Peptides derived from alpha s1- and beta-caseins by the Lactobacillus helveticus CP790 proteinase were investigated for their inhibitory activities against angiotensin I-converting enzyme. The antihypertensive effect of casein hydrolysates in strain SHR spontaneously hypertensive rats was also investigated. Both alpha s1- and beta-casein hydrolysates inhibited this enzyme. Some of these peptides showed enzyme inhibitory activity, and one of them from beta-casein inhibited the enzyme greatly; the concentration of an angiotensin I-converting enzyme inhibitor needed to inhibit 50% of the enzyme activity was 4 microM. The hydrolysate of casein demonstrated antihypertensive activity in spontaneously hypertensive rats at an orally administered dosage of 15 mg/kg of body weight. MILK fermented with L. helveticus CP790, containing about .3% peptides, also showed antihypertensive activity in SHR rats with 5 ml/kg of body weight (15 mg of peptide/kg); however, the milk fermented with L. helveticus CP791, a variant defective for proteinase activity, did not show this activity. Results suggested that the peptides liberated from casein by the proteinase in the culture medium showed antihypertensive effect in SHR rats.

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A cell-wall-associated proteinase from Lactobacillus helveticus CP790, grown in skim milk, was purified to homogeneity by DEAE ion exchange chromatography in the presence of EDTA. Its molecular weight was estimated to be 45,000 by SDS-PAGE and also by the recovery of proteinase activity only from this fraction of SDS-PAGE gel slices of crude extract. It had maximum activity at pH 6.5 and 42 degrees C. Since the activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), it was suggested to be a serine-type proteinase. It preferentially hydrolyzed alpha- and beta-casein but did not hydrolyze kappa-casein by the purified enzyme. The 10 main peptides liberated from alpha-casein, and the 15 peptides liberated from beta-casein, were isolated by reversed phase HPLC. These peptides were hydrolyzed by HCl and their amino acid compositions were analyzed and identified. Many of peptides were located in the C-terminal parts of alpha- and beta-casein. Peptide bonds cleaved by the proteinase had no clear specificity but Leu-X, Phe-X, Ser-X, Lys-X, Glu-X, and Gln-X sequences frequently appeared.

Monoclonal antibodies against a cell wall-associated 45-kDa proteinase from Lactobacillus helveticus CP790 were prepared and used for an immunoblotting analysis of the cell wall extract of CP790. They were found to react with an unidentified 46-kDa protein as well as the 45-kDa proteinase. The 46-kDa protein was copurified with the 45-kDa proteinase by affinity column chromatography using antibody-fixed Sepharose and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted from the gels. An elution profile of the cyanogen bromide digest of the purified 46-kDa protein obtained by reversed-phase high-performance liquid chromatography was identical to that of the 45-kDa proteinase except for one peak. An analysis of the N-terminal 21-amino-acid sequence revealed that the 46-kDa protein possesses an extra 7 amino acids at the N terminus of the 45-kDa proteinase. The 46-kDa protein was produced at constant levels during fermentation in a skim milk medium, while the 45-kDa protein was mainly observed in the middle of the exponential phase of growth and was produced in proportion to the proteinase activity. Moreover, only the 46-kDa protein was detected in the crude extract of L. helveticus CP791, a variant strain of CP790 defective in proteinase activity. These data strongly suggest that the 46-kDa protein is a precursor, inactive form of the 45-kDa proteinase.