Efstathios Alichanidis

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.

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.

Suitability of some microbial coagulants for Feta cheese manufacture

Etude comparative de succedanes de presure obtenus a partir de Mucar pusillus, M. miehei et Endothia parasitica avec la presure de veau

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.

Effect of refrigerated storage of milk on the manufacture and quality of Teleme cheese

Teleme cheese made from milk stored at 4–5 °C for 1–5 d and then pasteurized was compared with that made from unstored control milks. In the raw milks titratable acidity, tyrosine value and acid degree value increased more rapidly in stored milks than in the controls. With pasteurized milks, variations in coagulation times were attributable to levels of psychrotrophs in the corresponding raw milks. Differences were minor for milk fat and N content between cheeses. Curds made from milks stored > 4 d differed markedly from the controls in pH, moisture, N soluble at pH 4·6 and trichloroacetic acid (TCA)-soluble N, but only small differences in levels of casein fragments were detected. Proteolysis in mature cheese (60 d old) made from milk stored > 3 d differed significantly from the controls, and casein fragments were detectable. Off flavour related to excessive protein breakdown developed only in cheese made from milk stored > 4 d. In 60 d old cheeses there was significantly more rancidity in those made from milk stored for > 3 d than in those made from control milk. Unacceptable rancidity became evident at 60 d in cheeses produced from milk stored > 4 d. For Teleme cheesemaking psychrotrophic bacteria multiplying in the milk to levels likely to be encountered in commercial practice are of far greater importance for their lipolytic than for their proteolytic enzymes.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

An extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43,000 and contained 2 g atom Ca/mol. It was active over a pH range 4.8-9.5 and had optimum activity on casein at pH 7.0 with Km = 0.17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 degrees C; above 50 degrees C activity declined rapidly, but significant activity persisted at 4 degrees C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

Ripening changes in Kopanisti cheese.

Changes in a Greek traditional soft cheese, Kopanisti, were followed during ripening. The a s -casein content was hydrolysed faster than the β-casein so that in mature cheese only 23% and 35% respectively of these proteins remained intact. Leucine, γ-aminobutyric acid, valine and alanine were the dominant free amino acids in the mature cheese. Lipolysis was intense. The characteristic rich flavour and peppery taste appeared after 16 d ripening and the best overall cheese quality was produced after 32 d maturation

Glycoproteins in the heat- and acid-stable fraction of ovine milk

Affinity chromatography on a concanavalin A-Sepharose support was used to isolate two glycoprotein fractions from a heat- and acid-stable fraction of ovine milk. One of these glycoprotein fractions was purified by rechromatography on DEAE-cellulose to essentially a pure protein yielding a single band on gel electrophoresis. The apparent Mr of this glycoprotein (GP2) as estimated by electrophoresis was 5,500. It contained 8.88% carbohydrate and 0.61% P. The other glycoprotein fraction (GP3) contained 0.53% P and 17.76% carbohydrate including sialic acid, mannose, galactose, fucose, galactosamine and glucosamine. It appeared on electrophoresis in acrylamide gels as a slow-moving broad band. On similar treatment in the presence of sodium dodecyl sulphate it revealed four glycoprotein zones with apparent Mr of 15,200, 18,300, 23,500 and 25,300. Both GP2 and GP3 contained low amounts of aromatic and sulphur-containing amino acid residues and large amounts of Asp, Glu, Ser and Leu. GP3 is similar in some respects to the bovine milk heat-and acid-stable fraction constituent, component 3.