Fractionation of cell-free extracts of Lactobacillus casei NCDO 151 (grown in Man's et al. (1960) broth) by polyacrylamide disc electrophoresis showed the presence of 4 constitutive peptidases. The enzymes appear to be a tripeptidase with a narrow substrate specificity, two true dipeptidases with identical broad-specificity, and a probable carboxypeptidase with broad-specificity. A probable amino peptidase could also be isolated from the cell-free extract by density gradient electrofocusing. Growth of the organism in skim milk resulted in the formation of two inducible dipeptidases. Examination of 5 other strains of L. casei for the presence of the two constitutive dipeptidases and the carboxypeptidase confirmed the results obtained for the species. Some strains, however, had one more peptidase. The peptidases of the strains differed relatively in the activity and substrate specificity from strain to strain. On polyacrylamide gel, the peptidase activity of L. plantarum appeared as one band only, probably of a carboxypeptidase.

Individual milk samples of 50 goats, 50 ewes and 50 cows were examined for the total viable count, coliform bacteria, staphylococci and salmonellae. Growth of enterotoxin A producing Staphylococcus aureus NCTC 10652 in the milk of the three animal species was also studied. The average total count was 1.9 X 10(7) cells/ml for cow's, 7.7 X 10(6) for goat's and 2.7 X 10(6) for ewe's milk with micrococci staphylococci, rods and streptococci being the predominant organisms in the three milks, respectively. Goat's milk contained the lowest numbers of coliforms and ewe's milk the highest numbers. Staphylococcus aureus could not be detected in goat's milk, whilst 16 and 26% of the ewe's and cow's milk samples contained 100 and 80 cells/ml, respectively. Out of 39 coagulase positive staphylococci, 27 were thermonuclease positive, 18 produced lecithinase and 15 fermented mannitol. Red blood cells of sheep origin were much more resistant to lysis by ewe's strains compared to bovine strains. Growth curves of Staphylococcus aureus were nearly linear at 17 degrees C but exponential at 31 degrees C without lag phase. Hazardous numbers of about 10(6) cells/ml were readily reached at 31 degrees C after 6 h and at 17 degrees C after 18 h. Salmonellae could not be detected in any of the samples examined. Out of 19 enterobacteria suspected to be salmonellae 11 proved to be Proteus and 8 Citrobacter.

Crude cell-free extracts of some strains of each L. casei and L. plantarum were assayed for their amino-, imino- and endopeptidase as well as the caseinolytic activity. L-alanine-, L-phenylalanine- and L-leucine-p-nitroanilide but not L-glutamic acid-p-nitroanilide, were hydrolyzed by all the strains indicating an amino-peptidase activity. The activity of proline iminopeptidase was very low compared to that of the aminopeptidase. L. casei could be distinguished from L. plantarum by its high endopeptidase activity against succinyl-phenylalanine- and glutaryl-phenylalanine-p-nitroanilide. The caseinolytic activity of cell-free extract of L. casei ATCC 393 was about one seventh the caseinolytic activity of intact cells, suggesting that the bulk of the cellular proteinase activity is located in the cell wall. It appears that a metallo aminopeptidase and a probable cysteine one are involved in the hydrolysis of amino acid-p-nitroanilide, whereas the endopeptidase activity appears to be related to a cysteine enzyme. Incubation of gels with L-leucine-p-nitroanilide after electrophoresis allowed the revealing of 2 zones of aminopeptidase activity in a strain of L. casei and only one in two others, but in L. plantarum it did not allow the revealing of any. The high proteolytic activities of L. casei compared to those of L. plantarum may explain its more frequent occurrence in cheese and its probable role in the ripening of many cheese varieties.

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