Proteinase activity response in Lactobacillus delbrueckii subsp. bulgaricus CRL 656 under different stress factors

PESCUMA, M.; HEBERT, E.M.; MOZZI, F.; FONT DE VALDEZ, G.

Simposio; IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications.; 2013

Lactic acid bacteria (LAB) play an important role in food fermentation by reducing pH through organic acid production and releasing small peptides and amino acids, among other features. In LAB, the cell wall-associated proteinase (CEP) is the main enzyme responsible for protein degradation. It has been established that CEP expression is repressed when LAB grow in peptide rich-media. Whey, the main by-product of cheese manufacture, is widely used as additive in several food products mainly because its high nutritional value. However, the major whey protein B-lactoglobulin (BLG) is highly resistant to hydrolysis by digestive and non-digestive enzymes. Interestingly, this protein was efficiently degraded by the L. delbrueckii subsp. bulgaricus CRL 656 CEP when the strain was grown in a chemically defined medium (CDM), free of peptides. In contrast, when the strain was grown in a medium containing peptides such as whey, BLG was scarcely hydrolyzed. It has been postulated that certain stress growth conditions (i.e. high osmolarity, high temperature) can de-repress several proteolytic enzymes. The aim of this work was to evaluate the CEP activity of L. delbrueckii subsp. bulgaricus CRL 656 grown in CDM, CDM supplemented with whey protein hydrolyzate (CDMB), and in CDMB submitted to osmotic or heat shock. The strain was grown in CDM or CDMB at 40°C and samples collected every hour were analyzed for growth (OD560), pH and proteolytic activity (using S-Ala as chromogenic substrate or by quantifying whey protein degradation by SDS-PAGE). Osmotic stress was applied by adding 0.6 M NaCl to cells in log growth phase while heat stress was induced by incubating the cells at 55°C during the first 30 min of the assay. L. delbrueckii subsp. bulgaricus CRL 656 showed higher cell growth rate (0.24 times) in CDMB than in CDM; a decrease in culture pH to a value of 5.0 was observed at 6 h of incubation while the same pH value was attained after 24 h in CDM. In contrast, CEP activity was repressed between 20 and 45 times in CDMB respect to CDM. When osmotic stress was applied, cell growth and CEP activity diminished (8.7 and 3.6-6.1 times, respectively). When cells were subjected to heat stress, cell growth rate was higher (1.1 times) than that observed without stress; interestingly, CEP activity increased 1.5 times. These results showed that peptides present in whey protein hydrolyzate effectively repressed CEP expression in L. delbrueckii subsp. bulgaricus CRL 656 while high temperature stress showed a positive effect on CEP derepression. The mechanisms of CEP expression in L. delbrueckii subsp. bulgaricus CRL 656 remain still to be elucidated.