Influence of pH on the proteinase activity of thermophilic lactobacilli.

ESPECHE TURBAY, B.; SAVOY DE GIORI, G.; HEBERT, E.M.

San Miguel de Tucumán

Simposio; II SIMPOSIO INTERNACIONAL DE BACTERIAS LACTICAS; 2006

CERELA-CONICET

Lactobacillus (L.) helveticus and L. delbrueckii subsp lactis (L. lactis) are thermophilic lactic acid bacteria (LAB) used as starter cultures for the manufacture of hard cheeses. The proteolytic system of LAB is not only vital for growth in milk, but also contributes to the formation of flavor and texture of fermented products. The first step in the use of caseins is the degradation of these proteins to oligopeptides by an extracellular proteinase (Prt). Previously, we demonstrated that cell enveloped-associated proteinases (Prt) of L. helveticus CRL 1062 and L. lactis CRL 581 remained associated with the cells even after treatment with lysozyme. The aim of this study was to analyze the influence of pH in the proteinase stability of L. helveticus CRL 1062 and L. lactis CRL 581. Working cultures of lactobacilli were propagated in chemically defined medium until exponential growth phase, washed and resuspended in 0.85% saline solution. Proteinase activity was measured at optimum pH (pH 7) using the chromogenic substrate Succinyl-Ala-Ala-Pro-Phe-pNA and corroborated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using alpha- and beta-casein as substrates. The effect of pH in Prt stability was analyzed by incubating the enzyme at 40°C during 4h in a pH range between 4.5 and 8.0. The Prt activity of L. lactis CRL 581 was 10 times higher than that of L. helveticus CRL 1062. No extracellular caseinolytic activity was detected in culture supernatant or was released by washing CRL 1062 cells with a calcium-free buffer. The stability of this enzyme was higher than 90% in all pH assayed. L. helveticus CRL 1062 hydrolyzed both alpha- and beta-casein, alpha-casein being degradated more rapidly than beta-casein. The caseinolytic specificity was not affected by the pH of the medium. By contrast, L. lactis CRL 581 hydrolyzed mainly beta-casein and the enzyme was released to the extracellular medium at pH higher than 6.0. These differences in the caseinolytic activity and the stability at acid pH suggest that L. helveticus CRL 1062 and L. lactis CRL 581 could be used as starter cultures for the manufacture of fermented products. The released of CRL 581 Prt to the extracellular medium would allow their purification in order to use as additive in functional foods. Lactobacillus (L.) helveticus and L. delbrueckii subsp lactis (L. lactis) are thermophilic lactic acid bacteria (LAB) used as starter cultures for the manufacture of hard cheeses. The proteolytic system of LAB is not only vital for growth in milk, but also contributes to the formation of flavor and texture of fermented products. The first step in the use of caseins is the degradation of these proteins to oligopeptides by an extracellular proteinase (Prt). Previously, we demonstrated that cell enveloped-associated proteinases (Prt) of L. helveticus CRL 1062 and L. lactis CRL 581 remained associated with the cells even after treatment with lysozyme. The aim of this study was to analyze the influence of pH in the proteinase stability of L. helveticus CRL 1062 and L. lactis CRL 581. Working cultures of lactobacilli were propagated in chemically defined medium until exponential growth phase, washed and resuspended in 0.85% saline solution. Proteinase activity was measured at optimum pH (pH 7) using the chromogenic substrate Succinyl-Ala-Ala-Pro-Phe-pNA and corroborated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using alpha- and beta-casein as substrates. The effect of pH in Prt stability was analyzed by incubating the enzyme at 40°C during 4h in a pH range between 4.5 and 8.0. The Prt activity of L. lactis CRL 581 was 10 times higher than that of L. helveticus CRL 1062. No extracellular caseinolytic activity was detected in culture supernatant or was released by washing CRL 1062 cells with a calcium-free buffer. The stability of this enzyme was higher than 90% in all pH assayed. L. helveticus CRL 1062 hydrolyzed both alpha- and beta-casein, alpha-casein being degradated more rapidly than beta-casein. The caseinolytic specificity was not affected by the pH of the medium. By contrast, L. lactis CRL 581 hydrolyzed mainly beta-casein and the enzyme was released to the extracellular medium at pH higher than 6.0. These differences in the caseinolytic activity and the stability at acid pH suggest that L. helveticus CRL 1062 and L. lactis CRL 581 could be used as starter cultures for the manufacture of fermented products. The released of CRL 581 Prt to the extracellular medium would allow their purification in order to use as additive in functional foods.