Rodney J. Brown

Plasmin in milk and dairy products: an update

Abstract Plasmin is secreted as plasminogen that is activated in blood and milk. Its role in blood is to proteolytically break down blood clots. The enzyme has affinity for lysine and arginine residues and preferentially cleaves Lys-X and Arg-X bonds. Bovine plasminogen is different from human plasminogen because it is not activated by streptokinase. Plasmin purified from bovine milk is identical to that from blood when compared kinetically, immunologically and by partial sequencing (15% of the total amino acids sequenced). Plasmin purified from milk has optimum activity at pH 7.5 and 37 °C. Methods for determining plasmin activity in milk have been developed. Synthetic chromogenic substrates are often used because they are sensitive and require little sample preparation. Plasmin is associated with casein micelles in milk and degrades β-, α s1 - and α s2 -caseins to γ-caseins, proteose-peptones and possibly λ-casein. Peptide bonds in β-, α s1 and α s2 -caseins that are sensitive to plasmin in buffered model systems have been identified. Some of these bonds are hydrolysed in milk and cheese but more work is required to link work in model systems to milk and dairy products. Enzyme activity increases in milk with stage of lactation, severity of mastitic infection and lactation number. Plasmin contributes to proteolysis during ripening of some cheese varieties depending on cooking temperature and pH during ripening. The enzyme is heat resistant and survives many UHT treatments, but its role in the gelation of UHT-treated milk is not fully understood.

Use of amino acid analysis for estimating the individual concentrations of proteins in mixtures.

The concentrations of five individual proteins in a mixture were determined from one amino acid analysis of the mixture by solving for each protein using simultaneous equations. Dried casein and whey were separated into five individual protein components using reversed-phase HPLC. Individual proteins were collected and analyzed for amino acid composition. These data were used as standards. Mixtures of purified proteins were analyzed for total amino acid composition and the concentrations of individual proteins in the mixtures were determined by solving simultaneous equations based on the amino acid analysis composition of the standards.

Development of surface functionality of casein particles as the controlling parameter of enzymic milk coagulation

Abstract Changes in a colloidal milk system were monitored by measuring changes in turbidity and the modulus of the resultant milk gel. Turbidity was measured in milk of normal concentration and in milk that had been diluted to eliminate the effects of multiple light scattering. The relationship between true gelation time and observed coagulation time, and how gelation time might affect the theoretical modelling of enzymic milk coagulation are discussed. The turbidity of milk at normal concentration cannot be directly related to particle size but it could be correlated with gel modulus measurements. True gelation time was calculated by fitting gel modulus data to an exponential equation. The result coincided with the inflexion point in the turbidity plots. Surface functionality of casein particles in relation to the collision area and the extent to which k -casein macropeptide must be removed from the particle surface for successful collisions are discussed.

Purification of Monospecific Polyclonal Antibodies from Hyperimmune Bovine Whey Using Immunoaffinity Chromatography

Whey, a by-product of cheese production, is a potential source of proteins. Immunization of dairy cows in mid-lactation with mouse IgG and dinitrophenyl-keyhole limpet hemocyanin resulted in the formation of antibodies to these antigens in both blood serum and milk. The antibodies remained in whey during cheese making, and were isolated by immunoaffinity chromatography on matrices with immobilized antigens. The isolated monospecific antibodies were pure and retained their reactivity to antigens.