1. Milk Proteins
By:
Antima Gupta

2. What is Milk ?
Milk may be defined as the whole, fresh, clean, lacteal secretion obtained by the complete milking of one or more healthy milch animals excluding that obtained 15 days before or 5 days after calving.

3. Milk composition

4. Composition of Milk of Different Species Percentage Composition
Name of Species
Percentage Composition
Water
Fat
Protein
Lactose
Ash
Buffalo
84.2
6.6
3.9
5.2
0.8
Cow
86.8
4.6
3.4
4.9
0.7
Goat
86.5
4.5
3.5
4.7
Human
87.7
3.6
1.8
6.8
0.1
Deficient in iron and vitamin C

5. Milk Constituents Milk Water Total Solids (TS) Fat True Fat
Associated substances
Phospholipids
Cholesterol
Carotene
Vitamins (A,D,E,K)
Solid-not-fat (SNF)
Lactose
Nitrogenous substances
Non-Protein
Protein
Casein
(α, β, γ)
Lactalbumin
Lactoglobulin
Protease-Peptones
Mineral matter
Other constituents
Pigments
Dissolved gases
Vitamins (B complex, E)
Enzymes

6. Proteins Proteins are the organic substances.
Proteins are the polymers of amino-acids, some are ‘essential’ and others ‘non-essential’.
Protein content of milk of different species:
Name of the species
Protein (%)
Buffalo
3.9
Cow
3.4
Goat
3.5
Human
1.8
Camel
4.0

7. Milk proteins Total protein content in milk – 2-4%
Two major types of milk protein
Caseins
Whey proteins
The ratio of casein : whey varies:
Cow, buffalo, goat, sheep- 80:20
Human milk- 40:60
Whey (milk serum) is the liquid remaining after milk coagulation and curd removal. It contains water, lactose and soluble non-casein proteins.

8. Functions of milk protein in body
In addition to meeting the requirements for various nutrients, milk supplies:
Protective factors, e.g. immunoglobulins, lactoferrin and some enzymes, e.g. lysozyme and lactoperoxidase;
Growth factors and other hormones;
Digestive/nutritional aids, e.g. vitamin- or metalbinding proteins and lipase.

9. Classification of Milk Proteins
Casein
α-Casein
αs-Casein
αs1-Casein
αs2- Casein
κ- Casein
β- Casein
γ- Casein
Whey Proteins
α- lactalbumins
β- lactoglobulins
Blood Serum Albumins
Immunoglobulins
Lactoferrin
Transferrin
Protease-peptones
Non-Protein Nitrogen

10. Fractionation of Milk Proteins
Skim Milk
Heated at 95°C, 10 min, cool, acidify to pH 4.6, filter
Acidify to pH 4.6, filter
Filtrate
(Whey Proteins)
Precipitate
(Casein)
Filtrate
Precipitate
(Whey proteins and Casein)
Neutralize and saturate with MgSO4, filter
12% TCA
Precipitate
(Proteose- Peptone)
Filtrate
(NPN)
Precipitate (globulins)
Filtrate (albumins)

11. Difference between CASEIN and WHEY proteins
Casein precipitate at pH 4.6.
Casein is very stable to high temperature. Ex- Milk heated at its natural pH at 100°C for 24 h without any coagulation.
Casein are phosphoproteins, on average : 0.85% phosphorous.
Casein is low in sulphur (0.8%)
Casein is synthesized in mammary glands.
Casein exist in milk as large colloidal aggregates.
Whey protein do not precipitate at pH 4.6.
Whey proteins are heat labile, completely denatured by heating at 90°C for 10 min.
Whey proteins contains no phosphorus.
Whey proteins are relatively rich in sulphur (1.7%).
Whey proteins is synthesized in mammary gland and some are derived from blood.
Whey proteins molecularly dispersed in solution.

12. Casein
Casein are phosphoproteins precipitated from raw milk at pH 4.6 at 30°C.
They comprise almost 80% of the total protein content in milk.
Casein exists only in milk and is found in the form of a calcium caseinate-phosphate complex.
It is present in the colloidal state.
Casein proteins are mainly classified as:
α Casein → 75%.
β Casein → 22%.
γ Casein → 3%.

13. Amino acid Composition
All caseins have high content (35-45%) of apolar amino acids.
All caseins have a very high content of proline due to which it is having low content of α-helix or β-sheet structures in the casein (susceptible to proteolysis without prior denaturation- important for neonatal nutrition).
Caseins are deficient in sulphur containing amino-acids which limits their biological value i.e. 80. But αs2-casein, are rich in lysine, an essential amino acid in which many plant proteins are deficient. Consequently, casein are very good nutritional supplements for cereal proteins.

14. α-caseins are the major casein proteins
α-caseins are the major casein proteins. Its containing phosphate groups. It is poorly soluble in water.
β- caseins contains about 5 phosphate residues, it is more hydrophobic than α-caseins and κ-casein. It is poorly soluble in water.
Because α-caseins and β-caseins are highly phosphorylated, they are very sensitive to the concentration of calcium salts, that is, they will precipitate with excess Ca2+ ions.

15. κ-caseins are glycoproteins
κ-caseins are glycoproteins. They contain carbohydrate part which is negatively charged.
Do not precipitate in the presence of Ca2+ ions.
Protect other caseins from precipitation and make casein more soluble forming casein micelles.
In milk, κ-casein combine with α-caseins and β- caseins to form micelle.

16. Casein structure is introduced by Morr in 1967.
The word “micelle” describes structure not a single compound. Casein micelles consist of water, protein (94%), and salts (6%) principally calcium (37.5%), phosphate (50%), magnesium and citrate and referred to as colloidal calcium phosphate (CCP).
Casein micelle under Microscope

17. Casein Micelle structure

18. Casein micelle are generally spherical in shape with diameter 50-500 nm and mass 106-109 Dalton.
In milk micelle are present per ml.
Micelle are of colloidal dimension capable of scattering light which give milk its white color.
Micelle are linked together by calcium phosphate giving micelle open and porous structure.
If CCP is removed by acidification or if κ-casein removes by rennet, colloidal stability get disturbed and it results in coagulation or precipitation.

19. (Acid precipitation method)
Production
ACID CASEIN
(Acid precipitation method)
RENNET CASEIN
(Enzymatic method)
Skim milk
Precipitation /coagulation (using HCl)
Cooking
Dewheying
Washing
Dewatering
Drying, Tempering, Grinding
Skim milk
Precipitation /coagulation (using Rennet)
Cooking
Dewheying
Washing
Dewatering
Drying, Tempering, Grinding

20. Casein Based Product
Caseins
Caseinates
Milk protein concentrates

21. Milk Protein Concentrate
Caseinate
Milk Protein Concentrate
Caseins
Skim milk
Coagulant/precipitant
Cooking
Dewheying
Washing
Drying
Casein
Acid casein curd
Mincer
Colloid Mill
Mixer
Heat Exchanger
Balance tank
Spray Drier
Caseinate
Skim milk
Ultrafiltration
Milk proteins
Evaporator
Spray dried
Milk protein concentrate

22. Whey proteins
Also known as whey or serum protein or non-casein nitrogen.
Whey proteins comprise almost 20% of the total protein content in milk.
Whey proteins are mainly fractionated using 50% saturated MgSO4 into insoluble lactoglobulins and soluble lactalbumins.

23. Lactalbumin fraction of bovine milk contains three main proteins β-lactoglobulin, α-lactalbumin and blood serum albumin and several minor proteins such as lactoferrin, serotransferrin and several enzymes.
Lactoglobulin fraction consists of immunoglobulins (Ig), esp. IgG1, with lesser amount of IgG2 IgA and IgM.

24. β-Lactoglobulin
It is a major protein in bovine milk representing about 50% of total whey protein and 12% of total protein of milk.
It is rich in sulphur amino acids which give it a higher B V of 110.
Responsible for cooked flavour of milk.
Bind retinol
It is a globular protein
Soluble protein.
Easily digested.

25. α- Lactalbumin
It represents about 20% of the proteins of bovine whey and 3.5% of the total milk proteins.
It is a metallo-protein.
Bind Calcium ions (Ca++)
Rich in tryptophan
Important role in lactose synthesis

26. Blood serum albumin
Milk contains very low level of BSA ( % of total N).
Bind lipids and metal ions
Lactotransferrin
It contains iron which is bounded to a glycoprotein.
It facilitates iron transport and storage.
Found in high concentration in human colostrums and milk.

27. Immunoglobulins (Ig)
Mature milk contains g Ig/l but colostrum contains up to 100 g Ig/l.
Five classes of Ig: IgA, IgG, IgD, IgE and IgM.
Out of this IgA, IgG and IgM are present in milk.
The principle Ig in bovine milk is IgG1 while in human milk it is IgA.
They provide immunity to the body.

28. Products from whey proteins
Whey protein concentrates:
Ultrafiltration/diafiltration of acid or rennet whey to remove varying amount of lactose, and spray-drying is done (30-80% protein).
Whey protein isolates:
Proteins are adsorbed on an ion exchanger, washed free of lactose and salts and then eluted by pH adjustment. The eluted is freed of salts by ultrafiltration and spray-drying is done (95% protein).
Whey protein hydrolysates

29. Whey Protein Concentrate (liquid)
Ultrafiltration
Whey Protein Concentrate (liquid)
Evaporation/
Drying
Dialysis
Enzyme treatment
Evaporation/
Drying
Drying
Whey Protein Concentrate
Whey Protein Hydrolysate
Whey Protein Isolate

30. Whey protein Isolate (WPC- 95%)
Whey protein Hydrolysate
Whey protein Concentrates (WPC %)

31. Functional properties of milk proteins

32. Application of casein S. No. Product Function 1
Bakery Products (Bread, biscuit, breakfast cereals, cake mixes, frozen cakes, pastries)
Nutritional, sensory, emulsifier, dough consistency, texture, volume 2
Dairy products (Imitation cheese, coffee creamer, yoghurt, milk beverages, butter like spreads, milk shakes)
Fat and water binding, texture enhancing, shredding properties, emulsifier, increase gel firmness, nutritional, foaming properties 3
Beverages (Drinking chocolates, fruit beverages, cream liqueurs, wine and beer industry)
Stabilizer, whipping and foaming properties, emulsifier, clarification, reduce color and astringency 4
Dessert products (Ice-cream, frozen desserts, mousses, instant puddings, whipped toppings)
Whipping properties, impart body, texture, film former, emulsifier 5
Meat products (Comminuted meat products)
Emulsifier, water binding, improves consistency, releases meat proteins for gel formation 6
Pasta products (imitation pasta)
Nutritional, texture, freeze-thaw stability 7
Textured products (Protein-enriched snack, meat extender)
Structuring, texturing, nutritional

33. Application of Whey proteins
S. No.
Product
Function 1
Bakery Products (Bread, cakes, muffins, croissants)
Nutritional, emulsifier, egg replacer 2
Dairy products (Yoghurt, Quarg, Ricotta cheese, cream cheeses, cheese filling and dips)
Nutritional, consistency, curd cohesiveness, emulsifier, gelling, sensory properties 3
Beverages (Soft drinks, fruit juices, powdered or frozen orange beverages)
Nutritional, viscosity, colloidal stability 4
Dessert products (Ice-cream, frozen juice bars, frozen dessert coatings)
Skim-milk solids replacement, whipping properties, emulsifying, body/texture 5
Meat products (Frankfurters, luncheon meats, injection brine for fortification of whole meat products)
Pre-emulsion, gelation, yield 7
Textured products (Puffed snack foods, protein-enriched snack-type products, meat extenders)
Structuring, texturing, nutritional

36. Modification
Proteins are modified to improve their functional properties.
Proteins are modified by two ways:
Chemical Modification
Enzymatic Modification
Modification include either introduction of a new functional group to the protein or removal of component part from the protein.

37. Chemical Modification
Primary structure of proteins can be chemically modified in order to improve their functional properties.
Main classes of reactions used to chemically modify the side chain of amino acids are acetylation, glycation, alkylation, oxidation, reduction and esterification.

39. Changes after modification
BLG showed better emulsifying properties, after glycation with glucose-6-P and better heat stability, foaming properties and solubility after glycation with glucose, mannose or galactose.
Esterification improves the solubility, emulsifying activity of milk proteins by blocking carboxylic group and increasing the net positive charge.

40. Advantage Disadvantage
Chemical modification is not very desirable for food applications
Harsh reaction conditions
Non-specific chemical reagents
Difficulty to remove residual reagents from the final product
Advantage
Enzyme modification provide several advantages over chemical such as:
Fast reaction rates
Mild conditions
High specificity.

41. Enzymatic modification
Enzymatic methods are consequently more attractive to the food processor.
Commercially modifying enzymes are: Proteases, transglutaminase, protein kinase, and peptidoglutaminase.
Hydrolysis of peptide bonds causes several changes in proteins:
The NH3+ and COO– content of the protein increases, increasing its solubility;
The molecular mass of the protein decreases;

42. Enzymatic modification increases the foam expansion and stability.
- The globular structure of the protein is altered, exposing the previously hidden hydrophobic groups.
Emulsification activity to decrease with increasing net charge and with the decreasing hydrophobicity due to proteolysis, but it increases the stability of emulsion.
Enzymatic modification increases the foam expansion and stability.
Gelation decreases on enzymatic modification

43. Biologically active peptide
Bioactive peptides are specific protein fragments that have a positive impact on body functions and may ultimately influence health.
Biologically active peptides can be prepared in the following ways:
Enzymatic hydrolysis by digestive enzymes
Fermentation of milk with proteolytic starter cultures
Proteolysis by enzymes derived from microorganisms or plants.

44. Biological effects in maintenance of health, disease and performance
Antihypertensive
Immunomodulating
Bioactive peptides
Opioid
Antimicrobial
Antioxidant
Antithrombotic

45. Examples of Biologically Active Peptide
S. No.
Bioactive peptide
Function 1
Lactenin
Antibacterial peptides 2
αs1-Casokinin-5
Antihypertensive peptides 3
β-Lactorphin 4
Casoplatelin
Antithrombotic peptides 5
Caseinophospho-peptide
Calcium binding and transport 6
Casoxin C
Opioid antagonist 7
Lactoferroxin A

46. Toxicological aspects of Milk proteins
Milk protein intolerance (MPI): abnormal reaction by the body’s immune system to protein.
MPI can be divide into: IgE- immediate reaction (within 2 hours) and non-IgE delayed reaction (48 hours to 1 week).
Symptoms: vomiting, abdominal pain, blood in stools, hives, rashes, wheezing, irritability, facial swelling and poor growth due to poor absorption of the nutrient.

47. Suggested Readings
Fox, P F and McSweeney, P L H Dairy Chemistry and Biochemistry. Kluwer Academic Publisher, New york.
Jean-Marc Chobert Milk protein tailoring to improve functional and biological properties. J. BioSci. Biotech. 1(3):
Dominic W.S. Wong , Wayne M. Camirand , Attila E. Pavlath , Dr. Nicholas Parris & Dr. Mendel Friedman (1996) Structures and functionalities of milk proteins , Critical Reviews in Food Science and Nutrition, 36:8,
Bartłomiej Dziuba, Marta Dziuba Milk Proteins-Derived Bioactive Peptides in dairy products: Molecular, Biological and methodological aspects. Acta Sci. Pol., Technol. Aliment. 13(1) , 5-25

48. Thank you...