2. Definition of Milk?
Definition - Normal secretion of the
mammary gland of mammals
More precisely it is a very complex
product that contains more than
100,000 chemicals that are either in
solution, suspension or emulsion with
water.
3. WHY MILK IN DAILY DIET?
• Milk contains more of the known essential nutrients required
for health than any other single food.
• Milk contains more utilizable calcium and good quality
protein.
• Casein major milk protein has unique property of influencing
its own digestion as well as other proteins fed with it.
• Conjugated linoleic acid of milk fat supresses cancer,
atheroselerosis and fat deposition in the body.
• Milk is an ideal food for elderly due to its high nutritive value.
• Milk is both a fast and conveinent food no preparation,
immediately ready for consumption.
4. A glass of LOW FAT MILK Regular Soft drink
( 250 ml ) Rs. 10.00 ( 300 ml ) Rs.20. 00
Milk Soft drink
5. Main Ingredients
Milk
Total Milk Solids
Water 87.3 %
100-87.3= 12.7 %
Fat 3.9 % Solids-not-fat 8.8 %
6. Composition of Milk
Water 87.5%
Lactose 4.9%
Protein 3.2%
Casein 2.9%
Lactalbumin 0.52%
Lactoglobulin 0.20%
Fat 3.7%
Minerals 0.72%
Calcium 0.12%
Phosphorus 0.11%
Chlorine 0.11%
7. Other Compounds Normally Found in Milk
• Acids
– Citrate, Formate, Acetate, Lactate, Oxalate
• Enzymes
– Peroxidase, Catalase, Lipase, Phosphatase
• Gases
– Oxygen, Nitrogen, Carbon dioxide
• Vitamins
– A, C, D, B Complex
9. • Milk has well defined physical equilibrium -
Three Phase
• Emulsion
• Colloids
• Solution
10. Milk Composition
• Solution - dissolve to the molecular level
– Lactose
– Minerals
• Suspension - Colloid, particles so small they will
not settle out
– Proteins - Casein
• Emulsion - compounds that don't mix with water
– Butterfat
15. Fat Globule
Fat Globule membrane
Globule range in diameter ( 0.1 - 20µm);
Av= 3.5µm.
Size & size distribution may be
measured by light microscopy, light
scattering, electronic counting devices TGS
(Coultercounter)
98-99%
About 75% of globules are < 1µm dia.
Thickness of FGM= 15nm (10-20nm)
16. Milk fat Globules
• Covered by a thin membrane from secretory cells (Protein and
Phospholipids)
• Helps prevent coalescence and flocculation
• Protects fat from enzymatic action
• Properties enable the production of butter, whipped cream and
ice cream
• Homogenization
– Decreases diameter making them more stable in suspension
18. Major Fatty Acids of Bovine Milk
• Milk Fat consist of >200 different FA , only 15 fatty
acid occur in proportion greater than 1% of milk fat
• Saturated fatty acids (65%)
– No double bonds
– Related to some health concerns
• Monounsaturated fatty acids (25%)
– One double bond
– Thought to be generally healthy
• Polyunsaturated fatty acids (10%)
– Greater than one double bond
19.
20. Fatty Acid Profile of milk fat
• High conc. of short chain fatty acids (C4:0–C8:0 carbon atoms)
Help to maintain a degree of liquidation in the relatively
saturated milk fat at body temp, and this may be important for
efficient secretion
• Appreciable amount of medium chain fatty acids. (C10:0–
C14:0 carbon atoms)
Absorbed directly via portal blood stream rather than through the lymphatic
system, so direct contribution to energy metabolism
( especially brain) of new born.)
• Difference in long chain fatty acids (14 – 26 carbon atoms)
ability to oxidize long chain fatty acids is not developed fully at birth
21. Importance of Lipids
1. Economics: Price of milk, because it is the costliest
component of milk.
2.Nutritional Function:
Rich source of energy. Highest caloric values per weight
(9Kcal/gm)
Carrier of fat soluble Vitamins – A,D,E,K
Source of essential fatty acids – linoleic & Arachidonic acid.
Source of prostaglandins – Earlier sexual maturity, increased
work capacity.
22. • Milk fat contain lactones which contribute to
flavour of milk 70-120mg/kg milk fat
• Milk fat is regarded as the most digestible of
the various dietary fats and oils
• Digestibility of milk fat is 99% whereas palm
oil show 91%
23. Why digestibility of milk fat is good
• Most of the FA present in milk are liquid
• The digestibility of fat whose melting point is
below 450C is 95% or better
• Positioning of the fatty acid in TG
• Smaller fat globules are absorbed directly
24. Benefits of milk Fat
• Antimicrobial activity- by reducing surface tension & by
decreasing pH
• SCFA- butyric acid promote the growth of Bifidobacterium in colon
• Anticarcinogenic role-CLA
CLA-antioxidant, boost immune system, reduce risk of heart disease by
reducing levels of prostaglandins, CLA also improve bone density
increase insulin sensitivity
• Sphingolipids- ceramides-regulate cell function, protective effect
against colon cancer
25. Milk Fat and CVD
• Milk contain relatively low cholesterol
Cow-2.8mg/g
Buffalo-1.9 mg/g
• Exp conducted on animals can not be extrapolated
to humans
• Exp animals have low cholesterol levels and
absorption is more
Rat- 50-80%
Monkey & dog- 40-75%
Rabbits –upto 90%
• Lipid Hypothesis Stearic & palmitic-36%-hypercholesterolemic
26. Non – nutritional Functions:
Improve the physical properties in:-
Baked goods – Bread, Cakes,Biscuits Cookies,
Pasteries, crackers by trapping air in dough.
Lubricating action provides characteristic form
and texture.
Frying Medium.
Flavour & Palatability.
28. Protein – Wide Range of Functions
in Living Organisms
Cells, Tissues & Muscles
P
Blood
R
O
T Enzymes & Hormones
EI
N
Antibodies
Recovery of Wear & Tear
29. Protein – In Milk Pricing
Price of Raw Milk to Producers – Traditional basis
• Fat and / or Solids not fat
Demand of Milk Fat Preference for Milk Protein
• Component Pricing based on Protein –
Already Popular in Western Countries
31. Protein Composition of Milk
Approx. % of skim milk
Protein protein
Isoelectric point Molecular weight
alpha-Casein 45-55 4.1 23,000
kappa-Casein 8-15 4.1 19,000
B-Casein 25-35 4.5 24,000
gamma-Casein 3-7 5.8-6.0 30,650
alpha-Lactalbumin 2-5 5.1 14,437
B-Lactoglobulin 7-12 5.3 18,000
Blood serum albumin 0.7-1.3 4.7 68,000
Lactoferrin 0.2-0.8 87,000
Immunoglobulins
IgG1 1-2 160,000
IgG2 0.2-0.5 160,000
IgM 0.1-0.2 ~1,000,000
IgA 0.05-0.10 ~400,000
Proteose peptone fraction 2-6 3.3-3.7 4,100-200,000
32. Various proteins in milk
• s1-Casein,s2-Casein,-Casein,
-Casein and K casein
• -lactoglobulin
• -lactalbumin
• Immunoglobulin
• Proteose peptone
• Serum albumin
• Lactoferrin
33. • Casein micelles-
70-110 nm CM
110-160nm BM
• Soluble casein
• Casein-Acid, Rennet
• Caseinates
• Whey protein concentrates, Whey
protein Isolates, Whey powders
34. Preparation of casein and whey proteins
Acid (isoelectric) precipitation
Centrifugation
Centrifugation of calcium-supplemented milk
Salting-out methods
Ultrafiltration
Gel filtration (gel permeation chromatography)
Precipitation with ethanol
Cryoprecipitation
Rennet coagulation
35. Microheterogeneity of the caseins
Each of the four caseins, αs1 -, αs2 -, β- and κ –caseins, exhibits variability,
which refers to as microheterogeneity, arising from five causes :-
1. Variability in the degree of phosphorylation
Each of the 4 caseins is phosphorylated to a characteristic but variable level
Casein Number of phosphate residues
αs1 8, occasionally 9
αs2 10, 11, 12 or 13
β 5, occasionally 4
κ 1, occasionally 2 or perhaps 3
The number of phosphate groups in the molecule is indicated as αs1-CN 8P or αs1-CN
9P, etc. (CN = casein)
2. Disulphide bonding
αs1 & β no cysteine or cystine
αs2 & κ each contains two cysteines per mole
It normally exist as intermolecular disulphide bonds
36. 3. Hydrolysis of primary caseins by plasmin
γ-Caseins are produced from β-casein by proteolysis by plasmin, an indigenous
proteinase in milk.
Corresponding N-terminal fragments are the principal components of the proteose-
peptone (PP) fraction, i.e. PP5 (β-CN fl-105/107), PP8 slow (β-CN f29-105/107) and
PP8 fast (β-CN fl-28).
Normally, the γ-caseins represent only about 3% of whole casein but levels may be
very much higher (up to 10%) in late lactation and mastitic milks
37. 4. Variations in the degree of glycosylation
κ -Casein is normally glycosylated containing about 5% of carbohydrate (NANA or
sialic acid, galactose and N-acetyl galactosamine
Carbohydrates are located towards C-terminal of the molecule, attached through an 0-
threonyl linkage, mainly to Thr131 of κ –casein
Number of oligosaccharides per κ -casein molecule varies from 0 to 4
κ -casein in colostrum is even more highly glycosylated; more sugars are present & the
structures are more complex and uncertain.
Carbohydrate bestows on κ -casein quite high solubility and hydrophilicity.
5. Genetic polymorphism
Aschaffenburg & Drewry (1956) discovered that the whey protein, β-lactoglobulin (β-
lg), exists in two forms, A & B - differ from each other by only a few amino acids.
Milk of any individual animal may contain β-lg A or B or both, & the milk is indicated
as AA, BB or AB with respect to β -lg. This phenomenon was referred to as genetic polymorphism
Occurs in all milk proteins; a total of ~30 variants have been demonstrated by PAGE
42. Casein
• Phosphoproteins – about 80% of all milk proteins
• Lack Disulfide bonds and has little secondary and tertiary structure
• Form a micelle (140nm) - hydrophobic
– Calcium and phosphates stabilize structure
• Several forms
– Alpha - phosphorylated form S1 and S2 variants (39%)
– Beta - major (28%)
– Kappa - glycoprotein (10%)
• Stabilizes the micelle
– Gamma - fragments of beta (3%)
• Precipitated by
– pH below 4.6 – Low solubility – Clots in stomach
– Ultracentrifugation
– Ultrafiltration - reduce trucking costs of milk used for cheese.
– Rennin - cheese
• Cleaves Kappa casein
43.
44. Whey proteins
About 20% of the total protein of bovine milk belongs to a group of proteins
generally referred to as whey or serum proteins or non-casein nitrogen
Whey proteins, as a group, are –
1. the proteins remaining soluble at pH 4.6;
2. soluble in saturated NaCl;
3. soluble after rennet coagulation of the caseins;
4. by gel permeation chromatography;
5. by ultracentrifugation, with or without added Ca2+.
On a commercial scale, whey protein-rich products are prepared by:
Ultrafiltration/diafiltration of acid or rennet whey
Ion-exchange chromatography
Demineralization by electrodialysis and/or ion exchange, thermal
evaporation of water and crystallization of lactose
Thermal denaturation, recovery of precipitated protein by
filtration/ centrifugation and spray-drying, to yield lactalbumin
45. β- lactoglobulin
Occurance
Major whey protein (WP)-50% of total WP & 12% of total protein of milk
Principal WP in bovine, ovine caprine & buffalo milks
Does not occur in human, rat, mouse or guinea-pig milks
Contains 4 genetic variants – A, B, C & D in bovine milk
Amino acid composition
Amino acid sequence of bovine β-lg consists of 162 residues per monomer
Physiological function
Acts as a carrier for retinol (vit. A)
Binds retinol in a hydrophobic pocket, protect it from oxidation
Transport it through stomach to small intestine where it is transferred to a retinol
binding protein, which has similar structure to β-lg
β-lg also binds FFA & thus it stimulates lipolysis
46. Whey Protein: -Lactoglobulin
Most abundant whey protein (approximately half of the
total protein in bovine whey) (not in humans)
Structure
162 amino acids, 9 beta strands, alpha-helix, 2 disulfides,
1 free sulphhydryl group (-SH)
β-barrel domain
hydrophobic pocket
Stability
pH
temperature
surface activity
46
47. • source of essential and branched chain amino acids. A retinol-
binding protein also exists within the β-lactoglobulin structure
having potential to modulate lymphatic response.
• Various peptides derived from proteolytic digestion of β-Lg by
enzymes have shown inhibitory activity against Angiotensin-
Converting Enzyme (ACE) which plays a major role in the
regulation of blood pressure.
• The active peptides were usually short (< 8 amino acids) and
could be enriched from a mixture of proteins and other peptides
using UF with low molecular mass cut-off membranes.
• β-Lg exert an antimicrobial function in vivo after its partial
digestion by endopeptidases of the pancreas, and small
targeted modification in the sequence of these peptides could
be useful in expanding their anti-microbial function.
47
48. α -lactalbumin
Occurance
Represents about 20% of bovine whey protein & 3.5% of total milk protein
Principal protein in human milk
Small protein of molecular mass 14 kDa
Amino acid composition
Contain 123 residues per monomer & is rich in tryptophan & sulphur
Principal α-la contains no phosphorus or carbohydrate
Metal binding and heat stability
α-la is a metallo-protein & binds Ca2+ per mole in a pocket containing 4 Asp residues
Ca2+ containing protein is quite heat stable & hence imparts high heat stability to α-la
(the protein renatures after heat denaturation)
Metal free protein is denatured at quite low temperatures & does not renature on
cooling
50. • α-Lactalbumin (α-La) is one of the main proteins found in human
and bovine milk comprising approximately 20-25% of whey
proteins.
• The peptide with the amino acids sequence Tyr-Gly-Leu-Phe (f 50-
53), released from α-La by pepsin treatment is shown to inhibit
ACE.
• Recently, a folding variant of α-La named HAMLET/ BAMLET for
human/bovine α-La made lethal to tumour cells has been
discovered, which selectively enters tumour cells and induces an
apoptosis like mechanism.
• α-La was observed to improve cognitive performances in stress-
vulnerable individuals by increased brain tryptophan and
serotonin activity.
• Other clinical trials suggested that α-La could be used to improve
sleep in adults suffering from nutritional disturbances.
50
51. • Purified α-La is most readily used in infant formula
manufacturing, as it has the most structurally similar
protein profile compared to human.
• α-La is rich in essential amino acids, this protein is
ideally suited to fortifying infant formulae. Clinical
trials with α-La enriched infant formula have also been
shown to exhibit antimicrobial activity.
• At a standard pasteurization temperature of 72oC for
15 s, the bioactive whey proteins retain most of their
activity.
51
52. Biological function
α-la plays imp. role in lactose synthesis
UDP-D –Galactose + D-glucose Lactose - Lactose + UDP
synthetase
Lactose synthetase
consists of dissimilar A (UDP-galactosyl transferase)
protein subunits
B (α-la)
In the absence of B protein, the A protein acts as a non-specific galactosyl
transferase but in the presence of B protein, it becomes very specific & transfers
galactose only to glucose to form lactose
α-la is therefore a ‘specifier protein’
Concentration of lactose in milk is directly related to the concentration of α-la in
milk
53. Bovine Serum Albumin
Normal bovine milk – 0.1 – 0.4 gl-1; 0.3 – 1.0 % of total N
Large molecule of molecular mass 66 kDa; 582 amino acids
Contains 17 disulphides & 1 sulphydryl
BSA in milk is a result of leakage from blood
Binds metals and fatty acids and hence stimulate lipase activity.
Immunoglobulins
Mature milk – 0.6 – 1.0 g Igl-1 (3.0 % of total N), Colostrum -100 gl-1
Complex protein consisting of 5 classes – IgG, IgA, IgD, IgM & IgE
IgG, IgA, & IgM are present in milk & IgG occurs as IgG1 & IgG2
IgG consists of two long (heavy) & two shorter (light) polypeptide chains
linked by disulphides
Principal Ig in bovine milk is IgG1 while in human milk it is IgA
Physiological function – provides various types of immunity in the body
54. Lactoferrin
• Lactoferrin is an iron-binding glycoprotein of the
transferrin family which was first fractionated as an
unknown “red fraction” from cows’ milk by Sørensen
and Sørensen (1939).
• Bovine and human lactoferrins has a sequence identity
of 69%.
• Human milk has the highest LF concentrations (1.5 to
2.0 mgmL-1) of the mammalian milks compared with the
average LF concentration in bovine milk of 0.1 mgmL-1.
• Lactoferrin can bind the free ferric iron with high
affinity, and thus function as a local antioxidant,
protecting the immune cells against the free radicals
produced by them.
54
55. Immunoglobulins
• An immunoglobulin (Ig) is an antibody or gamma-globulin. There are
five classes of antibodies-IgA, IgD, IgE, IgG, and IgM. The IgG class
antibodies comprise 80-90% of total Igs (about 50 g L-1) in the early
bovine colostrum from the milking of the first day after parturition.
• The biological function of immunoglobulins (Igs) in bovine milk and
colostrums is to protect mammary gland against pathogens and to
provide the calf with an immunological protection against surrounding
pathogens.
• Immunoglobulins, together with lactoferrin, lacto-peroxidase and
lysozyme form the very important anti-microbial system of bovine
lacteal secretions.
55
56. • A treatment with specific colostral Igs reduced the
degree of gastric inflammation and Helicobacter
colonization in mice and the degree of inflammation in
children too.
• When used as a mouth rinse for 3 days a immune
preparation resulted in a higher resting pH in dental
plaque of adults as compared with the control groups.
• Infants receiving the formula containing the immune
preparation (0.5 g per kg body weight for 7 days) had a
significantly lower incidence of diarrhoea than those
given a formula containing the control preparation
during the 6 months follow-up period
• There is evidence suggesting that shigellosis could be
prevented by using colostral Ig preparations from
cows immunized against certain Sh. flexneri antigens
56
57. • In clinical trials, the Ig preparations have seldom caused
any undesired symptoms and the health risks are mainly
due to allergenic properties of whey proteins.
• Bovine Ig for human application is only in its infancy. There
are uncertainties w.r.t. dosage and timing of administration.
• Immunoglobulins are heat-sensitive molecules subject to
human digestive action and may present difficulties with
respect to palatability or shelf life of food products due to
contaminating enzymes of a proteolytic nature.
57
58. Major differences between the caseins and whey proteins
1. Caseins precipitate from the solution when the pH of milk is adjusted to 4.6
where as the whey proteins do not precipitate and remains in solution
2. Chymosin and some other proteinases (known as rennets) produce a very slight,
specific change in casein, resulting in its coagulation in the presence of Ca2+,
whereas the whey proteins undergo no such alteration
3. Casein is very stable to high temperatures; milk may be heated at its natural pH
6.7 at 100°C/24h without coagulation and it withstand heating at 140°C/20min.
The whey proteins, on the other hand, are relatively heat labile, being
completely denatured by heating at 90°C for 10min
4. Caseins are phosphoproteins, containing, on average, 0.85% phosphorus, while
the whey proteins contain no phosphorus
59. 5. Casein is low in sulphur (0.8%) while the whey proteins are relatively rich (1.7%)
6. Casein is synthesized in the mammary gland while some of the whey proteins (β-lg
and α-la) are also synthesized in the mammary gland, while others (e.g. bovine
serum albumin and the immunoglobulins) are derived from the blood
7. The whey proteins are molecularly dispersed in solution or have simple
quaternary structures, whereas the caseins have a complicated quaternary
structure and exist in milk as large colloidal aggregates, referred to as micelles,
with particle masses of 106-109 Da
Other protein fractions
Milk contains two other groups of proteins or protein-like material,
proteose-peptone fraction and
non-protein nitrogen (NPN) fraction
60. Major bioactive protein components in
bovine colostrum and milk
Values expressed as weight of Colostrum Milk
protein L-1
Immunoglobulin (Ig) A 3.2-6.2 g 0.1 g
IgG1 48-87 g 0.4 g
IgG2 1.6-2.9 g 0.05 g
IgM 3.7-6.1 g 0.05 g
Insulin like growth factor (IGF) -I 0.1-2 mg 25 µg
IGF-II 0.1-2 mg 2 µg
Transforming growth factor (TGF) -β 20-40 mg 1-2 µg
EGF 4-8 mg 2 µg
Lactoferrin 1.5-2 mg 0.1µg
Lysozyme 0.1-0.7 mg 0.1-0.3 mg
Lactoperoxidase 30 mg 20 mg
60
61. Lysozyme
• Lysozyme is an important antimicrobial agent in milk, which
kills bacteria by cleaving the β-1,4-glycosidic bond between
N-acetylglucoseamine residues of the peptidoglycan in the
bacterialcell wall.
• LZ is a major component of the whey fraction in human milk
(0.4 g L-1) although its concentration in bovine milk is
several orders of magnitude lower (0.13 mg L-1).
• Lysozyme in both cow and buffalo milk exhibited maximum
activity at pH 7.4.
61
62. Lactoperoxidase (LP)
• This enzyme system catalyzes peroxidation of thiocyanate and some
halides (such as iodine and bromium), which ultimately generates
products that inhibit and/or kill a range of bacterial species.
• The important biological function of this enzyme is the bactericidal
affect against gram –ve as well as gram +ve bacteria in the presence of
hydrogen peroxide and SCN or halogens.
• Lactoperoxidase is able to form oxidized halides and pseudohalides,
which are potent biocidal small molecules.
• Many attempts have been made to use LP as an ingredient for extending
the shelf life of milk and milk products.
62