Peptide Hydrolases of Lactobacillus helveticus and Lactobacillus delbrueckii ssp. bulgaricus

Lactobacillus helveticus CNRZ 32 had more aminopeptidase and dipeptidase activity than did Lactobacillus helveticus ATCC 10797 or Lactobacillus delbrueckii ssp. bulgaricus ATCC 12278, whereas L. helveticus ATCC 10797 was most proteolytic (100%), followed closely by L. helveticus CNRZ 32 (92%), and L. delbrueckii ssp. bulgaricus ATCC 12278 (50%). Crude cell-free extracts of the three lactobacilli lacked carboxypeptidase and endopeptidase activities. Lactobacillus helveticus CNRZ 32 had two aminopeptidase-active bands, whereas L. helveticus ATCC 10797 and L. delbrueckii ssp. bulgaricus ATCC 12278 each had only one active aminopeptidase band; relative mobility values of bands differed among strains. The three lactobacilli each had two active dipeptidase bands with different relative mobilities. Intracellular protease, aminopeptidase, and dipeptidase activities of L. helveticus CNRZ 32 increased gradually at the beginning of the log phase and reached a maximum at the beginning of the stationary phase, but after 24h, enzyme activity was slightly reduced. Freezing cells at –96°C and holding them at that temperature for 1 wk reduced aminopeptidase and dipeptidase activities 10% compared with those of unfrozen cells. The freezing process also reduced the population of viable cells grown in MRS broth or skim milk by 50 or 75%, respectively. Aminopeptidase activity of cells grown in sterile skim milk or a whey-based medium did not vary markedly from that of cells grown in MRS broth, but dipeptidase activity increased 1.5-fold for cells grown in the whey-based medium. Proteolysis of skim milk by whole cells required up to 145h, whereas only 54h was needed by cell-free extracts. Extracts of L. helveticus ATCC 10797 were most proteolytic, and completely hydrolyzed αs1-, β- and κ-caseins without preference, whereas an extract of L. delbrueckii ssp. bulgaricus ATCC 12278 hydrolyzed β- and κ-caseins completely, and partially hydrolyzed αs1-casein. Extracts of L. helveticus CNRZ 32 partially hydrolyzed β-casein, while αs1-casein resisted hydrolysis. The pH of milk at the end of proteolysis was below 4.0 for L. helveticus CNRZ 32 and L. helveticus ATCC 10797, but above 4.0 for L. delbrueckii ssp. bulgaricus ATCC 12278.