Pages 1881-1894 MILK PROTEINS Heterogeneity, Fractionation and Isolation

MILK PROTEINS/Heterogeneity, Fractionation and Isolation
1881
Contents
K F Ng-Kwai-Hang, McGill University, Quebec,
Canada
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Copyright 2002, Elsevier Science Ltd. All Rights Reserved
Introduction
stated, the term `milk proteins' in this article refers to
the bovine species.
Milk protein is a very heterogeneous group of
molecules and, for ease of description, could be
classi®ed into ®ve main categories: caseins, whey
proteins, milk fat globule proteins, enzymes and
other miscellaneous minor proteins. Among the
several factors which contribute to the heterogeneity
of milk proteins, this article will focus on methods
used for isolation, molecular structure, degree of
posttranslational modi®cation, self-association and
association between different types of protein, differences in the amounts and relative proportions of
individual proteins, origin of the proteins, diversity
of functions, and the presence of homologues across
species. The heterogeneity of milk proteins is further
complicated by the presence of genetic variants which
have been identi®ed in several species apart from
bovines. With the developments in molecular biology
and the improvements in cloning techniques, it is
possible to increase further the heterogeneity of milk
proteins by site-directed mutagenesis, controlling
the levels of expression of proteins indigenous to
milk and of novel proteins that are foreign to the
mammary gland.
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Heterogeneity, Fractionation
and Isolation
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Heterogeneity, Fractionation and Isolation
Casein Nomenclature, Structure and Association Properties
Caseins, Micellar Structure
Caseins, Functional Properties and Food Uses
Caseins, Industrial Production and Compositional Standards
Alpha-Lactalbumin
Beta-Lactoglobulin
Minor Proteins, Bovine Serum Albumin and Vitamin-Binding Proteins
Lactoferrin
Immunoglobulins
Whey Protein Products
Bioactive Peptides
Analytical Methods
Functional Properties
Protein Coprecipitates
Nutritional Quality of Milk Proteins
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MILK PROTEINS
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The milk from approximately 150 of the estimated
4000 mammalian species has been analysed in different degrees of detail and, in all cases, this secretion
has been shown to contain a protein component
which varies from 1% in human to >20% in rabbit
milk. Berzelius, in 1814, described the ®rst method
for the separation of casein, the major protein component of cows' milk. With the development of more
sophisticated analytical techniques over the years,
more than 200 types of protein have been characterized in bovine milk. The proteins in cows' milk
are the most widely studied in terms of their isolation, characterization, structural properties, functions
and biosynthetic pathways. Hence, unless otherwise
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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For a better appreciation of this article, some
familiarity with the nomenclature used for the milk
protein system, as proposed by a committee of the
American Dairy Science Association, is recommended.
Classification and Nomenclature of
Milk Proteins
When raw skim milk is adjusted to pH 4.6, a precipitate containing approximately 80% of the protein
of cows' milk is formed. This precipitation procedure
has been used as the basis for the classi®cation of
milk proteins into two main groups, with casein in
the precipitate and whey protein (noncasein protein)
in the supernatant. Table 1 gives a summary of some
of the characteristics of the four types of casein and
the four major proteins in the whey protein fraction.
The primary structure (amino acid sequence) and the
gene sequences of the four caseins, ranging in molecular mass from 19 038 for k-casein to 25 388 for
aS2-casein, have been established. Among the major
whey proteins, a-lactalbumin is the smallest (molecular mass 14 175 Da) and the immunoglobulins are
the largest (143 000±1 030 000 Da). The complete
amino acid sequences of b-lactoglobulin, a-lactalbumin and serum albumin, with molecular masses
of 18 277, 14 175 and 66 267 Da, respectively, are
also known. Unlike b-lactoglobulin and a-lactalbumin, serum albumin and some of the immunoglobulins are not synthesized in the mammary gland.
The immunoglobulins are extremely heterogeneous
and their identities are based on immunochemical
properties. Five classes of immunoglobulins (IgG,
IgA, IgM, IgE, IgD) have been identi®ed in bovine
milk. Their basic structure is similar to other immunoglobulins in that they have two heavy and two
light polypeptide chains covalently linked by disulphide bonds. The molecular mass varies from 50 to
70 kDa for the heavy chains, depending on the type,
and is about 25 kDa for the light chains. In addition to
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The heterogeneity of milk proteins has contributed
to dif®culties associated with their fractionation and
isolation and, as we acquire more information about
the macromolecules involved, these problems are being
overcome. For more than 50 years, it was thought
that the casein fraction prepared by Hammarsten in
1883 was a pure protein. The application of movingboundary electrophoresis by Mellander in 1939 demonstrated the heterogeneity of the casein fraction
with three electrophoretic components denoted as a-,
b-, and g-. Present-day knowledge con®rms that each
of the three components represents more than one
protein. The primary structure and gene sequence
of the four caseins (aS1-, aS2-, b-, k-) and three of
the whey proteins (b-lactoglobulin, a-lactalbumin,
bovine serum albumin) are now established. The
fractionation and isolation of protein components
depend on the intrinsic physicochemical properties of
the individual proteins. Because some of the proteins
tend to self-associate or associate with other proteins,
a denaturing reaction is required prior to the fractionation stage. Techniques of ultracentrifugation,
size-exclusion chromatography, ultra®ltration and
sodium dodecyl sulphate±polyacrylamide gel electrophoresis (SDS±PAGE) could be adapted for the
separation of certain proteins on the basis of molecular mass differences. Variability in net electrical
charge, sensitivity to ions, e.g. calcium, and solubility
in the presence of denaturing agents, e.g. urea, could
be exploited for the isolation of some proteins by
precipitation with different concentrations of salt
(ammonium sulphate, calcium chloride), solutions of
alcohol under different pH and temperature conditions. Based on the charge and ionic strength properties of the proteins, several chromatographic and
electrophoretic procedures have been developed for
their fractionation and isolation. The most appropriate procedures for the isolation of milk proteins
depends on the level of purity and the amount (analytical, preparative, industrial) of protein required.
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Table 1 Characteristics of the major proteins in cows' milk
Protein
a
23 164
25 388
23 983
19 038
18 277
14 175
66 267
1 430 000±1 030 000
No. of AA residues
Total
Pro
Cys
199
207
209
169
162
123
582
17
10
35
20
8
2
28
8.4%
0
2
0
2
5
8
35
2.3%
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aS1-Casein
aS2-Casein
b-Casein
k-Casein
b-Lactoglobulin
a-Lactalbumin
Serum albumin
Immunoglobulin
Molecular mass a
No. of Presence of Concentration Genetic variants
(g l ÿ1)
detected
PO4
CH2O
8
10±13
5
1
0
0
0
Ð
0
0
0
‡
0
0
0
‡
10
2.6
9.3
3.3
3.2
1.2
0.4
0.8
A, B, C, D, E, F, G, H
A, B, C, D
A1, A2, A3, B, C, D, E, F, G
A, B, C, E, Fs, FI, GS, GE, H, I, J
A, B, C, D, E, F, H, I, J
A, B, C
Ð
Ð
Molecular mass is for genetic variants in bold.
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
Heterogeneity of Milk Proteins
± His
± Pro
± Ile
± Lys ± His
± Gly ± Sj er ± Glu ± Sj er
P
P
Glu ± Ala ± Glu ± Sj er ± Ile ± Sj er ± Sj er ± Sj er
P
P
P
P
Ile ± Gln ± Lys ± Glu ± Asp ± Val ± Pro ± Ser
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Leu ± Lys ± Lys ± Tyr ± Lys ± Val ± Pro ± Gln
His ± Ser ± Met ± Lys ± Glu ± Gly ± Ile ± His
Glu ± Leu ± Ala ± Tyr ± Phe ± Tyr ± Pro ± Glu
Ser ± Gly ± Ala ± Trp ± Tyr ± Tyr ± Val ± Pro
Asp ± Ile
10
± Gln ± Gly
30
± Pro ± Gln
50
± Thr ± Glu
70
± Glu ± Glu
90
± Glu ± Arg
110
± Leu ± Glu
130
± Ala ± Gln
150
± Leu ± Phe
170
± Leu ± Gly
190
± Glu ± Asn
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Leu ± Arg ± Phe ± Phe ± Val ± Ala ± Pro ± Phe
Ser ± Lys ± Asp ± Ile
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Caseins
Bovine milk contains four types of casein denoted as
aS1-casein, aS2-casein, b-casein and k-casein, all of
which are products of speci®c genes. However,
alkaline PAGE of whole casein preparations resolves
in excess of 20 protein bands. Several of the electrophoretic bands represent posttranslational products
of one of the four caseins (see below).
The caseins, which are synthesized in the
mammary gland, are proteins containing ester-bound
phosphate and due to their relatively high content of
proline (see Table 1), they tend to have very little
secondary structure. The primary structure of aS1casein, containing 199 amino acid residues, is shown
in Figure 1. It has no cysteine residue and eight
phosphates attached to serines. A minor aS1-casein
with a faster electrophoretic mobility, denoted earlier
as aS0-casein, has been characterized and is slightly
different from the former by having nine phosphorylated serines and hence contain one extra
negative charge at alkaline pH. Three hydrophobic
regions are located in the sequences residues 1±44,
90±113 and 132±199. The sequence of residues
41±80 is very polar due to the presence of seven seryl
phosphates, eight glutamates and three aspartates.
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In addition to the high degree of heterogeneity which
exists between the ®ve different classes of milk proteins, there is also heterogeneity among proteins
within each class. This is to be expected when one
considers that milk proteins range from 10 to
H.Arg ± Pro ± Lys
>1000 kDa in molecular mass and have different
amino acid compositions and sequences which ultimately determine the structures and physicochemical
properties of the molecules. The degree of posttranslational modi®cation (proteolysis, phosphorylation,
glycosylation, formation of disulphide bridges) and
the existence of genetic variants further contribute to
the observed heterogeneity.
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differences in the organizational structure, there are
also differences in amino acid sequences and carbohydrate groups present in the immunoglobulin molecules. More than 60 enzymes have been identi®ed
in milk and, as a group, they account for less than
1% of the total milk protein. Enzymes are heterogeneously distributed in milk, e.g. catalase, lactoperoxidase, ribonuclease and glucosaminidase are
found mainly in the whey fraction while proteinases
and lipase are associated with the casein micelles,
and xanthine oxidase is found in the milk fat globule
membrane. The fat globules of milk are stabilized by
a complex membrane consisting of proteins and
phospholipids; these proteins are designated as milk
fat globule membrane proteins. The major proteins
of the milk fat globule membrane were recently reviewed. Proteins that do not fall into the classi®cation of caseins, major whey proteins, enzymes or
milk fat globule membrane proteins are categorized
as miscellaneous minor proteins and include transferrin, lactoferrin, ceruloplasmin, lactollin, glycoprotein-a, kininogen, M-1 glycoprotein, epidermal
growth factor, glycolactin, angiogenin, etc. A wide
range of biological functions has been assigned to
those minor proteins present at concentrations in the
mg kgÿ1 range.
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± Pro ± Asn ± Pro ± Ile ± Gly ± Ser
20
± Leu ± Pro ± Gln ± Glu ± Val ± Leu ± Asn ± Glu ± Asn ± Leu ±
40
± Val ± Phe ± Gly ± Lys ± Glu ± Lys ± Val ± Asn ± Glu ± Leu ±
60
± Asp ± Gln ± Ala ± Met ± Glu ± Asp ± Ile ± Lys ± Gln ± Met ±
80
± Ile ± Val ± Pro ± Asn ± Sj er ± Val ± Glu ± Gln ± Lys ± His ±
P
100
± Tyr ± Leu ± Gly ± Tyr ± Leu ± Glu ± Gln ± Leu ± Leu ± Arg ±
120
± Ile ± Val ± Pro ± Asn ± Sj er ± Ala ± Glu ± Glu ± Arg ± Leu ±
P
140
± Gln ± Lys ± Glu ± Pro ± Met ± Ile ± Gly ± Val ± Asn ± Gln ±
160
± Arg ± Gln ± Phe ± Tyr ± Gln ± Leu ± Asp ± Ala ± Tyr ± Pro ±
10
± Thr ± Gln ± Tyr ± Thr ± Asp ± Ala ± Pro ± Ser ± Phe ± Ser ±
199
± Ser ± Glu ± Lys ± Thr ± Thr ± Met ± Pro ± Leu ± Trp .OH
Figure 1 Primary structure of bovine aS1-casein B. (Reproduced with permission from Mercier JC, Grosclaude F and RibadeauDumas B (1971) Structure primaire de la caseÂine aS1-bovine. European Journal of Biochemistry 23: 41±51.)
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
± Glu ± Glu ± Ser ± Ile
± Ser ± Lys ± Glu ± Asn
± Glu ± Tyr ± Ser ± Ile
± Ile ± Thr ± Val ± Asp
20
± Ile ± Sj er ± Gln ± Glu ± Thr ± Tyr ±
P
40
± Leu ± Cys ± Ser ± Thr ± Phe ± Cys ±
60
± Gly ± Sj er ± Sj er ± Sj er ± Glu ± Glu ±
80
P
P
P
± Asp ± Lys ± His ± Tyr ± Gln ± Lys ±
100
± Tyr ± Leu ± Gln ± Tyr ± Leu ± Tyr ±
120
± Asn ± Ala ± Val ± Pro ± Ile ± Thr ±
140
± Ser ± Lys ± Lys ± Thr ± Val ± Asp ±
160
± Glu ± Glu ± Glu ± Lys ± Asn ± Arg ±
180
± Ala ± Leu ± Pro ± Gln ± Tyr ± Leu ±
200
± Gln ± Pro ± Lys ± Thr ± Lys ± Val ±
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10
H.Lys ± Asn ± Thr ± Met ± Glu ± His ± Val ± Sj er ± Sj er ± Sj er
P
P
P
30
Lys ± Gln ± Glu ± Lys ± Asn ± Met ± Ala ± Ile ± Asn ± Pro
50
Lys ± Glu ± Val ± Val ± Arg ± Asn ± Ala ± Asn ± Glu ± Glu
70
Sj er ± Ala ± Glu ± Val ± Ala ± Thr ± Glu ± Glu ± Val ± Lys
90
P
Ala ± Leu ± Asn ± Glu ± Ile ± Asn ± Glu ± Phe ± Tyr ± Gln
110
Gln ± Gly ± Pro ± Ile ± Val ± Leu ± Asn ± Pro ± Trp ± Asp
130
Pro ± Thr ± Leu ± Asn ± Arg ± Glu ± Gln ± Leu ± Sj er ± Thr
150
P
Met ± Glu ± Sj er ± Thr ± Glu ± Val ± Phe ± Thr ± Lys ± Lys
170
P
Leu ± Asn ± Phe ± Leu ± Lys ± Lys ± Ile ± Ser ± Gln ± Arg
190
Lys ± Thr ± Val ± Tyr ± Gln ± His ± Gln ± Lys ± Ala ± Net
207
Ile ± Pro ± Tyr ± Val ± Arg ± Tyr ± Leu.OH
(aS5- is a dimer of aS3- and aS4-). The 13 phosphates
(12 on serine, one on threonine) are located in three
regions of the molecule: residues 7±31, 55±66 and
129±143. Among the caseins, aS2-casein is the least
hydrophobic with regions of hydrophobicity located
at residues 90±120 and 160±207.
The sequence of the 209 amino acids in b-casein is
shown in Figure 3. It is the most hydrophobic casein,
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As shown in Figure 2, aS2-casein contains 207
amino acids. It has 10 prolines, more phosphoserines
(see Table 1) and more lysines than the other caseins,
and has two cysteines at positions 36 and 40. Several
forms of aS2-casein are discernible by PAGE due to
different degrees of phosphorylation which range
from 10 to 13 phosphate groups. These forms have
been identi®ed as aS2-, aS3-, aS4-, aS5- and aS6-casein
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± Lys ± Phe ± Pro ± Gln
± Gln ± Val ± Lys ± Arg
± Sj er ± Glu ± Glu ± Asn
P
± Thr ± Lys ± Leu ± Thr
± Tyr ± Gln ± Lys ± Phe
± Lys ± Pro ± Trp ± Ile
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Figure 2 Primary structure of bovine aS2-casein A. (Reproduced with permission from Brignon G, Ribadeau-Dumas B, Mescier JC,
PeÂlissier JP and Das BC (1977) Complete amino acid sequence of aS2-casein. FEBS Letters 76: 274±279.)
H.Arg ± Glu ± Leu ± Glu ± Glu ± Leu ± Asn ± Val ± Pro ±
Glu ± Ser ± Ile ± Thr ± Arg ± Ile ± Asn ± Lys ± Lys ±
Thr ± Glu ± Asp ± Glu ± Leu ± Gln ± Asp ± Lys ± Ile ±
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Pro ± Phe ± Pro ± Gly ± Pro ± Ile ± Pro ± Asn ± Ser ±
Pro ± Val ± Val ± Val ± Pro ± Pro ± Phe ± Leu ± Gln ±
Ala ± Met ± Ala ± Pro ± Lys ± His ± Lys ± Glu ± Met ±
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Glu ± Ser ± Gln ± Ser ± Leu ± Thr ± Leu ± Thr ± Asp ±
Gln ± Ser ± Trp ± Met ± His ± Gln ± Pro ± His ± Gln ±
Ser ± Val ± Leu ± Ser ± Leu ± Ser ± Gln ± Ser ± Lys ±
Pro ± Gln ± Arg ± Asp ± Met ± Pro ± Ile ± Gln ± Ala ±
209
Val ± Arg ± Gly ± Pro ± Phe ± Pro ± Ile ± Ile ± Val.OH
10
20
Gly ± Glu ± Ile ± Val ± Glu ± Sj er ± Leu ± Sj er ± Sj er ± Sj er ± Glu ±
30
40
P
P
P
P
Ile ± Glu ± Lys ± Phe ± Gln ± Sj er ± Glu ± Glu ± Gln ± Gln ± Gln ±
50
60
P
His ± Pro ± Phe ± Ala ± Gln ± Thr ± Gln ± Ser ± Leu ± Val ± Tyr ±
70
80
Leu ± Pro ± Gln ± Asn ± Ile ± Pro ± Pro ± Leu ± Thr ± Gln ± Thr ±
90
100
Pro ± Glu ± Val ± Met ± Gly ± Val ± Ser ± Lys ± Val ± Lys ± Glu ±
110
120
Pro ± Phe ± Pro ± Lys ± Tyr ± Pro ± Val ± Gln ± Pro ± Phe ± Thr ±
130
140
Val ± Glu ± Asn ± Leu ± His ± Leu ± Pro ± Pro ± Leu ± Leu ± Leu ±
150
160
Pro ± Leu ± Pro ± Pro ± Thr ± Val ± Met ± Phe ± Pro ± Pro ± Gln ±
170
180
Val ± Leu ± Pro ± Val ± Pro ± Glu ± Lys ± Ala ± Val ± Pro ± Tyr ±
190
200
Phe ± Leu ± Leu ± Tyr ± Gln ± Gln ± Pro ± Val ± Leu ± Gly ± Pro ±
Figure 3 Primary structure of bovine b-casein A2. (Reproduced with permission from Ribadeau-Dumas B, Brignon G, Grosclaude F
and Mercier JC (1972) Structure primaire de la caseÂine b bovine: seÂquence compleÁte. European Journal of Biochemistry 25: 505±514.)
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
Whey Proteins
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polarity and high content of negative charges. The
glycosyl moieties are bound to the caseinomacropeptide by O-glycosidic linkages with threonine at
positions 131, 133, 135 or 142. The two cysteine
residues of k-casein are located at positions 11 and
88 and the serine residue at position 149 and sometimes that at position 127 is phosphorylated. The
amphipathic character of k-casein encourages it to
form micelles in solution. Unlike other caseins, kcasein does not bind calcium extensively and is not
sensitive to precipitation by Ca2‡.
Heterogeneity of the caseins also arises from their
interactions with one another and with other proteins and small ions. With the calcium phosphate
present in milk, the caseins exist as micelles.
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has no cysteine, and a high proportion of proline (35
residues), which has a profound effect on its structure. At milk pH, the N-terminal 21-residue segment
is highly negatively charged while the rest of the
molecule, which is very hydrophobic, has no net
charge. The amphipathic nature of the b-casein
molecule is the reason why it forms micellar aggregates in solution. Beta-casein has been shown to
consist of six proteins with an identical amino acid
sequence, but with 0±5 phosphate groups attached to
serine residues. The g-caseins, which have been
known for a long time, are the hydrolytic products
of b-casein, produced by the action of plasmin.
Gamma-caseins corresponding to residues 29±209,
106±209 and 108±209 of b-casein are present in
the precipitate during isoelectric precipitation of
whole casein at pH 4.6. Other fragments of b-casein
(residues 1±28, 1±105 and 1±107) are found in the
whey and they constitute part of a fraction formerly
known as `proteose peptone'.
Kappa-casein is the only protein of the casein
family that is glycosylated. The different degrees of
glycosylation, as revealed by up to seven bands
during PAGE, are related to the number of negative
charges on the N-acetylneuraminic acid residues. The
primary structure of the carbohydrate-free portion of
the 169 amino acid-containing protein is shown in
Figure 4. Kappa-casein is the target for chymosin and
it is the most extensively studied milk protein. It
stabilizes casein micelles against calcium precipitation and loses this protective role when the
Phe105±Met106 bond is cleaved by enzymes to form
two peptides: para-k-casein (residues 1±105), which
is the hydrophobic portion and precipitates with the
casein micelles, and caseinomacropeptide (residues
106±169), which remains in solution due to its high
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The whey protein fraction, which accounts for approximately 20% of total protein, comprises the
noncasein proteins that remain soluble when caseins
have been isoelectrically precipitated at pH 4.6.
Whey proteins are an even more heterogeneous
group of compounds than the caseins and share few
common characteristics, other than being soluble
under conditions that render the caseins insoluble.
Unlike the caseins, which lack secondary structures,
the whey proteins have more organized secondary
and tertiary structures and most are globular proteins. Four major proteins, denoted as b-lactoglobulin,
a-lactalbumin, serum albumin and immunoglobulins, as shown in Table 1, account for >95% of the
noncasein proteins. Both b-lactoglobulin and a-lactalbumin are synthesized in the mammary gland
whereas serum albumin is transported to the
mammary gland via the blood serum.
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10
20
PyroGlu ± Glu ± Gln ± Asn ± Gln ± Glu ± Gln ± Pro ± Ile ± Arg ± Cys ± Glu ± Lys ± Asp ± Glu ± Arg ± Phe ± Phe ± Ser ± Asp
30
40
Lys ± Ile ± Ala ± Lys ± Tyr ± Ile ± Pro ± Ile ± Gln ± Tyr ± Val ± Leu ± Ser ± Arg ± Tyr ± Pro ± Ser ± Tyr ± Gly ± Leu ±
50
60
Asn ± Tyr ± Tyr ± Gln ± Gln ± Lys ± Pro ± Val ± Ala ± Leu ± Ile ± Asn ± Asn ± Gln ± Phe ± Leu ± Pro ± Tyr ± Pro ± Tyr ±
70
80
Tyr ± Ala ± Lys ± Pro ± Ala ± Ala ± Val ± Arg ± Ser ± Pro ± Ala ± Gln ± Ile ± Leu ± Gln ± Trp ± Gln ± Val ± Leu ± Ser ±
90
100
Asp ± Thr ± Val ± Pro ± Ala ± Lys ± Ser ± Cys ± Gln ± Ala ± Gln ± Pro ± Thr ± Thr ± Met ± Ala ± Arg ± His ± Pro ± His ±
110
120
Pro ± His ± Leu ± Ser ± Phe ± Met ± Ala ± Ile ± Pro ± Pro ± Lys ± Lys ± Asn ± Gln ± Asp ± Lys ± Thr ± Glu ± Ile ± Pro ±
130
140
Thr ± Ile ± Asn ± Thr ± Ile ± Ala ± Ser ± Gly ± Glu ± Pro ± Thr ± Ser ± Thr ± Pro ± Thr ± Ile ± Glu ± Ala ± Val ± Glu ±
150
160
Ser ± Thr ± Val ± Ala ± Thr ± Leu ± Glu ± Ala ± Sj er ± Pro ± Glu ± Val ± Ile ± Glu ± Ser ± Pro ± Pro ± Glu ± Ile ± Asn ±
P
169
Thr ± Val ± Gln ± Val ± Thr ± Ser ± Thr ± Ala ± Val.OH
Figure 4 Primary structure of bovine k-casein B. (Reproduced with permission from Mercier JC, Brignon G and Ribadeau-Dumas B
(1973) Structure de la caseÂine k b bovine: seÂquence compleÁte. European Journal of Biochemistry 35: 222±235.)
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
Trp
Val
Lys
Cys
Lys
His
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Val
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10
20
± Val ± Thr ± Gln ± Thr ± Met ± Lys ± Gly ± Leu ± Asp ± Ile ± Gln ± Lys ± Val ± Ala ± Gly ± Thr ± Trp ± Tyr ±
30
40
± Leu ± Ala ± Met ± Ala ± Ala ± Ser ± Asp ± Ile ± Ser ± Leu ± Leu ± Asp ± Ala ± Gln ± Ser ± Ala ± Pro ± Leu ± Arg ±
50
60
± Tyr ± Val ± Glu ± Glu ± Leu ± Lys ± Pro ± Thr ± Pro ± Glu ± Gly ± Asp ± Leu ± Glu ± Ile ± Leu ± Leu ± Gln ± Lys ±
70
80
± Glu ± Asn ± Gly ± Glu ± Cys ± Ala ± Gln ± Lys ± Lys ± Ile ± Ile ± Ala ± Glu ± Lys ± Thr ± Lys ± Ile ± Pro ± Ala ±
90
100
± Phe ± Lys ± Ile ± Asp ± Ala ± Leu ± Asn ± Glu ± Asn ± Lys ± Val ± Leu ± Val ± Leu ± Asp ± Thr ± Asp ± Tyr ± Lys ±
110
120
± Tyr ± Leu ± Leu ± Phe ± Cys ± Met ± Glu ± Asn ± Ser ± Ala ± Glu ± Pro ± Glu ± Gln ± Ser ± Leu ± Ala ± Cys ± Gln ±
130
140
± Leu ± Val ± Arg ± Thr ± Pro ± Glu ± Val ± Asp ± Asp ± Glu ± Ala ± Leu ± Glu ± Lys ± Phe ± Asp ± Lys ± Ala ± Leu ±
150
160
± Ala ± Leu ± Pro ± Met ± His ± Ile ± Arg ± Leu ± Ser ± Phe ± Asn ± Pro ± Thr ± Gln ± Leu ± Glu ± Glu ± Gln ± Cys ±
162
± Ile.OH
H.Leu ± Ile
Ser
of mannose, galactose, fucose, N-acetylglucosamine,
N-acetylgalactosamine and N-acetylneuraminic acid.
Milk serum albumin is physically and immunologically identical to blood plasma albumin.
The sequence of the 582 amino acids in serum
albumin is shown in Figure 7. All of the 35 cysteine
residues (except Cys34) form intrachain disulphide
bonds. The molecule is considered to have three
major domains, each consisting of two large double
loops and a small double loop, and assumes an
ellipsoidal shape. The N-terminal region is more
compact than the C-terminal region. There are different domains, varying in hydrophobicity, net charge
and ligand-binding sites. Isoelectric focusing patterns
of serum albumin show evidence of considerable
microheterogeneity of the protein.
The largest (molecular mass >1000 kDa) and most
heterogeneous of the major whey proteins belongs to
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More than half of the whey protein is b-lactoglobulin; its primary structure of 162 amino acids is
shown in Figure 5. It has ®ve cysteine residues capable of forming disulphide bonds between residues
66 and 160, 119 and 121, or 106 and 119. At pH 3 to
7, it self-associates to form dimers. Alpha-lactalbumin is the smallest of the major whey proteins,
with a molecular mass of 14 175 Da. It plays a very
important role in the biosynthesis of lactose by being
the modi®er in the lactose synthetase complex. The
sequence of the 123 amino acids in a-lactalbumin is
shown in Figure 6. All the eight cysteine residues in
a-lactalbumin are connected by disulphide bridges
between positions 6 and 120, 28 and 111, 61 and 77,
and 73 and 91. Several studies indicate that a-lactalbumin binds calcium, phospholipids and membranes. Minor forms of a-lactalbumin have been
shown to contain a carbohydrate moiety consisting
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Figure 5 Primary structure of bovine b-lactoglobulin B. (Reproduced with permission from Braunitzer G, Chen R, Schrank B and
Strangl A (1972) Automatische sequenzanalyse eines Proteins (b-lactoglobulin AB). Hopper-Seyler's Zeitschrift fuÈr physiologische
Chemie 353: 832±834.)
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10
H.Glu ± Gln ± Leu ± Thr ± Lys ± Cys ± Glu ± Val ± Phe ± Arg
30
Val ± Ser ± Leu ± Pro ± Glu ± Trp ± Val ± Cys ± Thr ± Thr
50
Ile ± Val ± Glu ± Asn ± Asn ± Gln ± Ser ± Thr ± Asp ± Tyr
70
Cys ± Lys ± Asn ± Asp ± Gln ± Asp ± Pro ± His ± Ser ± Ser
90
Leu ± Asn ± Asn ± Asp ± Leu ± Thr ± Asn ± Asn ± Ile ± Met
110
Ile ± Asn ± Tyr ± Trp ± Leu ± Ala ± His ± Lys ± Ala ± Leu
123
Glu ± Lys ± Leu.OH
20
± Glu ± Leu ± Lys ± Asp ± Leu ± Lys ± Gly ± Tyr ± Gly ± Gly ±
40
± Phe ± His ± Thr ± Ser ± Gly ± Tyr ± Asp ± Thr ± Glu ± Ala ±
60
± Gly ± Leu ± Phe ± Gln ± Ile ± Asn ± Asn ± Lys ± Ile ± Trp ±
80
± Asn ± Ile ± Cys ± Asn ± Ile ± Ser ± Cys ± Asp ± Lys ± Phe ±
100
± Cys ± Val ± Lys ± Lys ± Ile ± Leu ± Asp ± Lys ± Val ± Gly ±
120
± Cys ± Ser ± Glu ± Lys ± Leu ± Asp ± Gln ± Trp ± Leu ± Cys ±
Figure 6 Primary structure of a-lactalbumin B. (Reproduced with permission from Brew K, Castellino FJ, Vanaman TC and Hill RL
(1970) The complete amino acid sequence of a-lactalbumin. Journal of Biological Chemistry 245: 4570±4582.)
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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20
Lys ± Asp ± Leu ± Gly ± Glu ± Glu ± His ± Phe ± Lys ±
40
Gln ± Gln ± Cys ± Pro ± Phe ± Asp ± Glu ± His ± Val ±
60
Thr ± Cys ± Val ± Ala ± Asp ± Glu ± Ser ± His ± Ala ±
80
Asp ± Glu ± Leu ± Cys ± Lys ± Val ± Ala ± Ser ± Leu ±
100
Glu ± Lys ± Glu ± Gln ± Pro ± Glu ± Arg ± Asn ± Glu ±
120
Leu ± Pro ± Lys ± Leu ± Lys ± Pro ± Asp ± Pro ± Asn ±
140
Lys ± Phe ± Trp ± Gly ± Lys ± Tyr ± Leu ± Tyr ± Glu ±
160
Glu ± Leu ± Leu ± Tyr ± Ala ± Asn ± Lys ± Tyr ± Asn ±
180
Lys ± Gly ± Ala ± Cys ± Leu ± Leu ± Pro ± Lys ± Ile ±
200
Ala ± Arg ± Gln ± Arg ± Leu ± Arg ± Cys ± Ala ± Ser ±
220
Trp ± Ser ± Val ± Ala ± Arg ± Leu ± Ser ± Gln ± Lys ±
240
Leu ± Val ± Thr ± Asp ± Leu ± Thr ± Lys ± Val ± His ±
260
Ala ± Asp ± Asp ± Arg ± Ala ± Asp ± Leu ± Ala ± Lys ±
280
Lys ± Leu ± Lys ± Glu ± Cys ± Lys ± Asp ± Pro ± Cys ±
300
Glu ± Lys ± Asp ± Ala ± Ile ± Pro ± Glu ± Asp ± Leu ±
320
Asp ± Val ± Cys ± Lys ± Asn ± Tyr ± Gln ± Glu ± Ala ±
340
Tyr ± Ser ± Arg ± Arg ± His ± Pro ± Glu ± Tyr ± Ala ±
360
Glu ± Ala ± Thr ± Leu ± Glu ± Glu ± Cys ± Cys ± Ala ±
380
Phe ± Asp ± Lys ± Leu ± Lys ± His ± Leu ± Val ± Asp ±
400
Gln ± Phe ± Glu ± Lys ± Leu ± Gly ± Glu ± Tyr ± Gly ±
420
Lys ± Val ± Pro ± Gln ± Val ± Ser ± Thr ± Pro ± Thr ±
440
Gly ± Thr ± Arg ± Cys ± Cys ± Thr ± Lys ± Pro ± Glu ±
460
Ser ± Leu ± Ile ± Leu ± Asn ± Arg ± Leu ± Cys ± Val ±
480
Thr ± Lys ± Cys ± Cys ± Thr ± Glu ± Ser ± Leu ± Val ±
500
Asp ± Glu ± Thr ± Tyr ± Val ± Pro ± Lys ± Ala ± Phe ±
520
Cys ± Thr ± Leu ± Pro ± Asp ± Thr ± Glu ± Lys ± Gln ±
540
Lys ± His ± Lys ± Pro ± Lys ± Ala ± Thr ± Glu ± Glu ±
560
Phe ± Val ± Asp ± Lys ± Cys ± Cys ± Ala ± Ala ± Asp ±
580
Lys ± Leu ± Val ± Val ± Ser ± Thr ± Gln ± Thr ± Ala ±
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10
Asp ± Thr ± His ± Lys ± Ser ± Glu ± Ile ± Ala ± His ± Arg ± Phe ±
30
Gly ± Leu ± Val ± Leu ± Ile ± Ala ± Phe ± Ser ± Gln ± Tyr ± Leu ±
50
Lys ± Leu ± Val ± Asn ± Glu ± Leu ± Thr ± Glu ± Phe ± Ala ± Lys ±
70
Gly ± Cys ± Glu ± Lys ± Ser ± Leu ± His ± Thr ± Leu ± Phe ± Gly ±
90
Arg ± Glu ± Thr ± Tyr ± Gly ± Asp ± Met ± Ala ± Asp ± Cys ± Cys ±
110
Cys ± Phe ± Leu ± Ser ± His ± Lys ± Asp ± Asp ± Ser ± Pro ± Asp ±
130
Thr ± Leu ± Cys ± Asp ± Glu ± Phe ± Lys ± Ala ± Asp ± Glu ± Lys ±
150
Ile ± Ala ± Arg ± Arg ± His ± Pro ± Tyr ± Phe ± Tyr ± Ala ± Pro ±
170
Gly ± Val ± Phe ± Gln ± Glu ± Cys ± Cys ± Gln ± Ala ± Glu ± Asp ±
190
Glu ± Thr ± Met ± Arg ± Glu ± Lys ± Val ± Leu ± Thr ± Ser ± Ser ±
210
Ile ± Gln ± Lys ± Phe ± Gly ± Glu ± Arg ± Ala ± Leu ± Lys ± Ala ±
230
Phe ± Pro ± Lys ± Ala ± Glu ± Phe ± Val ± Glu ± Val ± Thr ± Lys ±
250
Lys ± Glu ± Cys ± Cys ± His ± Gly ± Asp ± Leu ± Leu ± Glu ± Cys ±
270
Tyr ± Ile ± Cys ± Asx ± Asx ± Glx ± Asx ± Thr ± Ile ± Ser ± Ser ±
290
Leu ± Leu ± Glu ± Lys ± Ser ± His ± Cys ± Ile ± Ala ± Glu ± Val ±
310
Pro ± Pro ± Leu ± Thr ± Ala ± Asp ± Phe ± Ala ± Glu ± Asp ± Lys ±
330
Lys ± Asp ± Ala ± Phe ± Leu ± Gly ± Ser ± Phe ± Leu ± Tyr ± Glu ±
350
Val ± Ser ± Val ± Leu ± Leu ± Arg ± Leu ± Ala ± Lys ± Glu ± Tyr ±
370
Lys ± Asp ± Asp ± Pro ± His ± Ala ± Cys ± Tyr ± Thr ± Ser ± Val ±
390
Glu ± Pro ± Gln ± Asn ± Leu ± Ile ± Lys ± Gln ± Asn ± Cys ± Asp ±
410
Phe ± Gln ± Asn ± Ala ± Leu ± Ile ± Val ± Arg ± Tyr ± Thr ± Arg ±
430
Leu ± Val ± Glu ± Val ± Ser ± Arg ± Ser ± Leu ± Gly ± Lys ± Val ±
450
Ser ± Glu ± Arg ± Met ± Pro ± Cys ± Thr ± Glu ± Asp ± Tyr ± Leu ±
470
Leu ± His ± Glu ± Lys ± Thr ± Pro ± Val ± Ser ± Glu ± Lys ± Val ±
490
Asn ± Arg ± Arg ± Pro ± Cys ± Phe ± Ser ± Ala ± Leu ± Thr ± Pro ±
510
Asp ± Glu ± Lys ± Leu ± Phe ± Thr ± Phe ± His ± Ala ± Asp ± Ile ±
530
Ile ± Lys ± Lys ± Gln ± Thr ± Ala ± Leu ± Val ± Glu ± Leu ± Leu ±
550
Gln ± Leu ± Lys ± Thr ± Val ± Met ± Glu ± Asn ± Phe ± Val ± Ala ±
570
Asp ± Lys ± Glu ± Ala ± Cys ± Phe ± Ala ± Val ± Glu ± Gly ± Pro ±
582
Leu ± Ala.OH
1887
Figure 7 Primary structure of bovine serum albumin. (Reproduced with permission from Brown JR (1975) Structure of bovine serum
albumin. Federation Proceedings 34: 591.)
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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belong to a separate group known as milk fat globule
membrane proteins. These proteins were traditionally classi®ed on the basis of their relative mobility
on SDS±PAGE. The protein bands were numbered
consecutively from the point of sample application in
order of increasing mobility. Confusion arose with
regard to the naming of a speci®c protein because of
inconsistencies in the use of the numbering system
and different electrophoretic procedures used by
various researchers. It has been recommended that
the practice of identifying the milk fat globule
membrane proteins by number be abandoned and be
replaced by speci®c names because they are characterized by molecular cloning and sequencing techniques. For example, the term `butyrophilin' is used
to denote a protein which has been known under
the following names: band IV/glycoprotein E, component IV, component V/glycoprotein 6, component
12/IV, Band 4/I and CB5/PAS 5.
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a group collectively known as the immunoglobulins,
which have been classi®ed as IgG, IgA, IgM, IgE and
IgD. They all exist either as monomers or polymers
of a basic unit made up of two light and two heavy
chains, each containing about 200 and 450±600
amino acid residues, respectively. The various classes
of immunoglobulins found in milk are similar to
immunoglubulins from other sources in terms of
structures and functions. The N-terminal half (residues 1±115) of the light chain is known as the
variable region because of its highly variable amino
acid sequence. The C-terminal half is known as the
constant region because the sequence of amino acids
is not as variable. The heavy chains also contain a
variable segment (residues 1±115) and a constant
region (residues 310±500). The variable regions of
the light and heavy chains are responsible for antigenbinding, whereas functions of complement ®xation,
membrane transport, catabolism and mediation of
immediate-type hypersensitivity have been attributed
to the constant region of the heavy chains.
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1888
Enzymes in Milk
A group of biologically signi®cant milk proteins,
which do not belong to the above four groups of
protein, are classi®ed separately in this category.
Two iron-binding proteins, transferrin and lactoferrin (formerly known as `red protein'), have been
identi®ed in milk. Lactoferrin is a nonhaem glycoprotein consisting of a single polypeptide chain of
690 amino acid residues with a molecular mass of
about 80 kDa. Ceruloplasmin is a copper-binding
protein which has been detected in milk, colostrum
and blood serum. Also present in milk are several
proteins that bind other nutrients and components,
such as folate, vitamin B12, corticosteroids and immunoglobulins; b2-microglobulin, also known as
lactollin, is part of the histocompatibility complex.
The 98 amino acids of b2-microglobulin, with a
molecular mass of 11 636 Da, have been sequenced.
With the development of more sensitive analytical
techniques, other proteins are being identi®ed and
added to the list of minor proteins. Recently, it was
reported that a novel protein, denoted as `glycolactin' and with a molecular mass of about 64 kDa,
has been isolated from milk.
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While a large number of enzymes have been isolated
in milk, only a relatively small number have been
characterized in detail. It is not known whether some
of the enzymes found in milk are truly indigenous or
may have been introduced due to microbial contamination. The ®rst reported enzyme in milk was
lactoperoxidase and, to date, over 60 enzymes have
been reported as being indigenous to milk. In certain
instances, the use of the pre®x `lacto' is confusing
because it implies that the enzymes occur only in
milk, e.g. lactoperoxidase refers to a speci®c enzyme
irrespective of its source. Nomenclature of the enzymes in milk does not pose any problems because
the standard nomenclature and classi®cation numbers are assigned. Milk enzymes are associated with
different milk components, e.g. catalase and ribonuclease are found in the whey protein fraction,
proteinases are associated with the casein micelles,
and xanthine oxidase is a major component of the fat
globule membrane. Many enzymes, e.g. catalase, are
present predominantly in one component of milk,
but are not con®ned exclusively to that component.
This may be due to redistribution of the enzymes in
the milk system. Alpha-lactalbumin, a major whey
protein, may be classi®ed as an enzyme because of its
involvement in lactose synthesis.
Minor Miscellaneous Proteins
Milk Fat Globule Membrane Proteins
A large number of proteins are present in milk at
relatively low concentrations and are not isolated
with either the casein or whey protein fractions but
Variability in Protein Concentration
The large variations in the concentrations and relative proportions of proteins in milk could contribute
to heterogeneity in properties and functionalities of
the protein component. For the milk of over 150
species that have been analysed, the data indicate
that total protein concentration varies from 1% in
humans to 24% in the whitetail jackrabbit and the
casein to whey protein ratio varies from 0.25 : 1 in
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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associated with no aS1-casein in the milk. Milk from
all the species analysed (except the Californian sea
lion) contains lactose and, because a-lactalbumin is
involved in the synthesis of lactose, one can assume
that the presence of a-lactalbumin is universal. Betalactoglobulin is the protein present at highest concentration in the whey of ruminant milks. Homologous b-lactoglobulin has been characterized in the
milk of cows, goats, water buffaloes and sheep.
Apparently, milk from humans and the kangaroo,
camel, guinea pig, rat and mouse are devoid of blactoglobulin. There are indications that mare's milk
contains two proteins that are very similar to bovine
b-lactoglobulin and can be classi®ed as b-lactoglobulin. A protein isolated from pigs' milk is somewhat
similar to ruminant b-lactoglobulin in size and composition, although it lacks a free sulphydryl group.
A cysteine-rich protein denoted as whey acid protein
(WAP) has been identi®ed in the milk of rat and
mouse, with 137 and 134 amino acid residues,
respectively. This protein is not found in ruminant
and human milk. Analogues of serum albumin and
immunoglobulin are present in the milk of all species
and are representatives of the proteins circulating in
the blood of the species. It is not known whether all
the milk fat globule membrane proteins and enzymes
associated with milk are present in all species because
those milk samples have not been analysed as extensively as bovine milk. The activities of milk enzymes are species-dependent. Ribonuclease activity is
higher in cows' milk than in human milk whereas
human milk has 1000 times more lysozyme activity
than cows' milk. Lactoferrin has been identi®ed in
the milk of humans, mouse, guinea pig, horse, pig,
cow and goat, but is absent from the milk of rabbit,
rat and dog.
Homology of Milk Proteins
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human milk to 6.3 : 1 in goats' milk. In many instances, a limited number of samples have been
analysed for most of the species compared to cows'
milk and, hence, the values obtained are not reliable.
Also, the nonbovine proteins have not been
analysed in great detail, as in the bovine milk. Within
the bovine species, the protein content and composition of the milk vary widely depending on genetics, health conditions and a whole host of
environmental factors, including nutrition of the animal, stage of lactation, age of the cow, season and
geographical location. Genetic polymorphism of milk
proteins (see below) also contributes to large variations in the concentration of certain milk proteins.
An example of differences according to the breed of
cow is demonstrated by comparing the average
protein content, of 3.42, 3.61, 3.58, 3.86 and 4.02%,
respectively, in the milk of Holstein, Brown Swiss,
Ayrshire, Jersey and Guernsey cows. In ®ve regions
of California, the milk protein varies from 3.32% to
3.82%, with the caseins representing 76±78% of the
total protein. Total protein, casein and whey protein
content are highest during the winter and the early
stage of lactation. Reports show that the ratio of
casein to whey protein decreases as cows become
older or with higher incidence of mastitis, and that
herd of cow, parity number, month of test, stage of
lactation and mastitis had signi®cant effects on the
concentrations and relative proportions of individual caseins, b-lactoglobulin, a-lactalbumin, serum
albumin and immunoglobulins. Some minor proteins, e.g. lactoferrin and transferrin, occur at higher
concentrations in colostrum than in mature milk.
1889
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The wide range of pro®les obtained when milk proteins from different species are subjected to PAGE on
the same gel slab clearly demonstrates heterogeneity
across species. In several instances, the nomenclature
developed for bovine milk proteins has been wrongly
applied to proteins of other species. This extension of
name use is justi®ed only if homologous proteins are
solidly established on a basis of structural and functional properties. Skim milk from all observed
species has a chalky-white or `milky' appearance due
to the scattering of light by casein micelles. Hence it
can be assumed that all milks contain a casein component. However, the four different types of caseins
are present in different proportions and, in some
cases, one or more of the caseins may be absent.
There is evidence that human milk is devoid of aS1and aS2-caseins, i.e. the casein micelles are made up
entirely of b- and k-caseins. Goats' milk represents
an interesting situation where the aS1-Cn0 allele is
Biological Roles of Milk Proteins
Milk is intended to be the sole source of food for the
suckling newborn. Milk proteins are a heterogeneous
group of compounds with a wide range of molecular
structures and properties. Likewise, they serve many
important biological roles, some of which have not
yet been determined. As discussed earlier, milk from
all species contains casein micelles. The caseins have
a loose structure due to a high proportion of proline
and hence they are very susceptible to hydrolysis
by the digestive enzymes to yield a well-balanced
mixture of amino acids for the nutrition of the neonate. Because the caseins are associated with high
concentrations of calcium and phosphorus, the digestion of these proteins releases signi®cant amounts of
these minerals, which become available to the young
animal.
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
Genetic Polymorphism of Milk Proteins
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the deletion of a segment in the aS1-casein. The
variants A and H of aS1-casein differ from variant B
by the absence of amino acid sequences 14±26 and
51±58, respectively. The deletion of residues 51±59
in aS2-casein A leads to the occurrence of variant D.
The remaining 41 genetic variants involve amino
acid substitutions. For example, k-casein A is different from k-casein B by having a threonine at position
136 and an aspartic at position 148, instead of an
isoleucine and an alanine at the two respective positions. The difference between variants A and B of blactoglobulin is due to two amino acid substitutions
at positions 64 (glycine for aspartic acid) and 118
(alanine for valine). In both cases, the presence of an
extra aspartic acid residue in the A variant renders
the proteins more negatively charged and hence the
A variants of k-casein and b-lactoglobulin have faster
electrophoretic mobilities under alkaline conditions
than their B homologues.
The subject of genetic polymorphism of milk
protein polymorphism has generated a lot of interest
among several groups of researchers in different
®elds. Some of the genetic variants of milk protein
are associated with the production, composition and
industrial properties of milk. Because the genetic
variants are inherited according to the simple Mendelian mode of inheritance, it is possible to breed for
speci®c variants of milk proteins. Such a scheme of
selection is being practised in certain countries to
favour higher frequencies of k-casein B and b-lactoglobulin B in the dairy cattle population. The B
variants of the two proteins are associated with
higher levels of fat and casein in the milk, better
coagulating properties on renneting, higher cheese
yield and better quality. Beta-lactoglobulin A milk
has a higher content of protein, due mainly to increased production of b-lactoglobulin, but a lower
concentration of casein than b-lactoglobulin B milk.
At the k-casein locus, the B variant is associated with
a higher concentration of k-casein, total casein and
total protein compared to the A variant. These are
only two examples that serve to illustrate how
genetic polymorphism could contribute to variability
in the amounts and relative proportions of milk
proteins.
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Alpha-lactalbumin is also of universal occurrence
and is required for the biosynthesis of lactose.
According to the osmoregulatory theory, the production of milk depends on the presence of lactose.
No biological roles have yet been assigned to blactoglobulin, although there are indications that it is
a retinol-binding protein and could also be involved
in the activation of lipase. Serum albumin and
the heterogeneous immunoglobulins are involved in
protection against infection and provide passive
immunity. The roles of many enzymes are not known.
In general, their main signi®cance seems to be the
defence against intestinal infection. Lysozyme acts as
a bactericidal agent by degrading the bacterial cell
wall to enhance the activity of immune antibodies.
Protein kinases are present in the milk fat globule
membrane and catalyse the phosphorylation of other
proteins and hence regulating the biological activities
of the latter. Mammary-derived growth inhibitor
appears to regulate cellular growth and function. The
iron-containing proteins (lactoferrin and transferrin),
folate-binding protein and copper-binding protein
serve to convey their prosthetic groups, which are
essential for the nutrition of the growing sucklings.
Lactoferrin also acts as a selective antibiotic because
it chelates iron and renders it unavailable to the bacterial population in the gut; in the mammary gland it
may bene®t the lactating cow by reducing mastitic
infection.
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A very signi®cant variability in milk proteins, leading
to further heterogeneity, is due to genetic events
leading to the occurrence of genetic variants. Since
the discovery in 1955 of the presence of two forms of
b-lactoglobulin, several groups around the world
have been actively engaged in research covering various aspects of genetic polymorphism of milk proteins. Several electrophoretic and isoelectric focusing
methods have been modi®ed and adapted for the
identi®cation of genetic variants of milk proteins. At
the latest count (see Table 1), 44 genetic variants
have been identi®ed among the four caseins, blactoglobulin and a-lactalbumin, and that number
will undoubtedly rise as more sophisticated methods
for detection are developed.
Genetic polymorphism of milk proteins is due to
either substitutions or deletions of amino acids in the
proteins as a consequence of mutations causing
changes in base sequences in the genes. This type of
polymorphism should be distinguished from that
caused by posttranslational modi®cation, such as the
extent of phosphorylation and glycosylation of the
molecule, as discussed previously. Among the 44
genetic variants presented in Table 1, two are due to
Fractionation and Isolation
The heterogeneous mixture of proteins in milk can be
separated and characterized by various physicochemical methods. Comprehensive reviews on the
different procedures for the fractionation and isolation of milk proteins are available. Many of the
procedures depend on the solubility differences and
the isoelectric precipitation of the caseins. Some of
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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MgSO4. Alpha-lactalbumin and b-lactoglobulin were
later isolated in crystalline form from the albumin
fraction. Aschaffenburg and Drewry described a
classical method for the isolation of b-lactoglobulin.
The casein and fat were precipitated from whole milk
by the addition of Na2SO4 to a ®nal concentration of
20% at 40 C. By cooling the supernatant to 25 C
and adjusting the pH to 2.0, a precipitate containing
a-lactalbumin was formed. Beta-lactoglobulin was
crystallized out after adjusting the pH to 6.0 and the
addition of more Na2SO4. Over the years, several
methods for preparing crystalline b-lactoglobulin
and a-lactalbumin by the use of other precipitating
agents, such as trichloroacetic acid and (NH4)2SO4,
have been reported.
In most instances, the above procedures do not
give a ®nal product of high purity. Further fractionation and isolation steps involving different types of
electrophoresis and chromatography are necessary
to achieve the level of purity required. With electrophoretic methods, one could expect to obtain pure
proteins in analytical amounts only. Considerably
larger quantities of pure proteins may be obtained
by the use of chromatographic methods. The development of membrane ®ltration has revolutionized
the fractionation of milk components in the dairy
industry.
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the caseins are not precipitated at pH 4.6 while
certain noncasein proteins may be coprecipitated with
the caseins. There is no practical and absolutely clearcut method for simply fractionating casein, and only
casein, from noncasein proteins. Moving-boundary
electrophoresis was used to resolve whole casein into
three fractions, denoted as a-, b- and g-, none of
which represented a pure protein. The interaction of
casein with calcium ions was exploited by the use of
CaCl2 solution to precipitate a calcium-sensitive
fraction, aS-casein, which contained mainly aS1-, aS2and b-caseins, from k-casein, the fraction insensitive
to calcium precipitation. Milder conditions for the
precipitation of the caseins by high salt concentration, e.g. (NH4)2SO4, have been used. Not all the
caseins precipitate and some immunoglobulins and
glycoproteins may be included in the precipitate. In
1938, Rowland developed a chemical method to
quantify protein distribution in milk and was able to
partition the protein as casein, globulins (precipitated
by saturation with MgSO4), lactalbumin and proteose peptone. The rates of sedimentation of proteins
from skim milk after dialysis against phosphate buffer
were used as a method for the isolation of some proteins. Several casein components could be prepared,
starting from a 50% alcohol solution of casein in
the presence of ammonium acetate and followed by
precipitation by varying the pH, ionic strength and
temperature. The interactions between the caseins
have hampered their fractionation. Dissociating
agents such as urea and a disulphide-reducing agent,
e.g. b-mercaptoethanol, are routinely used to permit
better fractionation of the caseins. Individual caseins
could be obtained starting with a whole casein
solution in 6 mol lÿ1 urea and adding water to precipitate out the components. The a-casein complex
is insoluble in 4.5 mol lÿ1 urea at pH 4.6±4.8 and
contains mainly aS1-, aS2- and k-caseins. Beta-casein
is soluble in 3.3 mol lÿ1 urea, but insoluble in
1.7 mol lÿ1 urea. Urea fractionation of casein is not
precise because some k-casein remains in the urea
solution with b-casein. In the method of Zittle and
Custer, acid casein is dissolved in 6.6 mol lÿ1 urea
and acidi®ed with 3.5 mol lÿ1 H2SO4. On dilution of
the mixture with water, a precipitate rich in aS1- and
b-caseins forms slowly. The supernatant is a good
source of k-casein.
The noncasein component obtained in the Rowland method of protein fractionation was resolved by
moving-boundary electrophoresis into immunoglobulins, a-lactalbumin, b-lactoglobulin, serum
albumin and proteose peptones. The complexity of
the whey protein fraction was demonstrated over a
century ago with the precipitation of a globulin
fraction from an albumin fraction by saturation with
1891
Zone Electrophoresis
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The proteins in milk have a wide range of isoelectric
points and molecular mass; hence they could be separated by different electrophoretic procedures which
could be adapted for the fractionation and isolation
of milk proteins in microgram or nanogram amounts.
Electrophoretic separations of the proteins have
been performed on skim milk, whole casein and
whey protein samples. Among the various supporting
media, including ®lter paper, cellulose acetate,
starch, agarose and polyacrylamide gels, which have
been used for electrophoresis of milk proteins, polyacrylamide is the most convenient and widely used.
The concentrations of acrylamide and crosslinking
reagent can be modi®ed to prepare gels of different
pore size in order to enhance separation based on
molecular mass. Polyacrylamide gels can also be
made with a concentration gradient of acrylamide.
The inclusion of SDS in PAGE also resolves the
proteins, according to their molecular mass. Denaturing agents, such as sodium dodecylsulphate and
urea, chelating agents, e.g. ethylene diamine tetraacetic acid (EDTA) and reducing agents, e.g. mercaptoethanol or dithiothreitol, may be included in
the gel to enhance separation of the proteins. A wide
variety of gel and electrode buffers, differing in type,
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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Chromatography
and aS2-casein had the largest number (10±13) of
phosphate residues. The most commonly used buffer
is phosphate, pH 6.8, containing dithiotreitol and
urea. The higher the net negative charges on a protein,
the more tightly it is bound to the solid phase. Different proteins are separated by gradually increasing
the phosphate concentration in the eluting buffer.
Using such a method, whole casein can be fractionated into g-casein, k-casein with different degrees
of glycosylation, b-casein and aS-casein. The latter
fraction indicates poor resolution of aS1-casein from
aS2-casein.
Ion-exchange chromatography is by far the most
commonly used chromatographic method for the
fractionation of milk proteins. This method is based
on binding the mixture of electrically charged proteins to the matrix and eluting the components with
increasing salt concentration. Although several anion
and cation exchangers have been used to fractionate
milk proteins, the former is used more frequently.
In both instances, NaCl provides counter-ions, with
Na‡ as the cation and Clÿ as the anion. Fractionation of proteins from skim milk by ion exchange
chromatography is not as effective as from whole
casein or whey protein preparation. The presence of
urea and a reducing agent, such as 2-mercaptoethanol or dithiothreitol, is essential for the effective
fractionation of the caseins. Satisfactory fractionation of whole casein into g-, k-, b-, aS2- and
aS1-caseins could be obtained with a diethylaminoethylcellulose (DEAE-cellulose) column and eluting
with a NaCl gradient in Tris buffer, pH 8.6, containing 6 mol lÿ1 urea. Many commercial ion exchangers have been used to fractionate the caseins,
eg., DEAE-TSK-5PW, Mono Q HR 5/5, Protein Pak
DEAE-15HR, macro Q 50, to name just a few.
DEAE-cellulose chromatography gives satisfactory
separation of the whey proteins with the fractionation of b-lactoglobulin, a-lactalbumin, serum
albumin and immunoglobulins. The amount of
protein that can be fractionated by column chromatographic methods in the laboratory is relatively
small, in the microgram to milligram range, and depends on the size of the column. To overcome this
constraint, batch fractionation of whole casein on
DEAE-cellulose has been achieved. This method involves suspending whole casein and DEAE-cellulose
in 3.3 mol lÿ1 urea solution at pH 7.4 and desorbing
the caseins by successive extractions with 0.00,
0.035, 0.085, 0.12 and 0.175 mol lÿ1 NaCl and ®ltration to recover the desired fractions. Starting with
20 g of whole casein, the recoveries are: 2.2 g kcasein, 6.58 g b-casein, 3.80 g aS2-casein and 4.89 g
aS1-casein. Semi-preparative fractionation of casein
into pure k-, b-, aS2- and aS1-caseins is possible with
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concentration and pH, have been used for electrophoresis. Under alkaline conditions, and in the presence of urea, the mobility is in the following order:
aS1- > aS2- > b- > k-casein. Under acidic conditions,
the genetic variants of b-casein migrate in the order
C > B > A1 > A2 > A3. The order of separation of
the major whey protein is: b-lactoglobulin > alactalbumin > serum albumin > immunoglobulins.
Isoelectric focusing, which is considered to be a
modi®cation of electrophoresis, has also been applied
extensively for the resolution of milk proteins. The
more recent literature abounds with the application
of capillary electrophoresis for the resolution of milk
proteins. This latter technique is more applicable for
analytical work and may not be suitable for isolation
and fractionation of milk proteins.
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Different types of chromatography, based on electrical charge, molecular mass or hydrophobic interactions, have been developed for the fractionation
and isolation of milk proteins. Several review articles
dealing with the chromatographic separation of milk
proteins have been published.
Gel ®ltration (molecular sieving) chromatography
has been used to fractionate and isolate proteins from
skim milk, whole casein or the whey protein fraction.
The principle is based on the capacity of different
proteins, depending on their molecular mass, to
penetrate the pores of the gel stationary phase. Large
proteins are excluded from the gel and are eluted
rapidly. Chromatography of skim milk on a Sephadex G200 column with pH 7.0 phosphate buffer
resolved three fractions containing mixtures of k-, band aS-caseins, followed by b-lactoglobulin and alactalbumin. Gel permeation chromatography is not
suitable for fractionating the caseins because they
occur as micelles of different size. Removal of calcium
results in dissociation of the micelles into large
polydispersed aggregates containing various proportions of the different caseins. In the presence of urea
and reducing agents, the caseins dissociate to monomeric forms, ranging in molecular mass from about
19 to 25 kDa (see Table 1), which are too close for
an effective separation. Whey proteins have been
fractionated successfully into b-lactoglobulin, alactalbumin, serum albumin and lactoferrin by gel
®ltration chromatography. The use of a Superose 12
column gives satisfactory fractionation of whey
proteins in the order: immunoglobulins, serum
albumin, b-lactoglobulin and a-lactalbumin.
Chromatography on a hydroxyapatite column is
interesting for the fractionation of caseins because
the separation is based on phosphate content. As
shown in Table 1, k-casein has the least number (1)
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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b-lactoglobulin/k-casein complexes. Heat, combined
with pH adjustment, can be exploited to fractionate
b-lactoglobulin and a-lactalbumin from whey. The
manufacture of different types of whole casein is also
considered as a method of protein fractionation. The
whole casein can be further fractionated to yield
individual caseins.
Development in membrane technology has provided different physical methods for the fractionation
and isolation of milk proteins. Several review articles
give details of the applications of membrane ®ltration in the dairy industry. In theory, the milk
proteins can be separated according to their size in
the following increasing order of molecular mass:
a-lactalbumin < b-lactoglobulin < serum albumin <
lactoferrin < immunoglobulins < casein
micelles <
milk fat globule membrane. Reverse osmosis requires
pressures of 3 500±10 000 kPa to remove water. It is
conveniently used to concentrate the proteins in
whey or skim milk. Ultra®ltration involves the use
of membranes with a molecular cutoff range of
1±200 kDa and operating pressure of 140±700 kPa.
When applied to milk, it results in a retentate containing fat and proteins while water, minerals and
lactose pass through as the permeate. The making of
cheese from ultra®ltered milk includes the whey
proteins in the ®nished product, thereby increasing
yield. Micro®ltration (membranes with pore size
0.2±2.0 mm; molecular cutoff >200 kDa) has contributed signi®cantly to the industrial fractionation
of milk proteins. Because the fractionation process is
purely mechanical at a low temperature, there is no
negative impact on the functionalities of the proteins.
The differences between the size of casein micelles
and the whey proteins are such that these two components can be fractionated easily by membrane ®ltration, with the whole casein being retained and the
whey proteins passing through. Further fractionations could be achieved by application of other
technologies. Beta-casein can be isolated by micro®ltration of calcium caseinate at 5 C. Dilute solutions of sodium caseinate can be fractionated by
ultra®ltration into a permeate rich in b-casein and a
retentate rich in aS- and k-caseins. A heat treatment
and membrane separation process have been developed to separate b-lactoglobulin from whey. This
involves the reversible polymerization of a-lactalbumin by heating at 55 C for 30 min at a low pH
and separation of the resulting aggregates, consisting
of a-lactalbumin and whey proteins other than
b-lactoglobulin, by micro®ltration. The permeate is
then processed by ultra®ltration in combination
with dia®ltration to yield puri®ed b-lactoglobulin.
The a-lactalbumin can be recovered from the retentate after solubilization at neutral pH, followed
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high-performance liquid chromatography (HPLC)
using a DEAE±15 HR anion exchange with a NaCl
gradient in Tris±urea buffer, pH 7.0. Commercially
available cartridges containing anion exchangers,
e.g. diethyl(2-hydroxypropyl) quaternary aminocellulose, have been used successfully for the fractionation of individual caseins and whey proteins
in gram quantities.
Although reversed-phase HPLC (RP±HPLC) and
hydrophobic interaction HPLC (HI±HPLC) depend
on the hydrophobic interactions between a stationary
phase and the proteins to be fractionated, their applications are quite different. In RP±HPLC, adsorption occurs in an aqueous solution of low ionic
strength and elution of proteins is obtained by
increasing the hydrophobicity of the mobile phase.
With HI±HPLC, adsorption of proteins is carried out
in aqueous solution at high ionic strength and elution
is achieved by reducing the ionic strength of the
mobile phase. Whole casein and whey proteins can
be fractionated on a phenyl hydrophobic interaction
column. For whole casein, elution is done with 0.8 to
0.5 mol lÿ1, pH 6.0, sodium phosphate buffer containing 3.75 mol lÿ1 urea. For whey proteins, fractionation is achieved with a 1.5±0.05 mol lÿ1 gradient
of ammonium sulphate in 0.05 mol lÿ1 sodium phosphate, pH 7.0. Several studies have reported on the
use of RP±HPLC to fractionate whole caseins. In a
typical procedure, whole casein is dissolved in pH 7
buffer containing urea and a reducing agent and
applied to the column. The components are eluted by
increasing the concentration of acetonitrile (hydrophobicity) linearly over time.
In addition to the fractionation of the caseins and
the major whey proteins, all the above procedures
have been used, as in other biochemical preparation,
to isolate minor milk proteins, including enzymes, fat
globule membrane proteins and other miscellaneous
proteins.
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At the industrial level, proteins are fractionated for
various purposes. The making of cheese is a form of
protein fractionation where most of the casein, apart
from the macropeptide portion of k-casein, ends up
in the curd and most of the whey proteins are in the
whey. The whey can be further processed to give
different protein-enriched products. Different combinations of temperature and heating time give rise
to different types of cheeses, with the incorporation
of varying amounts of whey protein, especially
b-lactoglobulin. Heat-induced interaction between
b-lactoglobulin and k-casein can alter the distribution of proteins in milk powders. Low-heat milk
powders contain more soluble whey proteins and less
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MILK PROTEINS/Heterogeneity, Fractionation and Isolation
Conclusions
Eigel WN, Butler JE, Ernstrom CA et al. (1984)
Nomenclature of proteins of cow's milk: ®fth revision.
Journal of Dairy Science 67: 1599±1631.
Fox PF (ed.) (1992) Advanced Dairy Chemistry, vol. 1,
Proteins. London: Elsevier Applied Science.
IDF (1996) Advances in Membrane Technology for Better
Dairy Products. International Dairy Federation Bulletin
no. 311. Brussels: IDF.
Ima®don GI, Farkye NY and Spanier AM (1997) Isolation,
puri®cation, and alteration of some functional groups of
major milk proteins: a review. Critical Review in Food
Science and Nutrition 37: 663±689.
Ima®don GI and Ng-Kwai-Hang KF (1992) Isolation and
puri®cation of b-lactoglobulin. Journal of Dairy Research 59: 101±104.
Jenness R (1974) The composition of milk. In: Larson BL
and Smith VR (eds.) Lactation: A Comprehensive
Treatise, vol. 3, pp. 3±105. New York: Academic Press.
Mather IH (2000) A review and proposed nomenclature
for major proteins of the milk-fat globule membrane
Journal of Dairy Science 83: 203±247.
McKenzie HA (1970) Milk Proteins: Chemistry and
Molecular Biology, vol. 1. New York: Academic Press.
McKenzie HA (1971) Milk Proteins: Chemistry and
Molecular Biology, vol. 2. New York: Academic Press.
Ng-Kwai-Hang KF and Dong C (1994) Semi-preparative
isolation of bovine casein components by high-performance liquid chromatography. International Dairy
Journal 4: 99±110.
Ng-Kwai-Hang KF and Grosclaude F (2002) Genetic polymorphism of milk proteins. In: Fox PF and McSweeney
PLH (eds.) Advanced Dairy Chemistry, vol. 1, Proteins,
3rd edn, pp. 737±814. New York: Kluwer Academic.
Ng-Kwai-Hang KF, Hayes JF and Moxley JE (1982)
Environmental in¯uences on protein content and
composition of bovine milk. Journal of Dairy Science
65: 1993±1998.
Ng-Kwai-Hang KF, Hayes JF, Moxley JE and Monardes
HG (1987) Variation in milk protein concentrations
associated with genetic polymorphism and environmental factors. Journal of Dairy Science 70: 563±570.
Ng-Kwai-Hang KF and PeÂlissier JP (1989) Rapid separation of bovine caseins by mass ion exchange chromatography. Journal of Dairy Research 56: 391±397.
Ribadeau-Dumas B (1988) Structure and variability of
milk proteins. In: Barth CA and Schlimme E (eds.) Milk
Proteins: Nutritional, Clinical, Functional and Technological Aspects, pp. 112±123. New York: SpringerVerlag.
Rosenberg M (1995) Current and future application for
membrane processes in the dairy industry. Trends in
Food Science Technology 6: 12±19.
Swaisgood HE (1982) Chemistry of milk protein. In: Fox
PF (ed.) Developments in Dairy Chemistry, vol. 1,
Proteins, pp. 1±59. London: Elsevier Applied Science.
Wei TM and Whitney RM (1985) Batch fractionation of
bovine caseins with diethylaminoethyl cellulose. Journal
of Dairy Science 68: 1630±1636.
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With the progress being made in protein engineering
and in molecular genetic techniques, further heterogeneity of milk proteins, as outlined above, is expected by modi®cation of the milk protein genes and
controlling the amounts of speci®c genes being expressed. Just a few examples will serve to illustrate
this point. Breeding programmes are already in place
to reduce the frequency of the aS1-Cn0 allele in the
dairy goats and to increase the frequency of the kcasein B allele in the bovine population in order to
increase the amount of casein in milk and improve its
cheesemaking characteristics. The expression of alactalbumin, which is involved in lactose synthesis,
could be controlled so that lactose content of milk
can be modi®ed. The amount and type of k-casein
being expressed could be altered so as to improve
micellar structure and the cheesemaking properties of
the milk. The properties of b-lactoglobulin could be
modi®ed by altering its primary structure in such a
way as to increase the degree of glycosylation or
formation of disulphide bridges. In contrast to the
above modi®cations of milk proteins, which have
direct implications for the dairy industry, proteins
exogenous to the mammary gland have been successfully incorporated into milk, e.g. the expression
of b-lactoglobulin in mouse milk and of factor IX in
sheep's milk. The latter type of modi®cation of milk
proteins will be of great interest to pharmaceutical
companies. Isolation and fractionation of milk
proteins will still be based on the intrinsic physiochemical properties of the proteins. Different chromatographic techniques are more widely used for this
purpose and `in the future' better solid matrices will
be developed. On an industrial scale, membrane
®ltration techniques will play a major role in the
isolation of milk proteins.
Further Reading
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by ultra®ltration. Hydrophilic cellulose membranes
with a large pore size have been used to fractionate
whey proteins into a-lactalbumin plusb-lactoglobulin
and a higher molecular weight component containing bovine serum albumin, immunoglobulins and
lactoferrin.
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See also: Enzymes Indigenous to Milk: Lipases and
Esterases; Plasmin System in Milk; Phosphatases;
Lactoperoxidase; Xanthine Oxidase; Other Enzymes.
Human Milk. Mammals. Milk Proteins: Heterogeneity,
Fractionation and Isolation; Casein Nomenclature,
Structure and Association Properties; Caseins, Micellar
Structure; Alpha-Lactalbumin; Beta-Lactoglobulin; Minor
Proteins, Bovine Serum Albumin and Vitamin-Binding
Proteins; Lactoferrin.
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