Professor of Novosibirsk State Agrarian University Milk proteins Professor of Novosibirsk State Agrarian University Korotkevich O.S.

Proteins are an extremely important class of naturally occurring compounds that are essential to all life processes. They perform a variety of functions in living organisms ranging from providing structure to reproduction. Milk proteins represent one of the greatest contributions of milk to human nutrition. Proteins are polymers of amino acids. Only 20 different amino acids occur, regularly in proteins.

Phosphoproteins: Phosphate is linked chemically to these proteins examples include casein in milk and phosphoproteins in egg yolk. Lipoproteins: These combinations of lipid and protein are excellent emulsifying agents. Lipoproteins are found in milk and egg yolk.

The protein component of milk can be divided into two groups, the casein fraction and the whey proteins:

The casein proteins are the ones that will form the curd during cheese making. Casein proteins tend not to have a particularly compact globular structure and they tend to be rather susceptible to proteolysis. As they are all phosphorylated, they bind the calcium content of the milk and exist in the form of casein micelles.

Casein Casein was first separated from milk in 1830, by adding acid to milk, thus establishing its existence as a distinct protein. In 1895 the whey proteins were separated into globulin and albumin fractions. It was subsequently shown that casein is made up of a number of fractions and is therefore heterogeneous. The whey proteins are also made up of a number of distinct proteins as shown in the scheme in Figure.

Casein is easily separated from milk, either by acid precipitation or by adding rennin. In cheese-making most of the casein is recovered with the milk fat. Casein can also be recovered from skim milk as a separate product. Casein is dispersed in milk in the form of micelles. The micelles are stabilised by the Κ- casein. Caseins are hydrophobic but Κ-casein contains a hydrophilic portion known as the glycomacropeptide and it is this that stabilises the micelles. The structure of the micelles is not fully understood.

When the pH of milk is changed, the acidic or basic groups of the proteins will be neutralised. At the pH at which the positive charge on a protein equals exactly the negative charge, the net total charge of the protein is zero. This pH is called the isoelectric point of the protein (pH 4.6 for casein). If an acid is added to milk, or if acid- producing bacteria are allowed to grow in milk, the pH falls. As the pH falls the charge on casein falls and it precipitates. Hence milk curdles as it sours, or the casein precipitates more completely at low pH.

Chymosin Reaction The chymosin content of the rennin causes a specific and rapid cleaveage of the kappa- casein component of the casein micelles. This protein stabilises the micelles and after cleavage, the casein proteins precipitate under the influence of the calcium ions. Chymosin specifically recognises the sequence from His 98 to Lys 111 and cleaves the peptide bond between Phe 105 and Met 106 in the kappa- casein chain:

In addition to its role in milk clotting, chymosin is also involved in the proteolytic changes occurring during ripening.

Casein proteins contains phosphorus and will coagulate at pH 4.6. Casein proteins group together form sphere-like structures called micelles. Casein micelles are not solid throughout. Instead, the way the protein molecules group together leaves spaces, much like tunnels through the micelles. These spaces allow the liquid part of milk to flow in and out of the micelles.

Casein micelles

Casein Micelle Structure The first representation of casein micelle structure shown here is that of the "casein sub- micelle" model. It has evolved from several earlier models. It is very difficult to imagine exactly what the micelle looks like. However, scientists do know a great deal about the micelle from its behaviour, and any model needs to account for all of the known facts. This model, which nows dates to about 20 years old, is based on the inclusion of many known facts about casein micelle behaviour. See Walstra and Jenness for discussion of this model.

There is not universal acceptance of the above model among dairy scientists, in fact there is mounting evidence that well-defined casein submicelles do not exist, rather the structure is more open and fluid, perhaps a "bowl-of- spaghetti" type of model. The big problem with the earlier model was the distribution of calcium phosphate, and it is certainly apparent now that calcium phosphate exists more evenly distributed throughout the micelle, so based on the above model, both within and outside the submicelles.

The next model shown here has evolved recently, from work especially by Carl Holt and co-workers at Hannah Research Institute, Scotland. It shows a more or less spherical, highly hydrated, and fairly open particle. Polypeptide chains in the core are partly cross- linked by nanometer sized clusters of Ca phosphate; the internal structure gives rise to an external region of lower segment density known as the hairy layer, which confers steric and/or charge stability to native casein particles. This figure also shows equilibria between the micelle and the milk serum with acidification and with heating.

Casein micelles are too large to dissolve and form a solution, like salt in water does. Casein micelles are too small to sink, like sand in water does. Instead, the micelles float around in the milk. This type of mixture is called a colloid.

Milk Coagulation The coagulation of milk by chymosin includes two separate steps: proteolysis and aggregation.

Whey proteins After the fat and casein have been removed from milk, one is left with whey, which contains the soluble milk salts, milk sugar and the remainder of the milk proteins. Like the proteins in eggs, whey proteins can be coagulated by heat. When coagulated, they can be recovered with caseins in the manufacture of acid-type cheeses.

The whey proteins are made up of a number of distinct proteins, the most important of which are b-lactoglobulin and lactoglobulin. b-lactoglobulin accounts for about 50% of the whey proteins, and has a high content of essential amino acids. It forms a complex with Κ-casein when milk is heated to more than 75°C, and this complex affects the functional properties of milk. Denaturation of b- lactoglobulin causes the cooked flavour of heated milk.

Whey proteins, also called serum proteins, do not contain phosphorus and do not coagulate at pH 4.6. Whey proteins are individual protein molecules spread throughout the liquid part of the milk.

If casein micelles come apart, the proteins forming it are not soluble in the liquid. Thus they “come out of solution.” This means that these proteins separate from the liquid. These proteins now coagulate, which means they clump together into a mass called curd.

Casein proteins, unlike most proteins, is not denatured by heat Casein proteins, unlike most proteins, is not denatured by heat. For example, when an egg is heated, the protein making up the liquid surrounding the yellow yolk changes to a white solid. The protein has been denatured–changed in some chemical way. So, heating milk does not cause the casein proteins to be denatured– to coagulate. Casein proteins are denatured by a change in pH. The casein proteins will coagulate at pH 4.6.

Other milk proteins In addition to the major protein fractions outlined, milk contains a number of enzymes. The main enzymes present are lipases, which cause rancidity, particularly in homogenised milk, and phosphatase enzymes, which catalyse the hydrolysis of organic phosphates. Measuring the inactivation of alkaline phosphatase is a method of testing the effectiveness of pasteurisation of milk.

Peroxidase enzymes, which catalyse the breakdown of hydrogen peroxide to water and oxygen, are also present. Lactoperoxidase can be activated and use is made of this for milk preservation. Milk also contains protease enzymes, which catalyse the hydrolysis of proteins, and lactalbumin, bovine serum albumin, the immune globulins and lactoferrin, which protect the young calf against infection.

Milk Protein Allergy

"Milk protein allergy" is an allergic reaction to proteins commonly found in cows milk. It is caused by your immune system reacting because it believes the protein in the milk is a threat to your body. Your immune system will do it's best to get rid of the invader, just as it would a foreign virus or poison. During the allergic reaction your body releases histamine, a chemical which causes blood vessels to dilate and leak, mucous membranes to start producing and other effects. The leaking blood vessels causes redness and itching over certain parts of the body or even all of it. The increased mucous may make you congested. Other reactions include nausea, vomiting, diarrhoea and behavioural changes. It can also lead to anaphylaxis in which the patient's air passages swell and close and blood pressure falls abruptly, leading, if untreated to death.

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