Dairy Chapter 1 PDF

Laboratory of Food Technology and Engineering
Academic year 2004-2005
Milk and Dairy Technology

Prof. Dr. ir. K. Dewettinck (Koen.Dewettinck@UGent.be)
Prof. Em. Dr. ir. A. Huyghebaert
ir. R. Rombaut (Roeland.Rombaut@UGent.be)
Chemical and physical properties of milk __ 1

1.1. Introduction _________________________________________________ 1
1.1.1. Mean composition and microstructure _____________________________ 1
1.1.2. Different kinds of milk _________________________________________ 4
1.1.3. Influencing factors concerning composition ________________________ 6
1.2. Carbohydrates ______________________________________________ 12
1.2.1. Lactose ____________________________________________________ 12
1. Chemical and biochemical properties of lactose ___________________ 12
2. Physicochemical aspects of lactose _____________________________ 15
3. Fermentation of lactose ______________________________________ 18
1.2.2. Other carbohydrates __________________________________________ 19
1.3. Lipids ______________________________________________________ 20
1.3.1. Fat content _________________________________________________ 20
1.3.2. Chemical properties __________________________________________ 21
1.3.3. Physical properties ___________________________________________ 23
1. Melting range ______________________________________________ 23
2. Supercooling_______________________________________________ 25
3. Nucleation and crystal growth _________________________________ 25
4. Polymorphism______________________________________________ 25
1.3.4. Deterioration of milk fat _______________________________________ 26
1. Lipolysis __________________________________________________ 26
2. Oxidation _________________________________________________ 27
3. Fishy taste_________________________________________________ 28
1.3.5. Other lipid components _______________________________________ 28
1. Phospholipids ______________________________________________ 28
2. Sterols ____________________________________________________ 28
3. Other lipids ________________________________________________ 28
1.3.6. Fat globules ________________________________________________ 30
1. Fat globule distribution_______________________________________ 30
2. Structure of fat globule_______________________________________ 31
3. Agglutination (Fig. 1.40 and Fig. 1.41) __________________________ 33
1.4. Proteins ____________________________________________________ 35
1.4.1. Introduction _______________________________________________ 35
1.4.2. Caseins ___________________________________________________ 37
1. αs1-Caseins ________________________________________________ 37
2. αs2-Caseins ________________________________________________ 38
3. β-Caseins _________________________________________________ 39
4. κ-Caseins _________________________________________________ 39
1.4.3. Casein micelles_____________________________________________ 40
1. Introduction _______________________________________________ 40
2. Structure __________________________________________________ 40
3. Stability of the casein micelles _________________________________ 41
1.4.4. Whey proteins______________________________________________ 42
1. β-Lactoglobulin ____________________________________________ 42
2. α-Lactalbumin _____________________________________________ 43
3. Bovine serum albumin _______________________________________ 44
4. Immunoglobulins ___________________________________________ 44
5. Lactotransferrin ____________________________________________ 45
6. Enzymes (See part 1.6.)______________________________________ 45
1.4.5. Milk Fat Globule Membrane Proteins ___________________________ 45
1.4.6. Non-protein nitrogen ________________________________________ 45
1.4.7. Enzymatic coagulation of caseins _____________________________________ 46
1. Introduction _______________________________________________ 46
2. Primary phase of rennet action _________________________________ 46
3. Secondary (non-enzymatic) phase of coagulation __________________ 48
4. Curd formation _____________________________________________ 48
5. Final stage_________________________________________________ 48
6. Rennets ___________________________________________________ 49
1.4.8. Heat-induced coagulation_____________________________________ 51
1.4.9. Acid-induced coagulation_____________________________________ 52
1.4.10. Ethanol coagulation _________________________________________ 52
1.4.11. Age-gelation of Sterilized Milks _______________________________ 52
1.4.12. Denaturation of whey proteins _________________________________ 54
1.5. Minerals and ionic equilibra ___________________________________ 55
1.5.1. Ash content ________________________________________________ 55
1.5.2. Composition of mineral fraction _______________________________ 55
1.5.3. Equilibra __________________________________________________ 55
1. Ca/P-equilibrum ____________________________________________ 55
2. Factors influencing equilibra __________________________________ 57
1.5.4. Trace elements _____________________________________________ 57
1. Content of trace elements _____________________________________ 57
2. Role of Cu and Fe___________________________________________ 57
1.6. Enzymes____________________________________________________ 59
1.6.1. Lactoperoxidase ____________________________________________ 59
1.6.2. Xanthine oxidase ___________________________________________ 59
1.6.3. Catalase___________________________________________________ 62
1.6.4. Lipase ____________________________________________________ 62
1.6.5. Phosphatase _______________________________________________ 62
1.6.6. Protease___________________________________________________ 62
1.6.7. Other enzymes _____________________________________________ 63
1.6.8. Reductase _________________________________________________ 63
1.7. Vitamins____________________________________________________ 64
1.7.1. Fat-soluble vitamins _________________________________________ 64
1. Vitamin A _________________________________________________ 64
2. Vitamin D _________________________________________________ 64
3. Vitamin E _________________________________________________ 64
4. Vitamin K _________________________________________________ 64
1.7.2. Water soluble vitamins _______________________________________ 64
1. Vitamin B1 ________________________________________________ 64
2. Vitamin B2 ________________________________________________ 65
3. Niacine ___________________________________________________ 65
4. Vitamin B6 ________________________________________________ 65
5. Pantothenic acid ____________________________________________ 65
6. Biotine ___________________________________________________ 65
7. Folic acid _________________________________________________ 65
8. Vitamin B12 _______________________________________________ 66
9. Vitamin C _________________________________________________ 66
1.8. Other compounds ____________________________________________ 67
1.8.1. Organic acids (Fig. 1.60) _____________________________________ 67
1.8.2. Gasses ____________________________________________________ 67
1.8.3. Biological factors ___________________________________________ 67
1.9. Physical properties ___________________________________________ 69
1.9.1. Density ___________________________________________________ 69
1.9.2. Dry matter_________________________________________________ 70
1.9.3. Freezing point______________________________________________ 70
1.9.4. Acidity (Fig. 1.61) __________________________________________ 71
1.9.5. Redox potential_____________________________________________ 71
1.9.6. NIR ______________________________________________________ 72
CHEMICAL AND PHYSICAL PROPERTIES OF MILK 1
Chapter 1:
Chemical and physical properties of milk
1.1. Introduction
1.1.1. Mean composition and microstructure
Milk has a variable composition, influenced by a number of factors. Some of

these factors are outlined in the following paragraphs. These factors influence
the composition mainly through quantitative variations, e.g. season, stage of
lactation, breed...
Milk can be defined as a polydispers system of water with:
Fat globules enveloped by a membrane, in emulsion Ø 0,1-10 µm

- Casein micelles Ø 10-100 nm
- Lipoprotein particles Ø 10 nm
- Soluble components e.g. globulins, sugars, minerals
Milk minus fat globules is called milk

plasma. It is almost but not quite equal to
separated milk (skimmed milk). Milk
serum is defined as milk plasma minus
casein micelles or milk minus fat, caseins
and insoluble salts. Dry matter contains
all dry milk components. Also fat-free dry
matter is defined (dry matter - fat).
In Fig. 1.1 and Fig. 1.2 the properties of

the principal structural components of
milk are shown.
Fig. 1.1: Milk viewed at different

magnifications. A: ×5, ×500, ×50 000
Fig.1.2: Composition and structure of milk

(approximate average quantities in 1 kg milk)
Fig.1.3: Properties of the main structural elements of milk, including

approximate numerical values
1.1.2. Different kinds of milk
Milk can be classified to its nature or to its technological process.

Fig. 1.4 and Fig. 1.5 show that the milks of species raised for milk production
differ markedly in gross composition, although fat and protein content are
positively correlated:
P = 1,93 + 0,42.F with P = protein content
F = fat content
Some of the properties in comparison with cow's milk are explained below.
human milk
- specific composition (Fig 1.6)
- more natural defense components
- (immunoglobulines, oligosaccharides, Bifidus factors)
- large amounts of lactose
- low in proteins (more whey proteins, fewer caseins)
goat's milk
- relatively more proteins and fat
- used for cheese manufacturing
sheep's milk
- rich in proteins and fats
- used for cheese manufacturing (Feta, Roquefort,…)
buffalo milk
- composition comparable to cow's milk
- high fat content (6-7%)
horse's milk
- low fat and protein content
- used for yogurt
- low nutritional value
- used in Eastern countries, Russia
Distinction is made between :

casein-rich milks :
- cow, sheep, goat, buffalo
- usable for cheese manufacturing; compact coagulation
- difficult digestion
albumin rich milks :

- humans, horse
- no cheese manufacturing possible; fine coagulation
- better digestion
In this course only cow's milk is discussed unless specified otherwise

Human Cow’s
g/100g
Milk Milk
Energy (kcal/110g) 74.7 66
Lactose 7.1 4.7
Lipids 4.54 3.8
Linolic acid 0.3-0.6 0.035
Cholesterol 0.014 0.021
Total protein 0.9 3.3
Ratio whey/casein 60/40 20/80
Casein 0.25 2.7
Whey 0.7 0.6
α-lactalbumine 0.26 0.11
lactoferrine 0.17 Traces
β-lactoglobulin none 0.36
lysozym 0.05 Traces
serumalbumin 0.05 0.04
IgA 0.1 0.003
Non-protein N 0.5 0.3
Vitamins (mg/100ml)
C 4 1
Fig.1.4: Relation of fat and protein of milks B1 0.01 0.03
B2 0.03 0.17
of hoofed mammals B3 0.2 0.08
B6 0.01 0.04
B9 (µg/100ml) 5 4
B12 (µg /100ml) 0.01 0.2
Biotine (µg/100ml) 0.7 2
A (IU) 190 102
D (IU) 2.4 1.4
E 0.3 0.07
K (µg/100ml) 1.5 5.8
Minerals (µg/100ml)
Ca/P ratio 2 1.2
Ca 30 125
P 15 90
Mg 3 12
Fe 0.1 0.03
K 50 150
Na 20 35
Zn 0.3 0.4
Cu 0.04 0.02
Fig.1.6: Comparison of cow’s and

mother’s milk
Fig.1.5: Gross composition of milks of various species

1.1.3. Influencing factors concerning composition
Variations in composition of the major and minor components are found. Even
when all influencing factors are kept constant, differences in milk composition of
different individuals are noticed.
1. Breed
A wide diversity of breeds of cows exists (Fig. 1.7). Breeds are predominately
the result of selection by people to obtain cattle suitable for the production of
milk, meat, or draught power and fit for local conditions, such as climate, feed,
terrain, and customs.
Ayrshir Brown Guernse Holstei Jersey Shorthor

e Swiss y n n
Fat 3.95 3.98 4.72 3.54 5.13 3.5
Crude
3.48 3.64 3.75 3.29 3.97 3.32
protein
Lactos
4.60 4.93 4.70 4.67 4.82 4.58
e
Ash 0.72 0.74 0.76 0.72 0.76 0.75
Total
12.77 13.07 14.04 12.16 14.42 12.27
solids
Fig.1.7: Different breeds of cows and the gross composition of the milk
2. Season / nutrition
Milk composition is influenced by seasons (Fig. 1.8 and Fig. 1.9). This is among
others the result of different feeding; silaged feed during winter months instead
of fresh grass during summer months. Differences are found in fat and protein
content. This has major implications for processing.
Fat
16 Protein Lactose Ash Total solids
% in milk
14
12
10
0
Jan feb mar april may june july aug sep oct nov dec
Fig.1.8: Influence of the season on milk composition
Fig.1.9: Examples of
seasonal variation in
properties of milk and
milk products
A. Firmness of butter (yield

stress)
B. Fat content of separated
milk
C. Quantity of citric acid
metabolized by
Leuconostoc cremoris in
milk
D. Heat coagulation time of
milk at 140°C
E. Percentage of herd
samples having fat acidity
>1.0mmol/100g
F. Color of milk fat (460 nm)
Because of homeostasis (i.e. the capability of an organism to maintain a

constant composition of body fluids and cells, despite gross differences in
food), nutrition has a comparatively small effect on most milk constituents.
Influencing factors are grass, ensilaged food, hay, ...
Fat content is affected distinctly by the feed, and fat composition even more. A
high-protein diet causes nonprotein N content of milk to increase. Several minor
components, such as carotene and fat-soluble vitamins, are strongly affected
by the content in the feed. This is the same for some trace elements.
Climate or weather conditions have little effect on milk composition. High

ambient temperatures (>30°C) tend to give higher fat, lower N and lower
lactose contents. At temperatures below freezing, fat and N contents both tend
to be higher.
3. Stage and number of lactation

The life cycle of a cow starts with a gestation period of 9 months. The period of
lactation or milk production is 305 days, in which the cow produces about 6000
to 8000 kg of milk. (note: the calf only needs about 1000 kg of milk for growth).
There is a peak in milk production around the third lactation. If the yield remains
good, the total amount of lactations is around 5-7. Three months after calving,
the next insemination is done, ensuring a yearly calving cycle. About 40 - 60
days before the next calving, the cow is dried off (lowering water
supplementation results in yield drop); there is no milking during this stage as
the fetus needs to grow and the udder needs regeneration in order to have high
yield in the next milking cycle
The life of a female cow can be summarized as follows:

Age
0 Birth of the calf
15 months Heifer inseminated for first calf
24 months Birth of the first calf: start of the milking cycle
27 months Insemination for second calf
34 months Drying off
36 months Second calf is born: start of second milking cycle
Cycle repeats for 5-7 lactations
The time elapsed after parturition or calving considerably influences milk

composition, presumably because the needs of the calve change with age.
Colostrum differs from normal milk; more salts, proteins (mainly serum
proteins) and less lactose. The composition evoluates towards its normal
composition within 4 days. After this period further changes occur: decrement in
protein content, casein, serum proteins, ashes, fat-free dry matter. At the end of
the lactation an increment occurs.
Lactose content is almost constant during lactation; fat content is correlated to
fat-free dry matter during lactation. Besides these structural elements, also pH
changes (in the beginning 6,6; for a period 6,7 and rises to 6,9 at the end)
The age of the cow has a very small but consistent effect on milk composition.
Fat and fat-free dry matter decrease slightly with each successive lactation.
Fig.1.10: approximate average milk Fig.1.11: Composition and early-

yield per cow and total dry matter lactation for successive milkings after
parturition
Fig.1.12: relations between milk components and stage of lactation

4. Mastitis
Several species of pathogenic bacteria may cause inflammation of the udder.
This is called mastitis. It causes a decrease in milk yield and a change in milk
composition (Fig. 1.13); the number of somatic cells also increases. The
variations in milk composition caused by mastitis are given in the table below:
Constituents Normal Abnormal Percent
milk (%) milk (%) Change
Solids non-fat 8.9 8.8 -1
Fat 3.5 3.2 -9
Lactose 4.9 4.4 -10
Total protein 3.61 3.56 -1
Casein 2.8 2.3 -18
Whey proteins 0.8 1.3 +62
Serum albumin 0.02 0.07 +250
Sodium 0.057 0.105 +84
Chloride 0.091 0.147 +61
Potassium 0.173 0.157 -9
Calcium 0.12 0.04 -66
Lactose and chloride content are negatively correlated. This is the result of the
activity of the udder, which maintains the milk isotonic. If lactose-production is
restricted by an affected udder, more chlorides are added to the milk by the
udder. This phenomenon can be used to detect mastitis infection. The number
of Koestler is a valuable parameter to differentiate normal from mastitis milk :
100 x % chlorides 100 x %chlorides
Normal milk = = 1,5 − 3,0 Mastitis milk = > 3,0
%lactose % lactose
Fig.1.13: Milk yield and concentration of

components of milk as a function of cell
count
5. Method of milking
During milking the fat content of the milk leaving the udder increases (e.g. from
1-10%), although there are marked differences among cows in this respect.
Nevertheless fat-free dry matter remains constant. The interval between milking
influences fat content, but not fat-free dry matter; longer intervals result in a
higher fat content and morning milk contains less fat then evening milk.
6. . Individual
Variations within breeds are the result of genetic and environmental factors. By
selection high-productive animals are obtained.
7. Quarters
Differences in composition in the milk of different quarters of the udder of one
cow mostly are negligible, unless a quarter is or has been affected by mastitis.
8. Other factors
Milk may vary, of course, in composition or properties because of contamination
and processing, but these aspects are not considered here.
1.2.
MILK AND DAIRY TECHNOLOGY

Carbohydrates
The most prominent carbohydrate of milks of most species is lactose. It is

virtually unique to milk, having been found elsewhere only in fruits of certain
members of the Sapotacea. Besides lactose, also its components (glucose and
galactose) and breakdown elements (lactic and butyric acid) are found in minor
quantities. Other carbohydrates are found only in small quantities.
1.2.1. Lactose
1. Chemical and biochemical properties of lactose

Chemically, lactose is a disaccharide consisting of one residue each of D-
glucose and D-galactose joined in a β-1,4-glycosidic linkage. Both moieties
occur predominantly in the pyranose ring form. So lactose can be called 1,4-β-
D-galactopyrasonyl-D-glucopyranose:
Lactose is a reducing sugar. This property is of importance in the analysis of

milk. This also causes non-enzymatic browning, (Maillard reaction). Amino
acids and reducing sugars react forming aroma products (HMF, aldehydes and
pyrazines) and brown components. Nutritional value of milk lowers during this
reaction.
A compound found in heated milk products is lactulose.
Concentrations of this compound up to about 1% may occur in commercial

evaporated milk. Lactulose is formed by the Lobry-De Bruyn transformation
(Fig. 1.14). Lactulose is sweeter than lactose and promotes the growth of
Bifidobacterium bifidum and thus beneficial in the diets of human infants.
Lactulose is also used as a parameter to evaluate the thermal treatment of milk
(sterilized versus UHT-milk). In UHT-milk, the maximum allowed concentration
is 600 ppm.

Fig.1.14: Lobry de Bruyn-Alberda van Ekenstein transformation of lactose into

lactulose and epilactose (Gal. = Galactose moiety)
The lactose content of milk is relatively constant at 4,8 to 5,2 % lactose

monohydrate. Lower levels occur in colostrum and mastitis milk to offset high
mineral levels and maintain osmotic balance. Lactose comprises about 52 % of
milk solids-non-fat, about 70 % of whey solids and > 90 % of the solids in milk
ultrafiltrate. The lactose concentration of other dairy products is given in figure
1.15.
Lactose intolerance is caused by a shortage of the lactase-enzyme (β-

galactosidase) and results in non-hydrolysis of lactose into glucose and
galactose.
Lactose is not as sweet as such other common sugars as sucrose, fructose and
glucose (Fig. 1.16). It is relatively sweeter at higher concentration. β-lactose is
sweeter than α-lactose, but this difference is not important since the small
difference between freshly prepared solutions is eliminated quickly by
equilibration of the anomers (see further).

Average
Dairy product lactose
concentration
Regular Whole Milk 4.8%
Light Cream 3.9%
Whipping Cream 2.9%
Sour Cream 3.9%
Buttermilk 4.3%
Yogurt, commercial lowfat 1.9-6.0%
Yogurt, commercial whole milk 4.1-4.7%
Sweet Acidophilus Milk 4.4%
Kefir, commercial part-skim 4.0%
Sweetened Condensed 12.9%
Ice Creams 3.1-8.4%
Butters 0.8-1.0%
Nonfat Dry Milk 51.3%
Dry Whole Milk 37.5%
Dry Whey (sweet-type) 63.0-75.0%
Dry Whey (sour-type) 61.0-70.0%
Blue 0.0-2.5%
Brie 0.0-2.0%
Gouda 0.0-2.2%
Cheddar (mild) 0.0-2.1%
Cottage Cheese (uncreamed) 0.0-3.5%
Quarg 3.0%
Fig.1.15: Lactose concentration of
some dairy products
Fig.1.16: Relative sweetness of sugars (percent concetration to give equivalent sweetness)

2. Physicochemical aspects of lactose
Mutarotation
Lactose exists in both α and β forms, which are indicated by interchanging the
OH and H on the reducing group. Lactose is optically active because of its
asymmetry and the α-form can be distinguished from the β form by its greater
rotation of polarized light in the dextro direction.
The α- and β-forms of lactose exist in solution in a temperature-dependent
equilibrium,following the next formula.
[β]/[α] = 1,64 - 0,0027.T with T = temperature in °C
This equilibrium is reached after 24 hours. E.g.: At 15 °C [β]/[α] = 1,59 or in this

solution 38 % of the lactose is present in the α form, 62 % in the β form.
Solubility
α- and β- lactose differ considerably in solubility and in the temperature
dependence of solubility. This factor is influenced by mutarotation. Lactose
solutions can be supersaturated easily, i.e. nucleation does not occur easily
(Fig. 1.17). At concentrations over 2,1 times the final solubility, spontaneous
crystallization occurs. At a relative supersaturation below 1,6 the solution
becomes metastable. Cristallization occurs only after a long time, or seeding
with lactose crystals is needed to induce crystallization.
Crystallization of lactose is of great practical importance, not only as a step in
the manufacture of lactose, but also because it may crystallize in some milk
products, notably sweetened condensed milk and ice cream.
During evaporation processes, the solubility is exceeded. In condensed milk, a
directed crystallization in the α-form is observed; crystals don't exceed 10 µm.
(Crystals of 30 µm give the defect sandy mouthfeel)
Fig.1.17: Lactose solubility curves

Crystal forms
Usually α-lactose crystallizes as a hydrate containing equimolar amounts of
lactose and water. The crystals are very hard, not hygroscopic and poorly
soluble (fig 1.18). Therefore, it may cristallize in some milk products, especially
ice cream and sweetened condensed milk (‘sandiness’ in milk products).
Above 93 °C, anhydrous β-lactose crystallizes. β-lactose dissolves much faster
than α-lactose hydrate at room temperature, as its solubility is about 10 times
higher and the crystals are usually smaller with a larger surface area.
Amorphous lactose is formed during rapid drying, like in a spray drier. This is
the glass state. Amorphous lactose quicky dissolves on addition of water, and
α-lactose may start to crystallize. It is very hygroscopic (it attracts moisture from
the air), which is of importance in milk or whey powder with amorphous lactose,
as it than can be converted α-lactose hydrate, resulting in hard lumps in the
powder and finally caking of the whole lactose mass.
Instant milkpowder has a better solubility by addition of moisture during the
drying process, resulting in a partial crystallisation into stable α-lacose hydrate.
As such, loose and spongy aggregates are formed that easily can be
redispersed in water. (Fig 1.19)
Fig.1.18: Lactose crystals

Fig.1.19: Modifications of lactose

3. Fermentation of lactose
Lactose can be fermented by bacteria that have a β-galactosidase (lactase)-
system.
- Lactic acid fermentation OH

OH
Lactose → glucose + galactose → 4 lactic acid
O
Lactic Acid
The fermentation of glucose follows the glycolitic or Embden – Meyerhof
pathway. Galactose-6-P is converted by the tagatose pathway. Dependent on
conditions and culture, the following reaction products can be formed:
OH OH
H COOH
COOH H
D (-) lactic acid L (+) lactic acid Racemic solution
When 1 % lactic acid is formed, the reaction process is slowed down (pH drop).
At this moment 20% of the lactose is metabolized (in yoghurt about 40%, as the
yoghurt bacteria cannot metabolize galactose)
Homofermentative bacteria transform lactose for 95 % into lactic acid and for 5
% into other components. Important aromatic compounds are:
O
- Diacetyl, the main aromacomponent in butter O
- Acetaldehyde, the main aromacomponent in Yoghurt O

Diacetyl Acetaldehyde
- Propionic acid formed out of lactic acid (Swiss type cheeses) O
OH
3 Lactic acid → 2 propionic acid + acetic acid + H2O + CO2
Propionic Acid
- Alcohol fermentation
OH
Lactose → acetaldehyde + CO2 → ethanol

Ethanol
This fermentation is performed by yeasts and is mostly undesired. In
some sour foaming milkdrinks it is desired (kefir, koumiss).
O
- Butyric acid fermentation
OH
Butyric Acid
2 Lactic acid → butyric acid + 2 CO2 + 2 H2
This fermentation is highly undesirable in cheese because of the excess

gas-production and off-flavor (late blow). It's characteristic for Clostridia
bacteria. (C. tyrobutyricum)

1.2.2. Other carbohydrates
Free glucose and galactose are detected readily in fresh milk, but there is a
lack of agreement on their concentrations.
Milk has no carbohydrates as glycogen or starch. Oligosaccharides are present
in small quantities, but are of great biological importance. Those
oligosaccharides are composed out of glucose, galactose and fukose (6-
deoxygalactose).
Some oligosaccharides are composed of hexosamines: N-acetylglucosamines,
a bifidus factor in human milk, have a serological activity (anti-hemaglutination).
Some of those oligosaccharides are made of neuraminic derivatives as N-acetyl
neuraminic acid (NANA). They are
bounded on κ-casein and have an anti-
bacterial activity.
1.3.

Lipids
1.3.1. Fat content
The fat content of milk is variable and dependent on a large number of factors.
This is caused by differences between individuals, but it is also influenced by
the season. Season variations are shown in Fig. 1.20 and Fig. 1.21
Fig.1.20: fatty acid composition of milk
Fig.1.21: Iodine value at different times of the year

1.3.2. Chemical properties
The global composition of milkfat is depicted in Fig. 1.22. Neutral lipids form
the major fraction, in particular the triglycerides. Polar lipids form a small
fraction but have an important structural function. In milk, these lipids are
present in the fat globule, in the fat globule membrane and in the plasma. Fig
1.23 and 1.24 illustrates this.
Fig.1.22: An overview of the lipids of fresh milk
Product (mass %) Total Fat Phospholipids Phospholipids Steroles FFA

(% on fat)
Milk 4.00 0.04 0.88 0.01 0.008
Skim Milk 0.06 0.02 25.00 0.00 0.003
Cream 10.00 0.07 0.65 0.03 0.017
Cream 20.00 0.12 0.60 0.06 0.032
Cream 40.00 0.21 0.53 0.11 0.06
Buttermilk (20%) 0.40 0.07 17.50 0.01 0.002
Buttermilk (40%) 0.60 0.13 21.67 0.01 0.002
Butter 81.00 0.25 0.31 0.21 0.12
Fig.1.23: Approximate content of various lipids in some dairy products

The major group, the triglycerides, consists of numerous different fatty acids.
This pattern is very specific for milk fat (Fig. 1.24). The following conclusions
can be drawn:
- Milk fat contains a relatively high proportion of short-chain fatty acids

(C4-C10). Butyric acid is specific for milkfat of ruminant species.
- The proportion of saturated fatty acids is high (63% w/w).
- Oleic acid is the most abundant of the unsaturated fatty acids residues.
- The other unsaturated fatty acids residues are present in a wide variety
of chain length, unsaturation and isomers.
- In the triglycerides, these fatty acids are stereospecifically distributed

within the molecule (Fig. 1.25). Milk fat has an asymmetric structure,
which influences the texture of derived products.
- There are several "odd" types of fatty acids (uneven, branched, keto,
hydroxy). This makes a very great range of some 250 different fatty acid
residues; to this must be added fatty alcohol and aldehyde residues.
Fig.1.24: Outline of fatty acid composition of milk lipids

Fluctuations in the composition occur, dependent on feed, season and breed.

These factors are of major technological importance as they influence the
physical properties of the milkfat (harder fat during winter periods).
Apart from triglycerides, mono- and diglycerides and even free fatty acids
occur, nevertheless in small quantities. This last group contains also the
hydrolytic products originating from lipolysis.
Fatty acid Average Sn-1 Sn-2 Sn-3

C4:0 11.8 - - 35.4
C6:0 4.6 - 0.9 12.9
C8:0 1.9 1.4 0.7 3.6
C10:0 3.7 1.9 3.0 6.2
C12:0 3.9 4.9 6.2 0.6
C14:0 11.2 9.7 17.5 6.4
C15:0 2.1 2.0 2.9 1.4
C16:0 23.9 34.0 32.3 5.4
C16:1 2.6 2.8 3.6 1.4
C17:0 0.8 1.3 1.0 0.1
C18:0 7.0 10.3 9.5 1.2
C18:1 24.0 30.0 18.9 23.1
C18:2 2.5 1.7 3.5 2.3
Of which is CLA 0.55
C18:3 Traces
Fig.1.25: Composition and stereospecific distribution of
fatty acids in milk fat triglycerides
1.3.3. Physical properties
1. Melting range
Melting characteristics are of major importance in the manufacturing of milk and
milk fat in particular. The melting point of milk fat is dependent of the fatty acids
present and its properties:
- Chain length and unsaturation
- Place of the double bound
- The presence of trans-isomers
- The fatty acid distribution within the triglycerides
In practice, butteroil is characterized by the solid fat content (SFC), which is

determined NMR (nuclear magnetic resonance). These characteristics can also
be found through DSC (differential scanning calorimetry) or DTA (differential
thermal analysis). In DSC the accumulated melting warmth is measured during
the transformation of solid to liquid state. In DTA the measurement is based on
temperature measurements (Fig. 1.26 to Fig. 1.29).

Fig.1.26: Solid fat curve of fractionated milk fat
Fig.1.27: DSC-curve of fractionated milk fat
Fig.1.28: Phase diagram

of a binary mixture. L is
the liquid line, S is the solid
line

Fig.1.29: Typical solid fat content curves of different fats
2. Supercooling
During cooling of liquid fat, supercooling occurs. Supercooling in fat in free form
is mostly less than 5°. Supercooling of fat in emulsion instead can be more
exquisite.
3. Nucleation and crystal growth

In milk fat, a supercooling of 5 K is sufficient to induce nucleation, and
crystallization of the highest melting triglycerides. As soon as crystals of these
have formed, they act in turn as catalytic impurities for the nucleation of other
triglycerides.
Milk fat in bulk shows little hysteresis between solidification and melting curves.
If the fat is finely divided, as in fat globules, the situation may be different. In
bulk fat, one catalytic impurity per milligram would suffice to ensure rapid
crystallization. But in milk 1 mg of fat is divided among some 108 globules, in
homogenized milk among at least 1011 globules. In each of those, at least one
nucleus must be formed, and now the number of catalytic impurities may easily
become limiting. Consequently, supercooling must be intensert and hysteresis
can be considerable.
4. Polymorphism
Triglycerides, like most molecules with long aliphatic chains, can crystallize in
three polymorphic modifications, denoted as α, β' and β. Each modification is
characterized by its crystal lattice type (mode of packing), not by its geometrical

form (habit). An important feature is the distance between the chains, the so-
called short spacings.
The melting points increase in the order α, β' and β, as do the melting heat and
the density of the crystals. This implies that closeness and intricacy of fit of the
molecules increase and their freedom of motion decreases.
The α and β' modifications are not stable. Translations can take place
according to Fig. 1.30 where solid arrows denote exothermal changes and
dotted arrows show endothermal changes. Nucleation of a fat usually occurs in
the α modification. Mostly, after a little while, transition to a stabler polymorph
occurs. In most fats, the α modification has only a short lifetime, while β' may
persist longer. In milk fat, α crystals can be very persistent.
Fig.1.30: Polymorphism of triacylglycerides
1.3.4. Deterioration of milk fat
1. Lipolysis
Hydrolysis of fatty acid esters by the action of lipases results in the common
flavor defect know as lipolytic or hydrolytic rancidity and is distinct from
oxidative rancidity. This lipolysis can occur either by lipases present in the milk
(mainly lipoprotein lipase) or by bacterial lipases. The properties of the fat
globule membrane are of major importance: reduced contents of phospholipids
or mastitis can increase the sensitivity of the fat globule for lipolysis. Other
factors that destabilize the fat globule membrane, especially agitation and
foaming, also promote lipolysis. Lipolysis is promoted in the manufacturing of
cheese.
Sensory perception of lipolytic rancidity is strongly affected by the pH of the
product, as at low pH, more free fatty acids are present in the aqueous phase,

where they are more readily tasted. In fresh milk threshold values corresponded
to acid degree values (ADV) of 4,1 to 4,5 mmol per 100 g of fat.
2. Oxidation
Oxidation of milkfat and other fats proceeds by the well-known autoxidation
reaction in three stages: initiation, propagation and termination (Fig. 1.31).
Unsaturated fatty acids are transformed to hydroperoxides, the primar
reaction products. During propagation, antioxidant 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, secondary reaction products, which are
responsible for the rancidic taste and odour.
Fig.1.31: Autooxidation mechanism of lipids
The factors that affect oxidation can be divided into 2 groups: intrinsic and
extrinsic factors.
Significant intrinsic factors affecting milk fat oxidation are:
- Metalloproteins such as milk peroxidase and xanthine oxidase

- Endogenous ascorbic acid, which acts as a cocatalyst with copper to
promote oxidation
- Endogenous copper content
- Endogenous antioxidants, mainly tocopherols

Important extrinsic factors include

- Contamination with metals
- Temperature of storage
- Oxygen tension
- Heat treatment
- Agitation
- Light
- Acidity
In the perspective of manufacturing, the heat treatment is very important.

Heating of 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. Heating can also cause denaturation of metalloproteins and
increase the availability of metals for oxidation. However high heat treatment
stabilizes milk against oxidation. This effect is probably due to exposure of
sulfhydryl groups of denaturated proteins and the release of hydrogen sulfide.
Homogenization drastically reduces the sensitivity of milkfat to both copper- and
light-induced oxidation, probably because oxidation sensitive membrane lipids
are displaced. Photooxidation is accompanied by depletion of riboflavin,
ascorbic acid and some amino acids. So-called sunlight-flavor is caused by
oxidation of methionine to methional.
3. Fishy taste
Fishy taste can be induced by transformation of fosfatidylcholine to
trimethylamine.
1.3.5. Other lipid components
1. Phospholipids
Phospholipids comprise about 1% of the fat content. The fatty acids are more
unsaturated than those of the triglycerides (Fig 1.32). Due to its hydrophilic and
lipophilic properties they occur as well in the membrane as in the plasma
(lipoprotein particles). During separation processes phospholipids follow the
specific way of membrane fragments and plasma particles. Cerebrosides are
glycolipids with similar properties as the phospholipids (Fig 1.33).
2. Sterols
Sterols form the largest fraction of the unsaponifiable lipids. They
consist largely of cholesterol. A small fraction of the
cholesterol is esterified (so this fraction is saponifiable).
Sterols can be found in the 3 fractions. HO
Cholesterol
3. Other lipids
- Pigments; mainly carotenoids (β-carotene = provitamin A)
- Its concentration is season-dependent; in summer double quantities
compared to winter. In sheep, goat and buffalo milk is no β-carotene
present.

- Vitamins (mainly A, D and E)

- Antioxidants (mainly tocopherols)
- Squalene
- Polar fatty acids: keto acids en HO acids, important for lacton formation.
Fig.1.32: Fatty acid profiles of milk triglycerides and milk

phosphatidylethanolamine
Phospholipids With X=
O Phosphatidyl- CH2CH2NH3+
O ethanolamine 33 %
O
NH3+
O O P O X Phosphatidylserine 7.5 % CH2HC
O COO-
O
HO OH
Phosphatidylinositol 7.5 % OH
HO OH
Me+
Phosphatidylcholine 27 % CH2CH2 N Me
Me
Sphingomyelin 20 %
OH
O Me
+
N Me
N O P O
H Me
O O
Glucosylcerebroside 1.5 % Lactosylcerebroside 3.5 %

O
O
N R
N R O O
O O O
O O O O
O O
O O O O
O O
O O
Fig.1.33: Phospholipids and cerebrosides present in bovine milk

1.3.6. Fat globules
Milk can be considered as an oil in water emulsion. A thin membrane, 8 to 10

nm in thickness, which reduces the lipid serum interfacial tension, covers the
lipid droplets (fat globules). The fat globules can be seen under a microscope.
1. Fat globule distribution

Thermodynamically, no emulsion is stable; thus the stability of the fat emulsion
is a kinetic time-dependent phenomenon. Milk separates or creams
spontaneously and rapidly and many processes involve manipulation of the
creaming phenomenon. Emulsion stability is largely dependent on the size
distribution of the globules. In raw milk, fat globules range in size from 0,1 to 15
µm. The milk emulsion has been found to contain three distinct populations of
fat globules (Fig. 1.34):
- Small globules (<1µm) that represent 80% of the total number of the
globules, but only a small fraction of the total milk fat
- Large globules (>12µm) that represent 2-3% of the total milk fat
- The medium group which represents 95% of the total milk fat
The following example indicates the large activity at the level of the membrane:
70 m2 per liter of milk (3,5 % fat).
The fat globule size distribution can be influenced by many factors:

- The individual
- The stage of lactation: shift to smaller globules during lactation (Fig.
1.35)
- The breed: in fat rich milk larger globules occur
The fat globules can be strongly modified by technological processes, e.g.

homogenization (Fig. 1.36). These items are discussed in other chapters.
2.
Fig.1.34: Size distribution Fig.1.35: Stage of lactation Fig.1.36: Effect of

of lipid globules in milk and breed differences in homogenization of milk fat
size distribution of milk on the volume frequency
lipid globules distribution of the fat
globules

Structure of fat globule

The fat globules are in fact glycerides surrounded by a fat globule membrane.
The composition of the membrane is dependent on the procedure of isolation,
making quantification of the membrane more difficult. In the membrane, the
following components are present (Fig. 1.37):
- Phospholipids and cholesterol

- Proteins, especially glycoproteins
- Enzymes (xanthine oxidase and alkaline phosphatase)
- High melting glycerides, which are probably crystallized on the
membrane during cooling.
Fig.1.37: Approximate content of various lipids in some dairy products
In earlier days the King membrane model was accepted; the membrane
consists out of several layers: a layer of proteins and a layer of phospholipids
(Fig. 1.38). The actual model that is accepted is the biological membrane
concept. The membrane consists out of 2 layers (Fig. 1.38):
1. The internal layer: a cellular membrane existing out of globular proteins
and phospholipids, that has no enzymatic activity and lays on a layer of
high melting glycerides;
2. The external layer with enzymatic activity. This layer determines
agglutination and adsorption phenomenons as well as the stability of the
globule. The membrane adsorbs metals Cu and Fe out of the plasma
(oxidation), and is also responsible for adsorption of microorganisms
(natural pasteurization).
The synthetic membrane, which is produced by homogenization and
recombination, is a casein membrane.

Fig.1.38: Milk fat globule membrane model
Fig.1.39: Milk fat globule with ruptured membrane

3. Agglutination (Fig. 1.40 and Fig. 1.41)

In undisturbed milk, lipid globules rise and form a cream layer. Centrifugation
causes decreaming. The creaming effect can be described by the law of Stokes
:
2r 2 (dp - df ) g
V =
9η
with V = speed of creaming g = gravity acceleration

r = radius η = viscosity
dp, df = density of plasma and fat
The speed of creaming is in reality much higher than this law predicts. Also an
adverse temperature effect is found (cold agglutination); at lower
temperatures creaming increases while V decreases as a result of a smaller
difference in density between plasma and fat. This effect can be explained by
flocculation. This floculles are formed by agglutination.
In heated milk, creaming doesn't often occur, as a consequence of denaturation
of agglutinins. The form of the flocules is temperature dependent. At room
temperature, only a small cream layer is produced, while at 7 or 8 °C, a
voluminous cream layer is formed. In homogenized milk no flocculation occurs,
but clustering.
Destabilization of the emulsion can be performed in different manners:
- Mechanical movement (free fat in cooled milk)

- Enzymes
- Churning (butter still contains fat globules; 10-50 % of the fat).
Fig.1.40: Agglutination processes

1.4.
Fig.1.41: Types of physical changes in oil-water emulsions

Proteins
1.4.1. Introduction
The proteins of milk are of great importance in human nutrition and influence
the behavior and properties of dairy products. Normal bovine milk roughly
contains 30-35 g protein/litre. The nitrogen content of milk is distributed among
caseins, whey proteins, milk fat globule membrane proteins and non-protein
nitrogen (NPN). (Fig. 1.42).
Fig.1.42: Milk proteins
The caseins, which account for 76-86% of the total protein, essentially all occur
in micelles. Most of these caseins can be represented by four gene products:
αs1 -, αs2 -, β- and κ-caseins in the approximate ratio 40:10:35:12. The casein
fraction also contains several minor proteins, most of which originate via post-
translational processing such as phosphorylation, glycosylation or limited
proteolysis.
The major whey proteins are β-lactoglobulins, α-lactalbumins, immunoglobulins,

and serumalbumin, in order of decreasing amounts. The amino acid
composition and properties of some milk proteins are summarized in Fig. 1.43
and 1.44.

Fig.1.43: Chemical composition of the major milk proteins
Fig.1.44: Properties of the major milk proteins

1.4.2. Caseins
Originally, the caseins were defined as the phosphoproteins that precipitate

from raw skim milk upon acidification to pH 4.6 at 20°C. The individual families
were identified by alkaline urea gel electrophoresis. With the resolution of their
primary structure, it became apparent that not all of the caseins contain
phosphorus. Some are also found in the acid whey after removal of the
precipitated caseins.
Caseins contain high numbers of proline residues (Fig. 1.43), which are
distributed relatively uniformly throughout the polypetide chains. Proline inhibits
formation of an ordered, stable α-helix. There is little evidence of tertiary
structure of caseins, which accounts for the stability of caseins against heat
denaturation, as 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. Significant effects of milk protein genetic variants on heat
stability, renneting properties, and concentration and distribution of milk
components have been reported.
Since the caseins contain negatively charged groups such as phosphates,
side-chain carboxyls, terminal carboxyls, and sulfhydryls, they bind a number of
different cations, such as calcium, barium, magnesium, potassium, sodium, etc.
The presence of highly charged segments facilitates electrostatic interactions.
The binding of calcium by the caseins is of primary interest because of its
involvement in micelle formation and its effect on the stability of the milk protein
system.
Due to the high content of phosphoseryl residues, αs1-, αs2- and β-caseins bind
polyvalent cations, principally Ca++, leading to charge neutralization,
aggregation and eventually to precipitation.
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
phosphorylation or formation of subfractions are indicated after the genetic
variant (e.g. αs2-CN B-13P).
1. αs1-Caseins
The αs1-caseins, along with the αs2-caseins, make up the previously designated
calcium-sensitive αs-casein fraction, precipitated with 0.4 M CaCl2 at pH 7 and
4°C.
At the present time, five genetic variants of αs1-caseins are known: A, B, C, D,
and E. The polymorphs are breed specific. The major B variant, predominant in
European cattle, has a molecular weight of 23614 Da.
The association of αs1-caseins is strongly dependent of pH, ionic strength and
kind of ion in the medium but essentially independent of temperature up to
30°C. At low Ca2+ concentrations, octamers are formed, which aggregate and
precipitate at higher Ca2+ concentrations.

A small amount of λ-caseins may be present in milk. These appear to be

fragments of αs1-caseins as the result of plasmin (an endogenous alkaline
proteinase present in milk) proteolysis. The corresponding hydrophillic N-
terminal fragments form a part of the so-called proteose-peptone fraction and
are recovered in the whey.
2. αs2-Caseins
The four recognized genetic variants of αs2-casein are designated A, B, C, and
D. The A and D variants have been observed in European breeds and have a
calculated molecular weight of 25230 Da.
αs2-casein self-associates at neutral pH via a series of consecutive steps similar
to αs1-casein; its association is strongly dependent on ionic strength. This
casein is even more sensitive to precipitation by Ca++ than αs1-casein. αs2-
casein has a remarkable dipolar structure with a concentration of negative
charges near the N-terminus and positive charges near the C-terminus (Fig.
1.45).
3.
Fig.1.45: Location magnitude and direction of charged residues (pH 6-7) in caseins.
(•) Pro
(S) Cys
(a) Location of glucide residues
(b) Point of cleavage by chymosin

β-Caseins
The β-casein family consists of one major component with at least seven
genetic variants (A1, A2, A3, B, C, D, and E) and eight minor components
which are proteolytic fragments of the major component. The A variants are the
predominant (nearly 100%) polymorphs in all species and strains of Bos and
have a calculated molecular weight of 23983 Da.
β-Casein has a strongly negatively charged N-terminal portion (Fig. 1.45), the
rest of the molecule has virtually no net charge. The β-casein molecule is
somewhat like that of an anionic detergent with a negatively charged head and
an uncharged hydrophobic tail.
The association of β-casein is dependent on ionic strength and pH but in
contrast to αs1- and αs2-caseins, its association is strongly temperature
dependent. β-Casein is monomeric at 4°C in the absence of Ca++ but
considerable association is apparent at 8.5°C.
The γ1-, γ2- and γ3-caseins, present in raw milk, are formed by the action of the
plasmin on β-casein in milk. The carboxyl terminal fragments of β-casein (γ-
caseins) remain associated with the casein micelle and are recovered by pH
4.6 precipitation. The N-terminal fragments (proteose-peptons) are hydrophilic
and appear as heat-stable fractions in whey.
4. κ-Caseins
κ-Casein occurs as a mixture of polymers held together by disulfide bonds. The
monomers consist of a major carbohydrate-free component and at least six
minor components. They possess considerable heterogenety arising from
several different sources:
- genetic differences
- variation in carbohydrate content and/or phosphate content
- a possible variation in the para-κ-portion of the molecule.
Two genetic variants of the κ-caseins are known: A and B. The A variant tends
to be the predominant variant in most breeds and has a calculated molecular
weight of 19007 Da.
The structure of the minor κ-casein components differ from the major
component in that while they have the same primary amino acid sequence, they
contain various amounts and types of carbohydrate (N-acetylneuraminic acid,
N-acetylgalactosamine and galactose) moieties attached to the polypeptide
chain by post-translational glycosylation.
κ-Casein forms trimers via intermolecular disulfide bonds; the trimers aggregate
further by hydrophobic bonding. κ-Casein, most of which contains only one
phosphoseryl residue, does not bind Ca++ strongly and is soluble in the
presence of high calcium concentrations. Further, κ-casein associates with αs1-
and/or β-casein, and in the presence of Ca++, κ-casein stabilizes these Ca-
sensitive caseins against precipitation by Ca++ with the formation of stable
colloidal particles.

1.4.3. Casein micelles
1. Introduction
In normal uncooled milk, ≈ 95% of the casein exists as coarse colloidal
particles, micelles, comprised of some 20 – 150.000 casein molecules with
molecular weights of ≈ 108 dalton and mean diameters`of ≈ 100 nm (range 50 -
500 nm) (Fig. 1.46). On a dry weight base, the micelles consist of ≈ 94% protein
and ≈ 6% of small ions, principally calcium, phosphate, magnesium and citrate,
referred to collectively as colloidal calcium phosphate (CCP). In milk the
micelles are highly hydrated, typically ≈ 2 g H2O/g protein. The approximate
composition of the casein micelle is given in Fig. 1.47.
Fig.1.46: Size frequency Fig.1.47: Composition of casein micelles

distribution of casein micelles in cow’s milk at room temperature. g/100g
micelles on a dry basis
Casein micelles have different functions:

- They form a coagulum in the stomach of the nursling, allowing a
slow release of nutrients
- They act as a way of transporting calcium and phosphate
- They are a source of essential amino-acids
- Enzymatic cleavage of the caseins produce various types of
biologically active peptides (casomorphines, …)
2. Structure
The detailed structure of the casein micelles is still not known but it is widely
accepted that the micelles are composed of spherical submicelles, 10 - 15 nm
in diameter, and have a porous structure. The stoichiometry of the four principal
caseins is believed to vary between indivual submicelles but various techniques
indicate that κ-casein, the principal micelle-stabilizing factor, is located
predominantly on the surface (Fig. 1.48).
Some of the casein fractions, particularly β-casein, are able to migrate out of
the micelle to the serum phase in a reversible manner without causing collapse
of the micellar structure. This migration is temperature dependent (higher at
lower T).

The very hydrophilic C-terminal part of most κ-casein molecules is sticking out
from the micelle core into the solvent as flexible "hairs". These hairs are
essential in providing stability against flocculation of the micelles.
The submicelles are linked together in the micelles by colloidal calcium
phosphate and hydrophobic bonds. The calciumphosphate acts as a
cementing agent.
Fig.1.48: Schematic presentation of the casein micelle
3. Stability of the casein micelles

- Heat stability. Limited heat treatment has no effect on the micelles.
Sterilization however can desintegrate the micelles into a fibrillar
structure. Homogenization and evaporation causes forming of larger
units. With fat globules, the micelles can form potein lipid structures. At
higher temperatures the micelles are stable for the following
temperature-time combinations: 10 minutes at 130 °C and 50 minutes at
115 °C.
- Lowering temperature. Lower temperatures can cause desintegration of

the micelles, in particular β-casein. This effect is important as milk is, for
primar conservation, cooled to below 5°C. However, this effect is
reversible, i.e. the micelles are re-integrated by increasing the
temperature.
- Change in mineral composition. Addition of CaCl2 raises the Ca-activity

and the colloidal calciumphosphate, causes a decrease of the pH and
the net charge, all of this being negative to the stability. Sodiumcitrate
and -phosphate give a more stable complex. The removal of calcium

ions from the micelle causes reversible dissociation of β- and κ-casein

from the micelles without micellar desintegration.
- Dehydration leads to aggregation of the micelles. Presumably, the

dehydration of the κ-casein causes a decrease in voluminosity and steric
repulsion. A similar effect occurs when large quantities of salts are
added. Aggregation of casein micelles in frozen milk products also
seems to be caused by salting out.
1.4.4. Whey proteins
Whey proteins are the major nitrogen compounds remaining in milk after
precipitation of the caseins by acid (pH 4.6) or by rennet (pH ≈ 6.7) and
represent ≈ 20% of the nitrogen in bovine milk. They possess a well-defined
tertiary structure and are heat-labile. The whey proteins include a
characteristic group of globular proteins, i.e. β-lactoglobulin, α-lactalbumin,
bovine serum albumin, the immunoglobulins, and some minor proteins, like
lactoferrin. They are synthesized in the mammary gland of the cow, e.g. β-
lactoglobulin and α-lactalbumin, or derived from the blood, e.g. bovine serum
albumin and immunoglobulins.
Some minor whey proteins have antimicrobial properties, e.g. lactoferrin,
lactoperoxidase, and lysozym.
The proteose peptone fraction occurs in acid and rennet whey. The
glycomacropeptides (originating from κ-casein) are only present in rennet whey.
Milk contains about 30 enzymes, derived mainly from blood and secretory cell
membranes. Some of these enzymes, especially lipoprotein lipase and
proteinase, are technologically important in milk and dairy products.
1. β-Lactoglobulin
β-Lactoglobulin is a major milk protein, representing about 50% of the whey
proteins or about 12% of total milk proteins. The monomeric molecular weight
of β-lactoglobulin is about 18300 Da. Eight genetic variants are known. The
high affinity of β-lactoglobulin for retinol has prompted speculations that its
biological function is related to vitamin A transport.
The conformation of β-lactoglobulin depends on the pH. At pH values between
6.7 and 5.2 (its isoelectric point), β-lactoglobulin exists at room temperature as
a stable noncovalently linked dimer. Between pH 5.2 and 3.5, the dimer of β-
lactoglobulin associates to form octamers of 147000 Da (especially the A
variant); octamerization is maximal from pH 4.4 to 4.7 at 0°C. Below pH 3.5, the
quaternary structures dissociate reversibly to monomers due to strong
electrostatic repulsive forces. The B variant (which is the predominant one in
Western cattle) octamerizes to a much smaller extend, possibly due to
increased electrostatic repulsion.
The free thiol group between Cys119 and Cys121 (Fig 1.49) is of great
importance for changes occuring in milk during heating, as it is involved in
reactions with other proteins, notably κ-casein and α-lactalbumin. β-
Lactoglobulin binds a variety of hydrophobic molecules.
2.

α-Lactalbumin
α-Lactalbumin represents about 20% of the proteins of bovine whey. It is a
small molecule with a molecular weight of about 14000 Da. The iso-electric
point of α-lactalbumin is pH 4.8. Three genetic variants, A, B, and C have been
identified in bovine milks. Only variant B is found in the milks of western breeds.
Several minor components have been observed in recrystallized preparations
of α-lactalbumin from bovine milk. These components have the same amino
acid composition as the major component but contain carbohydrates. These
glycosylated forms contain mannose, galactose, fucose, N-acetylglucosamine,
N-acetylgalactosamine, and N-acetyl-neuraminic acid.
The amino acid sequence of α-lactalbumin is similar to that of lysozyme. α-
Lactalbumin is necessary for the synthesis of lactose by its interaction with
galactosyltransferase, an enzyme that catalyzes the transfer of galactose.
Without α-lactalbumin, glucose is an extremely poor substrate for galactosyl-
transferase.
α-Lactalbumin contains eight cysteine groups, all of which are involved in
disulfide bonds (Fig. 1.49). α-Lactalbumin has a highly ordered secondary
structure, and hydrodynamic data indicate a compact, spherical tertiary
structure. Thermal denaturation of α-lactalbumin is accompanied by a release
of bound calcium, and α-lactalbumin is stabilized against heat denaturation and
aggregation in the presence of calcium.
α-Lactalbumin has good emulsifying and foaming properties.
Fig.1.49: Location magnitude and direction of charged residues (pH 6-7) in β-

lactoglobulin and α-lactalbumin
(•) Pro
(S) Cys
(a) Location of glucide residues
(b) Point of cleavage by chymosin
3. Bovine serum albumin

Serum albumin prepared from bovine milk is identical to the blood bovine serum
albumine (BSA), the major protein of plasma. Bovine milk contains ≈ 0.4 g
BSA/litre. It has a calculated molecular weight of 66267 D.
No specific function has been identified for BSA in milk; presumably, it is a
leakage protein. BSA is a well-known transport protein for insoluble fatty acids
in the blood circulatory system. The binding of fatty acids stabilizes the protein
molecule against heat denaturation. This ability to bind fatty acids may promote
lipolysis.
The tertiairy structure of BSA reveals three about equal-sized globular domains.
It has one free thiol and 17 disulfide linkages, which neatly hold the protein in a
multiloop structure.
BSA is highly soluble up to 35% (w/w) in pure water at 3°C, but it undergoes
extensive precipitation in the temperature range 40-45°C. This changed
solubility above 40°C parallels the reversible (partial) unfolding of BSA. On
acidification to pH 4.0, the BSA undergoes acid denaturation.
4. Immunoglobulins
Immunoglobulins are antibodies
synthesized in response to
stimulation by macromolecular
antigens foreign to the animal.
Bovine colostrum contains up to 100
g/litre immunoglobulins (Ig) but the
concentration decreases to < 1
g/litre within a week of parturition.
The primary function of Ig in milk is
to provide passive immunity for the
neonate.
The immunoglobulins are a very
complex, heterogeneous group of
proteins. Five principal classes of Ig
are recognized: IgG (80% of total),
IgA, IgM, IgD and IgE. The basic
sub-unit in each class consists of
four polypeptide chains, two heavy
(H, MW ≈ 50 – 70000 Da) and two Fig.1.50: Basic four-peptide chain
light (L, MW = 22400 Da) chains, structural unit of an immunoglobulin
linked by disulfide bridges (Fig.
1.50).
In the presence of other whey proteins, the Ig-fraction appears to be very

thermolabile, which may be related to the activity of thiol groups of β-
lactoglobulin and BSA.
The immunoglobulins have interesting, but so far not well understood,
technological properties such as cold agglutination of fat globules in milk and
binding of fatty solutes and bacteria in desalted, acidified whey.
5.

Lactotransferrin
The transferrins are a group of evolutionary related iron-binding proteins, the
best characterized members of which are:
- Serotransferrin (present in blood plasma and other extracellular fluids,

e.g. milk, spinal fluid, semen)
- Lactotransferrin (milk, pancreatic juice, tears, leucocytes).
Lactotransferrin, a monomeric glycoprotein with a molecular weight of about 80

kD, has the ability to bind reversibly two Fe+++ per molecule, and to suppress
bacterial activity by removing the iron that is required for bacterial growth.
Concentration of lactotransferrin in bovine colostrum and milk is ≈ 1 and 0.02 -
0.35 mg/ml, respectively. Because of the apparent physiological and nutritional
significance of lactotransferrins, several improved methods for their isolation
have been published.
6. Enzymes (See part 1.6.)
1.4.5. Milk Fat Globule Membrane Proteins

A thin membrane surrounds the fat globules in milk. It contains approximately
50% protein and accounts for about 1% of the total protein of the milk (Fig
1.37). The total protein complement of the membrane as observed is
dependent upon the past history of the membrane from its formation to its
analysis. Both the temperature and the time of storage before analysis can alter
the membrane composition and physical state. At the current moment, 8
proteins are well characterised (Mucin, xanthin dehydrogenase, adipophilin,
butyrophilin, Pas 3, Pas 6/7, Cd36, pp3 and FabP). Most likely, these proteins
are artefacts from the plasmamebrane from the udder.
1.4.6. Non-protein nitrogen
A large number of N-containing compounds of low molecular weight occur in

milk (250 - 350 mg of N per litre). The principal NPN components are listed in
Fig. 1.51. The wide variations in concentrations that have been reported for
these constituents probably arise from the fact that many of them are
metabolites of amino acids and nucleic acids and from the fact that their
concentrations in milk depend on the amounts of those substances consumed
by the cow.

Fig.1.51: Principal nonprotein nitrogen (NPN) compounds in milk
1.4.7. Enzymatic coagulation of caseins
1. Introduction
The clotting of milk by proteolytic enzymes represents one of the oldest
operations in food technology and milk-clotting enzymes have been used since
antiquity for the manufacture of cheese.
Milk-clotting is a complex process, involving
- a primary enzymatic phase in which κ-casein is altered and loses its

ability to stabilize the remainder of the caseinate complex
- a secondary non-enzymic phase in which aggregation of the altered
caseinate takes place,
- a third step where the aggregate of casein micelles forms a firm gel
structure
- a possibly seperate fourth step where the curd structure tightens and
syneresis (the expulsion of water by the curd arising from structural
rearrangements after formation) occurs.
2. Primary phase of rennet action

The primary phase of the rennet action involves the enzymatic cleavage of the
Phe105-Met106 linkage of κ-casein. This results in the formation of the soluble
glycomacropeptide (GMP), which diffuses away from the micelle and para-κ-
casein, a distinctly hydrophobic peptide that remains on the micelle (Fig. 1.52
and 1.53):
κ-casein para-
+ glycomacropeptide (106-169)
(1-169) κ-casein (1-105)
Strongly hydrophobic Hydrophilic

remains in micelle Soluble, Washed out

-1
This is a relatively quick reaction, the turnover being 100 s in milk, pH 6.7,
30°C, and seems independent of micelle size. Decreasing temperature by 10°C
reduces the enzymic phase by a factor of 2.
Fig.1.52: Properties of κ-casein and the polypeptides formed by chymosin action
3.
Fig.1.53: Renneting of milk. S: degree of splitting of κ-casein, A: aggregation of

paracasein micelles, F: firmness of the gel

Secondary (non-enzymatic) phase of coagulation

Hydrolysis of κ-casein during the primary phase of rennet action reduces the
intermicellar repulsive forces (electrostatic and steric) and the micelle stability.
Renneted micelles appear to be incapable of aggregating until about 80% of
their κ-casein has been destroyed. This behaviour can be explained either by
the loss of surface charge during renneting or by loss of steric stabilization.
During the last 20 % of the proteolytic reaction both the concentration of
micelles that are capable of aggregating and the rate at which they can
aggragate increase rapidly as the last of the stabilizing surface is removed.
The aggregation rate of the renneted micelles is unaffected by the
concentration of rennet or by the size of the casein micelles. It is however very
sensitive both to the concentration of calcium ions present in the solution and to
the temperature. Milk itself will not clot at less than about 15°C, and this can be
shown to be a direct consequence of the slowness of the aggregation reaction
at low temperatures. Low temperatures (less than 8°C) allow proteolysis of κ-
casein in the absence of coagulation, coagulation occurs on subsequent
warming. However, at temperatures above 45°C the aggregation is very
efficient and approaches the theoretical maximum rate at which particles can
collide. Addition of CaCl2 to milk decreases the coagulation time. At high
calcium chloride concentrations (0.4 M), the coagulation time is retarded
severely and only weak curd is obtained.
4. Curd formation
The curd starts to form at about the visually observed clotting time (RCT). It is
characterized by a steady aggregation of the rennet-treated casein micelles.
Chains of micelles are formed at first, these have begun to link into a loose
network. The network then extends and becomes more differentiated, with the
chains of micelles aligning together.
Renneting or clotting time is the resultant of two reactions. The enzymatic
reaction is largely determinant, as flocculation time is insignificant to splitting
time. At 4°C, the enzymatic reaction may be complete in about 3 h, while
clotting takes a week.
During this time, the linkage between the micelles also appear to strengthen.
Initially, many micelles are joined by bridges, but later these appear to contract,
bringing the micelles into contact and eventually causing partial fusion. The
chemical nature of the cross-links is not yet entirely clear, but the phosphoseryl
side chains of casein, especially β-casein, are probably involved. These may be
linked by Ca++ bridges.
5. Final stage
The final stage in the clotting of milk is not well defined and includes syneresis
and firming of the curd, a loss of paracasein micelle identity, and non-specific
proteolysis of caseins in the coagulum. The paracasein micelles fuse into larger
units as CCP rearranges throughout the micellar region, and this may be
analogous to binding between submicelles in a casein micelle.

Syneresis is enhanced by applying external pressure to the curd or deforming it

(stirring, cheddaring). Several factors affect the rate and extent of syneresis:
- Size of the curd pieces
- Pressure gradients`
- Ca-phosphate in the micelles decreases syneresis but promotes rigidity
- Lowering pH promotes syneresis (Ca-phosphate dissolves)
- Addition of Ca++ lowers syneresis
- At temperatures below 18°C no syneresis occurs
6. Rennets
Traditionally, a crude enzyme extract called rennet, prepared from calves’
stomachs, has been used in the milk clotting process. Recent shortages of this
material have led to the use of pepsins and acid proteases produced by other
animals, fungi and microorganisms. Presently, calf chymosin produced through
recombinant DNA techniques is also available. In more general use, any milk-
clotting enzyme preparation yielding a relatively stable curd is designated as
rennet.
Animal rennets.
The term "calf rennet" in the cheese industry, generally refers to an enzyme
extract obtained from the fourth stomach (abomasum) of 10- to 30-day-old
unweaned calves and used to coagulate milk for cheese production. The
purified milk-clotting enzyme present in crude rennet preparations is known as
chymosin (EC 3.4.23.4) with a molecular weight of 35,600 D. It belongs to the
group of aspartic proteases and is the standard against which all other types of
milk-clotting enzymes are compared. Chymosin coagulates milk rapidly at its
natural pH with little further degradation of the milk proteins.
As the calf ages, chymosin is replaced by pepsin, although in cattle, the
secretion of chymosin never comes to a complete stop. Although pepsin can
clot milk, its use as a 100% replacement for calf rennet in cheese making is
limited due to the following shortcomings:
- Set time for curd formation is extented

- The curd is softer than that obtained with calf rennet,
- Bitter peptides are formed,
- Fat loss is excessieve,
- The cheese flavor is generally bland
- Above pH 6.5 pepsin activity falls of so rapidly that its use is limited to
the production of only some sweet (e.g. swiss cheese) and some Italian
varieties of cheeses.
Plant rennets
Many proteolytic enzymes of vegetable origin are known to be milk coagulants,
such as papain (EC 3.4.4.10) from papaya, ficin (EC 3.4.4.12) from Ficus spp.,
and bromelain (EC 3.4.4.24) from pineapple. Unfortunately, most plant
coagulants have proved to be non-specific in their proteolytic activity and the
excessive proteolysis results in bitter products. At present there is no
commercially available rennet derived from a higher plant.

Bacterial rennets
A number of bacteria are reported to produce milk-clotting enzymes In spite of
some incentives (high titer of the active preparation, short generation times),
commercial production of rennets with bacteria has not been successful
because of the invariably strong and nonspecific proteolytic action of the milk-
clotting enzymes, resulting in loss of fat and nitrogen in the whey, reduced
yield, and poor quality of the aged cheese. Furthermore, many of the bacteria
are not approved for food use and some are known to be pathogenic. So, at
present there is no commercially available bacterial coagulant to substitute for
animal rennet. However, bacterial coagulants still remain promising, they may
find particular use for specialty cheeses or as partial substitutes in mixtures with
other enzymes.
Fungal rennets
Many species of filamentous fungi produce a chymosinlike enzyme but, in
general, most fungal coagulants are much too proteolytic for use as rennets.
The coagulants from three fungal species viz., Endothia parasitica
(Cryphonectria parasitica), Mucor miehei (Rhizomucor miehei) (thermophilics)
and Mucor pusillus (Rhizomucor pusillus) (mesophilic) have proved suitable for
large-scale commercialization.
Recombinant calf chymosin

Despite the efforts made in minimizing differences between microbial and calf
chymosin, variations still exist, making the latter the preferred enzyme for
cheese making. Some of these differences include:
- The ratio of proteolytic to milk-clotting activity is still a little higher for

microbial rennets, resulting in greater hydrolysis of cheese protein during
ripening and leading to soft body and texture
- Microbial rennets are more thermostable, cannot be inactivated at
normal pasteurization temperature, and therefore present problems in
processing of cheese whey,
- Change in milk-clotting activity from calf to microbial rennet will require
adjustment of the process parameters such as temperature, pH, calcium
concentration etc.
Because of the differences cited above, calf chymosin has been cloned in
suitable microbes. The calf chymosin produced by recombinant DNA
technology in Aspergillus niger var. awamori, Escherichia coli K12, and
Kluyveromyces lactis is now commercialized under the trade names
Chymogen® (Chr. Hansen), Chy-Max® (Pfizer), and Maxiren® (Gist-Brocades).
A variety of cheeses have been produced using recombinant chymosin and no
significant differences could be detected among cheeses made by rennet and
recombinant chymosin.

1.4.8. Heat-induced coagulation
The ability of bovine milk to withstand high processing temperatures is essential

for many modern milk-processing operations. While the non-casein proteins of
milk are relatively heat-labile, the caseins are remarkably heat-stable: sodium
caseinate in water can withstand heating at 140°C for >60 min at pH 6.7 and
typical bulk bovine milk is stable for ≈20 min at 140°C.
Most protein denaturation reactions are very pH dependent and the pH change
caused by heating is primarily responsible for heat coagulation (Fig. 1.54). The
decrease in pH upon heating is partially due to changes in the buffer capacity of
milk salts and the release of carbon dioxide. When milk at a pH of less than 6.5
is heated for 20 to 30 min at 100°C, it coagulates to form a gel. Casein micelles
isolated from such milk have denatured whey proteins attached to the micelle
surfaces. Such micelle surfaces aggregates of whey proteins and κ-casein may
serve to join the micelles to each other. When milk pH is greater than 6.7, more
heat treatment is required for precipitation and a gel does not form. At this pH,
the whey proteins are not denatured and coagulation occurs only when the
caseins in the micelles have changed enough to cross-link with each other.
Fig.1.54: Some heat-induced changes in milk likely to lead to

protein coagulation

1.4.9. Acid-induced coagulation
The four caseins are insoluble at their iso-electric points, i.e. ≈ pH 4.6, at
temperatures above 20 °C. The caseins remain more or less soluble if milk is
acidified to pH 4.6 at temperatures lower than 6°C; the precipitate formed
becomes progressively coarser and more rubbery as the temperature at
precipitation is increased. Raising the temperature gives an immediate
precipitation. Although the iso-electric point of the γ-caseins is about 6, they
also precipitate at pH 4.6. The proteose peptons are, by definition, soluble at
pH 4.6.
A pH-decrease causes release of Ca++ and decrease of micelle voluminosity.
So, micelles are demineralized during acidification. At pH 6.0 almost 50% of the
colloidal Ca is transformed to ionar Ca. Near pH 4.8 almost all phosphate is
dissolved and near pH 4.6 (the isoelectric point) casein does not contain Ca
anymore.
Acid coagulation is exploited in the manufacture of Cottage cheese, Quarg and
cultured dairy products, such as sour cream. In natural culturing processes
lactic acid produced by streptococci and lactobacilli causes coagulation by
lowering the pH near the isoelectric point of casein (pH 4.6). Cheeses can be
produced by adding rennet and acidulants, such as citric acid (queso blanco),
fumaric acid, glucono-delta-lacton, malic acid, phosphoric acid, succininic acid,
tartaric acid, and hydrochloric acid, to cold milk (4 - 8°C). Subsequent warming
of the milk (to 35°C) produces a uniform gel structure. Addition of acid to warm
milk results in a protein precipitate rather than a gel.
1.4.10. Ethanol coagulation

Milk coagulates on addition of ethanol, usually to ≈ 40%. The ability of milk to
withstand treatment with ethanol without coagulation (ethanol stability) has
been used as a not-very-effective selection test for milk for the manufacture of
sterilized concentrated milk. The ethanol stability of milk is strongly influenced
by the Ca++-concentration and the pH.
1.4.11. Age-gelation of Sterilized Milks

In-container sterilized evaporated milk occasionally gels during storage.
However, UHT-sterilized milks, especially concentrated milk, are very prone to
gelation during storage (age gelation, Fig. 1.55), limiting the usefulness of such
products. The cause(s) and mechanism of age-gelation have not been
definitively established, but there are probably two separate causes.
- Plasmin and extracellular proteinases secreted by psychotrophic

bacteria are major causative factors in the gelation of unconcentrated
UHT milk but physicochemical changes in the casein micelles appear to

be principally responsible for age-gelation of UHT-sterilized concentrated

milk.
- UHT sterile milk concentrate contains casein micelle-denatured whey

protein complexes (β-lactoglobulin/κ-casein interaction) that are about
double the size of native casein micelles. These complexes undergo
extensive aggregation during storage that eventually causes age
thickening.
Age thickening is promoted by high milk solids content, addition of alkali to raise
pH, and addition of citrate, phosphate, and other anions that lower Ca ion
activity. Conversely, addition of Ca++ improves stability of the product against
thickening.
Fig.1.55: Age thickening of concentrated milk (broken lines indicate milk with
added polyphosphate)

1.4.12. Denaturation of whey proteins
The whey proteins, which have typical globular conformations, are relatively
heat labile. Denaturation occurs upon heating, in figure 1.56 the difference in
heat sensitivity of the whey proteins is denoted. The susceptibility of whey
++
proteins to heat denaturation in whey is influenced by such factors as pH, Ca -
concentration, protein concentration and the presence of sugars, alcoholic
groups and protein modifying agents.
The ranking order of the heat sensitivity (denaturation temperature, determined
by DSC) of the individual proteins was α-lactalbumin > β-lactoglobulin > bovine
serum albumin > immunoglobulins.
Heat-induced interaction between β-lactoglobulin and κ-casein plays a major
role in determing the heat stability and rennet clotting behaviour of milk. β-
Lactoglobulin precipitates on casein micelles and is also precipitated by
destabilization of the micelles (β-lactoglobulin - κ-casein interaction).
1.5.
Fig.1.56: Denaturation of whey proteins at 30 min heating

Minerals and ionic equilibra
1.5.1. Ash content
The ash content of milk is about 0,70 - 0,85 % and is lower than the actual
present salt content. The ash comprises oxides of Na, K, Ca, Mg, Fe, P and S,
some chlorides, S and parts of P and Fe of organic origin.
1.5.2. Composition of mineral fraction

The composition of the mineral fraction of milk is outlined in Fig. 1.57. Three
families of salt constituents may be considered in milk. The first includes Na, K
and Cl, which exists almost entirely as free ions in milk and are readily diffusible
(present in milk ultrafiltrate). The concentrations of these 3 ions are negatively
correlated to lactose, as required to maintain osmotic equilibrium of milk with
blood.
A second family includes colloidal Ca, Mg, inorganic P and citrate. Total
concentrations of Ca, Mg, Pi and citrate in milk plasma are 30,3; 5,2; 21,4 and
9,5 mM.
A third family includes salts, whose concentrations are affected by the natural
pH of milk, namely, diffusible Ca, diffusible Mg, diffusible citrate, Ca2+ and
HPO42-.
1.5.3. Equilibra
The form in which the mineral occur, whether as ions, whether in colloidal form,
are of importance for the equilibra and as consequence for physicochemical
properties of milk, e.g. stability, heat stability, gelling, clotting.
1. Ca/P-equilibrum
Calcium is present in 4 forms:
- 20 % as organic Ca: Ca caseinate
- 80 % as inorganic Ca:
• 50 % as inorganic colloidal tricalciumphosphate
• 20 % as not ionized Ca salt: Ca citrate and phosphate
• 10 % ionic Ca
Phosphor is also present in different forms:

- 30 % as organic P
• 10 % in phopsholipids and other esters
• 20 % in caseins as phophoserine
- 70 % as inorganic P
• 35 % colloidal calciumphosphate
• 35 % soluble phosphate

Dialysis of milk doesn't result in removal of all Ca and P, which means that
these elements are partly bound. A part of the calciumphosphate is present in
an insoluble form, the colloidal calciumphosphate. Milk is supersaturated in
calciumphosphate; this is precipitated on the micelles. On the casein, cations
are bound as counter-ion for the negative charge of the protein.
Fig.1.57: Some minor components in fresh milk. Contents per liter

2. Factors influencing equilibra
ACIDIFICATION
During acidification more Ca will be present in its ionar form; the colloidal
calciumphosphate will be more soluble. By acidification micelles are
destabilized; the negative charge of the micelles decreases, which causes a
higher concentration of ionar Ca.
HEATING
During heating a part of the calciumphosphate will become insoluble and
precipitates on the micelles. This can be accompanied by a slight pH-drop.
Heating above 100°C causes splitting of phosphate from the caseins and
creates acids out of lactose. The result is a pH-drop that influences the
equilibria.
WATER REMOVAL
During evaporation processes, the concentration of solubilized components
increases, which can affect the equilibra.
ADDITIONS
Addition of CaCl2 causes an increase of Ca-activity and a decrease in the
stability of the micelles. Addition of sodiumcitrate and certain phosphates
betters the stability of the micelles.
1.5.4. Trace elements
1. Content of trace elements

In Fig. 1.58, the content of trace elements in milk is outlined. Some of these
trace elements have an important role in the activity of enzymes. The presence
of Manganese is also known to play an important role in the growth of lactic
acid bacteria.
2. Role of Cu and Fe
Cu and Fe have a technological significance as catalyzator of oxidation
reactions. Cu is present in fat globule, where it can oxidize (phospho)lipids.
Normally milk contains 20-25 mg.kg-1. Extra Cu can enter the milk through dust,
the apparatus and the water. This Cu is distributed between membrane and
serum.
Technological processes can influence the distribution of Cu-ions:
• Acidification causes a migration of Cu to the fat globule membrane;
• During cooling Cu migrates from the membrane to the plasma;
• During heating Cu migrates to the membrane, but high pasteurization
hinders this transport;
• The distribution of Cu, during decreaming, depends on the history of
milk (heating, cooling).

Fig.1.58: Trace elements in milk
1.6.

Enzymes
Milk contains both indigenous and exogenous enzymes, the latter being mainly
bacterial. With respect to dairy processing, the most significant bacterial
enzymes occurring in milk are heat-stable lipases and proteinases elaborated
by psychotrophic bacteria. Indigenous enzymes, the reactions they catalyze
and their location in milk are summarized in Fig. 1.59.
These enzymes need special attention as they cause several defects in milk but
also can be responsible for some beneficial effects; as lined out below:
- Desired transformations, e.g. ripening of cheese (natural proteases)

- Undesired degradation processes, e.g. lipolysis of raw milk
- Indicator enzymes for heating of milk
- Antimicrobial effects of certain enzymes
- Quality parameter
The most important of the indigenous enzymes present in milk and their activity
are explained in the following paragraphs.
1.6.1. Lactoperoxidase
Lactoperoxidase catalyzes oxidation by H2O2 of a long list of electron-donor

compounds, including aromatic amines, phenols, aromatic acids, leuko dyes,
tryosine and tryptophan, ascorbate, iodide, nitrite and thiocyanate. They
transfer oxygen in its atomar form from hydrogen peroxide to its substrates.
Lactoperoxidase may amount to as much as 1% of the total serum proteins of
milk (i.e. 60 mg.kg-1). The properties of this enzyme are well known (pH-
optimum 6,8; molecular weight 82.000). It is an indicator enzyme for high
pasteurization.
-
Lactoperoxidase catalyzes oxidation of thiocyanate (SCN-) to a product (OSCN
), which inhibits certain bacteria. Thiocyanate is a natural constituent of milk
and some bacteria produce hydrogenperoxide. Thus, in milk such bacteria
exhibit self-inhibition and are as such a natural anti-microbial constituent. In
some countries this effect is enhanced by further addition of hydrogen peroxide.
1.6.2. Xanthine oxidase

Xanthine oxidase is very prominent in milk. Most of it is associated with the fat
globule membrane. It catalyzes the reaction of purine bases (xanthine and
hypoxanthine) to urinic acid en hydrogenperoxide.
Xanthine oxidase can reduce NO3- (which occurs in milk only in trace quantities)
to NO2-, which is a powerful inhibitor of some bacteria. This property is put to
use in the manufacture of certain types of cheese, where a little nitrate is added
to the milk to prevent late blowing of the cheese by Clostridia.
The enzyme is especially activated by pasteurization and homogenization.

Fig.1.59: Enzymes of Bovine Milk

Fig.1.59: Enzymes of Bovine Milk (Continued)

1.6.3. Catalase
Catalase catalyzes the decomposition of H2O2 to H2O and O2. In milk its activity
parallels leukocyte count and is higher in mastitic milk and colostrum than in
mormal milk. It increases with the growth of bacteria.
The enzyme is inactivated at 65°C after 30 minutes and is used as a test to
control heat treatment of milk.
In this prospect the catalase procedure can be explained. H2O2 is added to milk
to inactivate sporeforming bacteria. The excess of H2O2 is decomposed by the
catalase.
1.6.4. Lipase
Lipase catalyzes the hydrolyzation of triglycerides to glycerol and fatty acids. In
milk, distinction can be made between plasma lipase and membrane lipase.
The membrane lipases are originally present on the casein micelles, but adsorb
irreversible to the fat gobule membrane during cooling of milk. These enzymes
are often called lipoprotein lipase. They are responsible for the rancid flavor of
raw milk.
The plasma lipase stays in the plasma during the cooling of milk. Nevertheless,
mechanical movement can activate the enzyme.
During cool conservation of milk, lipolysis can occur as a consequence of the
combination temperature - mechanical movement. The enzymes are
thermolabile and can be of bacterial origin.
1.6.5. Phosphatase
Phosphatases, also called phosphomonoesterases, are of natural origin and
are responsible for hydrolysis of phosphoesters.
A first phosphatase is the alkaline phosphatase. Its pH-optimum is about 9,6. It
is present in milk, but is destructed by pasteurization. It is an index for the
efficiency of pasteurization. Two major isoenzymes have been identified, α- and
β- phosphatase, mainly located in the milk plasma and fat globule membrane.
A second phosphatase present in milk is the acid phosphatase, with a pH
optimum of 4,5. This phosphatase is extremely heat stable, but is sensitive to
light and UV. It is present in low concentrations, particularly in skimmed milk.
1.6.6. Protease
The principal milk protease belongs to the alkaline serine proteinase class, and
is probably identical to the plasmin of blood and is present on the casein
micelles. It has alkaline properties, survives pasteurization and is relatively
resistant to UHT-treatment. This last characteristic can cause a bitter flavor
(hydrophobic peptides of low molecular weight) and changes in viscosity and
appearance. This is also of importance during the ripening of cheese.

A second protease, with maximal activity at pH 4,0, also occurs in milk. This
protease is very heat sensitive and hydrolyses preferably α-caseins. It is not yet
clear how this enzyme should be classified.
1.6.7. Other enzymes

Amylases catalyze the hydrolysis of starch to dextrines and maltose, dependent
on its nature (α- or β-amylases). The α-amylase is inactivated by heating 55°C
during 30 minutes, while the β-amylase keeps its activity after 30 minutes at
65°C.
Lysozyme is quantitatively an important fraction of the whey proteins of human
milk (400 mg.l-1) (less important in cow's milk). It is a powerful bactericide as it
attacks polysaccharides of the bacterial cell wall, causing lysis of the bacteria.
It releases bifidus factors.
Lactase normally doesn't occur, and if so only in small quantities.
1.6.8. Reductase
The reducing properties of microorganisms (and leukocytes) are used as
quality-parameter. It causes decoloration of methylene blue and other dyes.
Nowadays, this parameter has lost its importance as a result of better cooling
conditions, which has changed the bacterial flora in milk.
1.7.

Vitamins
Milk is a good source of diverse vitamins. Its mean composition is depicted in

Fig. 1.57. There is often a difference upon their solubility in fat or water.
1.7.1. Fat-soluble vitamins
1. Vitamin A
Vitamin A and its analogues are important for eye functioning. β-carotene, also
called provitamin A, is also present in milk. β-carotene is responsible for the
yellow colour of butter. The vitamin A content in whole milk is 0,4 mg per kg, in
skimmed milk only 20 µg per kg, in butter however 7,5 mg per kg.
During the summer, milk contains 50% more vitamin A than during winter
months. Pasteurization, sterilization and drying have nearly no effect on vitamin
A or β-carotene content.
2. Vitamin D
Vitamin D is important for the growth of the skeleton. Milk contains about 2 µg
vitamin D per kg. This value is very dependent on seasonal variations. Milk in
summer contains 2 to 3 times more vitamin D then milk in winter. Vitamin D is
very stable and its content is not affected by pasteurization or sterilization.
3. Vitamin E
Vitamin E (tocopherol) deficiency causes sterility in male and female animals.
The vitamin E content of human milk is about ten times that of cow's milk. The
need for vitamin E increase as the amount of unsaturated fatty acids in food
increases.
Milk contains only 1 mg vitamin E per liter (butter contains 23 mg per kg). The
vitamin E content is higher during summer than during winter. This is caused by
the higher tocopherol content of fresh grass. Heat or other processes don't
cause a significant decrease of the vitamin E content.
4. Vitamin K
Vitamin K is only in traces present in milk, if at all. Human needs for this vitamin
are supplied from consumption of plant materials containing it and by microbial
synthesis in the digestive tract.
1.7.2. Water soluble vitamins
1. Vitamin B1
Thiamine or vitamin B1 is essential for growth and metabolism of all animals but
also of many plants and microorganisms. In milk it is not only present in its free
form, but also phosphorylated (18-45%) and as a protein complex (5-17%). The
mean concentration in milk is about 0,43 mg per liter. De dairy requirement of a
grown person is about 1,0 to 1,4 mg.

Thiamine is more or less destroyed by heat, dependent on time-temperature

combination. In pasteurized milk and UHT milk the loss is about 3-4%, while in
sterilized milk this loss can mount up to 45%.
2. Vitamin B2
Vitamin B2 or riboflavin, is very important in hydrogen transport, carbohydrate
and protein metabolism, transformations of sugar and proteins into fatty acids,
and in eye-functioning. In cow's milk, riboflavin is mostly present in its free form.
The content in whole, skimmed and buttermilk is about 1,7 mg per liter. The
daily requirements of a grown person are 1,5 to 1,7 mg.
In the absence of oxygen, vitamin is very persistent during heat treatment. Thus
the effect of pasteurization and sterilization on the riboflavin is small.
3. Niacine
Niacine is a component of co-enzymes that are involved in citric acid cyclus,
metabolism of fatty acids and glycol acid cyclus. The niacine content of milk is
about 0,9 mg per liter, while in skimmed and butter milk, this content decreases.
The dairy requirement of a grown person mounts up to 13 to 18 mg.
4. Vitamin B6
Vitamin B6 is very important in protein metabolism. The main content in milk is
about 0,6 mg per liter. The daily requirement is about 1 to 4 mg per kg.
Pasteurization or homogenization gives no significant losses of vitamin B6
content, although great losses are caused by sterilization (up to 50%). Vitamin
B6 is also very sensitive to light.
5. Pantothenic acid
Pantothenic acid is involved in the metabolism of carbohydrates, fats and amino
acids. The mean content in milk is about 3,4 mg per liter. The daily
requirements for grown persons are about 10 to 15 mg. This vitamin is very
stable during a number of processes (e.g. manufacture of evaporated milk,
sterilized milk and milk powder).
6. Biotine
Biotine is involved in carbohydrate and fatty acids metabolism. Milk is a good
source of biotine. The mean content of whole milk is about 0,030 mg per liter, in
skimmed milk 0,016 mg and in buttermilk about 0,011 mg. The daily
requirement of a grown person is only 30 µg. Light and heat has only a small
effect on the vitamin content. In pasteurized and UHT the losses are less than
10 %, while in sterilized milk the losses can mount up to 10 to 15 %.
7. Folic acid
Folic acid is necessary for the normal development of red blood cells. Few data
about the content in milk are available; in raw milk it would be about 0,06 mg.
This value is also reached in cheese. Folic acid is very sensitive to heat,
oxygen and light. During sterilization, losses of 50 % can occur.

8. Vitamin B12
Vitamin B12 is the only vitamin that contains a metal (cobalt). Deficiencies of
this vitamin can cause anemia. B12 is only present in animal products and can
give problems for vegetarians. The mean content is about 4,2 µg per liter. A
grown person only needs 1 µg per day. Vitamin B12 is sensitive to heat and
oxygen. In strongly heated products, the losses can reach 100 %.
9. Vitamin C
Vitamin C is involved in the formation of intercellular material (collagen). Fresh
raw milk contains over 20 mg, but decreases to 16 mg in skimmed milk and 12
mg in buttermilk. The daily demands of a grown person are 55 to 60 mg.
Vitamin C is very sensitive to light, heat and metals (Cu, Fe). During
pasteurization 20 % is destroyed, during evaporation 75 % and during
sterilization 50 to 100 %.
1.8.

Other compounds
1.8.1. Organic acids (Fig. 1.60)
Citric acid is very important for the stability of milk. It is also a substrate for
organisms and is transformed to aromas.
Besides citric acid, also lactic acid, hippuric acid (benzoïc acid + glycocol) and
orotinic acid (precursor of nucleotides) are found.
Fig.1.60: Alcohols, carbonyls, acids and esters in milk
1.8.2. Gasses
After milking 8 % (volume) gasses, especially CO2 (6%), are present. During
processing (heating) and storage, the CO2 content decreases, while the O2 and
N2 content increase. Raw commercial milk contains at room temperature about
1,3 % N2 and 0,5 % O2 by volume, or about 15 and 6 mg.kg-1.
1.8.3. Biological factors

- Bifidusfactors
- Streptogenines
• Peptides formed out of casein
• Growth factors for lactic acid bacteria
- Lactenines
• Active against lactic acid bacteria
• L1, L3 agglutines
• L2 lactoperoxidase

1.9.

Physical properties
1.9.1. Density
The density of milk and milk products is used to convert volumetric

measurements to gravimetric and vice versa. Density can be expressed as
mass density or volumetric mass and is designated ρ. This parameter is
temperature-dependent. The density of milk at 20°C is on average about 1030
kg.m-3 and normally varies within the range of 1027 to 1033 kg.m-3.
The density of milk is dependent on composition and can be calculated from the
density and mass fraction of individual components. The following equations
have been cited to approximate the density of milk, skim milk, creams and
concentrated milks. At 20°C the densities of water, milk fat, protein, lactose and
other components are 998.2, 918, 1400, 1780 and 1850 respectively. As can be
calculated, fat content decreases the density of milk, while solubilized
components increase its density. As a consequence, the density of skimmed
milk is higher than that of whole milk. Addition of water causes a decrease of
the milk density.
⎛ ⎞
⎜ mx ⎟
∑
1
= ⎜ ⎟
ρ ⎜ ρx ⎟
⎝ ⎠
As previously designated, this parameter is temperature dependent. One must

take into account, that with a change in temperature, a slow stabilization of the
density has to be considered, because the physical properties of fat and the
hydratation of proteins can take time. This effect is often called the Recknagel
effect.
The density, expressed in kg.m-3, of some dairy products is outlined below:
- Fresh whole milk: 1030

- Heated standardized milk: 1030
- Skimmilk: 1035
- Sweet whey: 1025
- Light cream 20% fat: 1009
- Heavy cream 40% fat: 988
- Evaporated milk 26% solids: 1066
- Evaporated milk 32% solids: 1085
- Sweet condensed milk: 1310
- Buttermilk: 1029

1.9.2. Dry matter
Dry matter is in essence a chemical property, but has a major influence on

physical properties. Its mean value in milk is 12,50 %. Dry matter is determining
weight loss during heating.
The dry matter can be calculated from some characteristics by the Netherlands
formula:
100 (ρ 20 - 0, 9982)
D.M. = 1,23 F + 2,6
ρ 20
with ρ20 = volume mass at 20°C D.M. = dry matter F = fat content
Fat-free dry matter (F.F.D.M.) is defined as F.F.D.M. = D.M. - F
1.9.3. Freezing point
Freezing point is a colligative property that is determined by the molarity of

solutes rather than by the percentage by weight or volume. It is a constant that
is used to determine addition of water, which has a warmer freezing point as a
result. It is determined by some components in solution: lactose and salts.
Freezing point determinations may be done by the Horvet procedure, which
uses mercury in glass thermometer or, as in most modern instruments, by using
a termistor cryoscope. For scaling, solutions of sugar or NaCl are used:
7 % sucrose = -0,422°H (6,879 g NaCl diluted to 1000 ml)
10 % sucrose = -0,621 °H (10,175 g NaCl diluted to 1000 ml)
Conversion formulas are given :
m°H = 1,0356 °C
m°C = 0,9656 m°H
The freezing point of milk is usually in the range of -0,512 to -0,550°C with an
average of -0,522°C. If the freezing point of unwatered milk is known, the
relationship between added water and freezing point depression is given :
(C - D)(100 - S)
W=
C
with W = percentage (w/w) extraneous water in suspect milk

C = actual or reference freezing point of genuine milk
D = freezing point of suspect milk
S = the percent (w/w) of total solids in the suspect milk
Soured or fermented milk is suitable for added water testing because the
freezing points is lowered by lactic acid and increased concentrations of soluble
minerals. Several reports suggest that heat treatment of milk, including UHT
and retort sterilization causes little permanent effect on freezing points, but it
has also been suggested that freezing points are not a reliable index for added
water in processed milk.

1.9.4. Acidity (Fig. 1.61)
The acidity of milk is an important parameter for its quality, but is also a
characteristic for fermentation processes. Fresh milk normally has a pH of 6,6
to 6,8. This acidity is not the result of lactic acid, but a result of dry matter.
The acidity can be expressed in:

- D° (Dornic) # ml NaOH N/9 to neutralize 100 ml of milk
- N° (Normal grades) # ml NaOH 0,1N “ “ “ “
- S.H. (Soxhlet-Henkel) # ml NaOH 0,25N “ “ “ “
- % Lactic acid
Earlier, titration of milk at reception was based on the acidity: alizarol test,
bromocresol test, alcohol test, ...
Fig.1.61: Acidity of some milk products
1.9.5. Redox potential

The redox potential of milk is in the range of +0.2 to +0.3 V and is mainly
determined by dissolved oxygen. Milk is essentially oxygen free when excreted
but about 0,3 mM O2 is present after equilibrium with the surrounding air.
Removal of oxygen by nitrogen lowers the E° of milk to about -0,12 V.
Decreased oxygen tension by bacterial respiration is the basis of the methylene
blue reduction test for milk bacterial quality.
The other redox systems of significance in milk are ascorbate and riboflavin.
Ascorbate in freshly drawn milk is all in the reduced form, but oxidizes during
refrigerated storage. This process also produces singlet oxygen, which is
involved in lipid oxidation.
Riboflavin is important for photooxidation reactions in milk; methionine is
transformed into methional, which is the principal component of "sunlight" flavor
in milk.

Heat treatment is well known to increase the reducing capacity, mainly due to
activation of protein thiol groups and products of Maillard browning reactions.
Activated thiol groups cause cooked flavor, which decreases as cysteine bonds
reform on standing.
1.9.6. NIR
Near infrared spectroscopy (NIR) covers the range of the electromagnetic
spectrum between 780 and 3000 nm. In this range it is possible to study low
energy transitions, overtones and combinations of hydrogen stretching and
bending vibrations that have high frequencies and are well suited to quantitative
analysis applications.
First tests with this technique were carried out on milkpowders to analyze its
constituents (moisture, protein, lactose, fat and ash content). Later on also
whey powders were analyzed. Nowadays, routine analysis of cheese and liquid
milks are performed with NIR methods.

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Laboratory of Food Technology and Engineering

Academic year 2004-2005

Milk and Dairy Technology

Chemical and physical properties of milk __ 1

Chemical and physical properties of milk

1.1.1. Mean composition and microstructure

Milk has a variable composition, influenced by a number of factors. Some of

Fat globules enveloped by a membrane, in emulsion Ø 0,1-10 µm

Milk minus fat globules is called milk

In Fig. 1.1 and Fig. 1.2 the properties of

Fig. 1.1: Milk viewed at different

Fig.1.2: Composition and structure of milk

Fig.1.3: Properties of the main structural elements of milk, including

1.1.2. Different kinds of milk

Milk can be classified to its nature or to its technological process.

Distinction is made between :

albumin rich milks :

In this course only cow's milk is discussed unless specified otherwise

Fig.1.6: Comparison of cow’s and

Fig.1.5: Gross composition of milks of various species

1.1.3. Influencing factors concerning composition

Ayrshir Brown Guernse Holstei Jersey Shorthor

Fig.1.8: Influence of the season on milk composition

A. Firmness of butter (yield

Because of homeostasis (i.e. the capability of an organism to maintain a

Climate or weather conditions have little effect on milk composition. High

3. Stage and number of lactation

The life of a female cow can be summarized as follows:

The time elapsed after parturition or calving considerably influences milk

Fig.1.10: approximate average milk Fig.1.11: Composition and early-

Fig.1.12: relations between milk components and stage of lactation

Fig.1.13: Milk yield and concentration of

MILK AND DAIRY TECHNOLOGY

The most prominent carbohydrate of milks of most species is lactose. It is

1. Chemical and biochemical properties of lactose

Lactose is a reducing sugar. This property is of importance in the analysis of

A compound found in heated milk products is lactulose.

Concentrations of this compound up to about 1% may occur in commercial

MILK AND DAIRY TECHNOLOGY

Fig.1.14: Lobry de Bruyn-Alberda van Ekenstein transformation of lactose into

The lactose content of milk is relatively constant at 4,8 to 5,2 % lactose

Lactose intolerance is caused by a shortage of the lactase-enzyme (β-

MILK AND DAIRY TECHNOLOGY

Fig.1.16: Relative sweetness of sugars (percent concetration to give equivalent sweetness)

MILK AND DAIRY TECHNOLOGY

2. Physicochemical aspects of lactose

[β]/[α] = 1,64 - 0,0027.T with T = temperature in °C

This equilibrium is reached after 24 hours. E.g.: At 15 °C [β]/[α] = 1,59 or in this

Fig.1.17: Lactose solubility curves

MILK AND DAIRY TECHNOLOGY

Fig.1.18: Lactose crystals

MILK AND DAIRY TECHNOLOGY

Fig.1.19: Modifications of lactose

MILK AND DAIRY TECHNOLOGY

- Lactic acid fermentation OH

D (-) lactic acid L (+) lactic acid Racemic solution

- Acetaldehyde, the main aromacomponent in Yoghurt O

- Propionic acid formed out of lactic acid (Swiss type cheeses) O

Lactose → acetaldehyde + CO2 → ethanol

This fermentation is highly undesirable in cheese because of the excess

MILK AND DAIRY TECHNOLOGY

1.2.2. Other carbohydrates

MILK AND DAIRY TECHNOLOGY