CHEMISTRY OF MILK

Module 3. Milk proteins

Lesson 10
BOVINE SERUM ALBUMIN, IMMUNOGLOBULIN, PROTEOSE-PEPTONE, OTHER WHEY PROTEINS AND NON-PROTEIN-NITROGENOUS COMPOUNDS

10.1 Introduction

Whey being an important byproduct from cheese industry has a large number of important nutrients which must be utilized effectively and efficiently. It is also necessary to have some knowledge about these components for their incorporation in some of the dairy products or to be used independently.

10.2 Bovine Serum Albumin

This protein, a major component of blood serum, is synthesized in the liver and gains entrance to milk through the secretary cells, but it comprises only about 1.2% of the total milk protein. The protein as isolated from bovine milk could not be differentiated from that isolated from bovine blood by methods available in 1950. Since that time little work has been done on the serum albumin isolated from milk; it has been assumed to be identical to that in blood. The protein isolated from blood consists of a single polypeptide chain of 582 amino acid residues. Its tertiary structure reveals three equal-sized globular domains. It has one free thiol and 17 disulfide linkages, which neatly hold the protein in a multi loop structure. There is no specific role of this protein in the function of mammary gland. The behaviour of this protein in milk and milk products and its possible influence on their properties is not known.

10.3 Immunoglobulin

Immunoglobulins are antibodies synthesized in response to stimulation by macromolecular antigens foreign to the animal. They are polymers with two kinds of polypeptide chains, light chain (L) of MW 22,400, and heavy chain (H). The latter are of several types, including γ (MW 52,000), α (MW 52,000-56,000) and µ (MW 69,000).Each of the L and H chains consists of a relatively constant and highly variable sequence and appears to be coded for by two genes. The nomenclature of the classes of chains and of antibodies was developed for human antibodies and was extended to other species, including cattle, on the basis of homology of the heavy chains as revealed by immunological cross reaction with those of humans. IgG1 and IgG2 are each polymers of two light chains and two heavy chains of the γ type (γ₁and γ₂). The chains are joined by disulfide linkages to form two antibody sites, each consisting of the variable portion of an H and L chain. IgGI and IgG2 have about 2.9% bound carbohydrate and MW of about 1,50,000. They differ slightly in electrophoretic mobility.

IgA and IgM immunoglobulin likewise have the basic structure of two H and two L chains joined by disulfide bridges. In IgA, the H chains are of α type, and in IgM they are of µ type. IgA, is secreted as a dimer of two of the basic four-chain units joined by a polypeptide of MW about 25,000, called J-component, and associated with another called secretory component, SC. This complex is called secretory IgA (SIgA) and has a MW of about 3,85,000. The secretory component is a protein of MW about 75,000, consisting of a single polypeptide chain with number of internal disulphide salt bridges. The carbohydrate content is high consists of N-acetyl galactose amine, D-glucose, D-mannose, L-fucose and N-acetyl neuraminic acid. These sugars are bound to the SIgA. IgM consists of pentamer of the basic four chain units joined by J component and has molecular weight of 9,00 ,000 and carbohydrate content of 11-12%.

Fig. 10.1 Schematic conformation of immunoglobulin IgG, SIgA, and IgM. = di sulfide linkage. hatched = variable portion

(Source: Dairy chemistry and physics,Walstra and Jenness, 1984)

10.4 Proteose Peptone

Rowland has defined the conditions and procedures for obtaining the various fractions of protein in milk and to quantify them. It was observed by him that if milk is heated, about 80% of the whey proteins consisting mainly of α lactalbumin and β-lactoglobulins which precipitate with the casein when it is precipitated by aciditification to pH 4.6. The remaining 20% is a separate protein to which he applied the name proteose-peptone. Proteose and peptones are the polymers of amino acids which are of lower molecular weight than proteins. They often are formed by the partial hydrolytic degradation of proteins. They are usually not heat denaturable and hence it was easy for Rowland to reason by analogy that the proteins of milk which remain soluble in acid after heating are proteoses and peptones. The nature of proteins in this fraction has not been clearly established. The issue is complicated by the fact that the fraction may consist in part of native proteins and in part of breakdown products resulting from heat treatment.

10.5 Non-Protein-Nitrogenous Compounds in Milk

Addition of 12% TCA to milk would result in the precipitation of all the caseins, α lactalbumin and β lactoglobulins leaving the non protein nitrogenous compounds in the filtrate. The major compounds identified from this are uric acid, creatine, creatinine, orotic acid, α-aminonitrogen, hippuric acid, indicans,phosphoglyceroethanolamine, o-phosphoethanolamine and phenylacetylglutamine. The compounds present in the urine of dairy animals have a remarkable similarity between those present in non protein nitrogenfractions of milk. As a matter of fact the compounds or substances present in the urine of dairy animals are the resultant waste metabolites of dairy animal’s body. It is apparent that the bulk of these waste metabolites in the urine of dairy animals originate from the blood and hence this entry or appearance and levels in milk or urine are due to the protein metabolism of the animals. Intake of feed by the animals is directly proportional to the presence of these compounds or substances n milk. NPN in milk varies from season to season and has no biological value as protein. It cannot be utilized by the body as a substitute of protein nor can it increase the cheese yield. Pasteurization by itself has no effect. However pasteurization with homogenization causes an increase in the non protein and amino nitrogen content. Increase in NPN was observed when concentrated milk is stored.

10.6 Other Whey Proteins

10.6.1 Lactoferrin and transferrin

These are the two iron-binding proteins are found in milk. One of them, transferring (Tf), is a common blood plasmaprotein; the other, lactoferrin (Lf), is secreted not only by mammary glands but also by lacrymal, bronchial, and salivary glands and by kidney and endometrial mucosa. It also is found in specific granules in heterophilicleukocytes. Both Tf and Lf appear to be large single ­chain polypeptides of 600-700 amino acid residues. Reported molecular weights differ somewhat; recent work favours 75,000 to 77, 000 for Tf, but values for Lf are not so consistent, 77, 000 or 93,000 being reported. In both proteins about 4 mol % of the residues are Cys, and both have covalently linked carbohydrate consisting of N-acetylglucosamine, mannose, galactose, and N-acetylneuraminic acid. All transferrins and lactoferrins appear to bind 2 mol of Fe^{3 +} per mole. Tf and Lf differ markedly from each other in amino acid composition and in electrophoretic mobility. They can be detected readily in electrophoretic patterns by auto radiography with ⁵⁹Fe. Electrophoretic patterns of milk and blood preparations from individual an­imals reveal the occurrence of genetic variants of both proteins. No im­munological cross-reaction between Tf and Lf has been demonstrated even when both are from a single species. Amino acid analyses and partial se­quences of human Lf and Tf indicate some degree of homology between the two and some internal homology of peptide segments with in each; sequenc­ing is far from complete, however.

Both Tf and Lf can be determined quantitatively in a biological fluid by immuno diffusion method using a specific antiserum. The concentrations and ratios of Tf and Lf in milk vary greatly among species and with stage of lactation. The concentration of Lf in colostrums is about 1250 mg. kg^-I in mid-lactation, the concentration falls to less than 100 mg kg^-I. Concentrations of Tf in milk have not been determined accurately but may be similar to those of Lf. Lactoferr in is an inhibitor of bacteria because it deprives them of iron. The concentration of Tf in milk have not been determined accurately but may be similar to those of Lf. Lactoferrin is an inhibitor of bacteria because it deprives them of iron. The concentration of Lf in bovine milk is so low,however, that it does not exert any significant antibacterial effect.

10.6.2 Fat globules membrane proteins

The fat globulemembrane contains approximately 50% protein and accounts for about 1% of the total protein of the milk. Some of the protein constituents of the membrane are enzyme, but it is not possible at present to estimate the ratio of enzymatic and non-enzymatic components. The fat globule membrane proteins are difficult to resolve analytically and to separate preparatively because they interact strongly with one another and with lipids, and most of them are insoluble. Part is released from the globules by cooling the milk. Some success has been achieved in solubilizing the membrane proteins with detergents such as sodiumdodecyl sulfate coupled with reduction of disulfides.

The current nomenclature for milk fat globule membrane (MFGM) proteins recommended by the Milk Protein Nomenclature Committee of the American Dairy Science Associaion, the major proteins of MFGM in order of increasing mobility upon SDS-PAGE separation,are the mucin MUC-1, the redox enzyme, xanthine dehydrogenase/oxidase (XDH/XO),glycoprotein (PASIII), cluster of differentiation 36 (CD36), butyrophilin (BTN),adipose differentiation related protein (ADRP), and glycosylated variants of some polypeptide backbone designated PAS6/7 and fatty acid binding protein (FABP).

10.6.3 Acid glycoproteins

Formerly called orosomucoid. It has been isolated from human serum, colostrum, and milk, and from bovine serum and colostrum, but it has not been detected in bovine milk.It consists of a polypeptide chain of 181 residues to which five het­eropolysaccharide groups are linked to asparagines residues. The carbohy­drate constitutes about 45% of the total molecule. The function of this protein is not known. In any event, α₁-acid glycoprotein comprises only a small portion of the acid glycoproteins obtainable from fractionation of colostrum or milk on DEAE-cellulose.Five other fractions have been obtained in varying states of homogeneity. All contain carbohydrate and phosphate and promote the growth of Bifidobacteriumbifidum var. pennsylvanicus (for­merly Lactobacillus bifidus). The possibility that some of these glycoproteins represent partial degradation products of caseins or membrane materials has not been elucidated.

10.6.4 Folate binding protein

A specific protein that binds folate (FBP) has been isolated from milk. Affinity chromatography on Sepharose to which folate has been attached is especially effective in isolating this protein. Its concentration in normal milk is about8 mg/ 100 ml.

10.6.5 β₂-microglobulin

This protein consists of a single polypeptide chain of about 100 amino acid residues and MW of 11,800 daltons. It is present in several body fluids and in membranes of various types of cells. Its amino acid sequence indicates homology with the constant regions of immunoglobulin light and heavy chains. A protein that had long previously been crystallized from bovine milk and designated lactollin was shown in the year 1977 to be bovine β₂-microglobulin. It is a polypeptide of 98 amino acid residues,two of which are Cys. Direct quantification of microglobulin concentration have not been made in bovine milk; the amounts of lactollin that have been isolated from colostrum and milk are about 6 and 2mg per liter, respectively.