CHAPTER

1
Chemistry and Physics
H. D. GoffandA. R. Hill
1.1 Introduction, 2
1.2 Composition, 5
1.2.1 Proteins, 9
1.2.1.1 Caseins, 9
1.2.1.2 Whey Proteins, 14
1.2.1.3 Enzymes, 15
1.2.2 Lipids, 18
1.2.2.1 Chemical Properties, 18
1.2.2.2 Physical Properties, 19
1.2.2.3 Lipolysis, 22
1.2.2.4 Oxidation, 24
1.2.3 Lactose, 26
1.2.3.1 Biochemical Properties, 26
1.2.3.2 Physicochemical Properties, 26
1.2.4 Minor Components, 28
1.2.4.1 Vitamins, 28
1.2.4.2 Minerals, 29
1.3 Structure, 30
1.3.1 Casein Micelles, 30
1.3.1.1 Properties, 30
1.3.1.2 Stability, 35
1.3.1.3 Aggregation, 38
1.3.2 Fat Globules, 41
1.3.2.1 Native Fat Globule Membrane, 41
1.3.2.2 Recombined Membranes, 44
1.3.2.3 Stability, 46
1.3.2.4 Destabilization, 48
1.4 Physical Properties, 49
1.4.1 Density, 49
1.4.2 Viscosity, 50
1.4.3 Freezing Point, 52
1.4.4 Electrochemistry, 54
1.4.4.1 Electrical Conductivity, 54
1.4.4.2 Oxidation-Reduction Potentials, 55
 1.4.5 Surface Tension, 56
1.4.6 Acid-Base Equilibria, 57
L4.7 Heat Capacity and Thermal Conductivity, 60
1.4.8 Optical Properties, 60
1.5 Summary, 61
1.6 Future Developments, 62
1.7 References, 62

1.1 Introduction
A characteristic unique to mammals is their ability to secrete milk as a source of
nutrients and immunological protection for their young. Milk from domesticated
species has also been recognized since prehistoric times as a food source for hu-
mans.1 Some of the properties of milk that are still under study today, such as its
ability to clot with chymosin and the ability to turn milk into products such as cheese
and butter, have been known to humans for centuries.2 Consequently, the applica-
tions of chemistry and physical chemistry to milk are probably among the oldest
scientific disciplines and are still recognized as very important and integral parts of
the field of food science.
Today, the majority of milk for human consumption is secreted by the domesti-
cated cow, genus Bos, although milk from goats, buffaloes, and sheep, in addition
to human milk, is also consumed in significant quantity.
Milk is defined by the United States Code of Federal Regulations as "the lacteal
secretion, practically free from colostrum, obtained by the complete milking of one
or more healthy cows, which contains not less than 8.25% of milk solids-not-fat and
not less than 3.25% of milkfat".3 Reviews of the composition of goat's milk,4-5
ewe's milk,6 buffalo's milk,7 camel's milk,8 human milk,9 and the milk of other
species 1011 are available in the literature. This chapter is limited to a discussion of
cow's milk.
Milk is synthesized in the mammary gland. An average cow in North America
produces 5400 kg of milk in a 305-day lactation period.
The components of the mammary gland at various magnifications are shown in
Figure 1.1. The alveolus is the milk-producing unit within the gland. In the alveolus,
a single layer of epithelial secretory cells surrounds a central storage area, the lumen,
which is connected to a duct system. These secretory cells are, in turn, surrounded
by a layer of myoepithelial cells and blood capillaries. The raw materials for milk
production are transported via the bloodstream to the secretory cell. Within the cell,
components are synthesized mainly by the endoplasmic reticulum and its attached
ribosomes, which are supplied with energy from the mitochondria and then passed
along to the Golgi apparatus, which is responsible for their eventual movement out
of the cell. Vesicles containing many of the aqueous nonfat components are released
 SECMETOW
TISSUE ONt QUARTER

•LOOD
VESSEL

CAMlAMES

CtSTEWf

CONNCCTlVg
touct TISSUE

LARGE OUC f

VENOUS
OUCT BLOOO

CAPILLARIES LUMEN

MVOEPrrMEUAL
CELL
ARTEMAL LUMEN
BLOOO

ALVEOLUS I1WUTUN.

LMD MCtKMLU
OtKWLET ^ytfrtl
- S W
NUCLEUS MfTOCHQNOfOON
ENDOPIASMC
RETCULUM ,

SECRETORY CELL

Figure 1.1 Bovine mammary gland at various magnifications. (Reprinted from ref. 12,
p. 794, by courtesy of Marcel Dekker.)

by the Golgi apparatus, pass through the cytoplasm and the apical plasma membrane,
and are deposited in the lumen of the alveolus. Lipid droplets, synthesized by the
endoplasmic reticulum, also pass through the cytoplasm and the apical plasma mem-
brane and are deposited in the lumen.
As is discussed further in Section 1.3.2.1, it is believed that the milk fat globule
membrane (FGM) is comprised of the apical plasma membrane of the secretory cell,
which continually envelops lipid droplets as they pass into the lumen. The apical
cell membrane is continually being replaced from endomembrane material synthe-
sized in the endoplasmic reticulum and transported from the Golgi in the form of
vesicles containing aqueous nonfat components. The vesicle membrane fuses with
the apical cell membrane as the contents of the vesicle are released. Milk components
stored in the lumen of the alveolus are released into the duct system as a result of
hormonal stimulation. The duct systems within the mammary gland, a complex net-
work, flow into the teat cistern from which they are milked. Further details of milk
biosynthesis and mammary physiology are beyond the scope of this chapter and
have been reviewed extensively elsewhere. 13 " 15
Milk is estimated to contain more than 100,000 molecular species. However, the
average gross composition of milk can be simplified to 4.1% fat, 3.6% protein (75%
casein protein and 25% whey protein), 4.9% lactose, and 0.7% ash, with the balance
consisting of water.16 (Details of the composition of milk are covered in Section
1.2.) Variation in milk composition can be caused by inherited characteristics
(breed), physiological characteristics (stage of lactation, pregnancy, age, nutritional
balance, season, and udder health), and milking procedure (within milkings and
between milkings).3
Although milk is a fluid food, it has considerable structural organization (de-
scribed in further detail in Section 1.3). Milk can be described as:
• an emulsion of milkfat globules which contain the milk lipids, fat soluble vitamins,
and the components of the FGM;
• a colloidal suspension of casein micelles (which contain casein proteins, calcium,
phosphate, citrate and water), globular proteins, and lipoprotein particles; and
• a solution of lactose, soluble proteins, minerals, vitamins, acids, enzymes, and
other components.
Milk plasma is defined as milk minus the milkfat globules, which is close in
composition to separated or skim milk, although separation is never complete. Milk
serum is defined as milk plasma minus casein micelles, which is close to the com-
position of whey, except for the presence of some proteolytic products from chy-
mosin.16 The casein micelles and the milkfat globules are the principal structure-
forming constituents that form the basic structural elements of most dairy
products.17'18
Dairy foods make a significant contribution to the total nutrient intake of the
North American population, supplying, for example, one-fourth or more of individ-
uals' protein, calcium, phosphorus, and riboflavin requirements. Dairy foods are an
excellent source of vitamin B 12 as well as an adequate source of vitamin A, thiamine,
niacin, and magnesium. Vitamin D is added to most liquid dairy products; vitamin
A is added to most low-fat fluid products. Only iron, vitamin C, and folacin are
present in somewhat deficient amounts.1219 The nutrient composition of whole milk
is listed in Table 1.1.
From a nutritional viewpoint, milk has been described as nature's most nearly
perfect food, owing mainly to its biological role as the only source of nutrition for
the infant mammal. Milk proteins are slightly deficient in methionine and cysteine,
the sulfur amino acids. Milk lipids are slightly high in saturated fats and cholesterol
and thus may have an impact on cardiovascular disease. The nutritional significance
of milk proteins and lipids has recently been reviewed.19"21
A small but significant part of the population, particularly among African and
Asian peoples, produce less than average intestinal /3-galactosidase. This leads to
lactose intolerance, or malabsorption, which causes diarrhea, abdominal cramps, and
intestinal gas if dairy products are consumed. Lactose intolerance has recently been
reviewed.22
The purpose of this chapter is to serve as a reference for many of the processes
and technologies described in other chapters and volumes of this set. In this chapter,
we review the basics of milk composition and milk structure as they affect the
utilization of milk in industrial practice and provide a comprehensive bibliography
for further reading. This chapter is not designed to be a comprehensive review of
 Table 1.1 NUTRIENT COMPOSITION OF WHOLE MILK
(3.3% FAT)
Nutrient Amount in 100 g %RDAa in 250 ml

Protein 3.29 g 17.2

Vitamin A 31RE b 8.9
Vitamin C 0.94 mg 4.2
Thiamine 0.038 mg 8.2
Riboflavin 0.162 mg 30.0
Niacin 0.85 NEC 13.9
Vitamin B 6 0.042 mg 5.4
Folacin 5 |xg 3.2
Vitamin B 12 0.357 jig 30.7
Calcium 119 mg 32.0
Phosphorus 93 mg 25.0
Magnesium 13 mg 10.2
Iron 0.05 mg 0.9
Zinc 0.38 mg 6.5

From ref. 12, p. 822. Reprinted courtesy of Marcel Dekker.

a
Average Recommended Dietary Allowances for all males and females above
age 11.
b
Retinol Equivalents: 1 u,g retinol or 6 u,g ^-carotene.
c
Niacin Equivalents: 1 mg niacin or 60 mg dietary tryptophan. Only 10% of the
NE in milk corresponds to niacin.

the tremendously growing fields of dairy chemistry and physics. Several very recent
excellent reviews and monographs of aspects of dairy chemistry are available and
recommended for those seeking more detail.16^23"28

1.2 Composition
The gross composition of milk is defined as the fat, protein, lactose, ash, and total
solids content. Gross composition for large numbers of samples is determined by
indirect methods calibrated against chemical methods.29 The most common chemical
methods for milkfat determination are gravimetric (solvent extraction by the Mo-
jonnier or Roese-Gottlieb procedure) or volumetric (the Babcock or Gerber pro-
cedure).30 For raw milks, the Babcock procedure produces slightly higher results
(0.021% fat) than does the Mojonnier procedure and has significantly lower inter-
and intralaboratory repeatability.30
Total protein is generally determined as Kjeldahl nitrogen multiplied by the factor
6.38. This factor is still in common use, although a more representative one is 6.34.31
It is also common to report protein as crude protein (total N X 6.38), which over-
estimates true protein content (protein N X 6.38) by about 4 to 8%.3 The most
Table 1.2 GROSS COMPOSITION OF MILK OF VARIOUS BREEDS, g/100 g3
Breed Fat Protein Lactose Ash Total Solids

Holstein 3.54 3.29 4.68 0.72 12.16

Ayrshire 3.95 3.48 4.60 0.72 12.77
Guernsey 4.72 3.75 4.71 0.76 14.04
Jersey 5.13 3.98 4.83 0.77 14.42
Brown Swiss 3.99 3.64 4.94 0.74 13.08

common method of lactose analysis is polarimetric determination of lactose in a

clarified milk extract.32 Lactose is frequently reported (especially in the older liter-
ature) as lactose monohydrate, which overestimates the amount of lactose by 5.26%.3
Total solids of milk are most frequently determined by an oven method involving
initial drying on a steam bath followed by further drying in a forced air oven at 98
to 1000C,32 although a longer drying time in the oven without initial boiling off on
the steam bath may be more accurate.33 Ash content is normally determined by dry
ashing at about 5500C.32 Ash content is not equivalent to the total content of salts.
Milk salts are discussed in Section 2.4.
In the determination for payment purposes of the gross composition of producer
milk, the largest source of error is bulk tank sampling error. Standard deviations
associated with bulk tank sampling error of 0.01% for milk protein and 0.093% for
milk fat have been reported.34 Corresponding standard deviations associated with
laboratory analyses were 0.01% for both fat and protein. Milk analysis is discussed
in detail in Chapter 3.
Many factors affect the gross composition of milk. The factors most significant
to the processing of milk and milk products are breed, feed, season, region, and herd
health.35 In the short term, the only factors available to the farmer to alter milk
composition are selection of breed and feed.36 The gross composition of milk of
various breeds is listed in Table 1.2. Note that breeds producing high-fat milk also
produce milk with lower ratios of protein to fat. This is certainly significant to
multiple component pricing37"42 and suggests that genetic selection can achieve
relatively rapid increases in the ratio of milk protein to fat, provided the change is
achieved by lowering fat content.43 A large negative correlation between fat content
and protein/fat ratio but a small correlation between protein content and protein/fat
ratio have also been reported.44 Heritabilities (based on milk records of 32,000 first-
lactation cows) of percent composition of milk fat, protein, and protein/fat ratio were
0.61, 0.59, and 0.58.44
The effects of feed on milk composition have been reviewed.45'46 The most im-
portant dietary factors are the amount and type of roughage, the forage/concentrate
ratio, and the carbohydrate composition of the concentrates and lipids.46"49 Feeding
frequency does not affect milk composition, provided the total feed intake is con-
stant.50 The greatest effects of feeding are on the concentration of milkfat, with
smaller changes in protein concentration.
 Percent

Protein
Fat

Jan. Feb. Mar. Apr. May June July Aug.Sept.Oct. Nov. Dec.
1988
Figure 1.2 Seasonal variation of protein and fat content of Ontario milk. Primary standard
methods were Mojonnier for fat and semi-micro-Kjeldahl for protein. Protein is total nitrogen
X 6.38. Data represent means of 10,000 herds tested four times each month at the Ontario
Central Milk Testing Laboratory.

In the Northern Hemisphere, maximum annual fat contents occur during the win-
ter months, usually peaking in November or December; minimum fat contents occur
in August as shown in Figure 1.2.51 Seasonal trends in protein contents follow a
similar trend, with some significant differences: the seasonal variation is not as great,
the minimum occurs in July, and the maximum occurs in October (Fig. 1.2).51 These
differences cause seasonal variation of the protein/fat ratio of milk, which is of
significant economic consequence, especially to cheese manufacturing.51 Small sea-
sonal variations in lactose content have also been reported.52 Although there is some
evidence that climatic conditions affect milk composition, the principal effect of
climatic factors is on milk production.53 It is likely that the observed seasonal effects
on milk composition are primarily due to variations in feed and stage of lactation.3'54
Variations in feed and stage of lactation probably also account for most regional
variations in milk composition. Regional variations in the Ontario, Canada, milkfat
composition for the years 1978 to 1988 are shown in Figure 1.3. These data and
earlier unpublished data (Ontario Central Milk Testing Laboratory, Guelph) going
back to 1971 show a continual increase in average fat content of Ontario milk over
time, with little or no increase in protein content. The result is a significant decrease
in the protein/fat ratio of Ontario milk. There has also been a gradual increase in
average lactose content of Ontario producer milks, from 4.80% lactose monohydrate
(w/v) in 1970 to 5.2% (w/v) in 1988.
With respect to herd health, yield and compositional effects of greatest economic
Fat
%

WESTERN
SOUTHERN
NORTHERN
EASTERN
CENTRAL
ONTARIO

1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988
Year
Figure 1 3 Regional and annual variation of fat content of Ontario milk. Primary standard
method was Mojonnier. Data represent annual means within each region. Herds were tested
four times per month.

significance are due to mastitis.55 Average yield losses due to udder infection may
exceed 1 kg of milk per cow per day. 56 Somatic cell counts in excess of 300,000
indicate subclinical mastitis.57 In 1989, average somatic cell counts for all Ontario
producer milks were 350,000/mL. (Ontario Central Milk Testing Laboratory,
Guelph, Ontario, Canada). In the United Kingdom, the national average was 390,000/
mL. 58 Elevated somatic cell counts are correlated with reduced lactose content 52 and
a corresponding increase in mineral content to maintain osmotic equilibrium. Casein
content is reduced, but total protein content increases with increasing somatic cell
counts due to increased whey protein content.59 Modest levels of somatic cells may
affect cheese yield 60 due to increased proteolysis, 61 but effects of somatic cell counts
<2,000,000 ml" 1 on cheese texture and flavor are probably more significant than
yield effects. 58
Production aids may also affect milk composition. Supplementation of dairy ra-
tions with the antibiotic Flavomycin increases feed conversion efficiency, milk pro-
duction, and the percent composition of both fat and protein. 62 Like other factors
affecting milk composition, the effect on fat content is greater than on protein con-
tent. Numerous authors have reported minimal or no effects of bovine somatotropin
(BST) on gross composition of milk. 63 " 66
 1.2.1 Proteins
The nitrogen content of milk is distributed among caseins, whey proteins, and non-
protein nitrogen (NPN), excepting some minor proteins that are associated with the
FGM (Section 1.2.2).
Nitrogen distribution is normally determined by the classical Rowland fraction-
ation,67 which separates caseins from whey nitrogen by precipitation at pH 4.6 and
separates total proteins from whey NPN by precipitation with sodium acetate and
acetic acid at pH 5.0. Based on this procedure, average milk nitrogen distribution is
about 76% casein, 18% whey protein, and 6% NPN. This operational classification
of proteins is still used for both research and process control. However, a classifi-
cation system of milk proteins based on their amino acid sequences (Table 1.3) has
been developed by the American Dairy Science Association's (ADSA) Committee
on Milk Protein Nomenclature, Classification and Methodology.68 The amino acid
distributions of the principal milk proteins are summarized in Table 1.4.

1.2.1.1 Caseins
The casein content of milk is about 26 g/kg, representing about 80% of milk protein.
The principal casein fractions are asl-casein (10 g/kg), as2-casein (2.6 g/kg),
/3-casein (9.3 g/kg), y-casein (0.8 g/kg), and /c-casein (3.3 g/kg).16 These fractions
are all included in the pH 4.6 precipitate from milk, but y-caseins are now reclassified
as carboxyl terminal fragments of /3-casein. The corresponding N-terminal frag-
ments,—formerly classified as proteose-peptones70—are also classified as casein
subfractions.68 These fractions result from cleavage of ^-casein by the milk protease,
plasmin. The carboxyl terminal fragments (y-caseins) remain associated with the
casein micelle and are recovered by rennet coagulation and by pH 4.6 precipitation.
The N-terminal fragments are hydrophilic and appear as heat-stable fractions in both
cheese whey and the pH 4.6 supernatant. Carboxyl terminal fragments correspond
to /3-casein subfractions 2, 3, and 4; and the N-terminal fractions correspond to
/3-casein subfractions 5 to 9, as listed in Table 1.3. The N-terminal fractions do not
contain aromatic amino acids (Table 1.4) and, therefore, show no absorbency at
280 nm.
The nomenclature used for the caseins consists of a Greek letter with or without
a numerical subscript to identify the family of proteins; and an uppercase Latin letter
to indicate the genetic variant. Post-translational modifications such as phosphoryla-
tion or formation of subfractions are indicated after the genetic variant.68 For ex-
ample, the notation /3-casein B-5P (fl-105) indicates that the protein belongs to the
/3-family of caseins, is the B genetic variant, contains five phosphate groups, and
represents an N-terminal fragment of /3-casein B from amino acid residues 1 to 105.69
In most breeds of dairy cattle, a sl -casein is >90% variant B. Exceptions are
Guernsey and Jersey cattle, which produce about 75% variant B and 25% variant
C.71 The A variant of /3-casein occurs with nearly 100% frequency in most dairy
breeds, excepting Jersey and Brown Swiss, which produce significant levels of
/3-casein B. Significant effects of milk protein genetic variants on heat stability,72
TaWe 1.3 CLASSIFICATION AND DISTRIBUTION OF THE MILK PROTEINS-
GENUS BOS (30-35 g/L)69
I. Caseins (24-28 g/L)
A. ctsl-Caseins (12-15 g/L)
1. asl-Casein Xa-8P (genetic variants—A, B, C, D-9P, and E)
2. ctsl-Casein Xa-9P (genetic variants—A, B, C, D-10P, and E)
3. asl-Casein fragments0
B. <xs2-Caseins (3-4 g/L)
1. ots2-Casein XMOP (genetic variants—A, B, C-9P, and D-7P)
2. as2-Casein X M l P (genetic variants—A, B, C-10P, and D-8P)
3. as2-Casein XM2P (genetic variants—A, B, C-IlP, and D-9P)
4. as2-Casein XM3P (genetic variants—A, B, C-12P, and D-10P)
C. P-Caseins(9-ll g/L)
1. P-Casein Xa-5P (genetic variants—A1, A 2 , A3, B, C-4P, D-4P, and E)
2. p-Casein XMP (f 29-209) (genetic variants—A1, A2, A3, and B)
3. p-Casein Xa-(f 106-209) (genetic variants—A2, A3, and B)
4. P-Casein Xa-(f 108-209) (genetic variants—A and B)
5. p-Casein Xa-4P (f l - 2 8 ) b
6. P-Casein Xa-5P (f l-105) b
7. P-Casein Xa-5P (f l-107) b
8. p-Casein XMP (f 29-105) b
9. p-Casein XMP (f 29-107) b
D. K-•Caseins (2-4 g/L)
1. K-Casein XMP (genetic variants—A and B)
2. Minor K-Casein X M , -2, -3, etc. (genetic variants—A and B)
n. Whey proteins (5-7 g/L)
A. p-Lactoglobulins (2-4 g/L)
1. P-Lactoglobulins X a (genetic variants—A, B, C, D, Dr, E, F, and G)
B. ct-Lactalbumins (0.6-1.7 g/L)
1. a-Lactalbumin Xa (genetic variants—A and B)
2. Minor a-Lactalbumins
C. Bovine serum albumin (0.2-0.4 g/L)

(Continued)

renneting properties,73'74 and concentration and distribution of milk components

have been reported.75-76 The potential for genetic engineering of the caseins to mod-
ify the behavior of milk during processing has been reviewed.77 The milk proteins
of other species in comparison to bovine milk proteins have also been reviewed.11
Caseins are conjugated proteins with phosphate groups esterified to serine resi-
dues. The exceptions are some /3-casein fragments (Table 1.3) which contain no
phosphate. Phosphate groups are important to casein association and the structure
of the casein micelle (Section 1.3.3). Calcium binding by individual caseins is pro-
portional to phosphate content.71 In addition to phosphorylation, about one-third of
/c-casein monomers are glycosylated at threonine 133 (Fig. 1.4).69
Caseins contain high numbers of proline residues, which are distributed relatively
uniformly throughout the polypeptide chains (Fig. 1.4). Proline gives rise to a par-
ticular bending of the protein chain and inhibits formation of an ordered, stable
a-helix structure.78 Early literature suggests that caseins have little secondary struc-
Table 1.3 (Continued)
D. Immunoglobulins (0.5-1.8 g/L)
1. IgG immunoglobulins
a. IgG1 immunoglobulins
b. IgG2 immunoglobulins
c. IgG fragments
2. IgM immunoglobulins
3. IgA immunoglobulins
a. IgA immunoglobulins
b. Secretory IgA immunoglobulins
4. IgE immunoglobulins
5. J-chain or component
6. Free secretory component
in. Milk fat globule membrane (MFGM) proteins
A. Zone A (MFGM) proteins
B. Zone B (MFGM) proteins
C. Zone C (MFGM) proteins
D. Zone D (MFGM) proteins
IV. Minor proteins
A. Serum transferrin
B. Lactoferrin
C. P2-Microglobulin
D. Mrglycoproteins
E. M2-glycoproteins
F. OL1-AcId glycoprotein or orosomucoid
G. Ceruloplasmin
H. Trypsin inhibitor
I. Kininogen
J. Folate-binding protein (FBP)
K. Vitamin B12-binding protein
V. Enzymes
(See Table 1.5)
a
X represents the genetic variant.
b
Genetic variants of these fragments have not been specifically identified.
c
Nomenclature has not been established for these fragments.

ture.71 However, it has been reported that specified secondary structure in K-caseins
is in the range of about 50 to 75%,79 and there is evidence that native micellar caseins
may have as much as 14% helical structure, 27% /3-structure, and 41% turns, leaving
only 18% unspecified.80 However, there is little evidence of tertiary structure of
caseins, which accounts for the stability of caseins against heat denaturation because
there is little tertiary structure to unfold. Lack of tertiary structure also requires
considerable exposure of hydrophobic residues to water. This accounts for the strong
association reactions of caseins and their insolubility in water.
Both as2-casein and K-casein contain two cysteine residues, but other caseins have
no cysteine. Disulfide linked polymers of K-casein monomers, ranging from trimers
to very large polymers, exist naturally.71 Some covalent dimers (disulfide linked) of
as2- caseins also exist.16 Caseins differ greatly in charge distribution (Fig. 1.4) and
can be distinguished by their sensitivity to calcium precipitation.
Table 1.4 CHEMICAL COMPOSITION OF THE MAJOR PROTEINS
OCCURRING IN MILK68

P- Ot-
<*s2" K- P- 7r T2- Lacto- Lact-
Casein Casein Casein Casein Casein Casein Casein globulin albumin
Acid B A B A2 A2 A2 A A B

Asp 7 4 4 4 4 2 2 11 9
Asn 8 14 7 5 3 1 1 5 12
Thr 5 15 14 9 8 4 4 8 7
Ser 8 6 12 11 10 7 7 7 7
SerP 8 11 1 5 1 0 0 0 0
GIu 24 25 12 18 11 4 4 16 8
GIn 15 15 14 21 21 11 11 9 5
Pro 17 10 20 35 34 21 21 8 2
GIy 9 2 2 5 4 2 2 3 6
Ala 9 8 15 5 5 2 2 14 3
ViCys 0 2 2 0 0 0 0 5 8
VaI 11 14 11 19 17 10 10 10 6
Met 5 4 2 6 6 4 4 4 1
lie 11 11 13 10 7 3 3 10 8
Leu 17 13 8 22 19 14 14 22 13
Tyr 10 12 9 4 4 3 3 4 4
Phe 8 6 4 9 9 5 5 4 4
Trp 2 2 1 1 1 1 1 2 4
Lys 14 24 9 11 10 4 3 15 12
His 5 3 3 5 5 4 3 2 3
Arg 6 6 5 4 2 2 2 3 1
Pyr or GIu 0 0 1 0 0 0 0 0 0

The following summary of association characteristics and calcium sensitivites of

casein fractions is based largely on the discussion in ref. 16. The primary structure
of asl-casein consists of two hydrophobic regions (residues 1 to 44 and 90 to 199).
These regions contain all the proline residues, separated by a polar region (residues
45 to 89) that contains all but one of eight phosphate groups (Fig. 1.4). Association
of asl-casein at neutral pH is dependent on both ionic strength and temperature and
is mainly due to hydrophobic interactions and hydrogen bonding. asl-Casein can be
precipitated at very low levels of Ca 2+ (7 mM). as2-Casein has a concentration of
negative charges near the N-terminus and of positive charges near the C-terminus
(Fig. 1.4). It is similar to asl-casein with respect to association at neutral pH and
sensitivity to calcium precipitation.
/3-Casein has a highly charged N-terminal region and a hydrophobic C-terminal
region (Fig. 1.4), causing it to behave like a detergent. It is less sensitive to calcium
precipitation than are the a s l - and as2-caseins, and its association is very temperature
dependent, suggesting that hydrophobic interactions are most important. Association
does not occur if the hydrophobic portion of the molecule is cleaved. /3-Caseins are
the most water soluble of all caseins, especially at lower temperatures. /3-Caseins in
a si - en B

SS

as2-cn

p-cnA2

S S b a

K-en B

Residue Sequence Number

Figure 1.4 Location, magnitude (right ordinate), and direction ( ± ) of charged residues
(pH 6-7), Pro (.), and Cys (s), in caseins, (a) Location of glucide residue, (b) Point of cleavage
by chymosin. (Reprinted from ref. 16 by permission of John Wiley & Sons.)

milk also have a higher isoelectric point (about 5.2) than a sl -caseins (about 4.8),
which is important to the formation of acid gels (Section 1.3.1.3).81
Disulfide-linked polymers of K-casein further associate by noncovalent bonding
to form large polymers with molecular weights of 600,000 to 650,000. These poly-
mers are very stable at physiological pH and cannot be dissociated by changes in
ionic strength or temperature.71 K-Casein is extremely resistant to calcium precipi-
tation and is able to stabilize up to 10 times its own weight of a s - or /3-caseins
against calcium precipitation.16 This stabilizing ability is lost after rennet cleavage
of /c-casein at the Phe 1 0 5 -Met 1 0 6 bond, which results in the formation of a hydro-
phobic portion called para-K-casein (residues 1 to 105) and a hydrophilic portion
(residues 106 to 169). The hydrophilic fragment is referred to as K-casein glyco-
macropeptide (GMP), or caseinomacropeptide (CMP). The latter is a better term
because the predominant variants of K-casein are not glycosylated. 71 ' 82 CMP has an
apparent molecular weight of 33,000 by size exclusion chromatography, but disper-
sion and analysis of aggregates by sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) revealed size-heterogeneous peptides with molecular
weight <18,000. 83
In summary, all caseins self-associate and interspecies aggregation occurs in the
presence and absence of calcium. The order of decreasing sensitivity to calcium
precipitation is as2-, a sl -, /3-, and, finally, /c-, which stabilizes the casein system
against calcium precipitation. Caseins do not form aggregates with whey proteins
except during heat treatment (Chapter 2).

1.2.1.2 Whey Proteins

Proteins appearing in the pH 4.6 supernatant of milk are collectively referred to as
whey proteins. As noted earlier, this operational definition includes N-terminal frag-
ments of casein (Table 1.3), formerly known as proteose-peptone components 8-fast,
8-slow, and 5. A fourth proteose-peptone fraction, component 3, appears in whey
but is the antigenic equivalent of protein fractions isolated from the fat globule
membrane.84'85 Rennet whey also contains the CMP (C-terminal fragment of
/c-casein) which is cleaved by rennet.
The distribution of the principal whey protein fractions (/3-lactoglobulins,
a-lactalbumins, bovine serum albumin, and immunoglobulins) and the identification
of their genetic variants are listed in Table 1.3. Bovine /3-lactoglobulins of Western
breeds are almost exclusively A and B variants, with <i predominance of the B variant
in the most common breeds.71 In Western breeds, a-lactalbumins are almost exclu-
sively variant B. The total concentration of whey proteins in milk is 5 to 7 g/L, not
including about 1.1 g/L of proteose peptones.
/3-Lactoglobulin has two internal disulfide bonds and one free thiol group (Fig.
1.5). It has considerable a-helix and /3-structure and exists naturally as a noncova-
lently linked dimer. The dimers are further associated to octamers in the isoelectric
region (pH 3.5 to 5.2) but are dissociated to monomers at pH below 3.4.71 The
predominant B variant, which contains one more charged group (aspartic acid residue
64) than the A variant,68 forms octomers less readily, possibly due to increased
electrostatic repulsion. Association in the isoelectric region is preceded by a con-
formational change, which results in the binding of one proton group per monomer.71
A conformational change, known as the Tanford transition,86 takes place near pH
7.5 and results in the exposure of one previously untitrated carboxyl group and
increased reactivity of the free cysteine group,71 which is important to thermal in-
teractions of milk proteins.87"89 /3-Lactoglobulins may have a physiological role in
vitamin A transport.90
a-Lactalbumin contains eight cysteine groups, all of which are involved in di-
sulfide bonds (Fig. 1.5), and four tryptophan residues (Table 1.4), which account for
its high absorbency at 280 nm. Differences in aromatic amino acid contents permit
rapidfingerprintingof whey proteins by UV scanning spectroscopy.91 a-Lactalbu-
min has a highly ordered secondary structure, and hydrodynamic data indicate a
compact, spherical tertiary structure.71 At pH <4.0, a-lactalbumin undergoes a con-
formational change, which can be observed by circular dichroism92 and is accom-
panied by the release of bound calcium.93 A similar conformational change was
 S S S SH S

P-IgB

S S S SS S S S

a-Ia B

Residue Sequence Number

Figure 1.5 Location, magnitude (right ordinate), and direction ( ± ) of charged residues
(pH 6-7), Pro (.), and Cys (s), in /3-lactoglobulin and a-lactalbumin. (Reprinted from ref. 16
by permission of John Wiley & Sons.)

observed by circular dichroism after removal of calcium from a-lactalbumin at pH

7.5. 93 Magnesium 94 and other metal ions 93 - 95 are also bound by a-lactalbumin. The
conformational change at pH < 4 . 0 results in temperature- and concentration-
dependent aggregation of a-lactalbumin.96 Thermal denaturation of a-lactalbumin
is also accompanied by a release of bound calcium, and a-lactalbumin is stabilized
against heat denaturation and aggregtion in the presence of calcium. 97 ' 98

1.2.1.3 Enzymes
Milk contains both indigenous and exogenous enzymes, the latter being mainly bac-
terial. With respect to dairy processing, the most significant bacterial enzymes oc-
curring in milk are heat-stable Upases and proteinases elaborated by psychrotrophic
bacteria.99"101 Indigenous enzymes of milk, the reactions they catalyze, and their
location in milk are summarized in Table 1.5.
With respect to dairy processing and quality control, the most significant enzymes
are several hydrolases, namely, lipoprotein lipase, plasmin, and alkaline phosphatase.
The functions and significance of these enzymes are briefly described in this section.
Properties and functions of other indigenous milk enzymes have been reviewed. 1 6 1 0 2
Most milk enzymes have pH and temperature optima near physiological values,
with the notable exceptions of alkaline phosphatase and phosphoprotein phosphatase,
which have pH optima of 9.8 and 4.0 to 5.5, respectively. 16 Alkaline phosphatase
activity is used to distinguish raw milk from pasteurized milk because its heat sta-
Table 1.5 ENZYMES OF BOVINE MILK

ECNo. Enzyme Reaction Location

+
1.1.1.27 Lactate dehydrogenase L-lactate + NAD ^± pyruvate + Plasma
NADH 4- H +
1.1.1.37 Malate dehydrogenase L-malate 4- NAD + ^± oxaloacetate 4-
NADH 4- H +
1.2.3.2 Xanthine oxidase Xanthine 4- H2O 4- 2O2 ^ uric acid 4- MFGM
2O2 + 2H +
1.4.3.6 Amine oxidase (Cu RCH2NH2 + H2O 4- O2 ;± RCHO 4-
containing) H2O2 4- NH3
1.6.4.3 Lipoamide dehydrogenase NADH 4- H + 4- lipoamide ?± NAD +
(NAD + ) (diaphorase) + dehydrolipoamide
1.6.99.3 NADH dehydrogenase NADH 4- H + + acceptors NAD + 4- MFGM
(cytochrome c reductase) reduced acceptor
1.8 Sulfhydryl oxidase (not 2RSH + O2 ^ R-S-S-R 4- H2O2 Serum
1.8.3.2 thiol oxidase)
1.11.1.6 Catalase 2H2O2 ^± O2 4- 2H2O Leukocytes
1.11.1.7 Lactoperoxidase Donor 4- H2O2 ^± oxidized donor + Serum
2H2O
1.15.1.1 Superoxide dismutase 2O2 + 2 H + ^ ± O 2 + H2O2
2.3.2.2 7-Glutamyl transferase L-7-Glutamyl-peptide + amino acid ^ MFGM
peptide 4- L-glutamyl-amino acid
2.4.1.22 Lactose synthase UDP-galactose 4- D-glucose ^± UDP + Serum
lactose
2.4.99.1 CMP-A^-acetylneuraminate- CMP-7V-acetylneuraminate +
galactosyl-glycoprotein D-galactosyl-glycoprotein ?± CMP +
sialyl transferase N-acetylneuraminyl-D-galactosy-
glycoprotein
2.6.1.1 Aspartate aminotransferase L-Aspartate + 2-oxoglutarate ^± Plasma
oxaloacetate + L-glutamate
2.6.1.2 Alanine aminotransferase L-Alanine + 2-oxoglutarate ^± pyruvate
+ L-glutamate
2.7.1.26 Riboflavin kinase ATP + riboflavin ^± ADP + FMN
2.7.1.30 Glycerol kinase ATP 4- glycerol ^± ADP 4- glycerol-3-
phosphate
2.7.7.2 FMN adenyltransferase ATP 4- FMN ^± FAD 4- pyrophosphate
2.8.1.1 Thiosulfate sulfur transferase S 2 Oi" 4- CN- 5± SOf" + SCN-
(Rhodanase)
3.1.1.1 Carboxylesterase (B-esterase) R-COOR' 4- H2O ^ ROH + RCOOH
3.1.1.2 Arylesterase (A-esterase) A phenyl acetate 4- H2O ^ a phenol +
acetate
3.1.1.7 Acetylcholine esterase acetylcholine 4- H2O ^ choline MFGM
4- acetate
3.1.1.8 Cholinesterase An acylcholine 4- H2O ^ choline + Serum
carboxylate anion
3.1.1.34 Lipoprotein lipase Triglyceride 4- H2O ^ diglyceride 4- Casein
fatty acid
3.1.3.1 Alkaline phosphatase R-O-PO3H2 + H2O ^± ROH + H3PO4 MFGM
3.1.3.2 Acid phosphatase R-O-PO3H2 + H2O ^ ROH + H3PO4 MFGM

(Continued)
Table 1.5 (Continued)

ECNo. Enzyme Reaction Location

3.1.3.5 5'-Nucleotidase A 5' ribonucleotide + H2O ^ a MFGM

ribonucleoside + H3PO4
3.1.3.9 Glucose-6-phosphatase D-Glucose-6-phosphate + H2O ^± D- MFGM
glucose 4- H3PO4
3.1.3.16 Phosphoprotein phosphatase Protein phosphate -I- H2O ^ protein + Plasma
H3PO4
3.1.4.1 Phosphodiesterase A phosphoric diester + H2O ^ a MFGM
phosphoric monoester + alcohol
3.1.27.5 Ribonuclease (pancreatic) Endonucleolytic cleavage to 3' Serum
phosphomono- and oligonucleotides
ending in Cp or Up
3.2.1.1 a-Amylase Hydrolyzes a-1-4 glucan links in
polysaccharides at random
3.2.1.2 p-Amylase Hydrolyzes a-1-4 glucan links in Serum
polysaccharides by removing
successive maltose units from the non-
reducing end
3.2.1.17 Lysozyme Hydrolyzes the p-1-4 glycosidic bond
between N-acetylgucosamine and N-
acetylmuraminic acid units in Serum
mucopolysaccharides
3.2.1.24 a-D Mannosidase Hydrolyzes a-r>mannosides by removing
a-D mannose from the nonreducing
end
3.2.1.30 p-W-Acetyl- Hydrolyzes chitobiose and higher
D-glucosaminidase analogs and protein derivatives by
removing JV-acetyl-D-glucosamine
from the nonreducing end
3.2.1.31 p-Glucuronidase A p-D-glucuronide + H2O ^ alcohol +
D-glucuronic acid
3.4.21.7 Plasmin Hydrolyzes peptide bond, preferentially Casein
at Lys > Arg
3.4 Acid protease Hydrolyzes peptide bond
3.6.1.1 Inorganic pyrophosphatase H4P2O7 + H2O ;± 2H3PO4
3.6.1.3 Adenosine triphosphatase ATP + H2O ^± ADP + H3PO4 MFGM
(Mg 2+ activated)
3.6.1.9 Nucleotide pyrophosphatase A dinucleotide + H2O ^±
2 mononucleotides
4.1.2.13 Fructose-biphosphate r>Fructose-l,6-phosphate ^
aldolase dihydroxyacetone-phosphate +
D-glyceraldehyde-3-phosphate
4.2.1.1 Carbonic dehydratase H2CO3 s± CO2 + H2O
(carbonic anhydrase)
5.3.1.9 Glucose phosphate isomerase D-Glucose-6-phosphate ^± D-fructose-
6-phosphate

From ref. 16. Reprinted by permission of John Wiley & Sons.

bility is similar to the minimum conditions used for milk pasteurization.103 Two
isozymes, a- and /3-phosphatase, occur in milk. The latter is more abundant and
occurs mainly in the fat globule membrane. Interference by the heat-stable acid
phosphatase is avoided by performing the assay at pH near 10. The /3-isozyme of
alkaline phosphatase is also subject to renaturation, especially in creams, where the
enzyme is more concentrated.16 Residual phosphatase can be distinguished from
reactivated phosphatase by increased activity of the latter in the presence of mag-
nesium.104105 It was reported that heat inactivation of alkaline phosphatase was more
rapid in highly concentrated, ultrafiltered milk rententates.106
Milk lipoprotein lipase (LPL) has been well characterized.16'107"109 Milk LPL is
present mainly in the plasma in association with casein micelles. It does not attack
the fat globule unless the FGM has been damaged or if certain blood serum lipo-
proteins are present. These lipoproteins, acting as cofactors, enable LPL to attack
the lipoproteins of the FGM. Further discussion of lipolytic breakdown of dairy
products is presented in Section 1.2.2.3.
The principal milk protease is an alkaline serine protease, which is apparently
identical to blood plasmin.16'102 Plasmin is present mainly as plasminogen in fresh
milk, but, with time, it is converted to active plasmin. It has been indicated that the
plasmin content of milk is associated with the process of involution (i.e., the declin-
ing phase of milk production) and that administration of BST reduced levels of
plasmin and plasminogen in late lactation.61110 It is well known that increased pro-
teolytic activity is associated with high somatic cell counts, 111112 but the protease
associated with somatic cells is apparently not plasmin.113 Plasmin attacks both
/3-casein and as2-casein. As indicated previously, protein fractions formerly known
as y-caseins and proteose-peptones are plasmin produced fragments of /3-casein.
Plasmin has optimal activity at slightly alkaline pH and 37°C. The enzyme is ex-
tremely heat stable114 and is responsible for the development of bitterness in pas-
teurized and ultra-high-temperature processed milk. The distribution of plasmin be-
tween cheese and whey is dependent on the pH of whey separation; higher-running
pH causes increased retention of plasmin in the cheese.115

1.2.2 lipids
1.2.2.1 Chemical Properties
The milkfat of ruminants is very complex, due to the diversity of lipid species that
are produced by microbial activity in the rumen and are transported to the milk
secretory cells in the blood stream. 15116 " 118 Other lipids are produced by synthesis
in the secretory cells. 116119 Fatty acids found in milk fat include: (1) saturated even
and odd n-chain acids from 2 to 28; (2) at least 50 branched chain fatty acids; (3)
cis monoenoic fatty acids of 12 and 14 to 24 «-chain acids; (4) trans 16 to 24 n-
chain fatty acids; (5) various positional and geometric isomers of dienes and trienes
of 18, 20, 22, and 24 «-chain acids; and (6) small amounts of tetra- and pentanoic
acids (Tables 1.6 and 1.7).116 Short-chain fatty acids (butyric, caproic, caprylic, and
capric) comprise about 11 % by weight of total methyl esters. Quantitatively, the
Table 1.6 FATTY ACID COMPOSITION OF BUTTER OIL AS DETERMINED
BY GLC-MASS SPECTROMETRY (WEIGHT PERCENT)
OF TOTAL METHYL ESTERS116
Methyl Monoenes Branched
Ester
Carbons Saturates trans Iso Anteiso Other

4 3.25
6 2.32
8 1.85
10 4.02
11 0.16
12 4.15 0.03
13 0.03 0.01 Trace
14 11.05 0.47 0.08
15 0.95 0.08 0.23 0.42
16 26.15 1.25 0.03 0.32
17 0.70 0.32 0.01 0.33 0.40 DDL pristanate, 0.01
18 9.60 20.40 5.34 0.15
19 0.11 0.10 0.01 0.06 0.09 DDD pristanate, 0.01
20 0.19 0.15 0.01 0.04 DDL, DDD phytanates, 0.04
21 0.06 0.03 Trace Trace 0.01
22 0.10 0.02 Trace Trace
23 0.07 0.01 0.01
24 0.06 0.02 0.01
25 0.01
26 0.04

major fatty acids of milk fat are myristic (11%), palmitic (26%), stearic (10%), and
oleic (20%). Saturated fatty acids account for about two-thirds of milk fatty acids
(Table 1.8), with larger quantities of unsaturated fatty acids present during the sum-
mer months. The summer and winter mean iodine values of Finnish butter fat have
been reported as 36.1 and 27.6, respectively.120 Estimates of total trans fatty acids
vary widely within the range of 2 to 11%, expressed as elaidic acid {trans 18:1).121
The distribution in milk and some properties of the major milk lipids are sum-
marized in Table 1.8. Triglycerides account for 98.3% of milkfat. It is not known
whether small amounts of free fatty acids occurring in fresh milk are secreted from
the epithelial cell or are the product of early lipolysis. Phospholipids comprise about
0.8% of milk lipids and are mainly associated with FGM. About 0.3% of milk lipid
is sterols, mainly cholesterol, which is located mostly in the core of the fat globule.

1.2.2.2 Physical Properties

The physical properties of milkfat have been summarized as follows: density at 200C
is 915 kg m~ 3 ; refractive index (589 nm) is 1.462 and decreases with increasing
temperature; solubility of water in fat is 0.14% (w/w) at 200C and increases with
increasing temperature; thermal conductivity is about 0 . 1 7 J m - 1 S - 1 K " 1 at 200C;
Table 1.7 FATTY ACID COMPOSITION OF BUTTER OIL AS DETERMINED BY
GLC-MASS SPECTROMETRY (A CONTINUATION OF TABLE 1.6)116
Weight Percent of Total Methyl Esters

Methyl Ester Carbons Dienes Trienes Tetraenes Pentaenes

18
Positional isomers 0.14 0.02
2.30 0.60
Conjugated
cis, trans 0.70 di-0.03
trans, trans 0.05 tri-0.01
20
Positional isomers 0.03 0.01 0.10
Trace 0.13 0.02
0.02
22
Positional isomers 0.04 0.06 0.02
Trace 0.02 0.02
24
Positional isomers Trace 0.01
0.03
0.02

specific heat at 400C is about 2.1 kJ k g " 1 K" 1 ; electrical conductivity is <10~ 1 2
ohm" l cm" *; and the dielectric constant is about 3.1. 27 Crystallization of lipids and
the resulting effects on fat structure, melting range, and rheological properties have
been reviewed.122"124 The most complete review of the crystallization behavior of
milkfats is ref. 27. The following discussion is based largely on that reference.
The native mixture of milk lipids is solid at room temperature and is, therefore,
properly described as milk "fat" as opposed to "oil," which is liquid at room
temperature. The melting points of individual triglycerides in milk ranges from
— 75°C for tributyric glycerol to 72°C for tristearin. However, the final melting point
of milkfat is about 37°C because higher melting triglycerides are dissolved in liquid
fat.16 Milkfat crystals occur in a, /3^, /3J and /3 forms, although for slowly cooled
fat, the least stable a form is too transient to be observed.125 Crystal behavior and
melting curves of milkfat are complicated by the diverse lipid composition: trans
unsaturation increases melting points; odd-numbered and branched chains decrease
melting points because they are unable to form dense crystal structures; and varia-
tions in chain length also contribute to softer fats. Typical melting curves for summer
and winter milkfat are shown in Figure 1.6. Crystal structure and properties are also
dependent on the state of dispersion, so globular fat behaves very differently from
bulk fat and the crystal behavior of globular fat is affected by globule size. Ho-
mogenized recombined milkfat behaves differently because of its uniform lipid com-
position as opposed to natural fat, which shows wide variation in lipid composition
Table 1.8 AN OVERVIEW OF THE LIPIDS OF FRESH MILK
Component Percentage of
Fatty Acids the Lipid In
Percentage
in Core
Alcohol Other Milk Fat of Globule Milk
Lipid Class Residue Constituent MW Number X y (w/w) Globule Membrane Plasma
Neutral glycerides 98.7
Triglycerides Glycerol 728 3 14.4 0.35 98.3 -100
Diglycerides Glycerol 536 2 14.9 0.38 0.3 90? 10?
Monoglycerides Glycerol 314 1 15.0 0.36 0.03 +
Free fatty acids 253 15.8 0.36 0.1 60 10?
Phospholipidsa Phospho 0.8 65 35
group
Lecithin Glycerol Choline 764 2 17.2 0.60 0.26
Ph. ethanolamineb Glycerol Ethanolamine 742 2 17.9 1.00 0.28
Ph. serineb Glycerol Serine 784 2 17.8 0.80 0.03
Ph. inositide6 Glycerol Inositol 855 2 0.04
Plasmalogens Glycerol Choline6 -700 lf 0.02
Sphingomyelind Sphingosine Choline 770 1 19.0 0.20 0.16
Cerebrosidescd Sphingosine Hexose 770 1 20.0 0.20 0.1 70 30
Gangliosidescd Sphingosine Hexose8 -1600 1 0.01 70? 30?
Sterols 0.32 80 10 10
Cholesterol 387 0.30
Cholesteryl Cholesterol 642 1 16.0 0.40 0.02?
esters
Carotenoids + 0.002 95? 5?
vitamin A
From ref. 16. Reprinted by permission of John Wiley & Sons.
Note: Not complete: approximate average values from various sources, x — number of carbon atoms; y — number of double bonds.
a
A small fraction (e.g., 1%) is present as lysophosphatides.
b
Phosphatidyl ethanolamine + serine = cephalin.
c
Glycolipids.
d
Sphingolipids.
e
Or ethanolamine.
f
Also a fatty aldehyde residue.
8
Also neuraminic acid.
 1 2 3
Solid Fat
(%)

Temperature ( 0 C )
Figure 1.6 Melting curves of milk fat, determined by dilatometry. 1, summer fat, slowly
cooled before the experiment; 2, the same fat, rapidly cooled; 3, winter fat, rapidly cooled.
(From ref. 27 with permission of Pudoc, Wageningen, the Netherlands.)

and melting ranges of individual globules. For example, fat dispersed in natural
globules has a lower mean melting point than bulk fat but, because of widely varying
composition between globules, some dispersed fat has a melting point that is much
higher than the final melting point of bulk fat. These effects are summarized in
Table 1.9.

1.2.2.3 Lipolysis
Hydrolysis of fatty acid esters by the action of lipases results in the common flavor
defect known as lipolytic or hydrolytic rancidity and is distinct from oxidative ran-
cidity. A comprehensive review of flavor impairment of milk and milk products due
to lipolysis has been published by the International Dairy Federation.126
Lipases are unique among enzymes in that they are active at the lipid—serum
interface. In milk, lipases are ineffective unless the FGM is damaged or weakened
in some way. Lipolysis may be caused by the lipoprotein lipase (LPL), which is
endogenous to milk, or by bacterial lipases. The principal bacterial lipases that occur
in milk are heat-stable exocellular lipases of psychotrophic bacteria.99'127 However,
the principal psychotrophic bacteria of milk, Pseudomonas sp., do not elaborate
significant quantities of proteases or lipases until cell counts exceed 106 to 108
mL~ ] . 128 In practice, this means that significant elaboration of Pseudomonas lipases
is unlikely to occur except in improperly cleaned raw-milk-handling equipment; and
psychotrophic lipases should not be a serious problem, except possibly in ultra-high-
Table 1.9 FACTORS INFLUENCING CRYSTALLIZATION OF MILK FAT
Effect on

Crystallization
Factors Melting Range Amount of Solid Fat Characteristics

Fat composition Yes Yes 9

Lower temp, of Main melting at lower More
crystallization temp. Smaller
Rapid cooling Main melting at higher Usually more
temp. Smaller
Cooling in steps More than one melting Usually less Often larger;
max. spherulites
Preliminary cooling to Main melting at lower More Smaller
low temp. temp.
Prolonged at not too More even Usually more Larger; solid networks
low temp.
Fat in natural globules Somewhat higher final Less at high temp, Smaller; no networks
as compared to bulk melting point more at low temp.
fat
Smaller globules Still less at high Smaller
temp.

From ref. 27 with permission of Pudoc, Wageningen, Netherlands.

temperature processed milk, where low levels of heat-stable proteases and lipases
may cause deterioration.129
Cow's milk contains sufficient total lipase activity (mainly LPL) to release about
2 /imol of free fatty acids (FFA) per minute at 37°C, but the actual activity during
storage of raw milk at 4°C may be as low as 0.002 /imol of FFA min~ l 13° The
following conditions affect the rate of lipolysis in fresh milk: the pH of milk (6.6 to
6.8) and the storage temperature of raw milk (normally <4°C) are less than the LPL
optima of pH 8.3 and 37°C; about 80% of milk LPL is bound to micellar casein, 10
to 20% is present in the serum, and only 0 to 10% is associated with the fat globule;
milk plasma contains at least two inhibitors of lipolysis; and a lipoprotein is present
in milk, which acts as a cofactor to increase LPL activity.130 The inhibitory effect
of milk plasma is probably due to its effect on the distribution of LPL and can be
reversed by addition of heparin, which causes dissociation of LPL from the casein
micelles.131-132
The properties of the FGM are most important to lipolysis. The observed lactation
effects may be due to reduced contents of phospholipids in the FGM in late lacta-
tion.107 Mastitis, which alters milk composition, also increases sensitivity of the fat
globule to lipolysis.133 The lipoprotein cofactor, which is derived from blood, ap-
parently enables LPL to hydrolyse lipoproteins of the FGM and gives LPL access
to triglycerides in the fat globule.107 Other factors that destabilize the FGM, espe-
cially agitation and foaming, also promote lipolysis. Churned fat does not appear to
be a good substrate for LPL,134 but lipolysis is accelerated by the replacement of
the native membrane with surface active material (mainly casein micelles and whey
proteins) from the plasma.130 This effect is at least partly due to redistribution of
LPL from the plasma to the FGM and accounts for greatly increased lipolysis after
homogenization. Similarly, experiments with on-farm ultrafiltration demonstrate that
milk must be heated after ultrafiltration to inactivate LPL.135 Milking systems will
promote lipolysis to greater or lesser degrees, depending on the amount of agitation
and aeration that takes place during milking and milk transfer.130 Lipolysis can also
be induced in fresh milk by a temperature cycle of cooling to <5°C, warming to 25
to 35°C, and recooling to <10°C. 107 Such an effect may occur if a large amount of
warm milk is added to a small amount of cooled milk.
About 20% of cows produce milk in which LPL is activated by cooling to < 15°C
soon after milking. Lipolysis proceeds without subsequent thermal or mechanical
activation. This effect, frequently referred to as spontaneous lipolysis, is unlikely to
occur in herd milks or in pooled milks because it is prevented by mixing affected
milk with three to five times its volume of normal milk.136 The major conditions
that affect spontaneous lipolysis are: late-lactation milk is more susceptible than
early-lactation milk;137 fresh forage reduces the incidence of spontaneous lipolysis;
more lipolysis occurs during the winter months, but this effect may be related to
feed and lactation effects; and low-yielding cows are more likely to produce spon-
taneous milk. 107138139 Spontaneous vs. nonspontaneous milk may be determined by
differences in contents of lipolytic inhibitors and activators.
Sensory perception of lipolytic rancidity is strongly affected by the pH of the
product because, at low pH, more free fatty acids are present in the aqueous phase,
where they are more readily tasted.16 In fresh milk, sensory threshold values cor-
responded to acid degree values (ADV) of 4.1 to 4.5 mmol per 100 g of fat as
estimated by the Frankel and Tarassuk solvent extraction method and 1.85 to 2.05
as estimated by the Bureau of Dairy Industries (BDI) detergent extraction method.140

1.2.2.4 Oxidation
Oxidation of milk and other fats proceeds by the well-known autoxidation reaction124
in three stages: initiation, propagation, and termination. During propagation, antiox-
idant compounds such as tocopherols and ascorbic acid are depleted while peroxide
derivatives of fatty acids accumulate. Peroxides, which have little flavor, undergo
further reactions to form a variety of carbonyls, some of which are potent flavor
compounds, especially some ketones and aldehydes.
Most methods available to monitor lipid oxidation are unsuitable as an early index
of oxidized flavor development in milk: measurement of peroxides is not useful
because peroxides are unstable intermediates; tests based on colorimetric reaction of
thiobarbituric acid with malonaldehyde show some correlation to sensory values141
but are rather insensitive; and direct measurement of oxygen uptake is suitable only
for controlled experimental conditions.142
In milk, the initiation reactions involve phospholipids present in the FGM. Free
radicals formed from phospholipids are then able to initiate oxidation of triglycer-
ides, especially in the presence of copper and proteins.16 During the winter months
(October to May), when cattle are on dry feed, the incidence of oxidized flavor in
raw producer milks in Ontario is about 20% as determined for 2- to 3-day-old milk
by expert graders (unpublished data). The following summary of conditions affecting
oxidation of lipids and the development of oxidized flavor in milk is based mainly
on several significant reviews.16-128'142"146
Although all milk probably requires some extrinsic factor such as added copper
or light to initiate lipid oxidation, the milk of some cows and herds is said to oxidize
spontaneously, implying that oxidation results from factors intrinsic to this milk
whereas lipid oxidation in normal milk requires activation by extrinsic factors. Sig-
nificant intrinsic factors affecting milkfat oxidation include (1) metalloproteins such
as milk peroxidase and xanthine oxidase: (2) endogenous ascorbic acid, which acts
as a cocatalyst with copper to promote oxidation; (3) endogenous copper content;
and (4) endogenous antioxidants, mainly tocopherols. Fresh forage is well known to
control spontaneous oxidation, as indicated by obvious seasonal effects on the in-
cidence of oxidized flavor. This effect is probably due to increased levels of en-
dogenous antioxidants. However, attempts to supplement dry forage with tocopher-
ols have had limited success because only about 2% of added tocopherol is
transmitted to the milk. 147148 Most managers, therefore, have concentrated on the
control of extrinsic factors to minimize the extent of oxidation.
Important extrinsic factors include contamination with metals, temperature of
storage, oxygen tension, heat treatment, agitation, light, and acidity. Both copper
and iron may catalyze lipid oxidation, but probably only copper is significant in
milk. Added copper is much more potent than natural copper because a significant
portion of added copper goes directly to the fat globule.16 In some milks, 5 /jig k g " 1
of added copper is sufficient to induce lipid oxidation. Fortification149 or
contamination150 of milk with iron is reported to induce oxidized flavor development.
This seems surprising because iron should be inactivated by proteins in milk.16
Autoxidation of fats is generally increased with higher temperatures; but in raw
milk and low pasteurized milk, this trend is reversed,128 in spite of copper migration
from the fat globule to the plasma at low temperatures.16 Effects of low temperature
on oxidation are normal for processed dairy products. Heating milk causes migration
of copper from the plasma to the fat globule, indicating that oxidation of butter can
be reduced by separating cream before heat treatment and by separating to high fat
contents.16 Heating can also denature metalloproteins and increase the availability
of metals to catalyze oxidation; however, high-heat treatments (in excess of 800C)
stabilize milk against both copper- and light-induced oxidation.151 This effect is
probably due to exposure of sulfhydryl groups of denatured proteins and the release
of hydrogen sulfide, which binds copper as cupric sulfide. The oxygen tension re-
quired to permit lipid oxidation in milk is low (0.1 atmosphere oxygen pressure),
and bacterial respiration in normal fresh milk is of no consequence in decreasing
oxidation.152 De-aeration significantly reduces oxidation of packaged whole milk
powder.153
Homogenization drastically reduces the sensitivity of milkfat to both copper- and
light-induced oxidation, probably because oxidation-sensitive membrane phospho-
lipids are displaced. Ultrafiltered milks are also more resistant to oxidation.135
 Photooxidation of milk fat is accompanied by depletion of riboflavin, ascorbic
acid, and some amino acids.154"15® So-called sunlight flavor induced by photooxi-
dation is due to oxidation of methionine to methional by photoactivated riboflavin.
Sunlight flavor is, therefore, due to oxidation of proteins but is often confused with
lipid oxidation because the flavor notes are similar. Nonfat retail milks are reported
to show high incidence of oxidized flavor,145 but this effect is most likely sunlight
flavor.

1.2.3 Lactose
1.2.3.1 Biochemical Properties
The lactose content of normal milk is relatively constant at 4.8 to 5.2% lactose
monohydrate. Lower levels occur in colostrum and mastitic milk to offset high min-
eral levels and maintain osmotic balance.159 Lactose comprises about 52% of milk
solids-not-fat, about 70% of whey solids, and >90% of the solids in milk ultrafiltrate.
Several reviews have appeared on the utilization of lactose.160"162 In addition to
lactose (4-O-/3-D-galactopyranosyl-D-glucopyranose), fresh milk contains other car-
bohydrates in small amounts, including glucose (1 mg/ml), galactose (1 mg/ml), and
oligosaccharides (0.1 mg/ml).163
Lactose is synthesized in the mammary gland, where the final step is the transfer
of D-galactose to D-glucose by galactosyltransferase in the presence of a-lactalbu-
min, which acts as an enzyme modifier.164 Lactose is a reducing sugar with the
aldehydic group on the glucose residue. It exists in both a and /3 forms, which are
indicated by interchanging the OH and H on the reducing group. Lactose is optically
active because of its asymmetry, and the a form can be distinguished from the /3
form by its greater rotation of polarized light in the dextro direction.163 Optical
activity is the basis for polarimetric determination of lactose in fresh, nonfermented
dairy products.165 Polarimetry is not useful for fermented products due to interfer-
ence from lactic acid, which rotates light to the levo direction.
The most important function of lactose in milk and dairy products is its utilization
as a fermentation substrate. Lactose can be hydrolyzed to glucose and galactose by
/3-D-galactosidase (lactase). Elaboration of this enzyme gives lactic acid bacteria a
competitive advantage over many pathogenic and spoilage organisms. It is this prop-
erty that makes naturally fermented milk a relatively safe product and is the basis
for controlled fermentations in the production of cultured dairy products. Enzymatic
hydrolysis of lactose is used to reduce lactose crystallization in certain products and
to produce lactose-reduced products for persons who do not possess sufficient
lactase.166'167

1.2.3.2 Physicochemical Properties

There are several literature reviews on physicochemical properties of lac-
tose.16'160'163-166'168 Only those properties most important to dairy processing are
discussed here.
Figure 1.7 a-Lactose crystals prepared by scanning electron microscopy. (Courtesy of
A. Smith.)

The a and /3 forms of lactose exist in solution in a temperature-dependent equi-

librium, according to Eq. 1.1, where T is temperature in 0 C.
[/3]/[a] = 1.64 - 0.00277 (1.1)
Supersaturated solutions of lactose at temperatures >93.5°C crystallize as anhydrous
/3-lactose, which is sweeter than a-lactose monohydrate (a-hydrate).163 At temper-
atures <93.5°C, supersaturated solutions crystallize as a-lactose monohydrate. Mu-
tarotation from /3-lactose to a-lactose occurs as a-lactose is crystallized from solu-
tion. Crystals of a-lactose monohydrate form many different shapes, but all are
crystallographically equivalent to the most common "tomahawk" shape (Fig.
1.7).163 The different shapes arise from differential rates of crystallization on the
various crystal faces. The most important variables affecting the rate of crystal
growth and crystal shape is the degree of supersaturation (the ratio of actual con-
centration to the solubility) and the presence of inhibitors.
In milk, the most important inhibitor is /3-lactose, which inhibits crystallization
on some faces more than others and is largely responsible for the characteristic
tomahawk shape of crystalline a-lactose monohydrate.16 The final solubility of lac-
tose is 15.1 g/100 g of water at 100C and 11.9 g/100 g of water at 00C. In practice,
a-hydrate is likely to crystallize in refrigerated dairy products containing more than
about 13 g lactose per 100 g of water. Amorphous anhydrous lactose (lactose glass)
is formed by rapid drying (e.g., spray-drying of milk or whey) or by rapid freezing.
Lactose glass is extremely hygroscopic and accounts for the caking of skim milk
powder, which occurs at moisture contents greater than about 8%.163 When sufficient
Table 1.10 SOME MINOR COMPONENTS IN FRESH MILK. RESULTS ARE
EXPRESSED AS CONTENTS PER LITER 3'16'169>170

Minerals Vitamins

Sodium (mg) 350-900 A (jig RE) 400

Potassium (mg) 1100-1700 D(IU) 40
Chloride (mg) 900-1100 E(JJLg) 1000
Calcium (mg) 1100-1300 K(JJLg) 50
Magnesium (mg) 90-140 B 1 (jig) 450
Phosphorus (mg) 900-1000 B 2 (JJLg) 1750
Iron (|xg) 300-600 Niacin (jig) 900
Zinc (jig) 2000-6000 B 6 (M^g) 500
Copper (|xg) 100-600 Pantothenic acid (jxg) 3500
Manganese (|xg) 20-50 Biotin (jig) 35
Iodine (u,g) 260 Folic acid (jxg) 55
Fluoride (|xg) 30-220 B 12 (^g) 4.5
Selenium (|xg) 5-67 C(IiIg) 20
Cobalt ([Lg) 0.5-1.3
Chromium (|xg) 8-13 NPN Compounds
Molybdenum (jig) 18-120
Nickel (jLg) 0-50 Total NPN (mg) 229-308
Silicon ([ig) 750-7000 Urea-N (mg) 84-134
Vanadium (jxg) tr-310 Creatine N (mg) 6-20
Tin (jig) 40-500 Uric acid-N (mg) 5-8
Arsenic (|xg) 20-60 Orotic acid N (mg) 12-13
Peptides N (mg) 32
Selected Miscellaneous Compounds Ammonia N (mg) 3-14
Amino acid N (mg) 39-51
Ethanol (mg) 3 Choline (mg) 43-285
Formic acid (mg) 10-85 Carnitine (mg) 10-17
Acetic acid (mg) 3-50 Af-Acetylneuraminic acid (mg) 120-270
Lactic acid (mg) 34-104
Citric acid (mg) 1750
Phosphoric esters (mg) 300
Nucleic acids and Nucleotides (mg) 555

moisture is available, lactose glass forms a-hydrate crystals, which bind powder
particles together.

1.2.4 Minor Components

The contents of some minor components of milk are listed in Table 1.10, including
some vitamins, minerals, nonprotein nitrogenous compounds, phosphoric esters,
ethanol, and some acids.

1.2.4.1 Vitamins
The physiological functions and the activity in milk of vitamins have been re-
viewed. 1 7 0 1 7 1 Milk contains fat-soluble vitamins A, D, E, and K (Table 1.10). Milk
is an important source of dietary vitamin A; many Western countries require sup-
plementation of skim milk to replace vitamin A removed with the cream. U.S. Gov-
ernment Regulations require 2000IU of vitamin A per quart (1 U.S. quart = 0.95 L).
The actual amounts, however, have been reported to be less and are extremely var-
iable.172 Natural vitamin A activity is derived from retinol and /3-carotene and varies
with the season, due to seasonal variation in /3-carotene,170 which also accounts for
the seasonal variation in the color of milk fat. Vitamin D activity in milk is derived
from cholecalciferol (D3) and ergocalciferol (D2). Vitamin E occurs in milk as a-
tocopherol, an important natural antioxidant. Activities of vitamins D and E in milk
vary with the season or, more directly, with type of forage. Milk contributes a rather
small proportion of the dietary vitamin K in Western diets.
Milk is an important dietary source of water-soluble vitamins B 1 (thiamine), B 2
(riboflavin), B 6 (pyridoxine), B 12 (cyanocobalamin), niacin (nicotinic acid), andpan-
tothenic acid (Table 1.10). All the water-soluble vitamins are quite stable to milk
processing treatments, although riboflavin is extremely sensitive to degradation by
light of wavelengths <610 nm. Light-activated riboflavin is an agent in the devel-
opment of sunlight flavor in milk (Section 1.2.2.4) and also catalyzes the photodeg-
radation of ascorbic acid. Ascorbic acid is the most heat-labile vitamin in milk,
but this is of little consequence because milk is not an important source of dietary
vitamin C.

1.2.4.2 Minerals
Twenty-two minerals are considered essential to human nutrition. All of these are
present in milk, confirming milk's nutritional excellence. 169173174 However, nega-
tive factors may also exist. In particular, there is currently concern about iodine
concentrations, which may be elevated by disinfectant iodophors.175"180 There have
also been numerous recent investigations on the presence of radionuclides in milk,
especially 90Sr and 131 L 3 ' 181
Three families of salt constituents may be considered in milk.159 The first includes
sodium (Na), potassium (K), and chloride (Cl), which exist almost entirely as free
ions in milk and are readily diffusible (i.e., are present in milk ultrafiltrate). The
concentrations of these three ions are negatively correlated to lactose, as required
to maintain osmotic equilibrium of milk with blood. Thus, as compared to mid-
lactation, in early lactation, Na and Cl concentrations of milk are higher and lactose
concentration is lower.
A second family includes colloidal calcium (Ca), magnesium (Mg), inorganic
phosphorus (P1), and citrate. Total concentrations of Ca, Mg, P1, and citrate in milk
plasma are 30.3, 5.2, 21.4, and 9.5 mM, respectively, by calculation.159 On a molar
basis, about two-thirds of the calcium, one-third of the magnesium, one-half of the
inorganic phosphorus, and less than one-tenth of citrate in milk are colloidal (i.e.,
nondiffusible) and mainly present in the casein micelle (Section 1.3.1). Therefore,
concentrations of colloidal Ca, Mg, P1, and citrate are strongly correlated to the casein
content of milk.
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A third family includes salts, whose concentrations are affected by the natural pH
of milk, namely, diffusible Ca, diffusible Mg, diffusible citrate, Ca2+ , and HPO2T-
About 20 to 30% of diffusible Ca and Mg is present as free ions; the remainder
exists as citrate and phosphate salts. Diffusible Ca, Mg, and citrate concentrations
are positively correlated because Ca and Mg form strong complexes with citrate at
the pH of milk. A negative correlation between Ca 2+ concentration and pH and a
positive correlation between Ca 2+ and HPO 2 - relate to the solubility product of
micellar calcium phosphate. The degree of saturation of micellar calcium phosphate
and the concentration of H2POX are relatively constant. For example, increased
Ca 2+ causes formation of colloidal calcium phosphate, and the level of H2PO^ is
maintained by reaction of HPO2," with H + . Further discussion of acid-base equi-
libria is presented in Section 1.4.6.

1.3 Structure
1.3.1 Casein Micelles
1.3.1.1 Properties
In their classic monograph on dairy chemistry, Jenness and Patton in 1959 stated
that "Many of the problems of dairy technology revolve around the behaviour of
the (calcium caseinate-phosphate complex) and particularly the aggregation of the
particles by heat, salts, acid, and rennin. Therefore, a study of its composition and
properties is a most important phase of dairy chemistry".182 In the 30 years since
this statement was made, considerable effort has been put into studying the properties
of the casein micelle. The progress to date has recently been reviewed.183"185 The
intricacy of the micelles may be related to their biological functions in milk—to
carry a large amount of highly insoluble calcium phosphate to the mammalian young
in liquid form, and to form a clot in the stomach for more efficient nutrition.16'184
The micelle is extremely stable under some conditions of processing, for example,
concentration, ultrafiltration, pelleting, drying,183 but very unstable under others, for
example, acid, chymosin.16 A manipulation of micelle stability gives rise to many
traditional and nontraditional dairy products.17 A summary of the properties and
structure of the casein micelle will be presented here.
About 75% of the proteins in milk are classified as casein protein, that which
precipitates at pH 4.6.68 Most, but not all, of this casein protein exists in a colloidal
particle known as the casein micelle, which contains other components as well as
casein, including calcium, phosphate, citrate, minor ions, lipase and plasmin en-
zymes, and entrapped milk serum.16 This particle is a calcium-caseinate-calcium-
phosphate complex, and not a true micelle in the colloidal sense.16 The principal
casein proteins have been identified as asl~, as2-, /3-, and K-casein.184 Their properties
and primary structure have been discussed in Section 1.2.1.1. The identification of
two classes of milk proteins, y-casein and part of the proteose peptone fraction, has
been determined from primary sequencing to be degradation products or incom-
pletely synthesized precursors of /3-casein.68 The molar ratio of proteins within the