Investigating the genetic polymorphism of sheep milk proteins: an useful tool for dairy production

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Maria Selvaggi, Vito Laudadio, Cataldo Dario*, Vincenzo Tufarelli

Department DETO – Section of Veterinary Science and Animal Production, University of Bari ‘Aldo Moro’, 70010 Valenzano (BA) Italy Corresponding author: prof. Cataldo Dario tel.: +39.080.544.39.18 fax +39.080.544.39.25 e-mail address: [email protected] Abstract Sheep is the second most important dairy species after cow all over the world, especially in the Mediterranean and Middle East regions. In some countries, the hard environmental conditions require a peculiar adaptation and in these contexts, sheep are able to provide higher quality protein than cattle. In least developed countries, the number of dairy sheep and ovine milk production is progressively increasing. In order to improve dairy productions, in particular those with local connotation, it is necessary to deepen the current acquisitions on milk quality and rheological properties. The genetic polymorphisms of milk proteins are often associated to quantitative and qualitative parameters in milk being potential candidate markers that should be included in breeding strategies similarly to as experienced already in cattle. Due to the current and growing interest on this topic and considering the large number of new information, the aim of this study was to review the literature on sheep milk protein polymorphisms with a particular emphasis to recent findings to give scientists an useful support. Moreover, the effects of different protein variants on milk yield and composition were discussed. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6750 This article is protected by copyright. All rights reserved

Keywords: Gene Polymorphism, Milk, Casein, Whey protein, Sheep

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Introduction Milk and dairy products from cow, sheep, goat and buffalo are a significant part of the Mediterranean diet. Sheep dairy farming represents a crucial part of the agricultural economy in many countries, especially in the Mediterranean and Middle East regions. In the last years, interest concerning small ruminants milk has been increasing also to find a new exploitation for local breeds. For promoting the sheep dairy products it is very important to investigate the quality and technological aspects of milk. The composition of sheep milk is quite different if compared to cow milk. Generally, the composition and the physical characteristics of milk vary from species to species. As reported in Table 1, sheep milk has a higher level of total fat when compared with goat and cow milks (+51% and +54%, respectively). A similar trend has been observed for milk protein content; in fact, it is approximately 62.0 g/kg in ovine compared to 35.0 and 33.0 g/kg in caprine and bovine milk. The carbohydrate fraction of milk is lactose and its level in sheep milk is usually slightly higher than goat and cow milks. As a consequence, the level of solids-non-fat and total solids in sheep milk is strongly different from the considered counterparts. The total ash content of sheep milk is higher than those from ovine and bovine species. All these differences in milk composition were reflected in differences concerning the coagulation properties of milks. As for all mammalian species, the milk yield and composition is determined by genetic and non-genetic factors such as breed, diet, animals within breed, parity, litter size, stage of lactation, season, management and environmental conditions [1, 2]. Ovine milk have unique properties and specific technological destinations due to the peculiar milk protein composition (Table 2) and micellar structure. Sheep milk generally contains higher total solids than goat and cow milk with a greater cheese yield per unit than others. In fact, cheese curd contains primarily the fat and This article is protected by copyright. All rights reserved

casein from milk. Moreover, sheep milk is very sensitive to rennet because it has a higher β/αs-casein ratio and also coagulation in sheep milk proceeds faster than in cow milk. Casein

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micelle structure is similar in cow, goat and sheep milk, although sheep milk caseins are richer in calcium than cow casein. Since caseins play a key role in milk coagulation as responsible for primary structure of cheese curd, the clotting of milk would affect the cheese composition, texture and rheology. Ovine milk is an excellent source of high-quality protein, calcium, phosphorus and lipids. There is a good balance between the protein, fat and carbohydrate components [3]. Milk proteins include caseins and whey proteins [4]. The caseins are a family of phosphoproteins synthesized in the mammary gland in response to lactogenic hormones and other stimuli and secreted as large colloidal aggregates called micelles. The importance of milk proteins for food industry have made them a popular target for research becoming perhaps the most widely studied food proteins. Caseins are composed of calcium-sensitive caseins (αs and β) and of the calcium-insensitive κ-casein which is responsible for stabilizing the former against precipitation in the presence of calcium. Whey proteins are a group of proteins that remain soluble in milk serum or whey after acid precipitation of caseins at pH 4.6 and 20°C and after rennet precipitation [4]. The former whey protein source is known as acid whey, the latter is referred to as sweet or rennet whey. In Table 3, a list of milk protein genetic polymorphisms in sheep is reported. Considering the growing interest on milk protein polymorphisms, the aim of this review paper was to dissertate on the available literature on sheep milk proteins with a particular attention to recent findings. Moreover, the effects of different protein variants on milk yield and composition were discussed.

Caseins

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Initially, the caseins were considered as a homogenous substance. Subsequently, LindstromLang and Kodama [5] demonstrated that they were composed of two fractions: one which precipitated in the presence of calcium (calcium-sensitive caseins) and the other which was responsible for stabilizing the former against precipitation (calcium-insensitive caseins). Later, Mellander [6] demonstrated the presence of three different components designated as α-, β-, and γ-casein. Further studies revealed that an even greater number of components were present in the casein fraction [7] and much effort was dedicated to clarify these components. However, it is now possible to affirm that only four types of caseins exist and the earlier heterogeneity that was recognized by electrophoresis is due to effects of post-translational processing, alternative splicing of the gene product or genetic polymorphisms [8]. In 1984, the American Dairy Science Association Committee on Nomenclature and Classification proposed that the nomenclature developed for the bovine caseins, namely the αs1, αs2, β and κ-caseins, be adopted for investigations of milk proteins in other species. The caseins comprise the major protein fraction of the milk from most species. Their function is to transport calcium phosphate in milk and hence to provide the suckling infant with a source of calcium and phosphorus for bone formation as well as to contribute to the requirement for amino acids [9]. The caseins, being secreted proteins, possess N-terminal signal peptides that direct the passage of the newly synthesized polypeptides into the lumen of the endoplasmic reticulum, whereupon the signal peptides are removed from the primary translation products to yield the mature caseins. The four calcium-sensitive caseins show similar molecular weights (around 24 kDa), promoter regions, leader peptide sequences and locations of the major phosphorylation site. These data support the hypothesis of a common evolutionary origin of these genes from the duplications and modifications of a unique ancestral gene [10]. The remainder of the sequences appear to be subject to few functional constraints and have accumulated many nucleotide substitutions, as revealed by comparative sequence analysis

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[9]. The caseins concentration in ovine milk is higher than bovine and caprine counterparts [11]. Sheep and goat and milk have different proportions of the four major caseins compared

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to cow counterparts and there are great variations, especially between αs1-casein and αs2casein contents between individuals and breed, because of the occurrence of genetic polymorphisms for all milk proteins, which influence greatly their cheesemaking properties.

αs1- and αs2-caseins The αs1-casein, the primary protein in cow milk, is a structural component of the casein micelle and plays a functional role in cheese curd formation being characterized by a greater solubility in the presence of calcium. It is a highly phosphorylated protein [4]. The nucleotide sequence of the genes (CSN1S1) encoding for the αs1-caseins in many eutherian species shows the presence of a highly conserved signal sequence of 15 residues but quite divergent sequences for the mature proteins suggesting that they are encoded by a rapidly evolving gene family. The αs1-casein consists of 214 amino acids in each species [12]. Bovine αs1casein exists in two phosphorylated forms containing 8 and 9 phosphates/mol [13, 14]. Many αs1-caseins variants have been described in domestic species. In cattle, eight protein variants of the αs1-casein are known and classified from A to H; the two most common are B and C [4]. Many studies have been aimed at genetic polymorphisms of caseins in sheep milk. By using electrophoretic, immunochemical, and chromatographic methods, it has been observed that ovine αs1-casein exists as a number of distinct genetic variants both in the heterozygous and homozygous forms [15]. Several phenotypes (A, B, C, D, E, F, H and I) of αs1-casein in ovine milk have been discovered by protein electrophoresis [16-20]. The H variant was formerly defined as X variant. The Welsh variant, primarily identified by King [21] in Cluny Forest sheep the Welsh mountains, was identified as variant D [22, 23]. Primary structures have been determined only for variants A, C, D and E [24, 25]. The primary structure of A, C

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and D variants differed from each other by amino acid substitutions and phosphorylation degree [24]. Each variant consisted of three differently phosphorylated forms with 9, 10 and

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11 phosphate groups, respectively. The differences among these three genetic variants are silent substitutions determining the degree of phosphorylation. Variant C differs from variant A in the substitution Ser13→Pro13 which determines the loss of the phosphate group on site 12 of the protein chain. A substitution Ser68→Asn68 causes the loss of both phosphate groups in the residues Ser64 and Ser66 in variant D. As a consequence, variant D is the least phosphorylated variant. While αs1-casein C occurs in all sheep breeds with high frequency (0.485-0.890), variants E and F were identified at low frequencies in Italian breeds only [17, 26-29]. The high frequency of C variant is most probably the result of indirect selection in the past, as it showed a correlation with higher total protein and casein content, smaller micelle diameters and higher clotting capacity [17, 28, 30]. Conversely, the variant D, which showed negative effects on milk composition and cheese yield was detected in Sarda, Pinzirita, Massese, Appenninica and Sumava breeds [17, 29, 31-33]. Variant H seems to be specific for East Friesian sheep [18]. Samples of animals with αs1-casein H are also associated with a reduced protein expression level of this protein [19]. αs1-casein I variant was identified for the first time in Gray Horned Heath [34]. The αs2-like caseins represent a more disparate group of highly phosphorylated peptides than the αs1-caseins and have been identified from a number of eutherian species [9, 35, 36]. As in the αs1-caseins, the signal sequence is highly conserved but the mature proteins are quite divergent. αs2-casein (encoded by CSN1S2 gene) has a polypeptide chain of 207 residues with a large number of positively charged side chains, especially in the C-terminal segment [9]. There are no reports on the presence of αs2-casein in human milk. The role of αs2-casein in casein micelles has not been still studied in detail. This protein comprises up to 10% of the casein fraction in bovine milk; it consists of 2 major and several minor components

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exhibiting varying levels of post-translational phosphorylation as well as minor degrees of intermolecular disulfide bonding [4, 37]. In cattle, many protein variants of the αs2-casein are

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known (A-D) The protein variant A was originally determined chemically [38] and it is the most frequent in all breeds so far investigated and also almost fixed in most western breeds. Variants B and C are specific to zebu and yaks, respectively [39-41], whereas the D variant has been found at low frequencies in some European bovine breeds and in the African Namchi taurine breed [42-44]. Ovine αs2-casein is the most heterogeneous fraction having a multiphosphorylation level. Boisnard et al. [45] found two αs2-casein variants in sheep which differed at the amino acid level involving an amino acid replacement, Asn49 and Lys200 for Asp49 and Asn200, respectively. These variants exist as non-allelic long and short forms in the ratio 60-70/30-40%. The long and short forms of ovine αs2-casein are encoded by four mRNAs differing in the presence or absence of two stretches of 44 and 27 nucleotides in the 5'untranslated region and the coding frame, respectively. The physicochemical properties of the long and short αs2-caseins should be quite different and should confer unique properties to ovine milk. These four different transcripts probably arise from the incorrect processing of a unique pre-mRNA, resulting in the partial skipping of both the putative exons, with frequencies greater than 90% and 30%, respectively [45]. To date, the ovine αs2-casein presents seven variants recently named from A to G. Chianese et al. [46] identified three phenotypes, differing in electrophoretic behavior, discovering at the protein level an ovine αs2-casein low molecular weight variant in the Manchega breed. The phenotypic frequency of this new variant in the Manchega breed was 5.5%. Chessa et al. [47] called two phenotypes (identified by isoelectric focusing) A and B, which were characterized by amino acid exchanges at positions 75 and 105 of mature protein. The structural analysis give evidence that two amino acid exchanges account for the differences between the ancestral ovine αs2-casein A and the genetic variant B found in three Italian breeds: particularly a non-

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silent mutation, Asp75→Tyr75 and a silent one Ile105→Val105 [48]. αs2-casein A and B variants were the most frequent as observed by Chessa et al. [47] and Picariello et al. [48] in

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three Italian breeds such as Gentile di Puglia, Sarda and Comisana and subsequently confirmed by Giambra et al. [34] in different German breeds such as Gray Horned Heath, Merinoland, Merino Mutton and Rhön sheep. In addition, Giambra et al. [34] found two new variants (C and D) for the first time in German sheep breeds and their genetic control was confirmed. In particular, Giambra and Erhardt [49] reported three amino acid exchanges deduced from mRNA sequences: Val45→Ile45, Ala48→Ser48 (allele C) and Arg46→Ser46 (allele D). All the four variants were identified in Merinoland sheep, while A, B and C variants were observed in East Friesian, Gray Horned Heath and Rhön sheep breed and only A and B variants were found in Black Faced Mutton and Merino Mutton sheep breeds [34]. Amino acid substitutions responsible for E and F variants were firstly deduced from mRNA: Asp49→Asn49 and Lys200→Asn200 [45, 50] from the mature protein, respectively. Furthermore, cDNA sequencing led to detection of the Arg161→His161 substitution named as G variant; this variant is not detectable using isoelectric focusing [49]. However, with the exception of PCRRFLP test assessed to detect the G variant, DNA-based methods to quickly and accurately identify and genotype large population samples for the different variants of this gene are not yet available. The significance of the protein variation described at the genetic level needs further investigations; however, it was found that the αs2-casein genotype AB was significantly advantageous in comparison to AA in German East Friesian dairy sheep for milk and fat yield [18]. It is possible to suppose that the modification of the biochemical properties of the protein may affect the interactions among the four casein fractions within the micelle. Thus, the milk produced by different breeds may exhibit different technological quality.

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β-caseins β–caseins are described as “calcium-sensitive” because they precipitate in the presence of low

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concentrations of this cation. They are important in determining the surface properties of casein micelles and essential for rennet curd formation [51]. The β-casein family includes several eutherian and two marsupial proteins for which the amino acid and nucleotide sequence is known [9, 52-56]. Eutherian β-caseins sequences (encoded by CSN2 gene) are characterized by a highly conserved 15 residue signal sequence although the mature proteins show a degree of divergence across species in terms of amino acid identity similarly to that observed for the αs1-caseins [57]. The β-caseins are particularly rich in glutamines, the location of which is well conserved, as is the presence of a single major phosphorylation site near the N-terminus. The number of phosphorylation sites and the level of phosphorylation are less that observed for the αs1- and αs2-caseins. At least twelve bovine variants for βcasein, A1, A2, A3, B, C, D, E, F, G, H1, H2 and I have been reported [4]. The variant A2 is the most common variant used as reference. Lien et al. [58] studied the most common genetic variants in many Nordic breeds (A2, A1, B, A3). Recently, Jensen et al. [59] reported the variant I in Danish dairy breeds suggesting that it might be a relatively common variant. Ovine whole casein contains 45% β-casein, represented by two multiphosphorylated forms: β1 and β2-caseins which have similar amino acid compositions to bovine β-casein. Ovine β1casein has a higher phosphorus content and greater electrophoretic mobility at alkaline pH than ovine β2-casein; they have a common polypeptide chains and differ only in their degree of phosphorylation [60]. The occurrence of multiphosphorylated forms of β-casein might affect micelle stability and the availability and distribution of calcium in the milk. This depend on the capacity of single β-casein forms, phosphorylated to different degrees, to bind different amounts of calcium. On the other hand, the stage of lactation, health and age of individuals, as well as an altered availability of phosphate, play an important role in

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determining the level of phosphorylation of β-casein in individual milks [28]. Chianese [61] differentiated between three genetic variants of β-casein designated A, B and C using gel

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isoelectric focusing in Sarda breed. The only sequence difference found between A and C was the amino acid substitution of Glu2→Gln2 but no sequence data for the B variant is available yet. Moreover, an A→G transition leading to the Met183→Val183 exchange was detected at the DNA level by Ceriotti et al. [62]. These authors found that the allele A was the most frequent in three Italian sheep breed (Comisana, Sarda and Sopravissana). Subsequently, Corral et al. [63] observed a higher frequency of AA genotype in comparison to AG and GG ones in Merino population and, for the first time, they found that the GG genotype was associated with an increase in milk production, whereas the AA genotype was related to an increase in fat and protein percentage. Furthermore, Chessa et al. [50] found two new patterns, called X and Y; after sequencing, the same authors discovered that Y pattern was due to a silent mutation in the triplet coding for Gln at position 192 (G→A transition), whereas a C→A transversion, responsible for the amino acid exchange Leu196→Ile196, characterized the X variant. The CSN2 A allele was the most diffused in Massese, Garfagnina, Pomarancina and Zerasca breeds with frequencies ranging from 0.50 in the Garfagnina to 0.73 in the Massese breed. The X allele was found in all these breeds except Pomarancina, whereas CSN2 Y allele was found only in the Garfagnina and it could be a breed specific allele. The mutations found do not change the isoelectric point of the coded proteins confirming the monomorphic isoelectrophoretic pattern observed at the protein level [50]. Most probably, the ancestral variant of ovine β-casein is the A variant, from which the other allelic forms derived as a consequence of independent SNPs.

κ-casein

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The κ-casein constitutes about 15% of the total caseins and contains cysteine. The nucleotide and amino acid sequences of κ-casein from several eutherian species are known [64-68]. The

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κ-casein consists of 169 amino acids residues (19,023 Da) and occurs in polymeric form via disulfide bonds ranging in sizes from 60 to 600 kDa. This protein is the only casein soluble in the presence of calcium ions and has a much smaller phosphate component than other caseins. The phosphorylation sites are confined to the C-terminal region of the molecule and are present as single sites rather than the clusters found in the calcium-sensitive caseins. The signal peptide of κ-casein is 21 residues in length (compared to 15 in calcium-sensitive caseins). The κ-casein is the only eutherian casein that has conclusively been shown to contain carbohydrate moieties. These features have led to the conclusion that κ-casein is not related to the calcium-sensitive caseins [67, 69]. κ-casein is susceptible to cleavage by the aspartate protease chymosin. Jollés et al. [69] have shown that cleavage occurs at a specific Phe-Met bond in the C-terminal portion of bovine κ-casein. Nakhasi et al. [70] proposed that κ-casein be classified into two separate groups depending on the species of origin. Group I κcaseins (cow, sheep, goat, water buffalo) differ from group II κ-caseins (rat, mouse, pig, human) on the basis of hydrophobicity, carbohydrate content, amino acid composition and site of proteolytic cleavage. Group I κ-caseins contain the archetypal Phe-Met bond. In group II κ-caseins however, the cleavage site is specified by Phe-Ile or Phe-Leu. This divergence might reflect differences in the mechanism of clotting in ruminant and non-ruminant mammals. The κ-caseins are the only caseins from eutherian milk at least that are glycosylated. The carbohydrate groups are attached to κ-casein via O-glycosidic linkages to serine and threonine residues within the C-terminal portion of the molecule. Glycosylation occurs post-translationally and is catalysed by membrane bound O-glycosyl-transferases within the Golgi apparatus of mammary epithelial cells [71, 72]. Glycosylation increases during the colostral period and in response to mastitis but decreases with successive periods

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of lactation [73]. Human κ-casein is more highly glycosylated than that of the sheep or cow and carbohydrate residues may account for up to 55% of the weight of the molecule [74]. The

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carbohydrate portion of bovine κ-casein consists of galactose, N-acetyl galactosamine and Nacetyl neuraminic acid. Sheep κ-casein contains N-glycolyl-neuraminic acid in addition to these three sugars [71]. Hydrolitic action by chymosin at Phe-Met releases a hydrophobic Nterminal segment (1 to 105) and a hydrophilic soluble C-terminal segment (106 to 169) known as para-κ-casein and macropeptide, respectively [75]. The macropeptide is glycosylated with the carbohydrate moieties mostly linked to threonine, containing Nacetylneuraminic

acid,

galactose

and

galactosamine.

Para-κ-casein

possesses

no

carbohydrates. The uneven distribution of carbohydrates, plus the high content of aspartic and glutamic acid in the C-terminal sequence results in high net negative charges (3.5 at pH 6.8) that are involved in the stabilization of the κ-casein. The overall acidic character of the macropeptide is conserved in κ-caseins for several species, including cow, zebu, buffalo, goat, ewe, sow and human [76]. κ-casein is located mainly on the surface of the casein micelles and is responsible for their stability [77]. The presence of a glycan moiety in the Cterminal region of κ-casein enhances its ability to stabilize the micelle, by electrostatic repulsion and may increase the resistance by the protein to proteolytic enzymes and high temperatures [73]. The level of glycosylation does not affect micelle structure but it does affect the susceptibility of κ-casein to hydrolysis by chymosin, with susceptibility decreasing as the level of glycosylation increases [78-80]. The genetic polymorphism of the κ-casein gene (CSN3) has been well documented in cattle [81, 82], buffaloes [83-85], goat [86-88], sheep [89] yak [90] and equidae species [91-93]. Eleven variant alleles of the κ-casein gene have been identified in cattle, namely A, B, C, D, E, F, G, H, Al, and J [94]. However, only two alleles, namely A and B, are the most commonly identified in Bos taurus dairy cattle [95, 96]. The A and B variants differ in the amino acids 136 and 148 (A: Thr 36→Asp36; B:

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Ile148→Ala148) [8, 97]. The bovine protein variants have received considerable research interest in recent years and several studies on κ-casein variants resulted in its considerable

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association with lactation performance, milk composition, milk protein contents and rheological properties [98, 99]. Ovine κ-casein has been sequenced and was found to be formed by 171 amino acid residues [100, 101]. Despite the key-role played by κ-casein in the micellar structure and the clotting process, little is known about ovine κ-casein polymorphism. Alais and Jollés [102] isolated two protein fractions for κ-casein differing in glycosylation and phosphorylation degrees, but a genetic polymorphism was never suggested at protein level. So, κ-casein is considered to be monomorphic in sheep. Subsequently, molecular analyses of ovine CSN3 gene, mainly of exon 4, were conducted in different sheep breeds, and partly synonymous and non-synonymous sequence differences were found [62, 103-105]. In particular, Ceriotti et al. [62] identified a C→T-SNP in exon 4 at position 443 in Sopravissana sheep breed, leading to the amino acid exchange Ser104→Leu104. Both amino acids are neutral and, therefore, it is not possible to detect this substitution by protein electrophoresis. Feligini et al. [105] and Pariset et al. [106] identified a T→C SNP at position 237 of the mRNA sequence although the corresponding amino acid at position 35 remains a tyrosine. Within studies of Bastos et al. [104] on Portuguese indigenous breed, Sztankóová et al. [107] on Czech Sumava and Valachian breeds and Othman et al. [108] on Egyptian sheep breeds, the ovine CSN3 gene was found monomorphic. The κ-casein gene is the least studied with respect to its effect on milk yield and composition and to date, no association between CSN3 and milk yield has been reported [109].

Whey proteins The nutritive value of whey proteins is higher than caseins due to the peculiar composition of amino acids representing an important source of digestible proteins. At the same time, whey

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is a high-quality source of vitamins and minerals. Therefore, whey proteins are recommended for the formulation of milk products for replacement of bovine milk in infant nutrition [110].

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Whey proteins are globular molecules with a substantial content of α-helix motifs, in which the acidic/basic and hydrophobic/hydrophilic amino acids are distributed in a fairly balanced way along their polypeptide chains. About 75% of milk whey proteins are albumins (αlactalbumins and β-lactoglobulins) a valuable source of bioactive peptides with extra physiological activity. Whey proteins are also used as food ingredients due to their important functional properties such as gelling, film-forming, foaming and emulsifying [111]. Sheep milk whey proteins account for 17–22% of total proteins. Whey obtained from sheep milk is particularly rich in proteins with a high β-lactoglobulin and a low α-lactalbumin content [112114].

α-Lactalbumin The α-lactalbumin is a calcium metalloprotein with a single strong calcium binding site, being one of the major whey proteins in milk from ruminants [115, 116]. It stimulates lactose synthesis in the mammary gland by interacting with the enzyme UDP-galactosyl-transferase, giving rise to the heterodimer enzyme lactose synthase [117]. The α-lactalbumin is essential for the biosynthesis of lactose in the mammary gland and for the water movement into mammary secretory vesicles and then into the alveolar lumen; thus, the role of this protein is crucial for milk secretion [118]. Moreover, the α-lactalbumin is rich in essential and conditionally essential amino acids, it has a high content of lysine and cysteine and a particularly high content of tryptophan and it is a dominant protein in human milk [119]. The structure of the gene (LALBA) encoding this protein has been reported in human [120], bovine [121], caprine [122] and guinea pig [123] species, it has an organization similar to that of human lysozyme-encoding gene [124] suggesting a common ancestral origin. In cattle, two

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genetic variants of α-lactalbumin (A and B) have been most commonly found [4]. The B variant is present in milk of most Bos taurus cattle and both the A and B variants are found in

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the milk of Bos indicus cattle and in the droughtmaster (Bos indicus × Bos taurus) [125, 127]. Osterhoff and Pretorious [128] have been reported the presence of the A variant in South African cows of European descent. The A variant is present at a low frequency in the milk of some Italian and Eastern European Bos taurus breeds [129]; it contains glutamic acid at position 10 of the mature protein, while the B variant has an arginine at that position. A lot of investigations show that the SNP in α-lactalbumin change the gene expression and deal with differences in milk yield and composition [130, 131]. A third genetic variant, named C, has also been reported in Bali cattle (Bos javanicus) [132]. Until now, there is no evidence of this third allele neither in Bos taurus nor in Bos indicus. Recently, a new bovine milk protein variant was discovered (D variant) [133]. In ovine species, the LALBA gene is the less investigated among milk protein genes [134]. Two α-lactalbumin protein patterns (A and B) were made evident by starch gel electrophoresis in Sicilian breeds of sheep [135] with A variant being the most common one. On the contrary, B variant has rarely been detected and seems to be confined to specific breeds [136]. The same polymorphism was detected in the milk of Latvian Darkhead sheep by Stambekov et al. [137]. No studies regarding the influence of the ovine LALBA polymorphisms on milk production quantitative traits have been performed yet. However, due to the strong correlation between α-lactalbumin and the nutritional value and the functional properties of whey and whey products, to deeply investigate the eventual presence of protein variants and genetic polymorphisms could be an interesting perspective.

β-Lactoglobulin

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The β-lactoglobulin is a globular protein member of the lipocalin family, small proteins with many properties, such as the ability to bind small hydrophobic molecules. Although its

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biological function is still unclear, the β-lactoglobulin provides amino acids to the offspring and a possible role in the transport of retinol and fatty acids has been suggested. The βlactoglobulin is the main whey protein of ruminant milk; it is lacking in milk from rodents, rabbits and camels in which instead another major whey protein (whey acidic protein) is found [138]. Human milk is β-lactoglobulin free, while this protein is the major whey protein of cow, sheep, goat and mare milk. As reported by Businco and Bellanti [139], the βlactoglobulin is responsible for the onset of allergic forms to milk proteins affecting a high percentage of infants nourished with maternal milk replacements based on cow milk formula. β-lactoglobulin was the first protein in which polymorphism was found, it consists of 162 amino acids and forms stable dimmers in milk [140]. This protein is expressed in the mammary gland during pregnancy and lactation [141]. β-lactoglobulin is encoded by BLG gene. Its polymorphisms may be helpful as informative molecular markers for milk yield and composition as well as for rheological properties of milk. In cattle, twelve polymorphic variants of this protein are known; the two most frequent (A and B) were related with differences in milk protein yield and quality [8, 142, 143]. These two variants differ by two amino acid substitutions in the polypeptide chain arising from two single nucleotide substitutions in the β-lactoglobulin gene: Asp64→Gly64 and Val118→Ala118 [144, 145]. Investigations in many countries have shown that β-lactoglobulin is polymorphic in various breeds of sheep. Three co-dominant alleles (A, B and C) have been reported in this species differing by one or more amino acid changes. The genetic variant A differs from variant B in the amino acid sequence at position 20 (Tyr20→His20) [146- 149]; the rare variant C is a subtype of A with a single amino acid exchange at position 148 (Arg→Gln) as reported by Erhardt [136]. Variants A and B are the most common and have been detected in many

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breeds; various studies demonstrated a higher frequency of A allele than B as in Bavarian milk sheep [150], Latvian Darkhead [137], Massese [151], Pag Island sheep [152], Gentile di Puglia [47], Delle Langhe [153], some Polish and Egyptian sheep breeds [154, 155]. However, according to Arora et al. [156] the B allele was the most frequent in native Indian sheep breeds as reported also in Valle del Belice [157], Sarda [158] and in Latxa, Manchega and Churra breeds [159]. The variant C has been reported only in Merinoland, Hungarian Merino, Pleven [136] and in Serra da Estrela and Merino sheep [160, 161]. The influence of β-lactoglobulin variants on composition and cheese making properties is controversial, indicating superiority of either the A or B allele or no relationship with milk yield. Schmoll et al. [162] reported that East Friesian ewes with the AA genotype had the greatest milk yield in the first lactation, whereas the BB ones yielded more milk in the following lactations. Similarly, Giaccone et al. [163] found that the AA genotype was associated with greater milk yield in Valle del Belice ewes. On the other side, Pietrolà et al. [158] found no significant differences among genotypes in Sarda ewes and other studies point toward superiority of the B allele. Ramos et al. [164] observed higher milk yield in AB heterozygotes in Merino and Serra da Estrela sheep. In addition, the Serra da Estrela AA ewes presented lower milk yield when compared with AB animals with no significant difference AB and BB genotypes [161]. Recently, Kawecka and Radko [154] found no associations between β-lactoglobulin genotypes and milk yield and composition in some Polish sheep breeds. Yousefi et al. [165] revealed significant associations between AB genotypes and higher milk fat and lactose percentages in indigenous Zel sheep. Several studies have shown that ovine milk from AA animals seems to be more suitable for cheese making if compared to those from BB and AB individuals with a better renneting rate and cheese yield [28]. These results conflict with data reported by other authors; in fact, Pilla et al. [166] found that the B allele positively affected the rennet coagulation properties of milk, whereas Recio et al. [160] did not found any

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influence on milk rheology. Nevertheless, the available literature provides no conclusive evidence about the effect of different β-lactoglobulin genotypes on milk production traits in

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ovine species.

Minor whey proteins Milk minor proteins have a crucial role in the transmission of passive immunity to the offspring and in protection of the mammary gland [167]. This group of proteins include immunoglobulins, lactoferrin, transferrin, ferritin, lactoperoxidase, lysozyme, proteose peptone, calmodulin, prolactin, folate-binding protein and various growth factors. Immunoglobulins were among the first host protein defense systems due to their antimicrobial activity. The IgG dominate (~80%) in milk from ruminants, while in other milks, including human, IgA is the most abundant (~90%). Lactoferrin is a glycoprotein belonging to the transferrin family, which has a specific ability to bind iron. This protein is an essential element of non-specific innate immunity in mammals [168]. Among minor whey proteins, sheep milk also has proteose–peptones like caprine and other species milks. Proteose-peptones are characterized as a mixture of heat-stable, acid-soluble, phosphoglycol proteins [169]. Lysozyme is one of the mechanisms of non-specific, humoral immune response [170]; it is present at higher concentration in sheep milk compared to cow and goat milk. Antibacterial properties of lysozyme are of considerable interest for practical utilization in food industry; in fact, during cheese production lysozyme limits the growth of butyric fermentation bacteria [171]. Hormones, growth factors, and analogs are also present in sheep milk, and they could act as development and metabolic regulators. Although present at low concentrations, minor components of milk can influence metabolic, immunological and physiological processes and thus contribute to some peculiar characteristics of sheep milk and dairy products.

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Conclusions Sheep milk production is very important especially in Mediterranean and Middle Eastern

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countries. Although ovine milk is nutritionally richer if compared to goat and cow counterparts, it is rarely used as drinking milk but it is mainly used to produce a wide range of different cheeses and in some countries for yogurt or whey cheeses [2]. Thanks to the advance of knowledge it is now possible to deeply investigate the characteristics of sheep milk and the role of milk components during cheese making processes, that are essential key points for dairy industries development and enhancement as well as for promotion of dairy products. To improve local farms productivity and their livelihood in developing countries is a strategic purpose to avoid the off-farm migration and to safeguard the current wide sheep biodiversity in these areas; otherwise, it is important to highlight that to sustain the economy of rural and marginal areas is a crucial target also in developed countries. Milk production traits are under control of several genes; the genetic polymorphisms of milk proteins are strongly associated to quantitative and qualitative milk parameters [145, 169, 172]. Although association studies between milk protein polymorphisms and milk performance traits revealed controversial results in ovine species, to include molecular genetic markers in selection to improve milk production and composition is also a target in sheep breeding to enhance economy of dairy sheep production worldwide [28, 109, 159, 173]. Casein and whey protein polymorphisms may be implemented in genomic selection. Moreover, typing of autochthonous sheep breeds at milk protein loci gives the possibility to increase productivity and to avoid the loss of genetic variability preserving the biodiversity with a peculiar attention to endangered breeds carrying special milk protein variants. In addition, milk protein polymorphisms can be used for molecular tracing of typical cheeses, and for species identification of milk or dairy products [174-176]. This review paper represents a detailed and updated overview on polymorphisms of milk protein genes in sheep to offer a complete

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picture for those interested in these topics opening potential for new scientific discoveries and

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acquisition of knowledge.

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Accepted Article

Table 1 Composition of sheep, goat and cow milk (g/kg) Component Sheep Goat Cow Fat

79.0

38.0

36.0

Protein

62.0

35.0

33.0

Lactose

49.0

41.0

46.0

Solids-non-fat

111.0

76.0

79.0

Total solids

190.0

114.0

105.0

9.0

8.0

7.0

Total ash (g/kg)

Adapted from: Park [177]; Park et al. [169];

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Accepted Article

Table 2 Concentrations of protein fractions in sheep milk Total protein (g/kg)

45-66

Total casein (g/kg)

42-52

αs1-casein (% of total casein)

6.66

αs2-casein (% of total casein)

22.84

β-casein (% of total casein)

61.60

κ-casein (% of total casein)

8.90

Whey proteins (g/kg)

10-13

α−lactalbumin (% of total whey protein)

13.50

β-lactoglobulin (% of total whey protein)

46.70

Minor whey proteins (% of total whey protein)

39.80

Adapted from: Jandal [178]; Haenlein and Wendorff [2]; Degen [179]; Pandya and Ghodke [180]; Park et al. [169]

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Accepted Article

Table 3 Milk protein genetic polymorphisms in sheep Variants

References

αs1-casein

A, B, C, D, E, F, H, I

Chianese et al. [16]; Pirisi et al. [17]; Wessels et al. [18]; Giambra et al. [19, 20]

αs2-casein

A, B, C, D, E, F, G

Chianese et al. [46]; Chessa et al. [47]; Picariello et al. [48]; Giambra and Erhardt [49]

β-casein

A, B, C, X, Y

Chianese [61]; Chessa et al. [50]

κ-casein

monomorphic

Jollès et al. [100]; Bastos et al. [104]; Ceriotti et al. [62]; Feligini et al. [105]; Pariset et al. [106]

A, B

Chiofalo and Micari [165]; Erhardt [136]

A, B, C

Bell and McKenzie [146]; King [147]; Kolde and Braunitzer [148]; Erhardt [136]; Ali et al. [149]; Recio et al. [160]; Ramos et al. [161]

α-lactalbumin βlactoglobulin

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