4 Protein Components of Goat`s Milk

Protein
G.F.
Greppi
Components
et al.
of Goat’s Milk
4
Protein Components
of Goat’s Milk
G.F. GREPPI1,2, P. RONCADA2 AND R. FORTIN1
1Dipartimento
di Scienze Cliniche Veterinarie, Facoltà di Medicina
Veterinaria, Università di Milano, Via Celoria 10, 20133 Milan, Italy;
2Istituto Sperimentale Italiano ‘Lazzaro Spallanzani’, Località La Quercia,
260207 Rivotta D’Adda, Italy
Introduction
Milk is a complex biological fluid produced by the mammary gland which has
three main functions: (i) nutritional; (ii) immunological; and (iii) physiological.
Recently, the physiological role of milk has been emphasized in research, and
consumer demand for high-quality products has increased. Consequently, the
transformation industry has started to market ‘high-quality milk’ with particular
nutritional/dietary characteristics. In industrialized countries, milk proteins represent 30% of total dietary protein intake and 75% of Ca intake (Meisel, 1993a,b,
1997; Morand-Fehr, 1996; Reinert and Fabre, 1996; Spuergin et al., 1997; Restani
et al., 1999).
Several epidemiological studies have focused on the influence of cow’s milk
consumption on health, especially on cancer development. However, available
data are contradictory, due to the complex interactions among milk components.
Nevertheless, a positive linear relationship between cancer and energy intake or
fat-rich milk consumption has been suggested (Attaie and Richter, 2000).
Milk contains lipid components which are well-known anticarcinogenic
agents (Parodi, 1997). According to the World Cancer Research Fund, the lipid
components of milk and derived products increase the risk of two types of cancer
only (kidney and prostate) (WCRF, 1997).
In an early study on the use of goat’s and ass’s milk, high infant mortality
occurred due to the bad microbiological characteristics of the goat’s milk employed
(Michel, 1910). As a consequence, for more than half a century goat’s milk was
not recommended for human diets. However, since the 1980s several other
studies and epidemiological research have highlighted the nutritional properties
and the presence of minor components of pharmacological interest in goat’s
milk, thus recommending its usage in human feeding (Domeniconi and Balzola,
1980; Birkbeck, 1984; Parry, 1984; Darnton Hill et al., 1987; Awiatlo et al., 1990;
Park, 1991; Houdebine, 1995; MacDonald, 1995; Albertini et al., 1999).
© CAB International 2008. Dairy Goats Feeding and Nutrition
(eds A. Cannas and G. Pulina)
71
G.F. Greppi et al.
72
The US Food and Drug Administration has just concluded that food from
cattle, swine and goat clones and their progenies is as safe to eat as food from
animals bred conventionally (Rudenko and Matheson, 2007). This decision on
food safety will certainly be greatly discussed in the very near future.
Biochemical Characteristics
Milk characteristics can be specifically associated with the environment and species. For example, there is a positive linear relationship between the logarithm
of milk protein quantity and the body weight of the mother, which indicates
that some mammalian species may produce insufficient milk for the newborn
(Fig. 4.1).
Nowadays 4027 species are classified as mammals, but the milk composition
of only 200 species, 60 of these with fewer than ten samples, has been studied
(Jenness, 1974, 1980). High intraspecific variations in protein percentage are
commonly observed, probably due to differences in genotype and/or in environmental conditions. A high number of scientific studies have demonstrated that
milk protein content of 200 mammalian species can vary from 1% up to 20%.
Milk is a colloidal heterogeneous system where mineral salts, lactose and some
whey proteins dissolved in water represent the continuous phase, while casein
micelles represent the dispersed phase. Fat is present as tiny globules in this
cow
goat
Log of produced proteins (g/day)
horse
dog
rabbit
woman
rat
guinea pig
monkey
mouse
Body weight
Fig. 4.1. Relationship between body weight and produced proteins in mammals
(logarithmic scale).
Protein Components of Goat’s Milk
73
emulsion, the membranes of which are constituted mainly by a phospholipid–
protein complex. The biochemical composition and characteristics of milk vary
from species to species, reflecting the newborn’s nutritional needs (Jenness and
Sloan, 1970; Jenness, 1979; Davis and Collier, 1983; Targowski, 1983).
For a long time, the use of milk in the diet of adults and, in particular, of
infants has been studied. In comparison to the milk of other species, goat’s milk
has been considered a good source of proteins and amino acids (Tables 4.1–4.4).
In recent decades, milk has been considered as a message carrier to nursing
infants (Teschemacher et al., 1977; Teschemacher and Brantl, 1994; Teschemacher,
1995) and adults as well. In the past, very few studies indicated the presence of
neurotransmitters or hormonal substances in milk (Blanc, 1982). On the other
hand, in the last few years, hundreds of works have shown the presence of
prolactin, melatonin, oxytocin, growth hormone, luteinizing hormone-releasing
hormone, thyroid-stimulating hormone, vasoactive intestinal peptide, calcitonin,
neurotensin and cholecystochinin in milk, indicating that milk can be considered
an exogenous endocrine system for the newborn (Mackle and Bauman, 1998).
Table 4.1.
Milk composition of different species (g/l).
Primates
Human
Simian
Rodents
Rat
Ruminants
Goat
Cow
Cetaceans
Dolphin
Whale
Table 4.2.
Proteins
Lipids
Lactose
9–15
16
38–41
40
70–72
70
81
88
38
29–31
32–34
35–45
37–39
41–44
48
68
109
330
423
11
13
Composition of human, cow’s and goat’s milk.
Water (%)
Dry residue (g/l)
Dust (g/l)
Total proteins (g/l)
Carbohydrates (g/l)
Total lipids (g/l)
Non-protein N (g/l)
Ca (mg/l)
P (mg/l)
Human milk
Cow’s milk
Goat’s milk
87.6
11.7
2.0
10
70
38
3.2
33
15
87.3
12.5
8.0
34
48
37
2.5
125
96
87.5
13.6
8.9
33
51
29
3.2
124
105
G.F. Greppi et al.
74
Table 4.3.
(g/l).
Composition of the protein fraction of human, cow’s and goat’s milk
Total proteins
Caseins
aS1-Casein
aS2-Casein
b-Casein
k-Casein
Whey proteins
a-Lactoalbumin
b-Lactoglobulin
Serum albumin
Immunoglobulin
Lactoferrin
Lysozyme
Table 4.4.
Human milk
Cow’s milk
Goat’s milk
9–15
2.0–2.5
–
–
1.5
0.5
6.3
1.9–2.6
–
0.4
1.1
1.7–2
0.04–0.2
32–34
26–37
11–15
3–4
9–11
2–4
5.8–6.5
0.6–1.5
3–4
0.4
1.0
0.1
–
28–32
22–28
10
3
11
4
5.5–6.5
1.2
3.1
0.5
1.0
0.02–0.2
–
Protein amino acid composition of human, cow’s and goat’s milk.
Total proteins (g/l)
Essential amino acids
(mg/g protein)
Cysteine
Phenylalanine
Isoleucine
Histidine
Leucine
Lysine
Methionine
Tyrosine
Threonine
Tryptophan
Valine
Non-essential amino
acids (mg/g protein)
Aspartate
Glutamate
Alanine
Arginine
Glycine
Proline
Serine
Human milk
Cow’s milk
Goat’s milk
9–15
32–34
28–32
20
37
53
23
104
71
16
46
44
17
51
9
52
64
28
100
83
27
53
51
14
68
9
47
48
26
96
80
25
38
49
–
61
86
190
40
36
22
95
61
79
208
35
37
21
101
56
75
209
34
29
18
106
49
Protein Components of Goat’s Milk
75
These hormonal substances should be considered as passengers that use milk to
reach their target organ (Choick, 1998).
Knowledge of milk proteins has also increased enormously. For example,
the functional meaning of some caseins and whey proteins has been identified
and evidence for the importance of biopeptides (i.e. amino acid sequences in
alimentary proteins) has been found. These peptides are inactive when they are
present in their native protein, but they can be released in their active form after
proteolytic digestion (i.e. during gastric digestion or simply during any technological treatment). Since the 1980s, many authors have described active
biopeptides derived from milk proteins which, in contrast with endogenous
biopeptides, have many biological functions (Yoshikawa et al., 1988). Since
then, the nutritional importance of milk, such as in the diet of athletes, has
increased. Athletes need to consume proteins with high biological value, in order
to: (i) maintain a positive N balance during physical activities; (ii) allow an increase
of muscular mass; and, occasionally, (iii) repair muscular lesions after training
(Fig. 4.2). In these cases, since protein needs increase from twofold to threefold
with respect to normal needs, kidneys may suffer due to high protein intake. The
consequences from intense physical activity can be: (i) nutritional deficiencies; (ii)
formation of free radicals that could also lead to immunological deficiencies; and
(iii) a lack of Fe, during aerobic stress, with consequent limited performance. Iron
integration into the diet may have undesirable effects. Therefore, in all of these
cases, utilization of goat’s milk could be helpful (Domeniconi and Balzola, 1980).
Milk has two main protein fractions: caseins, which are predominant, and
whey proteins (Law and Brown, 1994). The qualitative and quantitative content
of each protein fraction depends on many factors: (i) physiological (lactation stage,
lactation order); (ii) environmental (climate, hygiene); (iii) genetic (breed, genealogy); and (iv) nutritional (Polidori et al., 1991). More precisely, six main proteins are present in milk: four caseins (aS1, aS2, b and k) and two whey proteins
(a-lactoalbumin and b-lactoglobulin) (Perez and Calvo, 1995). The protein fraction of goat’s milk, like that of other domestic ruminants, is constituted mainly by
caseins, which account for 80% of total proteins (Ambrosoli et al., 1988).
PROTEINS
40–110 g/day
CARBOHYDRATES
200–260 g/day
AMINO ACIDS
GLUCOSE
PROTEINS
13 kg
GLYCOGEN
0–0.5 kg
Fig. 4.2.
LIPIDS
65–95 g/day
FATTY
ACIDS
Acetyl-CoA
Energy
TRIGLYCERIDES
13 kg
Energy production and protein metabolism in the athlete.
G.F. Greppi et al.
76
It has been recently demonstrated that mRNA levels for the four caseins
produced in the mammary epithelial cells during lactation are almost equally
abundant (~25% each), while mature protein levels in milk differ greatly
(Bevilacqua et al., 2006). In particular, translational efficiency for aS1- and
b-casein is about four times higher than that for aS2- and k-casein, thus demonstrating that regulation of casein biosynthesis is also controlled at translational
level.
The most abundant milk proteins are the three caseins (aS1, aS2 and b),
which are sensitive to Ca. Casein biosynthesis is performed at cellular level under
the influence of external agents such as hormones (Figs 4.3 and 4.4). Casein is a
globular protein, present in milk as a colloidal suspension, having the aspect of
micelles. It is characterized by a hydrophobic core and a charged polar hydrophilic part. The anionic regions of the polar part are responsible for sensitivity to
Ca2+ and for some physicochemical properties of this protein (Jaubert et al.,
1999). Moreover, caseins are relatively hydrophobic, and under ionic conditions, such as those present in milk, they tend to associate tightly into their
typical colloidal form (micelles). Micelles are constituted by a protein fraction
and by a mineral component (Ca and phosphate). Caseins are a group of specific phosphoproteins in milk which precipitate: (i) when milk is acidified to a pH
Progesterone
Glucocorticoids
Insulin
Prolactin
P
P
P
DNA
(CHO)n
Ca2+
mRNA
Endoplasmic reticulum
Golgi
Fig. 4.3. Casein biosynthesis. Following external signalling (i.e. hormones) and
signal transduction inside the nucleus, casein genes are transcribed into mRNA
molecules, which undergo post-transcriptional processing once inside the cytosol.
Mature mRNA molecules are translated into single casein molecules which are
transferred inside the endoplasmic reticulum for post-translational modification.
Casein micelle formation begins in the cis-Golgi with condensation of casein
molecules; addition of Ca2+, possibly in the secretory vesicle, leads to maturation
of the casein micelles into particles sufficiently dense to be seen in the electron
microscope.
Protein Components of Goat’s Milk
77
Exon
Intron 18
size 81–8500 bp
Stop
Mature protein
Fig. 4.4.
Molecular aspects of biosynthesis.
of 4.6–4.7 at 20°C; (ii) by enzymatic treatment (chymosin); or (iii) by ultracentrifugation. 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 content 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 (Daddaoua et al., 2006).
Milk Protein Polymorphisms
Aschaffenburg and Drewry (1955) reported, for the first time, the presence of
two variants of b-lactoglobulin in cow’s milk. Since then, many other polymorphisms have been identified both in caseins and in whey proteins. In the past
decade, molecular and biochemical characterization of protein polymorphisms
has been performed and applied to the genetic improvement of animals and the
improvement of milk characteristics, as described in detail in the reviews of
Ng-Kwai-Hang (1997, 1998). Many protein variants were correlated with differences in casein or whey protein composition as well as in milk physicochemical
properties related to caseification (Martin et al., 2002).
In the last 20 years, electrophoretic methods have been widely used to identify genetic variants of proteins (Russo et al., 1981, 1986; Martin, 1993; Martin
et al., 1999). The molecular origin of milk protein polymorphisms is the substitution
G.F. Greppi et al.
78
Table 4.5.
Protein
Milk protein genetic variants.
Variant
aS1-Casein
A
B
C
D
E
aS2-Casein
A
B
C
D
Amino acid position
14–26
Misses
53
59
192
Ala
Gln
Gly
Glu
Thr
Lys
Gly
33–47
Glu
50–58
Ala
130
Thr
Gly
Misses
Thr
Ile
Ala, alanine; Gln, glutamine; Glu, glutamic acid; Gly, glycine; Ile, isoleucine; Lys, lysine; Thr,
threonine.
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 hydrophobicity and molecular size and shape (Russo et al., 1981, 1986; Addeo et al.,
1987, 1989; Grosclaude et al., 1987; Dall’Olio et al., 1988, 1989; Brignon et al.,
1989; Di Gregorio et al., 1989). Such modifications have been identified through
the use of electrophoretic and chromatographic techniques (Addeo et 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 (Parma et al., 1999a,b; Rando et 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 different localization of phosphorylation or glycosylation sites in the amino acidic
sequence of the primary structure (Bouniol et al., 1993, 1994; Ramunno et al.,
1994; Klose, 1999). Intrinsic electrophoretic heterogeneity of casein fractions is
due to the presence of incompletely phosphorylated or glycosylated sites in these
proteins (Bevilacqua et al., 2002; Martin, 1993; Ramunno et al., 2004).
The aS1- and aS2-caseins differ in their content of phosphoric groups, while
k-casein differs in its content of glucidic and phosphoric groups (Boulanger et al.,
1984; Leroux et al., 1992, 2003; Iametti et al., 1996). b-Casein heterogeneity
derives from incomplete saturation of its phosphorylation sites (Chianese et al.,
1993).
The most studied goat’s milk polymorphism has been regarding aS1-casein
(Bouniol et al., 1993; Remeuf, 1993; Jansa-Pérez et al., 1994; Remeuf et al.,
Protein Components of Goat’s Milk
79
aS2-Casein
b-Casein
k-Casein
aS1-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.
1995; Feligini et al., 1995, 1996, 1998; Chianese et al., 1996; Pena et 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 of aS1-casein in milk: (i) aS1-Cn A, aS1-Cn B and aS1-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 and aS1-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; Pierre et al., 1998a,b). Therefore, milk produced by homozygous
individuals AA, EE, FF and 00 would have 7.2, 3.2, 1.2 and 0 g aS1-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; Cattaneo et al., 1996; Recio et al.,
1997; Roncada et al., 1997, 2002a,b, 2003a,b,c, 2004; Murakami et al., 1998;
Bini and Roncada, 1999; Roncada and Greppi, 1999).
G.F. Greppi et al.
80
Table 4.6.
Individual casein content (%) per genotype.
Casein
Genotype
Medium
Weak
Null
a,b,cValues
aS1
aS2
b
k
Other
28.35a
5.20b
1.61c
0.62a
21.16b
23.98b
53.74b
49.01a
52.87b
15.76
19.15
17.07
1.62a
3.92b
4.46b
in a column with different superscript letters were significantly different (P ≤ 0.05).
Table 4.7.
Major whey protein content (%) per genotype.
Whey protein
Genotype
b-Lg
a-La
SA
Lf
Other
Medium
Weak
Null
43.8
45.8
50.0
21.8
23.3
23.1
9.0
7.7
7.0
2.6
1.5
1.5
22.8
21.7
18.4
b-Lg, b-lactoglobulin; a-La, a-lactoalbumin; SA, serum albumin; Lf, lactoferrin.
Fig. 4.6. Isoelectric focusing analysis of casein samples of goat’s milk (pH 7–4
from top to bottom) (in the low part, aS1 variants).
Protein Components of Goat’s Milk
81
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.
Compared with the other genotypes, milk with strong genotype at the
aS1-casein locus is well suited for dairy transformation due to the following properties: (i) higher Ca content, therefore higher propensity for enzymatic coagulation; (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 Luccia et al., 1990; Grousclaude et al., 1994; Noè, 1995;
Lamberet et al., 1996; Enne et al., 1997; Bramanti et al., 2003; Noè and Greppi,
2003), they might directly or indirectly influence milk technological and nutritional
properties (Ambrosoli et al., 1988; Addeo et al., 1989; Heil and Dumond, 1993;
Remeuf, 1993; Pirisi et al., 1994; Vassal et al., 1994; Delacroix-Buchet et al.,
1996; Buchin et 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), giving rise to two bands, b1 and b2. These two isoforms (b1 and b2) differ from each
other due to the presence of six and five phosphoric groups, respectively
(Galliano et al., 2004).
Some recent studies have demonstrated that the milk of Italian goat breeds
has a b-null allele (b-Cn 0) associated with the aS1-Cn A allele (Ramunno et al.,
G.F. Greppi et al.
82
c
d
b
Absorbance at 280 nm (AU)
a
20
40
Fig. 4.8. Reversed-phase high-performance liquid chromatograms of different
variants of aS1-casein (A, B and F); a = k-casein, b = aS2-casein, c = b-casein.
1996; Cosenza et al., 2003). This b-null allele reduces the content of b-casein,
but it is also associated with an increase of aS1- and total casein. Moreover, compared 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 (Chianese et al., 1993; Pena
et al., 1998).
Protein Components of Goat’s Milk
83
0.10
0.08
0.06
A214
b2
b1
k
0.04
aS1
aS2
0.02
0
0.06
g2
g3
k
0.04
A214
g1
0.02
aS1
b2
b1
S
S
S
0
Fig. 4.9 Capillary electrophoresis of goat’s milk (upper) and plasmin-treated goat’s
milk (lower); S indicates casein peptides.
Studies on aS2-casein are not as numerous as those on aS1-casein. aS2Casein 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 between aS1-Cn and k-Cn does not seem to be as strong as that between
aS1-Cn and b-Cn (Chang et al., 1993).
A genetic polymorphism at the k-Cn locus was also found in k-casein (Di
Luccia et al., 1990; Angiolillo et al., 2002; Yahyaoui et al., 2003; Prinzenberg
et al., 2005). This casein has two known variants, A and B, which probably differ
G.F. Greppi et al.
84
Table 4.8.
Allelic frequencies at aS1-Cn in different goat breeds.
Breed
Alpinea
Alpineb
Alpinec
Alpined
Alpinee
Alpinef
Alpineg
Blonde Ae
Canarianh
Corsei
Frisiane
Frisianc
Garganicb
Garganicd
Garganich
Malaguenianh
×Maltesiane
Maltesiand
Maltesianb
×Sardiniane
Ionicb
Marroche
Orobicf
Orobicc
Payoyah
Poiteviniani
Poitevinianj
Rovei
Saanena
Saanend
Saanene
Sardiniane
Sirianb
Tunisiane
Verzaschesee
Verzaschesek
Verzaschesec
Country
Obs.
Year
A
B
C
D
E
F
0
F
I
I
I
I
I
I
I
S
F
I
I
I
I
S
S
I
I
I
I
I
M
I
I
S
F
F
F
F
I
I
I
I
T
I
I
I
213
38
40
80
26
113
88
85
74
106
36
70
48
54
77
56
177
81
372
181
77
82
70
66
39
209
302
147
159
70
50
115
241
86
57
54
67
1994
1994
1993
1991
1995
1995
2005
1996
1991
1989
1995
2006
1994
1991
1991
1991
1995
1991
1994
1995
1994
1995
1995
2006
1991
1989
1995
1989
1994
1991
1995
1995
1994
1995
1995
1999
2006
14
3
–
–
13
11
43
1
28
6
–
13
39
61
8
–
21
33
46
20
39
21
4
–
4
5
4
12
7
5
10
11
50
31
4
3
–
5
26
–
–
15
29
20
3
32
13
3
0.7
41
–
25
25
38
–
16
53
27
52
–
0.8
14
35
36
5
6
35
8
59
17
41
34
30
4
1
1
–
–
–
–
0.6
–
–
–
–
–
5
37
–
–
–
28
1
–
8
4
–
–
–
–
–
0
0.3
–
–
–
–
1
–
–
–
5
–
–
–
–
0.6
–
–
–
–
–
4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0
–
–
–
–
–
–
–
2
–
34
42
–
35
9
16
11
5
20
14
5
20
–
–
62
70
4
–
5
5
5
9
2
0.8
82
45
–
62
49
45
18
–
–
5
–
5
20
41
10
–
59
40
14
23
33
–
59
37
56
–
2
5
5
11
11
14
14
1
7
47
96
–
14
43
10
46
14
10
7
6
5
7
49
75
5
8
23
6
21
30
1
58
20
8
55
11
–
–
–
–
27
27
3
8
4
6
45
2
–
–
9
11
–
–
54
14
16
17
48
11
0.7
F, France; I, Italy; M, Morocco; S, Spain; T, Tunisia.
Data source: aGrosclaude et al. (1994); bChianese et al. (1994, 1996); cCaroli et al. (1993, 2006);
dRamunno et al. (1991); eFeligini et al. (1998); fMeggiolaro et al. (1995); gBudelli et al. (2005);
hJordana et al. (1991); iMahé and Grosclaude (1989); jRicordeau et al. (1995); kIametti et al. (1999).
Protein Components of Goat’s Milk
85
in just a single amino acid residue. The physicochemical properties of k-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- and b-casein has not yet been demonstrated, it has been hypothesized that
many correlations exist between all casein loci for goats, as already found for cattle (Parma et al., 1999a,b, 2003a,b).
Allergic Reaction and Milk Intolerance
Alimentary intolerance is an abnormal reaction by the organism that occurs after
food ingestion, regardless of the pathogenetic mechanism involved. Allergy is
defined as the sum of clinical symptoms (e.g. rhinitis, asthma and eczema) which
appear after contact between an ‘antigen’ (e.g. dust mites, pollen, cow’s milk,
pig’s meat, barley) and a specific antibody of the ‘epsilon’ (IgE) class of
immunoglobulins. The IgEs are commonly attached to the surface of different
cell types such as mastocytes and polymorphonucleate basophiles. Contact
between an antigen and its specific IgE leads to the production of mediators,
such as histamine, which cause an excessive immune response, from which
derives the term hypersensitivity. Free histamine is present in considerable quantities in tuna, mackerel, other dark meat fish and in seasoned cheese. Other
foods, such as strawberries, tomato and albumen, cause the release of histamine
through particular mechanisms which are also valid for lecithins contained in
fruit, cereals and many vegetables (Carbonaro et al., 1996; Bellioni-Businco
et al., 1999; Cartoni et al., 1999).
If the above-mentioned and many other biologically active agents (e.g. antioxidants, antimicrobials, toxins, microorganisms) are present in great quantities,
they can potentially induce a great variety of symptoms, whose manifestation
depends on the sensitivity of each individual. For example, subjects with hyperactive gastrointestinal and respiratory mucus membranes or with irritable skin
have a low threshold and thus also react in the presence of low amounts of antigen (biologically active agent in food), showing symptomatology typical of alimentary allergy.
Cow’s milk contains at least 20 different proteins, but only five are related
to allergy: (i) serum albumin, g-globulin and a-lactoalbumin, which are thermolabile and thus easily deactivated; and (ii) b-lactoglobulin and casein, which are
thermostable and resistant to the proteolytic action of the digestive apparatus.
In the case of infants, the structure of any milk protein ingested would remain
unaltered, i.e. amino acids are not removed from its peptide chain, due to insufficient enzymatic activity of proteases or lack of a specific enzyme in the intestine.
Therefore, milk protein would be recognized as a foreign body (antigen). The
quantity of absorbed antigenic material depends on anatomic barriers (mucus,
epithelium) and/or its removal by immunological barriers, as a consequence of
the combination of antigens with secretory immunoglobulins (IgA) on the surface
of the epithelium. This is an important protective action which reduces the allergenic penetration, since antigens (milk proteins) do not have access to IgE
86
G.F. Greppi et al.
and IgG. If these barriers are weakened, by inflammatory processes or by lack of
IgA, the penetration of high quantities of antigens can cause sensitivity and
allergy in atopic individuals. In general, the newborn is not intolerant to maternal
milk, excluding those affected by serious metabolic diseases (phenylketonuria,
galactosaemia) or by diseases that may require interruption of milking (icterus, diarrhoea, constipation). However, in some cases cow’s milk is not tolerated by
breast-fed children as some of its thermostable protein components
(b-lactoglobulin and casein) are absorbed almost intact and recognized as ‘foreign’ proteins by hypersensitive subjects. Cow’s milk intolerance is conventionally defined as cow’s milk protein allergy (CMPA) or cow’s milk protein
intolerance, and can affect 2–4% of the population. In paediatrics, the estimated
frequency of cow’s milk intolerance ranges from 1 to 8%, while that of atopic
predisposition in the entire population varies between 15 and 20%. Cow’s milk
allergy is the first cause of digestive allergies in industrialized countries.
The CMPA can be of immunologic or non-immunologic origin. Among
non-immunologic intolerances lactose intolerance is the best known, being caused
by a lack of intestinal lactase. However, not all problems and symptomatologies
found in sensitive individuals following ingestion of certain foods are caused by
alimentary allergy (i.e. proved or likely to be of immunologic aetiology). In fact,
in many cases such disturbances are related to alimentary intolerance, pseudoallergy or idiosyncrasy.
CMPA encompasses different types of allergies: (i) type I, with immediate
hypersensitivity reaction which can lead to anaphylactic shock, nettle rash,
asthma crisis and serious digestive problems; (ii) type III, with complex immunologic reactions characterized by enteropathies and internal haemorrhages; and
(iii) type IV, with a retarded hypersensitivity which causes chronic diarrhoea.
Clinical symptoms of CMPA are very different. They can be acute or delayed,
and digestive or extradigestive. Even if the risk of anaphylactic shock is rare, it can
affect 10% of CMPA individuals. Digestive problems can be acute or chronic,
while described extradigestive symptoms are mainly related to the skin (dermatitis, nettle rash) or the respiratory tract (asthma). Nevertheless, renal problems,
blood alterations, and central nervous system or neurological alterations may
also occur, at a lower frequency.
The fact that the clinical symptomatology of CMPA is protein-related has
resulted in a shortage of specific diagnostic tools. For example, total and specific
IgE is a diagnostic tool only for type-I CMPA, while intestinal biopsy and permeability test are difficult to perform and are suitable only for enteropathic forms.
There are predisposing factors that can favour the appearance of CMPA symptoms, which are correlated with: (i) some degree of hereditability, i.e. allergic relatives; (ii) early feeding with cow’s milk (fresh or powdered), during the very first
days of a newborn’s life; and (iii) development of gastroenteritis within the first
3 months of life, after which individuals are considered to be at high risk. As
already noted, an inflammatory intestinal disease increases epithelial permeability and, as a consequence, the risk of reaction due to intolerance or allergy. More
than a quarter of newborns who suffered gastroenteritis in their first months of
life showed CMPA symptomatology afterwards. In addition, a relevant percentage of children who suffered from gastroenteritis within the first year and up to
Protein Components of Goat’s Milk
87
3 years of life also show CMPA symptoms later on. However, whereas milk is
essential in the first few months of life, it can be replaced by other food products
as children get older. The few studies conducted on the use of goat’s milk in
human feeding are encouraging (Dore et al., 1993). Moreover, goat’s milk has
been reported to be one of the best alternatives for the production of hypoallergenic formulas for infants in case of cow’s milk allergy (D’Auria et al., 2005).
Very recent studies in this field have demonstrated that goat’s and sheep’s milk
allergy differs from cow’s milk allergy because it affects older children and
appears later. Cow’s milk products do not cause any clinical manifestation in
patients allergic to goat’s and sheep’s milk, whereas goat’s and sheep’s milk
cause cross-reaction in patients allergic to cow’s milk (Ah-Leung et al., 2006).
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