Protein in Milk

Introduction

Normal bovine milk contains about 3.5% protein. Milk proteins are of two distinct types, whey proteins (serum proteins) and caseins. 
Caseins constitute over 80%  and whey constitute 20% of the total protein of milk, although the relative proportion of whey proteins to casein varies according to the stage of lactation. Milk produced in the first few days after calving and towards the end of lactation is of substantially higher whey protein content than that produced in mid-lactation. This increase is accompanied by elevated levels of blood serum proteins.

The natural function of milk proteins is to supply young mammals with the essential amino acids required for the development of muscular and other protein-containing tissues, and with a number of  biologically active proteins.

On acidification to pH 4.6 (the isoelectric pH) at around 30°C, about 80% of the total protein in bovine milk precipitates out of solution; this fraction is now called casein. The protein which remains soluble under these conditions is referred to as whey or serum protein or non-casein nitrogen.

The protein content in a given milk products influences the structure of that product.

Definition of Milk Protein

Proteins are defined as high molecular weight polymers of α-amino acids that are formed by living organisms. All these amino acids have L-configuration except glycine. The primary structure of proteins consists of a polypeptide chain of amino acids residues joined through peptide bonds.

Classification of Milk Proteins

1) Casein Protein

• Casein is the major protein of bovine milk and exists mainly as micellar form(or Colloidal State), where as in human milk major proteins are whey proteins. The Casein micelle consists of water, casein, salts and some minor components including lipase and proteinase. Casein micelles are built of smaller particles called sub-units or sub-micelles. Milk plasma free from casein micelles is known as milk serum.
• In its pure state it is snow-white,odorless, and tasteless.It contributes to whiteness of milk.In milk it is found in combination with calcium and is often designated as "Calcium caseinate".
• The caseins of milk may be subdivided into four main classes αs1,αs2, β - and κ-caseins . All four caseins have a distinctly amphiphatic character with separate hydrophobic and hydrophillic domains.  There are also several derived caseins, resulting from the action of indigenous milk proteinases, especially plasmin, on the main caseins. These are usually referred to as γ-caseins.
• Casein may be precipitated from milk by dilute acids, rennin, and alcohol. The casein precipitated with weak acids is free of calcium.The casein precipitated with alcohol is calcium caseinate. When precipitated with rennin, paracasein is formed.It contains more calcium than calcium caseinate. Pure casein is not precipitated by heat, but in fresh milk prolonged heating at high temperatures(100° C) for 12 or more  hours or 120° C under pressure will cause the precipitation of casein. Casein does not show denaturation. However, heating at temperatures above approximately 120°C causes the casein to slowly become insoluble due to chemical changes.The application of heat to milk that is slightly acid will cause the precipitation of casein.
• The caseins are all phosphoproteins with the phosphate groups being esterified to the serine residue in the protein chains. The phosphate groups bind large amounts of calcium, and they are important to the structure of casein micelles.
• The protein content of micelles is 92%, composed of αs1,αs2, β  and κ-caseins in an average ratio of 3: 1 :3: 1. The remaining 8% is composed of inorganic constituents, primarily colloidal calcium phosphate.

a) αs1-casein 

• The polypeptide chain of αs1-casein consists of two predominantly hydrophobicregions (residues 1-44, and 90-199) and a highly charged polar zone (residues 45-89).
• αs1-casein are sensitive to calcium due to presence of phosphate groups and precipitate in the presence of Ca2+ ions at a pH value of 7.0. 
• It aggregates and precipitates at very low concentrations of Ca2+ (7 mM Ca2+,28 mM NaCl). A small amount of peptides, sometimes called A-casein, is present in milk; these appear to originate from proteolysis of αs1-casein.
• αs1-casein contains seven to nine phosphoserine residues per mole. Phosphoserine residues are concentrated in clusters and are responsible for the existence of hydrophilic areas of strong negative charge.The molecules also contain blocks of hydrophobic residues.
• It  has 199 amino acids and its weight is app. 23.6 kDa.It has 17 proline residues that ultimately disrupt the formation of secondary structures, such as  α - helices and  β - sheets.

b) αs2 - 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. Its properties have not been investigated as thoroughly as those of the other caseins, but certainly it binds Ca strongly and is even more sensitive to precipitation by Ca2+ than αs1-casein. 
• It self-associates at neutral pH in the absence by Ca2+, and the association depends markedly on ionic strength and is at a maximum at an ionic strength of about 0.2. αs2-casein has two hydrophobic segments, 90-120 and 160-207.(207 amino acids) and weight is app. 25.4 kDa).It contains two cysteine residues.It contain two cysteines residue per mole which normally exist as inter-molecule disulphide bonds.

c) β-casein

• β-casein has a strong negatively charged sequence between residues 13 and 21, with four of the five phosphoserine residue and three Glu residue.The N- terminal 21- residue sequence of  β-casein has a net charge of -12.
• It contain no cysteine or cystine residue. Part of the β-casein is split by proteolytic enzymes into γ-casein and proteose peptone. The outstanding characteristics of the association of β-casein in both the absence and the presence of Ca2+ are its strong dependence on temperature.In the absence of Ca2+, only monomer is present at 4°C, but large polymers (20-24 monomers) are formed at room temperature.
• β-casein consists of 209 amino acids and is approximately 24 kDa. It is the most hydrophobic casein molecule. Similar to αs1-casein, β-casein has few secondary structures due to the presence of 35 proline residues.

d) κ-caseins

• About one-third of the κ casein molecules are carbohydrate-free and contain only one phosphate group (SerP-149).The N-terminal residue of κ-casein is glutamic acid. κ - casein consists of 169 amino acids and is approximately 19 kDa, and it contains both glycosylated and phosphorylated residues. 
• It can exist as a dimer up to a decamer with the subunits held together by disulfide linkages. Unlike the other caseins, it is not sensitive to calcium and surrounds the micelles, keeping them intact.There 5-65 and 105-115 segments of κ-casein are strongly hydrophobic.
• The κ-casein molecule appears to consist of a relatively stable, single disulphide bonded structure within which are both (α-helical and β-sheet regions). The chymosin-sensitive Phel05-Met106 bond is thought to protrude from the molecular surface. One third of the κ-casein molecule is represented by the strongly ionic C-terminal section, which contains the three oligosaccharide residues.

e) γ- casein

γ-caseins are derived by hydrolysis of  β-casein at positions 28/29, 105/106, and 107/108 by the  enzyme plasmin. The fragments 29-209, 106- 209, and 108 209 constitute the γ-caseins.

Structure of casein micelles 

• Casein micelles consist of a large portion (approximately 94%) of casein proteins that interact with each other and calcium. They vary in size from 80 to 1,000 nm with an average diameter of 150  nm in bovine milk.
• The casein micelle consisting of an aggregate of almost spherical sub-micelles, which inturn consist of more limited aggregates of casein molecules.The calcium phosphate and αs- and β-caseins are linked by the involvement of the phosphoserine residues in the structure of the calcium phosphate. 
• Kappa-casein is localized on, or very close to, the surface of the casein micelle. The hydrophobic part of the K-casein molecule is bound to the core of the micelle, while the hydrophilic macropeptide (C-terminal) forms a layer of highly hydrated 'hairs', which project(5-10 nm from micelle surface) into the aqueous phase. Kappa-casein hairs are responsible for the steric stabilization of casein micelles. 

• Bovine casein micelles contain water, protein (about 94%), salts (about 6%), including calcium, phosphorus, magnesium, citrate - colloidal calcium phosphate [CCP] other traces of metals, enzymes (lipases, esterases, proteases), and milk serum. 
• Casein micelles contain submicelles that range from 12 to 15 nm in diameter, Contain approximately 20 to 25 casein molecules and water (2 to 5  g/protein) with some submicelles containing  K - casein.
• Components of the casein micelle appear to be in equilibrium with the aqueous phase, and while freshly secreted milk contains only small quantities of soluble casein, some casein together with colloidal calcium phosphate dissociates from the micelle during storage at 0°C.

2) Whey Protein

• 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 are hydrophobic, globular,  highly ordered proteins that contain disulfie  linkages.  Whey proteins have well - developed secondary, tertiary, and quaternary structures. It has poor heat stability, it denature at temperatures greater than 75 ° C. It is green in color after extracting from milk. It is obtained by coagulating the milk such as during manufacturing of paneer, cheese and casein as by product. 
• Whey proteins comprise two gene products, β-Iactoglobulins and α-Iactalbumins,  and small quantities of the blood-derived proteins, serum albumin and immunoglobulins. 
• Several other proteins are found in small quantities in whey; these are β-microglobulin, lactoferrin and transferrin(both of which are iron binding proteins), proteose-peptones (partly derived from hydrolysis of β-casein) and a group of acyl glycoproteins.

a)  β-Iactoglobulin

• It is the most abundant whey protein and represents about 50% of the total whey protein in bovine milk. β-lactoglobulin consists of 178 amino acids with an approximate molecular weight of 18 kDa.
•  It exists in both the monomeric and dimeric form at equilibrium in bovine milk. Its association depends on temperature, pH, protein concentration, and ionic conditions.
• It contains two disulfide bonds and a single thiol group, which is of great importance for changes occurring in milk during heating. Location of one disulphide bond always occurs between Cys residues at 66 to 160 positions and the other link is between 106 and119 or 121. 
• The single free thiol appears to be equally distributed between Cys119 and Cys 121.
• There are eight known genetic variants of β-lactoglobulin :A,B,C,D,E,F,G and Dr. The A and B genetic variants are the most commomn and exist at almost the same frequency.

b) α-Lactalbumins

α-lactalbumin accounts for about 20% of the whey proteins and has three known genetic variants. It has a molecular weight of 14000Da and contains four interchain disulfide bonds.α-Lactalbumin binds two atoms of calcium very strongly, and it is rendered susceptible to denaturation when these atoms are removed.It is the principal protein in human milk.It consists of approximately 142 amino acids. 

α-lactalbumin’s biological function is as coenzyme in the synthesis of lactose. The protein is a small, compactly folded, more or less spherical molecule. It does not associate, except at low ionic strength.

c) 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 and represent about 5% of the total whey proteins.It has One free thiol and 17 disulfide linkages, which hold the protein in a multiloop structure.Bovine serum albumin consists of 582 amino acids and it is the longest protein. Its molecular weight is approximately 66 kDa.

d) Immunoglobulins

• 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) including γ (MW 52,000), α (MW 52,000-56,000) and μ (MW 69,000). 
• They account for up to 10% of the whey proteins.Immunoglobulins are large glycoprotein molecules of heterogeneous composition. They specifically occur in blood.The main immunoglobulins in milk are IgG1, IgG2, IgA, and IgM.Approximately 0.7 to 1mg/ml is present in bovine milk.IgG is the main immunoglobulin in milk.
• 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.
• 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.

               Schematic shape of immunoglobulins G1, secretory A1, and M. Disulfide linkages                         are designated by dashes. Variable portions are black

e) Proteose peptones 

Proteose peptone is defined as non-heat-sensitive, not precipitated at pH 4.6, and precipitated by 12% trichloroacetic acid. Derived from hydrolysis of β-casein. It is acid - soluble proteins and mainly responsible for the foaming of skim milk.It also Inhibit rancidity.

f) Lactoferrin and Transferring

• 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.
• Both Tf and Lf appear to be large single chain polypeptides of 600-700 amino acid residues. Lactoferrin is an inhibitor of some bacteria including Bacillus stearothermophilus and Bacillus subtilis. The inhibition is caused by removal of iron, more precisely Fe3+ ions, from the serum.Bovine milk contains lactoferrin app. 20 to 200 mg/L and Human milk contains 2 g/L.