role in livestock production, in fact it is uses and marketing exploitation strategies for goat milk are
estimated that over 80 % of the world’s goat population is also being investigated, for example, as a basis for nutra-
ceutical and infant foods. Dairy products are used as
vehicles for other functional ingredients such as phytos-
M. Selvaggi  V. Laudadio  C. Dario (&)  V. Tufarelli terols, fish fatty acids and many probiotic bacteria. Another
Department of DETO—Section of Veterinary Science and
aspect of demand for goat milk derives from the increasing
Animal Production, University of Bari ‘Aldo Moro’,
70010 Valenzano, BA, Italy number of people with cow milk allergies and other gastro-
e-mail: [email protected] intestinal diseases. Cow milk allergy is the most frequent

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allergy in the first years of life. In the absence of maternal content is in overall 5.8 % of total nitrogen. The main
milk, allergic subjects need an alternative protein source, components of the non-protein nitrogen fraction are urea,
which is usually based on hydrolyzed cow milk proteins free amino acids, nucleosides, nucleotides and polyamines
(caseins or whey proteins) or soybean-based formula. Milk [3, 4].
from various mammalian species (goat, donkey and horse) More than 95 % of the proteins contained in ruminant
has been suggested as a possible alternative to cow milk milk are synthesized from six structural genes that encode
also for infant-food formulae. That being so, quality and well characterized proteins, the whey proteins (b-lacto-
safety of goat milk must be necessarily improved to globulin and a-lactalbumin) and the caseins (as1, as2, b,
enhance the growing interest in goat dairy products all over and j-caseins).
the world. The caseins are a family of phosphoproteins synthesized
In order to promote the goat dairy products, there is a in the mammary gland in response to lactogenic hormones
clear need to know the quality and technological aspects of and other stimuli and secreted as large colloidal aggregates
milk produced. There are many qualitative differences termed micelles, which are responsible for most of the
between goat milk and milks produced by other domestic unique physical properties of milk. Their relevance for the
species not only in terms of composition. As a conse- dairy industry worldwide have made them an important
quence, goat milk have peculiar properties and specific target for study by many scientists leading to them
technological destinations; the milk protein composition in becoming perhaps the most widely studied food proteins.
toto and the unique micellar structure determine particular Initially, the caseins were considered as a homogenous
differences in the rheological proprieties of the curds pro- substance. Subsequently, Linderstrom-Lang and Kodama
duced after coagulation. [5] demonstrated that they were composed of two fractions:
Milk is a secretion of the mammalian gland, whose one which precipitated in the presence of calcium (cal-
composition and physical characteristics of vary from cium-sensitive caseins) and the other which was responsi-
species to species. It is a complex oil-in-water emulsion ble for stabilizing the former against precipitation
containing fat, protein, lactose, minerals as well as (calcium-insensitive caseins). Later, Mellander [6] dem-
enzymes, cells, hormones and immunoglobulins. The two onstrated the presence of three different components des-
major categories of milk proteins are: insoluble proteins ignated as a-, b-, and c-casein. Further studies revealed that
(the casein family) and soluble proteins (whey proteins) an even greater number of components was present in the
found in lactoserum. Hence, caseins include as1, as2, b, casein fraction [7] and much efforts were dedicated to
and j-caseins, while the whey proteins are a-lactalbumin clarify these minor components. However, with the
and b-lactoglobulin (Table 1). Milk also comprises expansion of molecular biology, it is now possible to affirm
important minor proteins, such as serum albumin, immu- that only four types of caseins exist and the earlier heter-
noglobulins, lactoferrin, transferrin, calcium-binding pro- ogeneity that was recognized by electrophoresis is due to
tein, prolactin, folate-binding protein and proteose- effects of post-translational processing, alternative splicing
peptone. The content of nitrogen components in goat milk of the gene product or genetic polymorphisms [8]. In 1984,
varies according to breed, genetics, season, stage of lac- the American Dairy Science Association Committee on
tation and feed. Goat milk has a natural whey protein to Nomenclature and Classification proposed that the
casein ratio of about 20:80. The non-protein nitrogen nomenclature developed for the bovine caseins, namely the
as1, as2, b and j-caseins, be adopted for investigations of
Table 1 Concentrations of protein fractions in goat milk proteins in other species.
Total protein (g/kg) 37.20
Caseins are the major protein fraction of milk in many
species and their function is to transport calcium phosphate
Total casein (g/kg) 24.00
in milk and hence to provide the newborns with a source of
as1-Casein (% of total casein) 5.60
calcium and phosphorus for bone formation as well as to
as2-Casein (% of total casein) 19.20
contribute to the requirement for amino acids [9]. The
b-Casein (% of total casein) 54.80
caseins, being secreted proteins, possess N-terminal signal
j-Casein (% of total casein) 20.40
peptides that direct the passage of the newly synthesized
Whey proteins (g/kg) 7.40
polypeptides into the lumen of the endoplasmic reticulum,
a-Lactalbumin (% of total whey protein) 24.00
whereupon the signal peptides are removed from the pri-
b-Lactoglobulin (% of total whey protein) 53.70
mary translation products to yield the mature caseins. The
Minor whey proteins (% of total whey protein) 22.30
calcium-sensitive caseins show similar molecular masses
NPN 9 6.38 (g/kg) 5.80
(around 24 kDa), promoter regions, leader peptide
Adapted from Park et al. [3], Barth and Behnke [11], Jenness [37], sequences and locations of the major phosphorylation site.
Mora-Gutierrez et al. [40] These data support the hypothesis of a common

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evolutionary origin of these genes from the duplications Caseins

and modifications of a unique ancestral gene [10]. The
remainder of the sequences appear to be subject to few as1- and as2-Caseins
functional constraints and have accumulated many nucle-
otide substitutions, as revealed by comparative sequence Among calcium-sensitive caseins, as1-casein has the
analysis [9]. In goat, a high polymorphism has been found greater solubility in the presence of calcium. It is a highly
at the four casein genes with several alleles associated with phosphorylated protein and it is a structural component of
null or reduced expression of the respective protein. the casein micelle playing a key role in cheese curd for-
Besides caseins nitrogen (76 % of total nitrogen content of mation [12, 20]. The gene encoding for the as1-casein is
milk), the nitrogen content of milk is also distributed known as CSN1S1 gene. In many eutherian species, the
among whey proteins (18 %) and non-protein nitrogen nucleotide sequence of this gene shows the presence of a
(6 %). As previously reported, the term whey proteins has highly conserved signal sequence of 15 residues even if
been used to describe the group of milk proteins that quite divergent sequences for the mature proteins suggest
remain soluble in milk serum or whey after precipitation of that they are encoded by a rapidly evolving gene family.
caseins at pH 4.6 and 20 °C and after rennet casein pre- The as1-casein consists of 214 amino acids in each species
cipitation [11, 12]. The former whey protein source is [21]. Bovine as1-casein exists in two phosphorylated forms
known as acid whey, the latter is referred to as sweet or containing 8 and 9 phosphates/mol [22]. Different variants
rennet whey [13]. Unlike casein, whey proteins contain no of the as1-caseins have been described within species. In
phosphorus and are characterized by a high content of cattle, eight protein variants of the as1-casein are known
sulfur-containing amino acids, mainly methionine and and classified from A to H. The two most common variants
cystine. This aspect is very important for the requirement (B and C) differ in the amino acid exchange Glu?Gly at
of newborns since they need 4–6 % of sulfur-containing amino acid position 192 of the mature protein [12].
amino acids (on total amino acids content) in order to well The as2-caseins are a more disparate group than the
support growth [14, 15]. as1-caseins and have been identified from a wide number
Whey proteins are globular molecules with a sub- of species such as ovine, caprine and bovine [9, 23, 24].
stantial content of a-helix motifs, in which the acidic/ The as2-casein is the least investigated protein probably
basic and hydrophobic/hydrophilic amino acids are dis- due to the difficult in isolation and purification. However, it
tributed in a fairly balanced way along their polypeptide is found throughout mammalian milks and, in some rare
chains [16]. About 75 % of all milk whey proteins are instances, contributes up to 29 % of the composition of the
albumins (a-lactalbumins and b-lactoglobulins) a valuable whole casein content. Furthermore, it is found in higher
source of bioactive peptides with extra physiological concentration in larger caprine casein micelles [25]. The
activity. From a nutritional point of view, milk whey as2-caseins are highly phosphorylated peptides. The com-
proteins have been considered superior to casein in vari- plete amino acid sequence of as2-casein consists 207 res-
ous aspects. They have a better amino acid profile, being idues with a large number of positively charged side
similar to those of human milk. Therefore, whey proteins chains, especially in the C-terminal segment [9]. Like j-
are recommended for the formulation of milk products for casein, it contains two Cys residues. However, it has a low
replacement of bovine milk in infant nutrition [17]. It was Pro content. No reports on the presence of as2-casein in
observed that the caseins undergo much lower digestion human milk are found [26]. The gene encoding for as2-
and absorption than the whey proteins [18]. Whey pro- casein (CSN1S2) has a signal sequence highly conserved
teins are used as food ingredients due to their important but the mature proteins are quite divergent as for as1-
functional properties such as solubility, viscosity, water- caseins. The role of as2-casein in casein micelles has not
holding capacity, gelation, adhesion, emulsification and been yet studied in depth. The as2-casein comprises up to
foaming [19]. 10 % of the casein fraction in bovine milk; it consists of
Due to the current and growing interest on this topic and two major and several minor components exhibiting
considering the large number of new information, the varying levels of post-translational phosphorylation as well
purpose of this study was to review the available literature as minor degrees of intermolecular disulfide bonding [12,
on goat milk proteins with a particular attention to recent 27]. Many protein variants of the as2-casein have been
findings on milk protein genetic variants to support scien- reported in bovine milk (A–D). The protein variant A was
tists in the acquirement of the new advances on this matter. initially determined chemically [28] and later by cDNA
Moreover, the effects of different protein variants on milk and genomic DNA sequencing [9, 29]. Variants B and C
yield and composition were discussed. are specific for zebus and yaks, respectively [30–32],

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whereas the D variant has been found at low frequencies in Table 2 Milk protein genetic polymorphisms in goat
some European bovine breeds and in the African Namchi Variant References
taurine breed [33–36]. The original variant A is the most
frequent in all breeds so far investigated and also almost as1-Casein A, B1, B2, B3, B4, C, Grosclaude et al. [41],
D, E, F, G, H, I, L, Moioli et al. [43], Rando
fixed in most western breeds. The different forms of the
M, N, O1, O2 et al. [44], Bevilacqua
gene and protein are caused by single nucleotide poly- et al. [45], Ramunno
morphisms that result in amino acid changes. et al. [46], Sacchi et al.
Until the late 1980s, it was believed that goat milk [47], Chianese et al. [49],
lacked the as1-casein [37]. Subsequently, many as1-casein Grosclaude and Martı̀n
[50]
variants have been found in goat milk with marked dif-
as2-Casein A, B, C, D, E, F, G, O Boulanger et al. [62],
ference in their level of expression [38–40]. In fact, the Bouniol et al. [63],
polymorphism of as1-casein is one of the most interesting Lagonigro et al. [65],
and extensively studied in goat and cow milk [41]. The Ramunno et al. [66],
types of mutations are single nucleotide substitutions, Ramunno et al. [68],
Erhardt et al. [67],
deletions or large insertions. The as1-casein shows great Marletta et al. [69]
individual quantitative variations due to the occurrence of b-Casein A, A1, B, C, D, E, O, Mahé and Grosclaude
genetic polymorphisms [41, 42] (see also Table 2). Nine of O’ [84], Neveu et al. [85],
the currently identified alleles in goat milk (A, B1, B2, B3, Ramunno et al. [87],
B4, C, H, L and M), identified as strong alleles, are asso- Persuy et al. [88],
Galliano et al. [89],
ciated with a high level of as1-casein in milk (*3.5 g/l), Cosenza et al. [90],
two (E and I) with medium levels (1.1–1.7 g/l), and three Caroli et al. [91]
(D, F and G; weak alleles) with low levels (*0.45 g/l). j-Casein A, B, B’, B’’, C, C’, Caroli et al. [140],
The O1, O2 and N are ‘‘null’’ alleles and produce no as1- D, E, F, G, H, I, J, Yahyaoui et al. [121],
casein in goat milk [41, 43–50]. Due to the important role K, L, M Jann et al. [141],
Prinzenberg et al. [122],
in cheese-making ability of as1-casein, the impact of low Caravaca et al. [142]
as1-casein content in milk can be significant [51–54]. a-Lactalbumin Three nucleotide Cosenza et al. [172], Lan
Optimization of curd firmness is crucial to cheese-makers substitutions et al. [173]
to optimize cheese yield. In addition, shorter coagulation b- Many nucleotide Chianese et al. [188],
time (the point at which coagulation appears firstly or the Lactoglobulin substitutions and Yahyaoui et al. [187],
time when casein micelles have aggregated sufficiently to one indel Pena et al. [190],
Graziano et al. [189],
form visible flocs) and higher coagulation rate (the measure Ballester et al. [191],
of how quickly the curd firms once coagulation has begun) Kumar et al. [192]
would shorten the processing time required for cheese
production. The potential to improve milk coagulation
properties by selecting for high as1-casein milk may be dairy farmers, in fact milk from animals possessing strong
significant. Research on milk from Saanen and Alpine goat alleles may be used for the dairy industry; while animals
breeds showed that as1-casein content is related with other with mild or null alleles can be reared to obtain milk for
milk components and coagulation properties [53]. In fact, allergic subjects [56–58]. Goat milk lacking as1-casein is
goat milks containing higher or intermediate as1-casein less allergenic than other goat milk. For goat breeding
had significantly higher levels of all milk components than programs, this new knowledge could be a challenge and
milks without as1-casein. Milks with higher solid not fat rewarding, especially since selection for or against as1-
and protein content began coagulating later than milk with casein is now practiced in some countries.
low solids, suggesting that protein delays the onset of It has been assessed that the variance in as2-casein con-
coagulation. Milks with high levels of as1-casein had long tent account for the unique physicochemical characteristics
coagulation time and formed firm curds probably due to the of goat caseins explaining a large part of differences in the
presence of higher amounts of as1-casein and as2-casein in technological properties between goat and cow milks [53,
the milk [55]. These two protein fractions may delay curd 59–61]. Concerning the polymorphism of as2-casein in goat,
formation by binding Ca2? ions, making fewer available at least eight alleles have been identified, associated with
for binding after proteolysis of j-casein by rennet [53]. three synthesis levels [47] (see Table 2). Boulanger et al.
Moreover, average coagulation rate values of milks con- [62] and Bouniol et al. [63] found three variants (A–C) dif-
taining high amounts of as1-casein were significantly fering by amino acid substitutions [64]. Variants A, B, C, E
higher than for milks lacking this protein [55]. To exploit [65], F [66] and G [67] are related to normal synthesis levels;
the genetic variability could be a good opportunity for whereas D and O are associated with lower and null synthesis

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level respectively [68]. The content of as2-casein was Two main phosphorylation levels (6 and 5P) corresponding
associated with allergenic properties of milk: variants A, B, to b1 and b2 proteins occur with not negligible abundance
C, E and F displayed a higher allergenic potency than did D [82] but other forms have also been reported (3 and 4P) by
and O [69]. D variant is a rare defective allele characterized Chianese et al. [81]. Caprine b-casein was initially con-
by a deletion, involving part of the exon 11 and of the fol- sidered as monomorphic; later, several studies showed the
lowing intron, that should results in the loss of the codons Pro presence of a null phenotype in different goat breeds [83,
(CCC122), Thr (ACC123) and Val (GTG124) and the substi- 84]. Subsequently, three variants A, B and C [84, 85],
tution Thr?Asn at position 121 [66]. The null allele shows a differing by a single amino acid substitution, were asso-
G?A transition at the 80th nucleotide in exon 11, creating a ciated with normal b-casein content. The C variant was
premature stop codon at position 110 [68]. Mass spectrom- found to be most frequent in Italian goat breeds [86].
etry studies revealed that in goat milk each as2-casein var- Moreover, two null alleles were characterized [87, 88]. To
iant can exist in two forms; the main component corresponds date, genetic polymorphism of the goat b-casein gene
to the full length of 207 residues, while the minor component, (CSN2) is characterized by extensive variability (Table 2),
caused by a skipping event, lacks the internal sequence which is under the control of at least 8 autosomal alleles:
Glu34-Glu42 or Lys35-Glu43 [70]. Recently, two new trun- A, A1, B, C, D, E, and 2 null alleles (O and O0 ) [84, 85, 89–
cated subvariants of goat as2-casein variants A and E have 91]; the genetic variants A, A1, B, C, D, and E being
been identified [70, 71]. An evolutionary pathway has been related to a normal b-casein content. Two null alleles (O
proposed [47] starting from the A variant and leading inde- and O0 ) of the CSN2 gene are both characterized by
pendently to the B, C, F alleles, each characterized by a mutations responsible for premature stop codons in exon 7
different amino acid substitution with respect both to goat [87, 88]. CSN2 variant O originates from a single deletion
variant A and to the bovine and ovine sequences. Thus, A of an A at position 16 of the 7th exon of the gene creating a
allele may be considered the ancestral variant. premature stop codon at position 73; whereas O0 allele is
characterized by a transition (C?T) at position 373 of the
b-Caseins 7th exon of the gene, leading to a premature stop codon at
position 182. These phenomena may determine non-func-
b-Caseins are described as ‘‘calcium-sensitive’’ because tional messengers and the absence of b-casein in milk [64].
they precipitate in the presence of low concentrations of Both the null alleles are associated with a non-detectable
this cation. They widely contribute to determine the surface amount of this protein in milk. The mRNA analysis
properties of casein micelles being essential for curd for- revealed that the transcript product amounts were almost
mation when milk is clotted by the enzyme chymosin [72]. 10 [87] and 100 [88] times smaller for the null alleles
The b-casein family includes several eutherian and two compared to the A variant. The alignment among the
marsupial proteins for which the amino acid and nucleotide mature b-casein sequence of different species suggests that
sequence is known [9, 73–77]. Eutherian b-caseins gene A is the ancestral allele in comparison with C [86]. In fact,
sequences are characterized by a highly conserved 15 the presence of Ala at position 177 of the mature protein
residue signal sequence although the mature proteins show coded by goat A allele has been found also in other
a degree of divergence among species in terms of amino ruminant species, whereas the C allele codes for Val at the
acid identity similar to that observed for the as1-caseins same amino acid position. Studies about the coagulation
[78]. The b-caseins are particularly rich in glutamines; the ability of individual milks with ‘‘null’’ b-casein showed
presence of a single major phosphorylation site near the that longer rennet coagulation times were needed in com-
N-terminus is well identified. These proteins have a lower parison with normal milk and weaker curd firmness
number of phosphorylation sites and a lesser phosphory- occurred [3]. The analysis of milk coagulation properties
lation degree compared to as1- and as2-caseins. At least 12 with and without b-casein demonstrates that milk without
bovine variants for b-casein, A1, A2, A3, B, C, D, E, F, G, b-casein shows longer coagulation times and low curd
H1, H2, I have been reported [12]. The variant A2 is the firmness. Moreover, it was found that cheese yield from
most common variant used as reference. Lien et al. [79] milk without b-casein is about 80 % of that obtained from
studied the most common genetic variants in many Nordic milk with a normal content of this casein [81].
breeds (A2, A1, B, A3). Recently, Jensen et al. [80]
reported the variant I in Danish dairy breeds indicating that j-Caseins
it might be a relatively common variant.
In goat milk, the b-casein is the most abundant protein The j-casein represents about 15 % of the total caseins.
[37]. The heterogeneity of polyacrylamide gel electropho- The amino acid and nucleotide sequence of j-casein from
resis and immunoblotting patterns was thought to be many eutherian species is already known [92–97]. The j-
originated only by a discrete phosphorylation rate [81]. casein consists of 169 amino acids residues (19,023 Da)

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and occurs in polymeric form via disulfide bonds ranging results in high net negative charges (3.5 at pH 6.8) that are
in sizes from 60 to 600 kDa. Compared to other caseins, j- involved in the stabilization of the j-casein. The overall
casein is the only one soluble in the presence of calcium acidic character of the macropeptide is conserved in j-
ions and has a much smaller phosphate component. The caseins for several species, including cow, zebu, buffalo,
signal peptide of j-casein is 21 residues in length (com- goat, ewe, sow and human [110]. j-Casein is mainly sited
pared to 15 in calcium-sensitive caseins). This casein is the on the surface of the casein micelles being responsible for
only eutherian casein that has been shown to contain car- their stability [111]. The presence of a glycan moiety in the
bohydrate moieties leading to the conclusion that this C-terminal region of j-casein stabilizes the micelle [104,
protein is not related to the calcium-sensitive caseins [95, 112]. The level of glycosylation does not influence the
98, 99]. j-Casein is susceptible to cleavage by the chy- micelle structure but it does affect the susceptibility of j-
mosin (rennin). Jollés et al. [98] have shown that cleavage casein to hydrolysis by chymosin, with a decreasing sus-
occurs at a specific Phe-Met bond in the C-terminal portion ceptibility as the level of glycosylation increases [113,
of bovine j-casein. Nakhasi et al. [100] proposed that j- 114].
casein be classified into two separate groups depending on The genetic polymorphism of the j-casein gene (CSN3)
the species of origin. Group I j-caseins (cow, sheep, goat, in several species: cattle [115, 116], buffalo [117–119],
water buffalo) differ from group II j-caseins (rat, mouse, goat [120–123], sheep [124], yak [125] and equidae species
pig, human) for hydrophobicity, carbohydrate content, [126–128]. Eleven variant alleles of the j-casein gene have
amino acid composition and site of proteolytic cleavage. been identified in cattle, namely A, B, C, E, F1, F2, G1, G2,
Group I j-caseins contain the archetypal Phe-Met bond. In H, I and J [12]. Initially, a variant called D was detected by
group II j-caseins however, the cleavage site is specified polyacrylamide gel electrophoresis analysis, however this
by Phe-Ile or Phe-Leu. This divergence might reflect dif- variant was later found to be identical to j-casein C [129].
ferences in the mechanism of clotting in ruminant and non- Nevertheless, only two alleles, namely A and B, are
ruminant mammals [101]. The j-caseins are the only gly- commonly identified in Bos taurus dairy cattle [130]. These
cosylated caseins from eutherian milk. The carbohydrate two variants differ in the amino acids 136 and 148 [131].
groups are attached to j-casein via O-glycosidic linkages Several studies on j-casein variants resulted in its signifi-
to Ser and Thr residues within the C-terminal portion of the cant association with lactation performance, milk compo-
molecule. Glycosylation occurs post-translationally and is sition, milk protein content and rheological properties
catalysed by membrane bound O-glycosyl-transferases [132–135].
within the Golgi apparatus of mammary epithelial cells Goat j-casein was isolated for the first time by Zittle
[102, 103]. Glycosylation increases during the colostral and Custer [136]. The complete amino acid sequences of
period and as a consequence of infections, but decreases 171 residues was determined by Mercier et al. [93]. Coll
with successive periods of lactation [104]. Human j-casein et al. [137, 138] reported the nucleotide sequence of cDNA
is more highly glycosylated than that of the sheep or cow and the promoter region of the gene. The gene comprises
and carbohydrate residues may account for up to 55 % of five exons which more than 90 % of the coding region for
the molecular weight [105]. The carbohydrate portion of mature protein (141 amino acids out of 171) is part of exon
bovine j-casein consists of galactose, N-acetyl galactos- 4 [121, 139]. Initially, the structural analysis of the goat j-
amine and N-acetyl neuraminic acid. The j-casein from casein locus indicated the presence of a biallelic poly-
sheep milk contains N-glycolyl-neuraminic acid in addition morphism (A and B variants) [140]. To date, based on the
to these three sugars [106]. Human j-casein is yet more published sequences, the CSN3 gene is considered highly
complex and contains fucose, galactose, N-acetyl gluco- polymorphic with up to 16 different alleles: A, B, B0 , B00 ,
samine, N-acetyl galactosamine and N-acetyl neuraminic C, C0 , D, E, F, G, H, I, J, K, L and M that yield 13 protein
acid [105, 107, 108]. Hydrolytic action by chymosin at variants [121, 122, 141, 142] (Table 2). Allele frequencies
Phe-Met releases a hydrophobic N-terminal segment studies indicate that A and B are the most frequent CSN3
(1–105) and a hydrophilic soluble C-terminal portion variants in Spanish, French, Italian and Egyptian goat
(106–169) known as para-j-casein and macropeptide, breeds [121, 122, 143, 144]. The F allele was described and
respectively [109]. The macropeptide is glycosylated, with found only in wild goat (Capra pyrenaica) while the G
the carbohydrate moieties mostly linked to Thr, containing have been found at relatively high frequencies only in
N-acetylneuraminic acid, galactose and galactosamine. Italian goats [121, 139]. The variants C, D, H, K, I, L and
Para-j-casein possesses no carbohydrates. Macropeptides M have been observed only in few breeds at low or
from cow and sheep contain only one glycosylated Thr, intermediate frequencies [122, 141]. Relevant surveys were
whereas the human one has ten sites of glycosylation [107]. carried out also by Angiolillo et al. [120] on the following
The uneven distribution of carbohydrates, in addition to the breeds: Spanish (Murciano-Granadina and Canaria),
high content of Asp and Glu in the C-terminal sequence French (Alpine and Saanen), Italian (Teramana, Garganica,

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Girgentana, Sarda, Cilentana Nera, Montefalcone). Prin- the water movement into mammary secretory vesicles and
zenberg et al. [122] analyzed the following populations: then into the alveolar lumen, the role of a-lactalbumin is
GermanWeisse Edelziege, Saanen, Nigeria Borno goat, crucial for milk secretion [150]. This protein is rich in
Cameroon Red Sokoto, West African Dwarf, Anatolian essential and conditionally essential amino acids and is a
hair goat and Angora at the same locus. These researches dominant protein in human milk [151]. The a-lactalbumin
demonstrated that the differences in the occurrence and has a high content of lysine and cysteine and a particularly
frequency of the alleles in these goat breeds reflect their high content of tryptophan [152].
geographic origin. The highest number of alleles was found The structure of the gene (LALBA) encoding this protein
in the goat breeds from the Near East. In particular, Ya- has been reported in rat [153], human [154], bovine [155],
hyaoui et al. [121] suggested that the distribution of such caprine [156] and guinea pig [157] species, it has an orga-
alleles has been influenced either by selection pressure for nization similar to that of human lysozyme-encoding gene
milk production or, more likely, by genetic drift. Moreover, [158] suggesting a common ancestral origin. The bovine a-
the same authors affirmed that A allele appear later in the lactalbumin has been localized on chromosome 5 [159, 160].
evolutionary pathway. In many breeds, the B allele was the The gene spans approximately 3,090 bp being composed of
most common suggesting that this variant may be the four exons and three introns [155, 161]. Two genetic variants
ancestral one [144]. The increasing interest on j-casein is of a-lactalbumin (A and B) have been most commonly found
mainly due to its role on technological properties of milk in cattle [12]. The B variant is present in the milk of most Bos
because of j -casein is located on the surface of the taurus cattle, and both the A and B variants are found in the
micelles. Goat CSN3 alleles A and B had different effects milk of Bos indicus cattle and in the droughtmaster (B.
on milk casein and protein contents. These two milk indicus 9 B. taurus) [162–165]. Osterhoff and Pretorious
components, together with fat content, determine the yield [166] have been reported the presence of the A variant in
as well as the organoleptic properties of cheese. In par- South African cows of European descent. The A variant is
ticular, the amount of casein associated with the BB and present at a low frequency in the milk of some Italian and
AB genotypes was greater than that found in AA goats; in Eastern European B. taurus breeds [167]; it contains a Glu at
the same way, the total protein content was higher for the position 10 of the mature protein, while the B variant has an
BB and AB goats compared with their AA counterparts. Arg substitution at that position [168]. A lot of investigations
Therefore, genotyping the CSN3 locus should be consid- show that the SNP in a-lactalbumin change the gene
ered when performing marker-assisted selection in dairy expression and deal with differences in milk yield and
goats [142]. More recently, it was reported that CSN3 composition [161, 169]. A third genetic variant, named C,
genotype has a significant effect on the rennet coagulation has also been reported in Bali cattle (B. javanicus) [170].
time being BB genotype better than AB without any Until now, there is no evidence of this third allele neither in
influence on cheese yield [145]. Together with the inves- B. taurus nor in B. indicus. Recently, a new bovine milk
tigations on variants associated with strong alleles at protein variant was discovered (D variant); the SNP causing
CSN1S1 and CSN1S2 loci, the inclusion of j-casein the amino acid change 65Gln[His is located in exon 2 of a-
genotypes, as an additional criterion, may supports the lactalbumin gene. This amino acid change is not expected to
selection for cheese-making properties. In fact, due to the affect protein function [171].
tight association among casein genes, the estimation of the The goat a-lactalbumin gene, located on chromosome 5,
relationship between casein variants and milk production is organized in four exons coding for a 123 amino acid
traits can be improved by considering the entire casein polypeptide chain. Vilotte et al. [156] isolated and char-
haplotype instead of single gene typing [47]. acterized a genomic clone encoding goat a-lactalbumin.
The sequence was aligned with that of its bovine coun-
terpart, finding that the 50 -flanking regions are highly
Whey proteins homologous, but two small insertions were detected in the
caprine sequence. One of these insertions fills the gap
a-Lactalbumin found in the so-called ‘milk-box’ consensus sequence of
the bovine gene. No further studies on the variability of this
The a-lactalbumin is a calcium metalloprotein with a single gene in goats are known. In contrast to numerous studies
strong Ca2? binding site, representing one of the major that had been carried out on goat casein genes and their
whey proteins in ruminant milks [146, 147]. It stimulates polymorphisms, studies on goat a-lactalbumin gene and its
lactose synthesis in the mammary gland by interacting with polymorphisms were not so frequent. It was only reported
the enzyme UDP-galactosyl-transferase, giving rise to the by Cosenza et al. [172] that this gene showed three dif-
heterodimer enzyme lactose synthase [148, 149]. Being ferent mutations in Italian goat populations: one in the first
this protein necessary for the production of lactose and for intron; one in the third exon and one in the 30 untranscribed

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region. The third exon mutation, a C?T transition identi- be linked with differences in milk protein yield and quality
fies a silent allele at position 80 of the mature protein. The [8, 180, 181]. These two variants differ by two amino acid
intronic mutation consists of a T?C transition in the 13th substitutions in the polypeptide chain arising from two
nucleotide of the first intron and in a C?G transversion single nucleotide substitutions in the BLG: Asp 64?Gly
located 187 nucleotides beyond the fourth exon. More and Val 118?Ala [182, 183].
recently, Lan et al. [173] found that a mutation in exon 3 The complete sequence of the goat BLG was described.
(M63868: g.1897T[C) identified an amino acid substitu- Similarities of this gene with ovine and bovine counterparts
tion Leu?Pro (p.L100P) of the a-lactalbumin. Here, the were 96.9 and 94.7 %, respectively [184, 185]. The b-
allele characterized by the presence of C was designated as lactoglobulin transcription unit spans 4.7 kb arranged in
A2, while that carrying a T was called A1. Phylogenetic seven small exons and six introns [186]. In goat, no vari-
analysis of LALBA gene in different species revealed that ants producing amino acid change have been characterized
this gene is highly conserved. The comparison of the gene at the DNA level. Genetic polymorphisms in the 50 -flank-
sequence has also highlighted the conservation of amino ing region were also observed by Yahyaoui et al. [187] in
acids with a functional roles [174]. A possible reason to Murciano-Granadina, Canaria, Payoya, Malaguena and
justify the highly conserved sequence may be the in a- Saanen breeds without any effect either on transcription
lactalbumin physiological functions, i.e. lactogenesis in the level and on the protein. However, Chianese et al. [188]
animal; however the mutations located in exonic region found an individual variability in b-lactoglobulin content in
may be linked to the adaptation of animals in different Girgentana goat breed; subsequently, the gene promoter
natural or conditioned environments [175]. Due to the region in individuals of this breed with reduced b-lacto-
strong correlation between a-lactalbumin and the nutri- globulin content was characterized and a new polymor-
tional value and the functional properties (i.e. gelling, film- phism located in the promoter region, not related to the b-
forming, foaming and emulsifying) of whey and whey lactoglobulin content was detected [189]. Moreover, dif-
products, to deeply investigate the eventual presence of ferent variants have been described by Pena et al. [190] and
protein variants and genetic polymorphisms could be an Yahyaoui et al. [187] in the 30 untranslated region (exon 7)
interesting perspective. and in the proximal promoter region of the gene in Spanish
and French goats. Ballester et al. [191] observed 15 b-
b-Lactoglobulin lactoglobulin polymorphisms in different goat breeds. Nine
polymorphisms are located in the promoter region and six
The b-lactoglobulin is a globular protein member of the in the exons of the gene. All polymorphisms are single
lipocalin family, small proteins with many properties, such nucleotide substitutions with the exception of one deletion/
as the ability to bind small hydrophobic molecules. insertion in the promoter region. Kumar et al. [192]
Although no clear physiological function has been yet observed two alleles having an effect on 90 days milk
defined for the b-lactoglobulin, beyond providing amino production in Jamunapari and Barbari goat breeds: animals
acids to the offspring, a role in the transport of retinol and carrying AA genotype yielded more milk than AB ones.
fatty acids has been suggested [176, 177]. The b-lacto-
globulin is present in the milk of many species, principally
in ruminants. It is absent in milk from rodents, rabbits and Minor proteins
camels in which instead another major whey protein (whey
acidic protein) is found [176]. Human milk is free of b- Milk protein fraction also includes other minor proteins
lactoglobulin, while this protein is the major whey protein such as immunoglobulins, lactoferrin, ferritin, transferrin,
of sheep, goat and mare milk. Businco and Bellanti [178] proteose-peptone, glycomacropeptides, calmodulin, lacto-
reported that b-lactoglobulin is responsible for the onset of peroxidase, lysozyme, prolactin and folate-binding protein
allergic forms to milk proteins that affect a considerable and various growth factors. The content of minor proteins
percentage of infants nourished with maternal milk is similar in goat and cow milk but the content of folate-
replacements produced with cow milk. b-lactoglobulin was binding protein is higher in goat than in cow milk (12 vs.
the first protein in which polymorphism was found. It is 8 lg/ml) [3]. Folate-binding protein is a glycoprotein with
well known that b-lactoglobulin gene (BLG) is located on about 22 % carbohydrates [37]. Lactoferrin is a glycopro-
chromosome 3 in ovine and chromosome 11 in goat and tein present in milk of all vertebrate species. This protein is
cattle [179]. Its polymorphisms may be helpful as infor- the major iron binding protein in human and mare milk,
mative molecular markers for milk yield and composition whereas transferrin is abundant in milk from rats and
as well as for rheological properties of milk. To date, rabbits [193]. Immunoglobulins classes (IgG, IgA and
twelve polymorphic variants of this protein are known in IgM) are also present in goat milk. Among minor whey
cattle, but the two most frequent, A and B, were shown to proteins, goat milk also has proteose-peptones like bovine

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and other species milks. Proteose-peptones are character- milk powder and goat milk-based infant and follow-on formu-
ized as a mixture of heat-stable, acid-soluble, phospho- lae. Int J Food Sci Nutr 59:123–133
5. Linderstrom-Lang K, Kodama SCR (1925) Studies on casein.
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minor components of milk can influence profound meta- Rendus des Travaux du Laboratoire Carlsberg 16:1–47
bolic, immunological and physiological processes and thus 6. Mellander O (1939) Elektophoretische untersuchung von casein.
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7. Wake RG, Baldwin RL (1961) Analysis of casein fractions by
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8. Ng-Kwai Hang KF, Grosclaude F (1992) Genetic polymor-
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technological characteristics as well as a potential use in as2- and b-casein cDNAs: comparisons with related sequences
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