CHEMISTRY OF MILK

Module 3. Milk proteins

Lesson 5
MILK PROTEINS: CLASSIFICATION, DISTRIBUTION, GENETIC POLYMORPHISM AND ITS IMPORTANCE

5.1 Introduction

In the earlier years proteins were classified largely on the basis of rather empirical fractionation procedures. In most cases the proteins were classified partly on the basis of their solubility and partly on the basis their composition. With the availability of better an alytical techniques it is established that the proteins which were earlier considered to be individual proteins are actually a mixtures of several other fractions.Presently the proteins in milk are classified on the basis of their fractionations and their behaviour during electrophoresis, difference in their solubility in various solutions, difference in their sedimentation rate etc.

5.2 Classification and Distribution of Milk Proteins

A common method for the classification and distribution of milk proteins is represented as follows

Genus Bos (30-35 g/L )

5.2.1 Caseins (24-28 g/L)

5.2.1.1 α_s1 -Caseins (12-15 g/L)

1. α_s1-Casein X-8P ( X = Genetic variants-A, B, C, D-9P, and E)

2. α_s1-Casein X-9P (X = Genetic variants-A, B,C, D-10P, and E)

3. α_s1-Casein fragments'

5.2.1.2 α_s2 caseins (3-4 g/L)

1. α_s2 -Casein X-l0P ( X =Genetic variants-A, B, C-9P, and D-7P)

2. α_s2 -Casein X-l1P (X =Genetic variants-A,B, C-10P, and D-8P)

3. α_s2-Casein X-12P (X =Genetic variants-A, B, C-11P, and D-9P)

4. α_s2 Casein X-13P (X =Genetic variants-A,B, C-12P, and D-10P)

5.2.1.3 β - Casein (9– 11 g/L)

• β-Casein X-5P (X =Genetic variants-A¹, A², A³,B, C-4P, D-4P, and E)
• β-Casein X-lP (f 29-209) (X=Genetic variants- A¹, A², A³,and B)
• β-Casein X-(f 106-209)(X =Genetic variants- A², A³, and B)
• β-Casein X-(f 108-209) (X =Genetic variants-(Fragments from A¹, A², A³ ) and B)
• β - Casein X-4P (f 1-28) ^*
• β -Casein X-5P (f 1-105) ^*
• β -Casein X-5P (f 1-107) ^*
• β -Casein X-lP ( f 29-105) ^*
• β Casein X-lP (f 29-107) ^*

5.2.1.4 κ -Caseins (2-4 g/L)

1.κ -Casein X-lP (X = Genetic variants-A andB)

2. Minor κ -caseins X -l, -2, -3, etc. ( x = Genetic variants-Aand B)

5.2.2 Whey proteins (5-7 g/L)

5.2.2.1 β Lactoglobulins (2-4 g/L)

β -Lactoglobulins X ( X = genetic variants-A,B, C, D, Dr, E, F, and G)

5.2.2.2 α-Lactalbumins (0.6-1.7g/L)

1. α-Lactalbumin X ( X = Genetic variants-Aand B)

2. Minor α-Lactalbumins

5.2.2.3 Bovine serum albumin (0.2-0.4 g/L)

5.2.2.4 Immunoglobulins (0.5-1.8 g/L)

a. IgGimmunoglobulins

• IgG₁, Immunoglobulins
• IgG₂, Immunoglobulins
• IgG fragments

b. IgMimmunoglobulins

c. IgA immunoglobulins

• IgA Immunoglobulins
• Secretory IgA Immunoglobulins

d. IgE Immunoglobulins

e. J-chaincomponent

f . Free secretory component

5.2.3 Milk fat globule membrane (MFGM) proteins

MFGM – A₁₅ – 127, C, S

Where MFGM indicates the protein satisfied the operational definition for a MFGM protein, A₁₅ designates the zone in a 15% Laemmliacrylamide gel, 127 is the “apparent molecular” weight in k-daltons, and C, S designates that the protein is both Coomassie brilliant blue and periodic acid /Schiff positive (PAS).

1. Six major proteins readily stained with Coomassie brilliant blue are XDH/XD; CD36, BTN,ADPH, PAS 6/7 And FABP.

2. Two stained by Periodic Acid/ Schiff (PAS) or silver stain are Muc-1 and PAS III.

(Source: Nomenclature of proteins of cows’ milk-5^th and 6^th revision, J. Dairy Sci., year: 1984 and 2004)

5.2.4 Minor proteins

• Serum transferrin

^* Genetic variants of these fragments have not been specifically identified

(Source: Fundamentals of Dairy chemistry, Wong etal., 1988)

5.3 Polymorphism in Milk Proteins

In 1956 As chaffenburg and Drewry discovered that the βlactoglobulin exists in two forms A and B which differs from each other by only a few amino acids. The milk of any few individual animal may contain β lg A orβ lg B or both and the milk is indicated as AA, BB or AB with respect to β lg.This phenomenon is referred as genetic polymorphism and has since been shown to occur in all the milk proteins. A total of about 30 variants have been demonstrated by Polyacrylamide Gel Electrophoresis (PAGE). Since PAGE differentiates on the basis of charge and molecular weight, only polymorphs which differ in charge alone are detected i.e a charged residue is replaced by a non charged or vice versa. Therefore it is likely that more than 30 polymorphs exist. The genetic variant present is indicated by a lain letter eg: α _s1 -CN A-8P,α _s1- CN B – 8P and α _s1 CN B – 9P etc.

The frequency with which certain genetic variant occur is breed specific and hence genetic polymorphism has been useful in phylogenic classification of cattle and other species various technologically important properties of the milk proteins are related to the genes that occur in the primary polypeptide chain of milk proteins, which will result in variation of the behaviour of that protein either on electrophoretic behaviour or its gel filtration pattern. This is usually due to the changes occurring in genetic code for that protein. This deviation or change is referred as polymorphism. There are several such variations observed in the milk proteins.

The casein genotype significantly influences the milk yield, fat yield and protein yield with highest yields obtained for the genotype BB. Cheese yield on a fixed amount of milk and fat yield were significantly related to κ polymorphism observed to be β lg genotype with highest estimates obtained for BB. Protein percentage was influenced by α_s1^– casein and κ casein with the genotype BC and BB respectively having the highest percentages

5.4 Distribution of Milk Proteins

The distribution of the proteins in milk is presented in a Figure 5.1. It could be observed that the proteins in milk are chiefly the caseins, whey proteins and some minor proteins. Since enzymes are protein in nature they are also grouped along with other proteins. Caseins being the major fraction of the entire milk proteins are present in micellar form while the whey proteins being considered as soluble proteins and which exists in colloidal form. Enzymes play a significant role in the raw milk storage and processing of the milk and also found to have industrial importance.

Fig. 5.1 Distribution of protein fractions in bovine milk

(Source: Developments in Dairy chemistry – 1, Proteins, Fox, 1982)

Minor proteins include vitamin binding protein, lactoferrin, metallo-protein, MFGM protein,Ceruloplasmin, etc.