CHAPTER 3
Chemistry of Milk Constituents
3.1 INTRODUCTION
Milk is a fluid secreted by the female of all
mammals, of which there are more than 4,000
species, for the primary function of meeting the
complete nutritional requirements of the neonate
of the species. It must supply energy (mainly
from fats and sugar [lactose]), amino acids (from
proteins), vitamins, and atomic elements (com-
monly but inaccurately referred to as minerals).
In addition, several physiological functions are
performed by milk constituents, including anti-
microbial substances (immunoglobulins, lacto-
peroxidase, and lactotransferrin), enzymes and
enzyme inhibitors, vitamin-binding carrier pro-
teins, and cell growth and control factors. Be-
cause the nutritional and physiological require-
ments of each species are more or less unique,
the composition of milk shows very marked
interspecies differences. The milks of only about
180 species have been analyzed, and the data for
only about 50 of these species are considered to
be reliable (sufficient number of samples, repre-
sentative sampling, and adequate coverage of
the lactation period). Not surprisingly, the milks
of the principal dairying species (i.e., cow, goat,
sheep, and buffalo) and Homo sapiens are
among those that are well characterized. The
gross composition of milks from selected spe-
cies is summarized in Table 3-1. Very extensive
data on the composition of bovine and human
milks are compiled by Jensen (1995).
In addition to the principal constituents listed
in Table 3-1, milk contains several hundred mi-
nor constituents, many of which have a major
impact on the nutritional, technological, and
sensoric properties of milk and dairy products
(e.g., vitamins, small inorganic and organic ions,
and flavor compounds).
Milk is a very variable biological fluid. In ad-
dition to interspecies differences, the milk of any
particular species varies with the breed (in the
case of commercial dairying species), health,
nutritional status, stage of lactation, and age of
the animal, the interval between milkings, and so
on. In a bulked factory milk supply, variability
due to many of these factors is reduced, but
some variability persists, and it can even be quite
large in situations where milk production is sea-
sonal. In addition to variations in the concentra-
tions of the principal and minor constituents due
to the above factors, the chemistry of some of
the constituents also varies (e.g., the fatty acid
profile is strongly influenced by diet). Some of
the variability in the composition and constitu-
ents of milk can be adjusted or counteracted by
processing technology but some differences
may persist. As will become apparent in later
chapters, the variability of milk composition
poses major problems in cheese production.
Physicochemically, milk is a very complex
fluid. The constituents of milk occur in three
phases. Most of the mass of milk is an aqueous
solution of lactose, organic and inorganic salts,
Table 3-1 Composition of the Milks of Some Species

Species Total Solids (%) Fat (%) Protein (%) Lactose (%) Ash (%)

Human 12.2 3.8 1.0 7.0 0.2

Cow 12.7 3.7 3.4 4.8 0.7
Goat 12.3 4.5 2.9 4.1 0.8
Sheep 19.3 7.4 4.5 4.8 1.0
Pig 18.8 6.8 4.8 5.5
Horse 11.2 1.9 2.5 6.2 0.5
Donkey 11.7 1.4 2.0 7.4 0.5
Reindeer 33.1 16.9 11.5 2.8
Domestic rabbit 32.8 18.3 11.9 2.1 1.8
Bison 14.6 3.5 4.5 5.1 0.8
Indian elephant 31.9 11.6 4.9 4.7 0.7
Polar bear 47.6 33.1 10.9 0.3 1.4
Grey seal 67.7 53.1 11.2 0.7

vitamins, and other small molecules. In this
aqueous solution are dispersed proteins, some at
the molecular level (whey proteins), others as
large colloidal aggregates ranging in diameter
from 50 to 600 nm (the caseins), and lipids,
which exist in an emulsified state as globules
ranging in diameter from 0.1 to 20 jam. Thus,
colloidal chemistry is important in the study of
milk, for example, in the context of surface
chemistry, light scattering, and rheology.
Milk is a dynamic system owing to the insta-
bility of many of its structures (e.g., the milk fat
globule membrane); changes in the solubility of
many constituents, especially the inorganic salts
and proteins, with temperature and pH; the pres-
ence of various enzymes that can modify con-
stituents through lipolysis, proteolysis, or oxida-
tion-reduction; the growth of microorganisms,
which can cause major changes either directly
through their growth (e.g., changes in pH or re-
dox potential [Eh]), or indirectly through en-
zymes they excrete; and the interchange of gases
with the atmosphere (e.g., CO2). Milk was in-
tended to be consumed directly from the mam-
mary gland and to be expressed from the gland at
frequent intervals. However, in dairy operations,
milk is stored for various periods, ranging from a
few hours to several days, during which it is
cooled (and perhaps heated) and agitated to vari-
ous degrees. These treatments will cause some
physical changes and permit some enzymatic
and microbiological changes that may alter the
processing properties of milk. It may be possible
to counteract some of these changes.
Although many of the minor constituents of
milk are important from a nutritional standpoint,
the technological properties of milk are deter-
mined mainly by its macroconstituents (pro-
teins, lipids, and lactose) and some of its low
molecular mass species, especially calcium,
phosphate, citrate, and pH. The properties of
these constituents, with emphasis on their sig-
nificance in cheesemaking, are discussed briefly
in this chapter. For a more thorough discussion,
the reader is referred to Cayot and Lorient
(1998); Fox (1982, 1983, 1985, 1989, 1992,
1995, 1997); Fox and McSweeney (1998);
Jenness and Patton (1959); Walstra and Jenness
(1984); Webb and Johnson (1965); Webb,
Johnson, and Alford (1974); and Wong, Jenness,
Keeney, and Marth (1988).
3.2 LACTOSE
Lactose is the principal carbohydrate in the
milk of all mammals, which is the only source.
Milk contains only trace amounts of other sug-
ars, including glucose, fructose, glucosamine,
galactosamine, neuraminic acid, and neutral and
acidic oligosaccharides.
The concentration of lactose in milk varies
widely between species (Table 3-2). The lactose
content of cow milk varies with the breed of
cow, individuality factors, udder infection, and
especially stage of lactation. The concentration
of lactose decreases progressively and signifi-
cantly during lactation (Figure 3-1); this trend
contrasts with the lactational trends for lipids
and proteins, which, after decreasing during
early lactation, increase strongly during the sec-
ond half of lactation. Lactose and soluble ions
(e.g., Na+, K+, and Cl~) are the compounds
mainly responsible for the osmotic pressure of
milk. During mastitis, the concentration of NaCl
in milk increases, resulting in an increase in os-
motic pressure. This increase is compensated for
by a decrease in the lactose content; that is, there
is an inverse relationship between the concentra-
tion of NaCl and lactose in milk, which partly
explains why certain milks with a high lactose
content have a low ash content and vice versa
(see Table 3-1). The inverse relationship be-
tween the concentration of lactose and chloride
is the basis of the Koestler's chloride-lactose test
for abnormal milk:
percentage of chloride x 100
Koestler number = percentage of lactose

Table 3-2 Concentration of Lactose in the Milks A Koestler number less than 2 indicates normal
of Selected Species milk while a value greater than 3 is considered
abnormal.
Species Lactose (%) Lactose plays an important role in milk and
milk products:
California sea lion 0.0
Hooded seal 0.0 • It is essential in the production of fermented
Black bear 0.4 dairy products, including cheese.
Dolphin 0.6 • It contributes to the nutritive value of milk
Echidna 0.9 and its products. However, many non-
Blue whale 1.3 Europeans have limited or zero ability to
Rabbit 2.1 digest lactose in adulthood, leading to a
Red deer 2.6 syndrome known as lactose intolerance.
Grey seal 2.6 Mature cheese is free of lactose, and hence
Rat (Norwegian) 2.6
cheese is suitable for inclusion in the diet of
Mouse (house) 3.0
Guinea pig 3.0 lactose-intolerant individuals.
Dog (domestic) 3.1 • It affects the texture of certain concentrated
Sika deer 3.4 and frozen products.
Goat 4.1 • It is involved in heat-induced changes in the
Elephant (Indian) 4.7 color and flavor of highly heated milk prod-
Cow 4.8 ucts.
Sheep 4.8
Water buffalo 4.8
Cat (domestic) 4.8 3.2.1 Structure of Lactose
Pig 5.5 Lactose is a disaccharide consisting of galac-
Horse 6.2
tose and glucose, linked by a P1-4 glycosidic
Chimpanzee 7.0
Rhesus monkey 7.0 bond (Figure 3-2). Its systematic name is O- (3-
Human 7.0 D-galactopyranosyl-(l-4)-a-D-glucopyranose
Donkey 7.4 (a-lactose) or 0-(3-D-galactopyranosyl-(l^)-p-
Zebra 7.4 D-glucopyranose ((i-lactose). The hemiacetal
Green monkey 10.2 group of the glucose moiety is potentially free
 Per cent
Week of lactation
Figure 3-1 Changes in the concentrations of fat (•), protein (•), and lactose (O) in milk during lactation.
(i.e., lactose is a reducing sugar) and may exist
as an a- or p-anomer. In the structural formula
of the a-form, the hydroxyl group on the Ci of
glucose is cis to the hydroxyl group at C2 (ori-
ented downward) and vice versa for the P-form
(oriented upward).
3.2.2 Biosynthesis of Lactose
Lactose is essentially unique to mammary se-
cretions. It is synthesized from glucose absorbed
from blood. One molecule of glucose is con-
verted to UDP-galactose via the 4-enzyme
Leloir pathway (Figure 3-3). UDP-galactose is
then linked to another molecule of glucose in a
reaction catalyzed by the enzyme lactose syn-
thetase, a 2-component enzyme. Component A
is a nonspecific galactosyl transferase that trans-
fers the galactose from UDP-galactose to a num-
ber of acceptors. In the presence of the B compo-
nent, which is the whey protein oc-lactalbumin,
the transferase becomes highly specific for glu-
cose (its KM decreases 1,000-fold), leading to the
synthesis of lactose. Thus, oc-lactalbumin is an
enzyme modifier, and its concentration in the
milk of several species is directly related to the
concentration of lactose in those milks; the milks
of some marine mammals contain neither oc-lac-
talbumin nor lactose.
The presumed significance of this control
mechanism is to enable mammals to terminate
the synthesis of lactose when necessary, that is,
to regulate and control osmotic pressure when
there is an influx of NaCl, such as during masti-
tis or in late lactation (milk is isotonic with
blood, the osmotic pressure of which is essen-
tially constant). The ability to control osmotic
pressure is sufficiently important to justify an
elaborate control mechanism and "wastage" of
the enzyme modifier.
3.2.3 Lactose Equilibrium in Solution
The configuration around the anomeric Ci of
the glucose moiety of lactose is not stable and can
readily change (mutarotate) from the a- to the
p-form and vice versa when the sugar is in solu-
tion as a consequence of the fact that the hemiac-
 Galactose Glucose
(a)

(b)

Anomeric carbon

(c)

(d)

Figure 3-2 Structural formulae of a- and (3-lactose: (a) open chains, (b) Fischer projection, (c) Haworth projec-
tion, and (d) conformational formula.
 hexokinase
GLUCOSE
ATP ADP
P-P
UDP glucose
UDP-glucose
pyrophosphorylase
UDP glucose-4-epimerase
UDP-galactose
galactosyltransferase
a-lactalbwnin
Glucose
Figure 3-3 Pathway for lactose synthesis.
etal form is in equilibrium with the open-chain
aldehyde form, which can be converted to either
of the two isomeric forms (see Figure
3-2). When either isomer is dissolved in water,
there is a gradual change from one form to the
other until equilibrium is established (i.e., mu-
tarotation). These changes are reflected by
changes in optical rotation from +89.4° for a-
lactose or +35° for p-lactose to a value of+55.4°
at equilibrium. These values for specific rotation
indicate that at equilibrium, a solution of lactose
consists of 62.7% p anomer and 37.3% a anomer.
The a and p anomers of lactose differ mark-
edly with respect to solubility, crystal shape, hy-
dration of the crystals, hygroscopicity, specific
rotation, and sweetness.
oc-Lactose is soluble to around 7 g/100 ml
H2O at 2O0C, and the solubility of p-lactose is
around 50 g/100 ml. However, the solubility of
oc-lactose is more temperature dependent than
that of (J-lactose, and their solubility curves in-
tersect at about 940C (Figure 3-4). Thus, oc-lac-
tose is the form normally produced by crystalli-
zation. oc-Lactose crystallizes as a monohydrate,
whereas crystals of P-lactose are anhydrous. Al-
though lactose has low solubility in comparison
with other sugars, once in solution it crystallizes
Glucose-6-phosphate
Phosphoglucomutase
Glucose-1 -phosphate
UTP ADP
UDP ATP
LACTOSE
slowly, and precautions must be taken in the
manufacture of concentrated and dehydrated
products; otherwise, hygroscopicity, caking, and
a grainy texture (due to the slow growth of lac-
tose crystals to a size greater than 15 [\m) will
ensue. These physicochemical properties of lac-
tose are of major concern to manufacturers of
concentrated, dehydrated, and frozen dairy
products, but problems can be avoided by proper
manufacturing procedures. Such properties are
of no consequence in cheese, in which all the
lactose is utilized either during manufacture or
early ripening; fresh curd contains about 1% lac-
tose. The behavior of lactose is of major concern
in the manufacture of whey powders, since
around 70% of the total solids in whey are lac-
tose, and hence the properties of whey concen-
trates and powders are strongly influenced by
the properties of lactose.
In cheese, lactose is fermented to lactic acid
by lactic acid bacteria, a process which has ma-
jor, indeed vital, consequences for the manufac-
ture and quality of cheese, as is discussed in
Chapters 5, 10, and 11.
For further information on the properties of
and the problems caused by lactose, the reader
is referred to Fox (1985, 1997), Fox and
 Final solubility at equilibrium
Solubility, g anhydrous lactose /100 g water
Usual range of
supersaturation
Initial solubility of a-lactose
Temperature, 0C
Figure 3-4 Solubility of lactose in water.
McSweeney (1998), Walstra and Jenness
(1984), and Wong et al. (1988).
3.3 MILK LIPIDS
The lipid content of milk varies more widely
than any other constituent; concentrations range
from around 2% to more than 50% (Table 3-3).
The average fat content of cow, goat, sheep, and
buffalo milk is 3.5, 3.5, 6.5, and 7 g/L, respec-
tively. Within any particular species, there are
considerable variations due to breed, individual-
ity, stage of lactation, age, animal health, nutri-
tional status, interval between milking, and so
on. Among the common breeds of dairy cattle,
Jersey cows produce milk with the highest fat
content (6-7%) and Holstein/Friesian, the low-
est. Within any breed, there are considerable in-
dividual cow variations. The fat content of milk
decreases for several weeks after parturition and
then increases, especially toward the end of lac-
tation (see Figure 3-1). If the interval between
milkings is not equal, the milk obtained after the
Initial solubility of (3-lactose
shorter interval has the higher fat content. The
synthesis of all milk constituents, including fat,
decreases during a mastitic infection, and the fat
content of milk decreases slightly as the animal
ages.
The lipids in milk are predominantly triglyc-
erides (triacylglycerols), which make up about
98% of the total lipid fraction; the remaining 2%
comprises diglycerides, monoglycerides, fatty
acids, phospholipids, sterols (principally choles-
terol), and trace amounts of fat-soluble vitamins
(A, D, E, and K). Typical values for the concen-
tration of the various lipids in milk are given in
Table 3-4.
3.3.1 Fatty Acid Composition
Ruminant milk fats contain a greater diversity
of fatty acids than other fats; about 400 fatty ac-
ids have been identified in bovine milk fat. The
predominant fatty acids have a straight carbon
chain with an even number of carbon atoms and
may be saturated or unsaturated (1,2, or 3 C = C
Table 3-3 Fat Content of Milks of Various Spe- droxybutyric acid) and its reduction to bu-
cies tanoic acid by bacteria in the rumen. The
high concentration of butanoic acid in rumi-
Species Fat Content (g/L) nant milk fats provides the basis for the
method commonly used to detect and quan-
Cow 33-47
tify the adulteration of milk fat with other
Buffalo 47
Sheep 40-99 fats, that is, the Reichert-Meissl number (ml
Goat 41-45 of 0.1 IyI KOH required to neutralize the
Musk ox 109 volatile water-soluble fatty acids released
DaII sheep 32-206 from 5 g fat upon hydrolysis).
Moose 39-105 • Ruminant milk fats, in general, and ovine
Antelope 93 milk fat in particular, contain relatively
Elephant 85-190 high concentrations of middle chain fatty
Human 38 acids (hexanoic [C6:0] to decanoic [Ci0:o]).
Horse 19 This is due to high thioacylhydrolase activ-
Monkeys 10-51 ity in the fatty acid synthetase complex,
Lemurs 8-33
which causes the early release of fatty acids
Pig 68
Marmoset 77 during the chain elongation process.
Rabbit 183 • The short and middle chain acids (C410-
Guinea pig 39 Cio:o) are relatively volatile and water
Snowshoe hare 71 soluble and have a relatively low flavor
Muskrat 110 threshold. They are esterified predomi-
Mink 134 nantly at the sn3 position of glycerol and
Chinchilla 117 hence are selectively released by lipases,
Rat 103 especially by the indigenous lipoprotein Ii-
Red kangaroo 9-119 pase in milk. In milk and butter, the release
Dolphin 62-330
of these highly flavored short chain fatty
Manatee 55-215
acids gives rise to off-flavors, referred to as
Pygmy sperm whale 153
Harp seal 502-532 hydrolytic rancidity. However, when
Bear (four species) 108-331 present at an appropriate level, these short
chain acids contribute positively to the fla-
vor of cheese, especially hard Italian and
blue-mold varieties.
double bonds). There are smaller amounts of • Ruminant milk fats contain low levels of
fatty acids with an uneven number of carbon at- polyunsaturated fatty acids (PUFAs; Ci8:2,
oms, branched or cyclic hydrocarbon chains, or Ci8:3), which are considered to be nutrition-
hydroxyl or keto groups. The principal fatty ac- ally desirable. However, the low level of
ids in the milk fat of a selection of species are PUFAs makes milk fat relatively resistant
listed in Table 3-5. to oxidative rancidity. The low concentra-
The fatty acid profile of ruminant milk fats tion of PUFAs in ruminant milk fats is due
has a number of interesting features: to the hydrogenation of dietary fatty acids
by bacteria in the rumen, although ruminant
• Ruminant milk fats contain a considerable feed usually contains high levels of PUFAs.
amount of butanoic acid (C4:0) and are in On the positive side, biohydrogenation of
fact the only fats that contain this acid. The PUFAs results in lower levels of trans iso-
high content of butanoic acid is due to the mers than chemical hydrogenation, such as
synthesis of 3-hydroxybutanoic acid ((3-hy- is practiced in the processing of vegetable
Table 3-4 Composition of Individual Simple Lipids and Total Phospholipids in Milks of Various Spe-
cies (Percentage of Total Lipids by Weight)
LIpId Class Cow Buffalo
Triacylglycerols 97.5 98.6
Diacylglycerols 0.36
Monoacylglycerols 0.027
Cholesteryl esters T 0.1
Cholesterol 0.31 0.3
Free fatty acids 0.027 0.5
Phospholipids 0.6 0.5
* T = trace
oils. Trans fatty acids are considered to be
nutritionally undesirable.
The concentration of PUFAs in ruminant milk
fats can be increased by including protected lip-
ids in the animal's diet. This involves encapsu-
lating the dietary lipids in a layer of polymerized
protein or using crushed vegetable seeds. Encap-
sulation protects the PUFAs against hydrogena-
tion in the rumen, but the capsule is digested in
the abomasum, liberating the encapsulated lip-
ids, which are then metabolized, as in non-
ruminants. Fat has a major effect on the rheo-
logical properties of cheese. Polyunsaturated
lipids, which have a low melting point, have an
undesirable effect on cheese texture, but a low
level is acceptable.
Although phospholipids are present at very
low concentrations in milk, they play an impor-
tant role in the emulsification of fat in milk. Milk
contains a relatively low concentration of cho-
lesterol, a high level of which in the diet is con-
sidered to be nutritionally undesirable. The cow
transfers dietary carotenoids to its milk, and
hence its milk fat has a yellow color, the inten-
sity of which depends on the concentration of
carotenoids in the animal's feed—fresh grass
and especially clover and lucerne are rich in
carotenoids (see Fox and McSweeney, 1998, for
the structures of the principal phospholipids,
cholesterol, and fat-soluble vitamins). Sheep
and goats do not transfer dietary carotenoids to
their milk, and consequently their milk fat and
Human Pig Rat Mink
98.2 96.8 87.5 81.3
0.7 0.7 2.9 1.7
T* 0.1 0.4 T
T 0.06 T
0.25 0.6 1.6 T
0.4 0.2 3.1 1.3
0.26 1.6 0.7 15.3
fat-containing products (including cheese) made
from ovine or caprine milk are much whiter than
their bovine counterparts. Products traditionally
made from ovine or caprine milk may be unac-
ceptable when made from bovine milk, owing to
their yellow color, especially if the cows are fed
on fresh grass. However, it is possible to bleach
or mask the color of carotenoids (e.g., using
H2O2, benzoyl peroxide, TiO2, or chlorophyll).
Some carotenoids are precursors of vitamin A
(retinol). Milk contains low levels of vitamin D,
and liquid milk products are commonly fortified
with vitamin D. Milk contains a substantial
amount of vitamin E (tocopherols), which is a
potent antioxidant. The tocopherol content of
milk may be increased by supplementing the
animal's diet with tocopherols; this may be done
for nutritional or stability reasons. However, lipid
oxidation is not a problem in cheese, probably
because of its low redox potential (Eh: -150 mV).
3.3.2 Milk Fat as an Emulsion
Lipids are insoluble in and less dense than
water (the specific gravity of fat and skim milk is
around 0.9 and 1.036, respectively), and hence
they would be expected to form a layer on the
surface of milk. Lipids in general can be made
compatible with water by forming an emulsion
through homogenization, in which the fat is dis-
persed as small globules, each of which is sur-
rounded by a layer of emulsifier. An emulsion is
defined as a two-phase system, one phase (the
Table 3-5 Principal Fatty Acids in Milk Triacylglycerols or Total Lipids of Various Species (Percentage of Total by Weight)

Species 4:0 6:0 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 C/20— U£2

Cow 3.3 1.6 1.3 3.0 3.1 9.5 26.3 2.3 14.6 29.8 2.4 0.8 T*
Buffalo 3.6 1.6 1.1 1.9 2.0 8.7 30.4 3.4 10.1 28.7 2.5 2.5 T
Sheep 4.0 2.8 2.7 9.0 5.4 11.8 25.4 3.4 9.0 20.0 2.1 1.4
Goat 2.6 2.9 2.7 8.4 3.3 10.3 24.6 2.2 12.5 28.5 2.2
Musk ox T 0.9 1.9 4.7 2.3 6.2 19.5 1.7 23.0 27.2 2.7 3.0 0.4
DaII sheep 0.6 0.3 0.2 4.9 1.8 10.6 23.0 2.4 15.5 23.1 4.0 4.1 2.6
Moose 0.4 T 8.4 5.5 0.6 2.0 28.4 4.3 4.5 21.2 20.2 3.7
Blackbuck antelope 6.7 6.0 2.7 6.5 3.5 11.5 39.3 5.7 5.5 19.2 3.3
Elephant 7.4 0.3 29.4 18.3 5.3 12.6 3.0 0.5 17.3 3.0 0.7
Human T T 1.3 3.1 5.1 20.2 5.7 5.9 46.4 13.0 1.4 T
Monkey (mean
of six species) 0.4 0.6 5.9 11.0 4.4 2.8 21.4 6.7 4.9 26.0 14.5 1.3
Baboon 0.4 5.1 7.9 2.3 1.3 16.5 1.2 4.2 22.7 37.6 0.6
Lemur macaco 0.2 1.9 10.5 15.0 27.1 9.6 1.0 25.7 6.6 0.5
Horse T 1.8 5.1 6.2 5.7 23.8 7.8 2.3 20.9 14.9 12.6
Pig 0.7 0.5 4.0 32.9 11.3 3.5 35.2 11.9 0.7
Rat 1.1 7.0 7.5 8.2 22.6 1.9 6.5 26.7 16.3 0.8 1.1
Guinea pig T 2.6 31.3 2.4 2.9 33.6 18.4 5.7 T
Marmoset 8.0 8.5 7.7 18.1 5.5 3.4 29.6 10.9 0.9 7.0
Rabbit T 22.4 20.1 2.9 1.7 14.2 2.0 3.8 13.6 14.0 4.4 T
Cottontail rabbit 9.6 14.3 3.8 2.0 18.7 1.0 3.0 12.7 24.7 9.8 0.4
European hare T 10.9 17.7 5.5 5.3 24.8 5.0 2.9 14.4 10.6 1.7 T
Mink 0.5 3.3 26.1 5.2 10.9 36.1 14.9 1.5
Chinchilla T 3.0 30.0 35.2 26.8 2.9
Red kangaroo 0.1 2.7 31.2 6.8 6.3 37.2 10.4 2.1 0.1
Platypus 1.6 19.8 13.9 3.9 22.7 5.4 7.6 12.2
Numbat 0.1 0.9 14.1 3.4 7.0 57.7 7.9 0.1 0.2
Bottle-nosed dolphin 0.3 3.2 21.1 13.3 3.3 23.1 1.2 0.2 17.3
Manatee 0.6 3.5 4.0 6.3 20.2 11.6 0.5 47.0 1.8 2.2 0.4
Pygmy sperm whale 3.6 27.6 9.1 7.4 46.6 0.6 0.6 4.5
Harp seal 5.3 13.6 17.4 4.9 21.5 1.2 0.9 31.2
Northern elephant seal 2.6 14.2 5.7 3.6 41.6 1.9 29.3
Polar bear T T 0.5 3.9 18.5 16.8 13.9 30.1 1.2 0.4 11.3
Grizzly bear T 0.1 2.7 16.4 3.2 20.4 30.2 5.6 2.3 9.5

* T = trace.
discontinuous, dispersed, phase) being dispersed
in the other (the continuous phase) and separated
by a layer of emulsifier. In milk and cream, fat is
the emulsified phase and water (or, more cor-
rectly, skim milk) is the continuous phase (i.e.,
milk is an oil-in-water emulsion). In butter (and
margarine), the situation is reversed: water drop-
lets are dispersed in a continuous oil/fat phase
(i.e., butter is a water-in-oil emulsion).
Emulsifiers are amphipathic molecules, with
hydrophobic (lipophilic, fat-loving) and hydro-
philic (water-loving) domains. The principal
natural emulsifiers are polar lipids and proteins;
in addition, numerous synthetic emulsifiers are
available and are used widely in the manufacture
of high-fat foods.
In milk, the fat exists as globules, 0.1-20 jam
in diameter (mean diameter, 3-4 urn). Numeri-
cally, most of the globules have a diameter less
than 1 jam, but these small globules represent
only a small fraction of the mass of milk fat. The
globules are surrounded by a structured mem-
brane, referred to as the milk fat globule mem-
brane (MFGM), consisting mainly of phospho-
lipids and proteins; the approximate composition
of the MFGM is summarized in Table 3-6. The
inner layers of the membrane are acquired within
the secretory cell (mammocyte) as the fat glob-
ules move from the site of biosynthesis (i.e., the
rough endoplasmic reticulum located toward the
base of the cell) toward the apical membrane,
through which they are expressed into the lumen
of the mammary alveoli by exocytosis. During
exocytosis, the fat globules become surrounded
Table 3-6 Gross Composition of the Milk Fat Globule Membrane
Amount in
Fat Globule
Component (mgWOg-1)
Protein 900
Phospholipid 600
Cerebrosides 80
Cholesterol 40
Neutral glycerides 300
Water 280
Total 2,200
by the apical cell membrane, which therefore
forms the outer layer of the MFGM of freshly
secreted milk fat globules. However, much of
this membrane, which has a typical trilaminar
fluid mosaic structure, is lost as the milk ages,
and much of it accumulates as lipoprotein par-
ticles, sometimes referred to as microsomes, in
the skim milk phase.
Many of the indigenous enzymes in milk are
constituents of the MFGM; consequently, iso-
lated membrane (prepared by de-emulsification
[churning] and washing) serves as the source
material for the isolation of many indigenous milk
enzymes. Xanthine oxidase is one of the principal
proteins of the MFGM. Two notable exceptions
are the principal indigenous proteinase plasmin
and lipoprotein lipase (LPL), which are associ-
ated mainly with the casein micelles. The MFGM
isolates and protects the triglycerides from LPL
but if the membrane is damaged (e.g., by agita-
tion), the enzyme and its substrate come into con-
tact, and lipolysis and hydrolytic rancidity ensue,
with undesirable consequences for the organolep-
tic quality of milk and many dairy products.
Although the milk fat emulsion is stable to
phase separation, it does exhibit rapid creaming
owing to the difference in density between the
phases (i.e., the fat globules rise to the surface
but remain discrete and can be redispersed by
gentle agitation). The rate of creaming is gov-
erned by Stokes' law:
Amount in
Fat Globule Percentage of Total
Surface (mg rrr2) Membrane by Weight
4.5 41
3.0 27
0.4 3
0.2 2
1.5 14
1.4 13
11.0 100
where v is the velocity of particle movement; r is
the radius of the globules; p1 and p2 are the den-
sities of the continuous and dispersed phases, re-
spectively; g is the acceleration due to gravity
(9.8 m s~2); and T] is the viscosity coefficient of
the emulsion.
For milk, the parameters of Stokes' law would
suggest that a cream layer would form after
about 60 hr, but in fact it forms in about 30 min.
This large discrepancy between the actual and
the predicted rate of creaming is due to floccula-
tion of the fat globules: the large globules rise
faster than and collide with smaller globules,
and the globules form clusters owing to the ag-
glutinating action of immunoglobulin M. This
protein is referred to as a cryoglobulin, since it
adsorbs onto the fat globules as the temperature
is reduced. The cluster then rises as a unit, col-
liding with other globules as it does so and there-
fore rising at an accelerating rate. The cryo-
globulins solubilize as the temperature is
increased and are fully soluble above 370C.
Consequently, creaming is promoted by low
temperatures and is very slow above 370C.
Cryoglobulins are denatured and inactivated by
heating at time-temperature treatments greater
than 740C x 15 s; hence, severely pasteurized
milk creams poorly or not at all. Sheep, goat, and
buffalo milks are devoid of cryoglobulins and
hence cream very slowly.
If the MFGM is physically damaged by high
temperatures and/or agitation, the globules coa-
lesce, and eventually phase inversion will occur
(i.e., an oil-in-water emulsion is converted to a
water-in-oil emulsion). Free (nonglobular) fat
will float on the surface. Such damage occurs to
at least some extent during cheesemaking; the
free fat is not incorporated into the coagulum
and floats as quite large masses on the surface of
the whey and is lost to the cheese. About 10% of
the fat in milk is normally lost in this way. It can
be recovered from the whey by centrifugation
and made into whey butter or other products.
Milk for many dairy products is "homog-
enized," usually by using a valve homogenizer.
Homogenization reduces the size of the fat glob-
ules (average diameter, less than 1 |um) and dena-
tures the cryoglobulins, and homogenized milk
does not cream owing to the combined effects of
globule size reduction and denaturation of cryo-
globulins. The membrane on the fat globules in
homogenized milk is mainly casein and does not
protect the triglycerides against lipolysis. Ho-
mogenized milk must therefore be pasteurized
before or immediately after homogenization to
prevent the occurrence of hydrolytic rancidity.
Milk for cheesemaking is not normally ho-
mogenized, because homogenized milk forms a
rennet coagulum (gel) with a lower tendency to
undergo syneresis upon cutting or stirring than
that from nonhomogenized milk. Homogeniza-
tion results in cheese with a higher moisture con-
tent. This situation arises because the casein-
coated fat globules behave somewhat like casein
micelles, but they limit the contraction of the
casein matrix. It may be advantageous to ho-
mogenize milk for low-fat cheese so as to obtain
a higher moisture content and thus soften the
texture of the cheese. In some cases, milk for
Blue cheese is separated and the cream homog-
enized to promote lipolysis (which is desirable
in Blue cheese). The lipolysed cream and skim
milk are then combined and pasteurized before
cheese manufacture. Milk for yogurt and cream
cheese is also homogenized to
• prevent creaming during the relatively long
gelation period
• increase the effective protein concentration
by converting the fat globules to pseudo-
protein particles, thereby giving a firmer
gel for a given level of protein
• minimize syneresis
Fat plays an essential role in cheese quality:
• It acts as a plasticizer and affects cheese
texture (low-fat cheese has a hard, crumbly
texture).
• It serves as a source of fatty acids, which
have a direct effect on cheese flavor and are
changed to other flavor compounds (e.g.,
carbonyls, lactones, esters, and thioesters).
• It acts as a solvent for flavor compounds
produced from lipids, proteins, or lactose.
With the objective of reducing the calorific
content of cheese, there is considerable commer-
Table 3-7 Protein Content in the Milk of Various one of the groups precipitates; these are known
Species as caseins. The proteins that remain soluble un-
der these conditions are known as serum or
Casein Whey Total whey proteins. Approximately 80% of the total
Species (%) Protein (%) (%) nitrogen in bovine, ovine, caprine, and buffalo
milks is casein, but casein constitutes only about
Bison 3.7 0.8 4.5
Black bear 8.8 5.7 14.5 40% of the protein in human milk. Both caseins
Camel (bactrian) 2.9 1.0 3.9 and whey proteins are heterogeneous and have
Cat - - 11.1 very different molecular and physicochemical
Cow 2.8 0.6 3.4 properties.
Domestic rabbit 9.3 4.6 13.9
Donkey 1.0 1.0 2.0 3.4.1 Caseins
Echidna 7.3 5.2 12.5
Goat 2.5 0.4 2.9 Bovine casein consists of four types of protein
Grey seal - - 11.2 with substantially different properties: asl-,
Guinea pig 6.6 1.5 8.1
ocS2-> P~» and K~; these make up approximately
Hare - - 19.5
38%, 10%, 34%, and 15%, respectively, of whole
Horse 1.3 1.2 2.5
House mouse 7.0 2.0 9.0 casein. The caseins are well characterized at the
Human 0.4 0.6 1.0 molecular level (some of the major properties are
Indian elephant 1.9 3.0 4.9 summarized in Table 3-8), and the amino acid
Pig 2.8 2.0 4.8 sequences are known (Figures 3-6 to 3-9). Some
Polar bear 7.1 3.8 10.9 of the more important properties of the caseins are
Red kangaroo 2.3 2.3 4.6 as follows:
Reindeer 8.6 1.5 10.1
Rhesus monkey 1.1 0.5 1.6 • They are quite small molecules, with mo-
Sheep 4.6 0.9 5.5 lecular masses of 20-25 kDa.
White-tailed • All are phosphorylated. Most molecules of
jack rabbit 19.7 4.0 23.7
ocsi-casein contain 8 mol PO4/mol of pro-
tein, but some contain 9 mol PO4/mol.
cial interest in the production of low-fat cheeses, (3-Casein molecules usually contain 5 mol
but the quality of such cheeses is reduced, and PO4/mol, but some contain 4 mol PO4/mol.
consequently they have had only limited market- ocs2-Casein molecules contain 10, 11, 12, or
ability. 13 mol PO 4 /mol. Most molecules of
K-casein contain 1 mol PO4/mol, but some
3.4 MILK PROTEINS contain 2 or perhaps 3 mol PO4AnOl.
• The phosphate groups are esterified as mo-
From a cheesemaking standpoint, the proteins noesters of serine and most occur as clus-
of milk are its most important constituents. The ters. The phosphate groups bind polyvalent
protein content of milk shows large interspecies cations strongly, causing charge neutraliza-
differences, ranging from about 1% for human tion and precipitation of ocsr, ocs2-, and (3-
milk to more than 20% for the milk of small caseins at greater than 6 mM Ca2+ at 3O0C.
mammals such as mice and rats (Table 3-7). K-Casein, which usually contains only 1
There is a good correlation between the protein mol PO4/mol, binds cations weakly and is
content of milk and the growth rate of the neo- not precipitated by them. It can stabilize up
nate of that species (Figure 3-5). to 10 times its weight of calcium-sensitive
The proteins of milk belong to two main cat- caseins via the formation of micelles (see
egories that can be separated based on their solu- Section 3.4.2). In milk, the principal cation
bility at pH 4.6 at 2O0C. Under these conditions, bound is calcium.
 Cat
Rat
Rabbit
Dog
Pig
Sheep
Goat
Calories from protein, %
Horse
Reindeer
Figure 3-5 Relationship between the growth rate (days to double birthweight) of the young of some species of
mammal and the protein content (expressed as the percentage of total calories derived from protein) of the milk of
that species.
• Only ocs2- and K-caseins contain cysteine,
which normally exists as intermolecular
disulphide bonds. as2-Casein usually occurs
as disulphide-linked dimers, but up to at
least 10 K-casein molecules may be di-
sulphide linked. The absence of cysteine or
cystine in asr and p-caseins increases the
flexibility of these molecules.
• All the caseins, especially P-casein, contain
relatively high levels of proline. In
p-casein, 35 of the 209 residues are proline,
and these are uniformly distributed through-
out the molecule. The presence of a high
level of proline prevents the formation of
secondary structures (ex-helices, p-sheets).
• Experimental techniques indicate that the
caseins have low levels of secondary and
tertiary structures, although theoretical cal-
culations indicate that they do have some
degree of higher structure. It has been sug-
gested that, rather than lacking secondary
Cow
Buffalo
Man
Days
structures, the caseins have very flexible
structures, which have been described as
rheomorphic. The lack of stable secondary
and tertiary structures renders the caseins
stable to denaturing agents (e.g., heat or
urea); contributes to their surface activity
properties; and makes them readily suscep-
tible to proteolysis, which is important in
cheese ripening.
• The caseins are relatively hydrophobic but
have high surface hydrophobicity (owing to
their open structures) rather than a high to-
tal hydrophobicity. The hydrophobic, polar,
and charged residues are not uniformly dis-
tributed throughout the molecular se-
quences but occur as hydrophobic or hydro-
philic patches (Figure 3-10), giving the
caseins strongly amphiphatic structures that
make them highly surface active. The
N-terminal 2/3 of K-casein, which is par-
ticularly significant in cheese manufacture,
Table 3-8 Amino Acid Composition of the Principal Proteins in Milk

Amino GVr OC52- P- K- 71- y2- 7 3_

p-Lacto a-Lact
Acid Casein B Casein A Casein A2 Casein B Casein A2 Casein A2 Casein A Globulin A Albumin B

Asp 7 4 4 4 4 2 2 11 9
Asn 8 14 5 7 3 1 1 5 12
Thr 5 15 9 14 8 4 4 8 7
Ser 8 6 11 12 10 7 7 7 7
SerP 8 11 5 1 1 O O O O
GIu 24 25 18 12 11 4 4 16 8
GIn 15 15 21 14 21 11 11 9 5
Pro 17 10 35 20 34 21 21 8 2
GIy 9 2 5 2 4 2 2 3 6
Ala 9 8 5 15 5 2 2 14 3
1/2 Cys O 2 O 2 O O O 5 8
VaI 11 14 19 11 17 10 10 10 6
Met 5 5 6 2 6 4 4 4 1
He 11 11 10 13 7 3 3 10 8
Leu 17 13 22 8 19 14 14 22 13
Tyr 10 12 4 9 4 3 3 4 4
Phe 8 6 9 4 9 5 5 4 4
Trp 2 2 1 1 1 1 1 2 4
Lys 14 24 11 9 10 4 3 15 12
O
His 5 3 5 ^ 5 4 3 2 3
Arg 6 6 4 5 2 2 2 3 1
PyroGlu O O O 1 O O O O O

Total residues 199 207 169 209 181 104 102 162 123
Molecular weight 23,612 25,228 19,005 23,980 20,520 1 1 ,822 11,557 18,362 14,174
HOave (kJ/residue) 4.89 4.64 5.12 5.58 5.85 6.23 6.29 5.03 4.68
 1 Glu-Val-Leu-Asn-Glu-Asn-Leu-
H. Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln-

21
Leu-Arg-Phe-Phe-Val-Ala-(Variants B, C, D, E)
-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys-Glu-Lys-Val-Asn-Glu-Leu
(Variant A)

41 Ala (Variants A, B, C, E) GIn (Variants A, B, C, D)

Ser-Lys-Asp-Ile-Gly-StrP-Glu-SerP-Thr-Glu-Asp-Gln- -Met-GIu-Asp-Ile-Lys- -Met
ThrP (Variant D) GIu (Variant E)
61
Glu-Ala-Glu-SerP-Ile-ScrP-SerP-ScrP-Glu-Glu-Ile-Val-Pro-Asn-SerP-Val-Glu-Gln-Lys-His-

81
Ile-Gln-Lys-Glu-Asp-Val-Pro-Ser-Glu-Arg-Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg-

101
Leu-Lys-Lys-Tyr-Lys-Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn-SerP-Ala-Glu-Glu-Arg-Leu-

121
His-Ser-Met-Lys-Glu-Gly-Ile-His-Aia-Gln-Gln-Lys-Glu-Pro-Met-Ile-Gly-Val-Asn-Gln-

141
Glu-Leu-Ala-Tyr-Phe-Tyr-Pro-Glu-Leu-Phe-Arg-Gln-Phe-Tyr-Gln-Leu-Asp-Ala-Tyr-Pro-

161
Ser-Gly-Ala-Trp-Tyr-Tyr-Val-Pro-Leu-Gly-Thr-Gln-Tyr-Thr-Asp-Ala-Pro-Ser-Phe-Ser-

181 GIu (Variant A, B, D) 199

Asp-Ile-Pro-Asn-Pro-Ile-Gly-Ser-Glu-Asn-Ser- -Lys-Thr-Thr-Met-Pro-Leu-Trp. OH
GIy (Variant C, E)

Figure 3-6 Amino acid sequence of asl-casein, showing the amino acid substitutions or deletions in the principal
genetic variants.

is strongly hydrophobic, whereas the C-ter- saccharides attached to the C-terminal of

minal 1/3 is strongly hydrophilic. The hy-
drophobicity of the caseins explains why
their hydrolysates have a high propensity to
bitterness, which is one of the principal de-
fects in many cheese varieties.
• K-Casein is glycosylated (asr, as2-, and p-
caseins are not). It contains galactose, ga-
lactosamine, and 7V-acetylneuraminic acid
(sialic acid), which occur as either trisac-
charides or tetrasaccharides attached to
threonine residues in the C-terminal re-
gion. K-Casein may contain O to 4 tri- or
tetrasaccharides moieties (i.e., 10 variants
of K-casein exist). The presence of oligo-
K-casein increases the hydrophilicity of
that region.
• All the caseins exhibit genetic polymor-
phism that involves the substitution of 1 or
2 amino acids and rarely the deletion of a
segment. The variant or variants present in
milk are determined by simple Mendelian
genetics. The presence of certain genetic
variants in milk has a significant effect on
the cheesemaking properties of the milk.
The preceding indicates that the casein system
is extremely heterogeneous and that a logical
nomenclature system is necessary. The follow-
 1
H. Lys-Asn-Thr-Met-Glu-His-Val-SerP-SerP-SerP-Glu-Glu-Ser-Ile-Ile-SerP-Gln-Glu-Thr-Tyr-

21
Lys-Gln-Glu-Lys-Asn-Met-Ala-Ile-Asn-Pro-Ser-Lys-Glu-Asn-Leu-Cys-Ser-Thr-Phe-Cys-

41
Lys-Glu-VaI-Val-Arg-Asn-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-SerP-SerP-SerP-Glu-Glu-

61
SerP-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys-Ile-Thr-Val-Asp-Asp-Lys-His-Tyr-Gln-Lys-

81
Ala-Leu-Asn-Glu-Ile-Asn-Gln-Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr-Leu-Gln-Tyr-Leu-Tyr-

101
Gln-Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp-Asp-Gin-Val-Lys-Arg-Asn-Ala-Val-Pro-Ile-Thr-

121
Pro-Thr-Leu-Asn-Arg-Glu-Gln-Leu-SerP-Thr-ScrP-Glu-Glu-Asn-Ser-Lys-Lys-Thr-Val-Asp-

141
Met-Glu-Sei-P-Thr-Glu-Val-Phe-Thr-Lys-Lys-Thr-Lys-Leu-Thr-Glu-Glu-Glu-Lys-Asn-Arg-

161
Leu-Asn-Phe-Leu-Lys-Lys-Ile-Ser-Gln-Arg-Tyr-Gln-Lys-Phe-Ala-Leu-Pro-Gln-Tyr-Leu-

181
Lys-Thr-Val-Tyr-Gln-His-Gln-Lys-Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys-Thr-Lys-Val-
(Leu)
201 207
Ile-Pro-Tyr-Val-Arg-Tyr-Leu. OH

Figure 3-7 Amino acid sequence of bovine ocs2-casein A, showing 11 of the 13 potential phosphorylation sites.

ing nomenclature has been adopted: The casein
family is indicated by a Greek letter with a sub-
script, if necessary: ocsr, ocs2-, P-, K-. The Greek
letter is followed by CN, and the genetic variant
is indicated by a Latin letter (A, B, C, etc.) with a
superscript, if necessary: asrCN B, p-CN A1.
Finally, the number of phosphate residues is in-
dicated: OC81-CN B-8P, p-CN Al-5P.
Minor components of the casein system are
the y-caseins, which are C-terminal fragments of
p-casein produced through the action of the in-
digenous proteinase plasmin. The N-terminal
fragments are included in the so-called proteose
peptone fraction of milk protein. These peptides
are summarized in Figure 3-11.
Ovine, caprine, and buffalo caseins generally
exhibit heterogeneity similar to that of the bo-
vine caseins.
3.4.2 Casein Micelles
As mentioned earlier, ocsr, ocs2-, and p-caseins,
which together constitute about 85% of whole
casein, are precipitated by concentrations of Ca
greater than 6 mM. Since bovine milk contains
about 30 mM Ca, it might be expected that these
caseins would precipitate in milk. However, K-
casein, which contains only 1 mol PCVmol, is
insensitive to Ca2+ and, moreover, can stabilize
up to 10 times its weight of the Ca-sensitive
caseins against precipitation by Ca2+. It does this
via the formation of a type of quaternary struc-
ture, referred to as the casein micelle.
The principal properties of the casein micelle
are summarized in Table 3-9. Many attempts
have been made to elucidate the structure of the
micelle. The most widely accepted view is that
 i
H.Arg-Glu-Leu-Glu-Glu-LeuAsn-Val-Pro-Gly-GIu-Ile-VaI"GIu-5erP-Leu5er/>-5erP-5erP-GIu-

21 *—^ Yi -caseins (Variant C)

GIu-Ser-Ile-Thr-Arg-Ile-Asn-Lyskys-Ile-Glu-Lys-Phe-Gln-Ser -Glu-LyS-GIn-Gln-Gln-
SerP GIu
(Variants A, B)
41
Thr-Glu-Asp-Glu-Leu-Gln-Asp-Lys-Ile-His-Pro-Phe-Ala-GIn-Thr-Gln-Ser-Leu-Val-Tyr-

61
Pro (Variants A2, A3)
Pro-Phe-Pro-Gly-Pro-Ile- -Asn-Ser-Leu-Pro-GIn-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr
His (Variants C, A1' and B)
81

Pro- VaI-VaI- Val-Pro-Pro-Phe-Leu-Gln-Pro-Glu- Val-Met-Gly- Val-Ser-Lys- Val-Lys-Glu-

^ Y3-caseins
101(Variants A1, A2, B, C) His I
Ala-Met-Ala-Pro-Lvsr -LyslGlu-Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe-Thr-
(VariantA5)|Gln
121
Ser (Variants A, Q ^2 -caseins
GIu- -Gln-Ser-Leu-Thr-Leu-Thr-Asp-Val-Glu-Asn-Leu-His-Leu-Pro-Leu-Pro-Leu-Leu-
Arg (Variant B)
141

Gln-Ser-Trp-Met-His-GIn-Pro-His-Gln-Pro-Leu-Pro-Pro-Thr-Val-Met-Phe-Pro-Pro-Gln-
161

Ser-Val-Leu-Ser-Leu-Ser-Gln-Ser-Lys-Val-Leu-Pro-Val-Pro-Gln-Lys-AIa-Val-Pro-Tyr-

181

Pro-Gln-Arg-Asp-Met-Pro-Ile-Gln-Ala-Phe-Leu-Leu-Tyr-GIn-Glu-Pro-Val-Leu-Gly-Pro-

201 209

Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-VaLOH

Figure 3-8 Amino acid sequence of bovine p-casein, showing the amino acid substitutions in the genetic vari-
ants and the principal plasmin cleavage sites (T).

the micelles are composed of submicelles of
mass around 5 x 106 kDa. The core of the
submicelles is considered to consist of the Ca-
sensitive asr, as2-, and p-caseins, with variable
amounts of K-casein located principally on the
surface of the submicelles. The K-casein-defi-
cient submicelles are located in the center of the
micelles, and the K-casein-rich submicelles are
concentrated at the surface. The hydrophobic N-
terminal segment of K-casein is considered to in-
teract hydrophobically with the Ca-sensitive
caseins, with the hydrophilic C-terminal seg-
ment protruding from the surface, giving the
whole micelle a hairy appearance (Figures 3-12
and 3-13). The colloidal stability of the micelles
is attributed to a zeta potential of about -20 mV
at 2O0C and the steric stabilization provided by
the protruding hairs. The submicelles are consid-
ered to be held together by microcrystals of cal-
cium phosphate and perhaps hydrophobic and
hydrogen bonds.
Although this model of the casein micelle is
not universally accepted, it is adequate to ex-
plain many of the technologically important
properties of the micelles, including rennet co-
agulation, which follows the specific hydrolysis
 1
Pyro-Glu-Glu-Gln-Asn-Gln-Glu-Gln-Pro-Ile-Arg-Cys-Glu-Lys-Asp-Glu-Arg-Phe-Phe-Ser-Asp-

21
Lys-Ile-Ala-Lys-Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg-Tyr-Pro-Ser-Tyr-Gly-Leu-

41
Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu-Ile-Asn-Asn-Gln-Phe-Leu-Pro-Tyr-Pro-Tyr-

61
Tyr-Ala-Lys-Pro-Ala-Ala-Val-Arg-Ser-Pro-Ala-Gln-Ile-Leu-Gln-Trp-Gln-Val-Leu-Ser-

81
Asn-Thr-Val-Pro-Ala-Lys-Ser-Cys-Gln-Ala-Gln-Pro-Thr-Thr-Met-Ala-Arg-His-Pro-His-

101 105|l06
Pro-His-Leu-Ser-Ph^Met-Ala-Ile-Pro-Pro-Lys-Lys-Asn-Gln-Asp-Lys-Thr-Glu-Ile-Pro-

121 He (Variant B)
Thr-Ile-Asn-Thr-Ile-Ala-Ser-Gly-Glu-Pro-ITir-Ser-T/ir-Pro-TTzr- -Glu-Ala-Val-Glu-
Thr (Variant A)

141 Ala (Variant B)

Ser-Thr-Val-Ala-Thr-Leu-Glu- - SerP-Pro-Glu-Val-Ile-Glu-Ser-Pro-Pro-Glu-Ile-Asn-
Asp (Variant A)

161 169
Thr-Val-Gln-Val-Thr-Ser-Thr-Ala-VaLOH

Figure 3-9 Amino acid sequence of bovine K-casein, showing the amino acid substitutions in genetic poly-
morphs A and B and the chymosin cleavage site, (T). The sites of posttransitional phosphorylation or
glycosylation are italicized.

of the micelle-stabilizing K-casein, as a result of heating (e.g., completely at 9O0C x 10 min).

which the stabilizing surface layer is lost.
As far as is known, the structure of the casein
micelles in bovine, ovine, caprine, and buffalo
milks is essentially similar.
3.4.3 Whey Proteins
The whey protein fraction of bovine, ovine,
caprine, and buffalo milk contains four main
proteins: (3-lactoglobulin (P-Ig, 50%), oc-lactal-
bumin (oc-la, 20%), blood serum albumin (BSA,
10%), and immunoglobulins (Ig, 10%; mainly
IgGi, with lesser amounts of IgG2, IgA, and
IgM). Human milk contains no p-lg and the prin-
cipal Ig is IgA.
The principal properties of the whey proteins
are listed in Table 3-8. In contrast to the caseins,
the whey proteins possess high levels of second-
ary, tertiary, and quaternary structures. They are
typical globular proteins and are denatured upon
They are not phosphorylated and are insensitive
to Ca2+. All whey proteins contain intramolecu-
lar disulfide bonds that stabilize their structure.
P-Lg contains one sulfydryl group that under
certain conditions can undergo sulfydryl-disul-
fide interactions with other proteins; the most
important of these interactions, with K-casein,
occurs upon heating at about 750C x 15 s. The
latter can markedly impair the rennet coagula-
tion properties of milk and alter the gel structure
and rheological and synertic properties of acid
gel-based products such as yogurt and fresh
cheeses.
The whey proteins are not directly involved in
cheese manufacture. However, they are indi-
rectly involved, as in these examples:
• Heat-induced interaction of whey proteins
with K-casein has undesirable effects on
rennet coagulation.
Figure 3-10 Schematic representation of the distribution of hydrophobic and charged residues in the principal milk proteins.
Figure 3-11 Principal peptides produced from p-casein by plasmin.
• Whey proteins are incorporated into cheese
made from milk concentrated by ultrafiltra-
tion.
• Whey proteins are heat-denatured in the
manufacture of some Quarg products.
• Valuable functional proteins are recovered
from whey.
3.4.4 Minor Proteins
Milk contains numerous minor proteins. These
are found mainly in the whey, but some are also
found in the fat globule membrane. These minor
proteins include enzymes (perhaps 60), enzyme
inhibitors, metal-binding proteins (especially
lactoferrin and osteopontin), vitamin-binding
proteins, and several growth factors. As far as is
known, most of these are of no consequence in
cheese. Some of the indigenous enzymes are ac-
tive in cheese during ripening, especially plasmin
and xanthine oxidase and possibly acid phos-
phatase. Lipoprotein lipase is probably quite im-
portant in raw milk cheese and perhaps even in
pasteurized milk cheese, since some probably
partially survives HTST (high temperature, short
time) pasteurization. The significance of other
indigenous enzymes in cheese has not been inves-
tigated and perhaps warrants study.
3.5 MILK SALTS
After milk has been heated in a muffle furnace
at around 60O0C for 5 hr, a residue (ash), repre-
senting roughly 0.7 g/100 ml of the mass of the
milk sample, remains. The ash contains the inor-
ganic salts present in the original milk plus some
elements, especially phosphorus, present origi-
nally in organic molecules, especially proteins
and phospholipids, and lesser amounts of sugar
phosphates and high-energy phosphates. The el-
ements in the ash are changed from their original
form; they are present, not as their original salts,
but as oxides and carbonates. Organic salts, the
most important of which is citrate, are lost on
ashing. Fresh milk does not contain lactic acid,
but lactic acid may be present in stored milk as a
result of microbial growth. Although the salts of
milk are quantitatively minor constituents, they
are of major significance to its technological
properties.
The typical concentration of the principal ele-
ments or compounds that constitute the salts of
Table 3-9 Average Characteristics of Casein Micelles

Characteristic Value

Diameter 120 nm (range: 50-500 nm)

Surface area 8 x 10-10cm2
Volume 2.1 x10-15cm3
Density (hydrated) 1 .0632 g cm-3
Mass 2.2x10- 15 g
Water content 63%
Hydration 3.7 g H2O g~1 protein
Voluminoslty 4.4 cm3 g-1
Molecular weight (hydrated) 1.3 x 1O9Da
Molecular weight (dehydrated) 5 x 1O8Da
Number of peptide chains 104
Number of particles per milliliter of milk 1014-1016
Surface area of micelles per milliliter of milk 5x10 4 cm 2
Mean free distance 240 nm

milk are summarized in Table 3-10. Some of
the salts are present in milk at concentrations
below their solubility limit and are therefore
fully soluble. However, others, especially cal-
cium phosphate, exceed their solubility and oc-
cur partly in solution and partly in the colloidal
phase, associated mainly with the casein mi-
celles. These salts are collectively referred to as
micellar or colloidal calcium phosphate (CCP),
although several other elements or ions are
present also. Several elements are also present
in the MFGM, mainly as constituents of en-
zymes. There are several techniques for parti-
tioning the colloidal and soluble salts (see Fox
& McSweeney, 1998). Typical distributions are
indicated in Table 3-10.
It is possible to either determine experimen-
tally or to calculate (after making certain assump-
tions) the concentration of the principal ions in
milk; these are also indicated in Table 3-10.
From a cheesemaking viewpoint, the most
important salts or ions are calcium, phosphate,
and, to a lesser extent, citrate. As shown in Table
3-10, bovine milk contains about 1200 mg Ca/L

Submicelle

Protruding
chain

Calcium
phosphate

Figure 3-12 Submicelle model of the casein micelle.

Figure 3-13 Model of the casein micelle.
(i.e., 30 mM). About 30% is soluble, most of
which occurs as un-ionized salts of citrate, but
about 30% exists as Ca2+, which means that 10%
of the total calcium exists as Ca2+ (2-3 mM). Al-
though present at low concentrations, Ca2+ are of
major significance in various aspects of the ren-
net coagulation of milk (see Chapter 6). The
[Ca2+] is inversely related to the citrate concen-
tration.
The insoluble calcium occurs mainly associ-
ated with the casein micelles, either as colloidal
calcium phosphate (CCP) or casein Ca. CCP
plays a major role in micellar integrity and has a
very significant role in rennet coagulation. The
precise composition and structure of CCP are
not known. The simplest possible structure is
tertiary phosphate, Ca3(PO4)2, but the form for
which the best experimental evidence exists is
brushite, CaHPO4.2H2O, which forms micro-
crystals with organic casein phosphate.
3.6 pH OF MILK
As will become apparent in subsequent chap-
ters, pH is a critical factor in several aspects of
the manufacture and ripening of cheese curd.
Table 3-10 Concentration and Partition of Milk Salts

Soluble

Species Concentration (mg/L) Percentage Form Colloidal (%)

Sodium 500 92 Completely ionized 8

Potassium 1,450 92 Completely ionized 8
Chloride 1,200 100 Completely ionized
Sulphate 100 100 Completely ionized
Phosphate 750 43 10% bound to Ca and Mg 57
51% H2PO4-
39% HPO42-
Citrate 1,750 94 85% bound to Ca and Mg
14%Citrate3-
1%H.citrate2-
Calcium 1,200 34 35% Ca2+ 66
55% bound to citrate
10% bound to phosphate
Magnesium 130 67 Probably similar to calcium 33

Table 3-11 Some Physical Properties of Milk

Property Value

Osmotic pressure - 700 kPa

Water activity, aw « 0.993
Boiling point -100.150C
Freezing point -0.5220C (approximately)
Redox potential, Eh (in equilibrium with air +0.25 to +0.35 V
at 250C and pH 6.6)
Refractive index, nD20 1 .3440 to 1 .3485
Specific refractive index - 0.2075
Density (2O0C) -1 030 kg x nrr3
Specific gravity (2O0C) -1.0321
Specific conductance - 0.0050 ohm-1 crrr1
Ionic strength - 0.07 M
Surface tension (2O0C) - 52 N m-1
Coefficient of viscosity 2.127 mPaxs
Thermal conductivity (2.9% fat) - 0.559 Wm- 1 ' K-1
Thermal diffusivity (15-2O0C) -1.25x10- 7 m 2 -s-1
Specific heat - 3.931 kJ • kg-1 • K~1
pH (at 250C) -6.6
Titratable acidity 1 .3-2.0 meq OH- per 100 ml
(0.14-0.16% as lactic acid)
Coefficient of cubic expansion (273-333 K) 0.0008 m3 • m-3 • K-1
 The pH of milk at 250C is usually in the range
6.5 to 7.0, with a mean value of 6.6. pH in-
creases with advancing lactation and may ex-
ceed 7.0 in very late lactation; colostrum can
have a pH as low as 6.0. The pH increases dur-
ing mastitic infection owing to the increased
permeability of the mammary gland mem-
branes, which permits greater influx of blood
constituents into the milk (the pH of cow's
blood is 7.4). The difference in pH between
blood and milk results from the active transport
of various ions into the milk; precipitation of
CCP, which results in the release of H+ during
the synthesis of casein micelles; higher concen-
trations of acidic groups in milk; and the rela-
tively low buffering capacity of milk between
pH 6.0 and 8.0.
One of the key events during the manufacture
of cheese is the production of lactic acid from
lactose by lactic acid bacteria (see Chapter 5).
Consequently, the pH decreases to about 5.0.
While lactic acid is primarily responsible for the
decrease in pH, the actual pH attained is
strongly affected by the buffering capacity of
the milk and curd.
Milk contains a range of groups that are effec-
tive in buffering over a wide pH range. The prin-
cipal buffering compounds in milk are its salts
(particularly soluble phosphate, citrate, and bi-
carbonate) and acidic and basic amino acid side-
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Cayot, P., & Lorient, D. (1998). Structures et techno-
fonctions desproteins du lait. Paris: Lavoisier.
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Vol. L Proteins. London: Applied Science Publishers.
Fox, P.F. (Ed.). (1983). Developments in dairy chemistry:
Vol. 2. Lipids. London: Applied Science Publishers.
Fox, P.F. (Ed.). (1985). Developments in dairy chemistry:
Vol. 3. Lactose and minor constituents. London: Elsevier
Applied Science Publishers.
Fox, P.F. (Ed.). (1989). Developments in dairy chemistry:
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Science Publishers.
Fox, P.F. (Ed.). (1992). Advanced dairy chemistry, Vol. 1.
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chains of proteins (particularly the caseins). The
contribution of these components to the buffer-
ing of milk is discussed in detail by Singh,
McCarthy, and Lucey (1997).
The buffering capacity of milk and curd is of
significance during cheesemaking, since it is the
factor that determines the rate of decrease in pH
caused by the production of lactic acid by the
starter. The buffering capacity of milk is low
near its natural pH but increases rapidly to a
maximum at about pH 5.1. This means that,
given a steady rate of acidification by the starter,
the pH of milk decreases rapidly initially and
later slows down. Since all of the soluble and
some of the colloidal calcium phosphate are lost
in the whey, it is not surprising that the buffering
properties of cheese differ from those of milk.
Cheddar and Emmental cheeses have maximum
buffering capacities at around pH 4.8.
3.7 PHYSICOCHEMICAL PROPERTIES
OF MILK
Information on the physicochemical proper-
ties of milk is important when developing and
processing dairy products, designing processing
equipment, and using dairy products in food
products. Some of the principal physicochemi-
cal properties are summarized in Table 3-11.
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Fox, P.F., & McSweeney, P.L.H. (1998). Dairy chemistry
and biochemistry. London: Chapman & Hall.
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chemical properties of milk. In P.F. Fox (Ed.), Advanced
dairy chemistry: Vol. 3. Lactose, water, salts and vita-
mins. London: Chapman & Hall.
Walstra, P., & Jenness, R. (1984). Dairy chemistry and phys-
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of dairy chemistry. Westport, CT: AVI Publishing.
Webb, B.H., Johnson, A.H., & Alford, J.A. (Eds.). (1974).
Fundamentals of dairy chemistry (2d ed.). Westport, CT:
AVI Publishing.
Wong, N.P., Jenness, R., Keeney, M., & Marth, E.H. (Eds.).
(1988). Fundamentals of dairy chemistry (3d ed.).
Westport, CT: AVI Publishing.