of 4.6–4.7 at 20°C; (ii) by enzymatic treatment (chymosin); or (iii) by ultracentri- fugation. Caseins are rich in proline, glutamate and glutamine, and poor in glycine and aspartate. A high amount of Ca and phosphate is required to make casein soluble in milk in a stable form, so that it becomes a nutritional source of amino acids.
A very important milk feature, especially in goat’s milk, is related to its con- tent of complex oligosaccharides, either in free form or conjugated to proteins. In fact, beneficial anti-inflammatory effects of such oligosaccharides, especially on inflammatory bowel disease, have been demonstrated (Daddaouaet al., 2006).
or deletion of amino acids in the protein chain (Table 4.5). Alleles associated with reduced protein synthesis have been reported by many authors (see the review of Valentine, 1998, for molecular aspects). These modifications result in changes of milk physicochemical properties such as charge, superficial hydro- phobicity and molecular size and shape (Russoet al., 1981, 1986; Addeoet al., 1987, 1989; Grosclaudeet al., 1987; Dall’Olioet al., 1988, 1989; Brignonet al., 1989; Di Gregorioet al., 1989). Such modifications have been identified through the use of electrophoretic and chromatographic techniques (Addeoet al., 1988).
Each protein fraction shows different genetic variants (Mercier and Vilotte, 1993). These differences in the primary structure of a protein can deeply modify its molecular characteristics. The ratios between various protein fractions can vary and are regulated by structural genes (Parmaet al., 1999a,b; Randoet al., 2000). Since each one of the four types of casein is polymorphic, different genetic variants can be identified. Polymorphisms can be attributed to two main factors: (i) genetic variants; and (ii) post-translational modifications, due to a dif- ferent localization of phosphorylation or glycosylation sites in the amino acidic sequence of the primary structure (Bouniolet al., 1993, 1994; Ramunnoet al., 1994; Klose, 1999). Intrinsic electrophoretic heterogeneity of casein fractions is due to the presence of incompletely phosphorylated or glycosylated sites in these proteins (Bevilacquaet al., 2002; Martin, 1993; Ramunnoet al., 2004).
TheaS1- andaS2-caseins differ in their content of phosphoric groups, while k-casein differs in its content of glucidic and phosphoric groups (Boulangeret al., 1984; Lerouxet al., 1992, 2003; Iamettiet al., 1996).b-Casein heterogeneity derives from incomplete saturation of its phosphorylation sites (Chianeseet al., 1993).
The most studied goat’s milk polymorphism has been regardingaS1-casein (Bouniol et al., 1993; Remeuf, 1993; Jansa-Pérezet al., 1994; Remeuf et al.,
Protein Variant Amino acid position
aS1-Casein 14–26 53 59 192
A Misses
B Ala Gln Glu
C Gly
D Thr
E Lys Gly
aS2-Casein 33–47 50–58 130
A Glu Ala Thr
B
C Gly Thr Ile
D Misses
Ala, alanine; Gln, glutamine; Glu, glutamic acid; Gly, glycine; Ile, isoleucine; Lys, lysine; Thr, threonine.
Table 4.5. Milk protein genetic variants.
1995; Feliginiet al., 1995, 1996, 1998; Chianeseet al., 1996; Penaet al., 1998;
Martin et al., 1999; Iametti et al., 2000). The gene encoding this protein is a major-effect gene, due to the presence of alleles which are responsible for high differences in the amount of such protein in milk (Parson and Heflick, 1997). At chromosomal level, seven alleles are associated with the codification of three different quantities ofaS1-casein in milk: (i)aS1-Cn A,aS1-Cn B andaS1-Cn C, so-called ‘strong alleles’, are associated with a production of 3.6 g/l per allele;
(ii)aS1-Cn E, so-called ‘medium allele’, is associated with a production of 1.6 g/l per allele; (iii)aS1-Cn D andaS1-Cn F, so-called ‘weak alleles’, are associated with a production of 0.6 g/l per allele; and (iv)aS1-Cn 0, which seems to be a ‘null allele’, whose homozygous form does not synthesize any kind of casein (Brignon et al., 1990; Pierreet al., 1998a,b). Therefore, milk produced by homozygous individuals AA, EE, FF and 00 would have 7.2, 3.2, 1.2 and 0 gaS1-casein/l, respectively (Tables 4.6 and 4.7). Some examples of the main electrophoretic techniques used in the analysis of the protein profile of goat’s milk are reported in Figs 4.6–4.9 (Jaubert and Martin, 1992; Cattaneoet al., 1996; Recioet al., 1997; Roncadaet al., 1997, 2002a,b, 2003a,b,c, 2004; Murakamiet al., 1998;
Bini and Roncada, 1999; Roncada and Greppi, 1999).
aS2-Casein
aS1-Casein b-Casein
k-Casein
Fig. 4.5. SDS-PAGE of milk caseins. Milk proteins can be separated and
identified by molecular mass (molecular weight; in kDa). One method of separating proteins is by polyacrylamide gel electrophoresis. This method essentially separates the proteins by molecular mass, with the largest proteins migrating more slowly in the gel and remaining nearer the top and the smaller proteins migrating more rapidly towards the bottom of the gel. The relative size of the caseins is ~25–35 kDa.
Casein
Genotype aS1 aS2 b k Other
Medium 28.35a 0.62a 53.74b 15.76 1.62a
Weak 5.20b 21.16b 49.01a 19.15 3.92b
Null 1.61c 23.98b 52.87b 17.07 4.46b
a,b,cValues in a column with different superscript letters were significantly different (P≤0.05).
Table 4.6. Individual casein content (%) per genotype.
Whey protein
Genotype b-Lg a-La SA Lf Other
Medium 43.8 21.8 9.0 2.6 22.8
Weak 45.8 23.3 7.7 1.5 21.7
Null 50.0 23.1 7.0 1.5 18.4
b-Lg,b-lactoglobulin;a-La,a-lactoalbumin; SA, serum albumin; Lf, lactoferrin.
Table 4.7. Major whey protein content (%) per genotype.
Fig. 4.6. Isoelectric focusing analysis of casein samples of goat’s milk (pH 7–4 from top to bottom) (in the low part,aS1variants).
Compared with the other genotypes, milk with strong genotype at the aS1-casein locus is well suited for dairy transformation due to the following prop- erties: (i) higher Ca content, therefore higher propensity for enzymatic coagula- tion; (ii) lower casein micelles diameter, therefore higher coagula consistency;
(iii) lower activation period of coagulation process, i.e. the time between curdle addition and coagula hardening; and (iv) faster formation of coagula.
Even if quantitative differences between genetic variants of single proteins are almost always small (Di Lucciaet al., 1990; Grousclaudeet al., 1994; Noè, 1995;
Lamberetet al., 1996; Enneet al., 1997; Bramantiet al., 2003; Noè and Greppi, 2003), they might directly or indirectly influence milk technological and nutritional properties (Ambrosoliet al., 1988; Addeoet al., 1989; Heil and Dumond, 1993;
Remeuf, 1993; Pirisi et al., 1994; Vassal et al., 1994; Delacroix-Buchet et al., 1996; Buchinet al., 1998; Trujillo et al., 1998; Clark and Sherbon, 2000). The main studies on goat casein polymorphisms are reported in Table 4.8.
b-Casein is the most abundant of the caseins on SDS-PAGE (Fig. 4.5), giv- ing rise to two bands, b1and b2. These two isoforms (b1and b2) differ from each other due to the presence of six and five phosphoric groups, respectively (Gallianoet al., 2004).
Some recent studies have demonstrated that the milk of Italian goat breeds has ab-null allele (b-Cn 0) associated with theaS1-Cn A allele (Ramunnoet al., Fig. 4.7. Electrophoretogram of goat’s milk in two-dimensional electrophoresis (pH 4–8 from left to right, molecular weight 220 kDa to 10 kDa from top to bottom).
Protein spots have been identified by matrix-assisted laser desorption/ionization–
time-of-flight mass spectrometry.
1996; Cosenzaet al., 2003). Thisb-null allele reduces the content ofb-casein, but it is also associated with an increase ofaS1- and total casein. Moreover, com- pared with heterozygous individuals, milk produced by homozygous b-Cn 0/0 individuals has: (i) coagulation time three times higher; (ii) coagula with reduced consistence; and (iii) lower yield of caciotta cheese (Chianeseet al., 1993; Pena et al., 1998).
20 40 a
Absorbanceat 280 nm (AU)
b
d c
Fig. 4.8. Reversed-phase high-performance liquid chromatograms of different variants ofaS1-casein (A, B and F); a =k-casein, b =aS2-casein, c =b-casein.
Studies on aS2-casein are not as numerous as those on aS1-casein. aS2- Casein has three known genetic variants, A, B and C (Bouniol et al., 1994), which have only recently been identified through electrophoretic techniques such as isoelectric focusing and SDS-PAGE. Alleles differ from each other only in a single amino acid substitution which has ties with a phosphorylation site. The link betweenaS1-Cn andk-Cn does not seem to be as strong as that between aS1-Cn andb-Cn (Changet al., 1993).
A genetic polymorphism at the k-Cn locus was also found in k-casein (Di Lucciaet al., 1990; Angiolilloet al., 2002; Yahyaouiet al., 2003; Prinzenberg et al., 2005). This casein has two known variants, A and B, which probably differ
aS2
A214A214 aS1
g1 g2 g3
S S S
b1 b2 k
b2 b1
k aS1 0.02
0.06
0.04
0.02
0 0.04 0.06 0.08 0.10
0
Fig. 4.9 Capillary electrophoresis of goat’s milk (upper) and plasmin-treated goat’s milk (lower); S indicates casein peptides.
Breed Country Obs. Year A B C D E F 0
Alpinea F 213 1994 14 5 1 34 41 5
Alpineb I 38 1994 3 26 1 5 42 10 8
Alpinec I 40 1993 – – – – – – 23
Alpined I 80 1991 – – – – 35 59 6
Alpinee I 26 1995 13 15 – – 9 40 21
Alpinef I 113 1995 11 29 – – 16 14 30
Alpineg I 88 2005 43 20 0.6 0.6 11 23 1
Blonde Ae I 85 1996 1 3 – – 5 33 58
Canarianh S 74 1991 28 32 – – 20 – 20
Corsei F 106 1989 6 13 – – 14 59 8
Frisiane I 36 1995 – 3 – – 5 37 55
Frisianc I 70 2006 13 0.7 – – 20 56 11
Garganicb I 48 1994 39 41 5 4 – – –
Garganicd I 54 1991 61 – 37 – – 2 –
Garganich S 77 1991 8 25 – – 62 5 –
Malaguenianh S 56 1991 – 25 – – 70 5 –
×Maltesiane I 177 1995 21 38 – – 4 11 27
Maltesiand I 81 1991 33 – 28 – – 11 27
Maltesianb I 372 1994 46 16 1 – 5 14 3
×Sardiniane I 181 1995 20 53 – – 5 14 8
Ionicb I 77 1994 39 27 8 – 5 1 4
Marroche M 82 1995 21 52 4 – 9 7 6
Orobicf I 70 1995 4 – – – 2 47 45
Orobicc I 66 2006 – 0.8 – – 0.8 96 2
Payoyah S 39 1991 4 14 – – 82 – –
Poiteviniani F 209 1989 5 35 – – 45 14 –
Poitevinianj F 302 1995 4 36 – – – 43 9
Rovei F 147 1989 12 5 0 0 62 10 11
Saanena F 159 1994 7 6 0.3 – 49 46 –
Saanend I 70 1991 5 35 – – 45 14 –
Saanene I 50 1995 10 8 – – 18 10 54
Sardiniane I 115 1995 11 59 – – – 7 14
Sirianb I 241 1994 50 17 – – – 6 16
Tunisiane T 86 1995 31 41 1 – 5 5 17
Verzaschesee I 57 1995 4 34 – – – 7 48
Verzaschesek I 54 1999 3 30 – 2 5 49 11
Verzaschesec I 67 2006 – 4 – – 20 75 0.7
F, France; I, Italy; M, Morocco; S, Spain; T, Tunisia.
Data source:aGrosclaudeet al. (1994);bChianeseet al. (1994, 1996);cCaroliet al. (1993, 2006);
dRamunnoet al. (1991);eFeliginiet al. (1998);fMeggiolaroet al. (1995);gBudelliet al. (2005);
hJordanaet al. (1991);iMahé and Grosclaude (1989);jRicordeauet al. (1995);kIamettiet al. (1999).
Table 4.8. Allelic frequencies ataS1-Cn in different goat breeds.
in just a single amino acid residue. The physicochemical properties ofk-casein are characterized by solubility in the presence of Ca2+ and sensitivity to chymosin. Its content in milk influences coagulation, since this phase starts only when all k-casein has been hydrolysed. Even if a strong association between aS1- andb-casein has not yet been demonstrated, it has been hypothesized that many correlations exist between all casein loci for goats, as already found for cat- tle (Parmaet al., 1999a,b, 2003a,b).