Anthony T. Andrews

Proteinases in normal bovine milk and their action on caseins

Native proteolytic enzymes in good quality normal bovine milk readily hydrolysed the caseins during incubation or storage, producing the gamma-caseins, proteose-peptone components 5 (PP5) and 8-fast (PP8F) and a considerable number of other unidentified fragments, many of which were also subsequently found in the proteose-peptone fraction. The rate of casein hydrolysis was greater in pasteurized than in raw milk, with beta-casein being slightly more susceptible to attack than alpha S1-casein. Measurements of gamma-casein and proteose-peptone formation have been made and it was found that PP5 was an intermediate product that was subject to further proteolysis while PP8F was a stable end-product. With the exception of component 3 (PP3), virtually all constituents of the proteose-peptone fraction increased during storage and appeared to be products of the action of proteolytic enzymes. Further evidence was obtained from the effects of various inhibitors that the principal proteinase of normal milk is plasmin, but slight differences were apparent between the protein breakdown patterns induced by storage and by added plasmin, which was consistent with the presence of more than one proteinase. Incubations in the presence of soya bean trypsin inhibitor to prevent plasmin action clearly revealed that another enzyme(s) was also involved.

Proteolysis of caseins and the proteose-peptone fraction of bovine milk

The proteolysis of highly purified samples of α s1 -, α s2 -, β-and κ-caseins by porcine plasmin and by bovine plasminogen with urokinase has been examined principally by gel electrophoresis. The resulting peptide band patterns were compared with those of total proteose-peptone (TPP) samples prepared from fresh and stored raw and pasteurized milk, and also with those obtained during the natural course of proteolysis by indigenous enzymes in milk during storage. TPP was found to contain at least 38 components detectable by a single electrophoresis run. Apart from residual traces of whey proteins and intact caseins nearly all of these components were fragments of caseins produced by indigenous plasmin, with products from the breakdown of α s1 - and β-casein predominating. Over 90 % of TPP has been accounted for in this way. A fragment consisting of residues 29–105 of β-casein was isolated and characterized from both stored milk and from plasmin digests of β-casein. This fragment was a relatively major product of the natural proteolysis occurring during storage of milk, but contrary to a report in the literature it was not the same as proteose-peptone component 8-slow. Since many of the components of TPP resulted from proteolysis, the composition of TPP was found to vary according to the time and temperature of storage of the milk from which it was prepared. Thus, while the proteose-peptone fraction of milk can easily be defined operationally it cannot be rigorously defined in terms of its composition unless the history of the milk is also defined.

Breakdown of caseins by proteinases in bovine milks with high somatic cell counts arising from mastitis or infusion with bacterial endotoxin.

Milk obtained from cows which were either infected by clinical mastitis or had been subjected to intramammary infusion of Escherichia coli endotoxin possessed high counts of somatic cells and very high levels of proteinase activity which hydrolysed the caseins almost completely in a few hours at 37 degrees C. The rate of hydrolysis of beta-casein was slightly greater than that of alpha S1-casein, but in both cases hydrolysis was enhanced by 6 cycles of freezing and thawing to disrupt somatic cell membranes. A study of the relationship between proteinase activity and cell count suggested that only some of the proteinase activity originated in the somatic cells and also that the identity of the cells making up the total cellular population was important. Maximum proteolysis occurred at 50-60 degrees C, but the temperature-activity curve was a broad peak. Likewise the pH versus activity plot was very broad and was almost flat over the pH range 6-9. Experiments with a number of inhibitors of proteinases failed to give a clear cut pattern of inhibition. All evidence obtained was consistent with the view that several different enzymes with different pH and temperature optima and different specificities contributed to the overall hydrolysis of caseins in these milks. From electrophoretic band patterns one of these enzymes was clearly plasmin, but in high cell count milks other proteinases also became significant.

A study of the heat stabilities of a number of indigenous milk enzymes

Heat stability profiles of a number of indigenous bovine milk enzymes were examined with the object of being able to monitor heat treatments slightly more severe than typical pasteurization conditions by measurements of residual enzyme activity after heating. Assay procedures were limited to simple fluorimetric, or preferably colorimetric, methods that would be most likely to form the basis of a quick, simple and inexpensive test. Both lipoprotein lipase and α-fucosidase were relatively sensitive to heat and were totally inactivated at temperature/time combinations below those of pasteurization, but the latter may be satisfactory for studying temperatures in the range 55–65°C. Rather more heat stable were N -acetyl-β-glucosaminidase and γ-glutamyl transpeptidase, which may be most appropriate for 65–75°C and 70–80°C respectively. Higher temperature treatments between 80 and 90°C could best be investigated by following α-mannosidase or xanthine oxidase activity.

Heat stability of plasmin (milk proteinase) and plasminogen

The effect of heating on plasmin activity in various media, including phosphate buffer pH 7.0, skim milk, blood plasma, solutions of casein and solutions of whey proteins were investigated. Plots of log residual activity v. heating time were linear at all temperatures from 63 to 143 degrees C. In buffer solutions the presence of casein led to substantial substrate protection, the Arrhenius plots being linear both in the presence and absence of casein. The activation energy, Ea, for the inactivation reaction, was 62.4 kJ/mol in buffer alone and 58.4 kJ/mol with casein present at 25 mg/ml. In skim milk, despite the presence of casein at a similar concentration, plasmin was no more stable to heat than in buffer alone, and a curved Arrhenius plot was obtained indicating a more complex inactivation mechanism. Heating in the presence of proteins having free -SH groups accelerated the inactivation of plasmin. The role of -SH groups was confirmed by experiments with added alpha-lactalbumin, in which no free -SH groups occur, and reduced carboxymethylated beta-lactoglobulin, both of which were without effect. In blood plasma, plasmin was less stable to heat than in buffer (pH 7.0) or in skim milk. Plasminogen behaved very similarly to plasmin either when activated to plasmin with urokinase before heating or when activated afterwards. A hypothesis is presented to describe the heat inactivation and denaturation of plasmin. Technologically important findings are that in skim milk plasmin was largely unaffected by pasteurization conditions and 30-40% of its activity remained even after ultra high temperature processing conditions.

Qualitative and quantitative determination of proteolysis in mastitic milks.

Proteolytic activity in mastitic skim-milk was often 5-10 fold higher than in normal milk, its level being related to somatic cell count but not precisely correlated with it. In milks with the highest levels of activity plasmin accounted for about one third of the total proteinase. A further third was sedimented with the micellar fraction together with the plasmin, but unlike plasmin, was not inhibited by addition of soyabean trypsin inhibitor (SBTI). The final third remained in the serum phase. Polyacrylamide gel electrophoresis (PAGE) showed that alpha-sl- and beta-caseins were degraded at about the same overall rate. The plasmin produced the usual readily identified fragments from beta-casein, but incubation of mastitic milk also produced changes in patterns in the gamma-casein region differing from plasmin-induced changes, which were also apparent when the micellar fraction was incubated. As they were inhibited by SBTI, a second trypsin-like enzyme in addition to plasmin may also have been present. Other proteinase(s) not inhibited by SBTI was also associated with casein micelles and produced at least 3 characteristic protein fragments seen on PAGE. The serum phase proteinase(s) was likewise not inhibited by SBTI, and did not produce any well-defined electrophoretic bands, suggesting a rather non-specific breakdown of caseins. After separation of mastitic whole milk, a considerable proportion of the proteolytic activity was found in the cream phase. The proportion was enhanced by freezing and thawing, and the enzyme appeared to be identical to the SBTI-resistant micellar proteinase. Because of the considerable proteolysis likely to occur under the time and temperature conditions involved, our results may provide some explanation for the problems encountered in cheesemaking with mastitic milks (e.g. yield losses, poor curd strength and off-flavour development).

Influence of storage of milk on casein distribution between the micellar and soluble phases and its relationship to cheese-making parameters.

The influence of storage time and temperature on the distribution of individual milk proteins between the micellar and soluble phases has been examined. Storage at 4 or 7 °C is accompanied by a dissociation of micellar caseins, particularly β-casein, into the soluble phase during the first 48 h, but on further storage there is a partial reversal of this process. At higher storage temperatures (10–20 °C) the contents of all the individual caseins in the soluble phase decrease throughout storage. During cheese–making, losses of fat and curd fines in whey were greater with increased soluble phase casein and clotting times were prolonged. Curd structure was weaker, curds were more moist and slightly lower cheese yields were obtained in stored milks with elevated soluble–phase casein. When milks were stored for up to 3 d at 4 °C cheese gradings were virtually unaffected by storage, but higher temperatures (10–15 °C) led to cheeses being downgraded, largely for body and texture defects but also for flavour after 3 d. Attempts to reverse the pattern of casein dissociation and to minimize the detrimental effects of milk storage on subsequent cheese-making showed that a heat treatment of 30 min at 60 °C was the most beneficial.

The roles of native milk proteinase and its zymogen during proteolysis in normal bovine milk

Proteolysis was measured quantitatively in normal bulk milk, either raw, pasteurized or heated (95 degrees C, 15 min). During incubation at 37 degrees C for 24 h, about 0.7 mM of peptide bonds were split in raw milk, and 1.8 mM after activation of the zymogen with urokinase. The same values were observed in pasteurized milk, and no significant activity was present in heated milk. When compared with a commercial plasmin preparation, these levels correspond to about 1.4 and 3.6 micrograms/ml of plasmin respectively. Most of this activity was separated in the micellar fraction, and it was suppressed by addition of soyabean trypsin inhibitor (SBTI). The remaining activity in the serum phase was not inhibited by SBTI and gave a rather non-specific breakdown with few well-defined casein fragments being produced. Upon further incubation, after the first 24 h, the activity increased, indicating that activation of the zymogen (plasminogen) occurred spontaneously. The rate of this activation was independent of the addition of more plasminogen and was higher in pasteurized than in raw milk. In pasteurized milk, all the native milk proteinase was in the form of the zymogen at the time of secretion. beta-Casein was the preferred substrate for the milk proteinase (plasmin) and produced gamma-caseins and proteose-peptone components 5 and 8-fast; other fragments were clearly visible on polyacrylamide gel electrophoresis, and included degradation products of alpha-sl-casein. The formation of all these fragments was enhanced by addition of urokinase alone, or of plasminogen and urokinase, or by increasing the incubation time. They were also produced by incubating the micellar fraction alone, but not the serum fraction. Additional fragments were produced when porcine plasmin was added presumably due to differences in specificity between the porcine and bovine enzymes or to contaminating enzymes. Proteolysis induced by additions of plasminogen alone, or of plasminogen plus urokinase, was closer to that observed for the native milk proteinase, and must be recommended for future work in which it is desired to enhance the level of proteinase without altering breakdown patterns, unless a very pure bovine plasmin is available.

The plastein reaction revisited: Evidence for a purely aggregation reaction mechanism

Abstract Various aspects of the plastein synthesis reaction were investigated using peptides derived from casein as substrate. With peptides obtained by partial acid hydrolysis a clear requirement for a proteinase to catalyse plastein synthesis was demonstrated and, although enzymes whose hydrolytic activity had been inhibited may act as rather inefficient catalysts, the native active enzymes were preferred. Blockage of peptide NH 2 or COOH groups reduced plastein yield but did not prevent synthesis. Results following addition of water-miscible organic solvents to reaction mixtures were more consistent with increased solubility of hydrophobic amino acids and peptides rather than with the influence of viscosity changes. Although plastein separates out of the reaction mixture in the form of a gel or precipitate and can be collected as an insoluble centrifuge pellet, on repeated washing and incubation it was gradually solubilised even in aqueous buffers, showing clearly that plastein formation is a reversible process. This was in effect confirmed by ionexchange chromatography and gel filtration experiments, in which there were some quantitative differences in peptide composition between peptide hydrolysate starting materials and resolubilised plastein pellets produced from them but no qualitative differences. This showed clearly that no appreciable amounts of new peptides were formed and ruled out covalent bond formation in a reversed hydrolysis or a transpeptidation pathway as the reaction mechanism. This conclusion was confirmed by SDS-PAGE and by preliminary small-angle neutron scattering experiments. We therefore conclude that the plastein synthesis reaction is a purely entropy-driven physical aggregation process.

The formation and structure of some proteose-peptone components.

Two constituents of the proteose-peptone fraction of bovine milk have been isolated and characterized. Component 5 (PP5) has been shown to represent residues 1-105 and 1-107 of the beta-casein amino acid sequence, while component 8-fast (PP8F) corresponds to residues 1-28 of beta-casein. Thus, these proteose-peptones represent the N-terminal portions of the beta-casein molecule, produced by proteolytic cleavages which form the gamma 1, gamma 2 and gamma 3-caseins from the C-terminal part. The continuing formation of the total proteose-peptone fraction, PP5, PP8F and the gamma-caseins during storage of raw milk at 18 or 37 degrees C has been also been demonstrated.