Proteinases of [i]Lactobacillus helveticus[/i] and their distinct capability to hydrolyze αs1 and -β-caseins at different NaCl concentrations

To control the organoleptic and techno-functional properties of dairy products requires to better understand how the lactic acid bacteria, as main actors involved in proteolysis can be influenced by micro-environment during processing, notably salting. In Swiss-type cheeses, salting occurs after the starter bacteria, streptococci and lactobacilli (Lactobacillus helveticus), have finished growing. Thus, salt can affect their survival during ripening and modulate the activity of their cell-wall proteinases (CEPs), which hydrolyze casein to supply the nitrogen requirements of the cells. The aim of this study was to evaluate how NaCl modulate the capability of L. helveticus CEPs to degrade s1 and -caseins. Five strains of L. helveticus were chosen, harboring one (PrtH3 or PrtH4) to four CEPs (PrtH, PrtH2, PrtH3 and PrtH4) or no CEP as a negative control. Resting cells from milk cultures of L. helveticus were incubated in the presence of 0.3mg/ml s1 or -casein, at NaCl concentrations ranging from 0% to 4.5% NaCl/moisture, at pH 5.2, corresponding to pH of the cheese before ripening, and pH 7.5, used as a reference. Casein degradation was monitored by Tris-tricine SDS PAGE. s1-and -Casein hydrolysis varied up to 100-fold depending on the strains, with a markedly higher hydrolysis for strains having the four CEPs or PrtH3 alone. As an example at pH 5.2 and 1.5% NaCl, activity of CEPs was 3.1% s1-casein degraded/min and 5.7% -casein degraded/min for strain having the four CEPs and 6.1% s1-casein degraded/min and 6.3% -casein degraded/min for PrtH3 alone while for the other strains CEP activity was ranging between 0.1-0.4% s1-casein degraded/min and 0.1-0.7% -casein degraded/min. A higher hydrolysis of both caseins was observed at pH 5.2 compared to 7.5. Strains reacted differently according to the change in NaCl concentrations. A marked decrease in CEP activity was observed for NaCl concentrations above or equal to 3%. This decrease was dependent on the strain as well as the type of casein. The profile of large peptides produced during hydrolysis differed according to the pH and NaCl concentrations suggesting either a different substrate conformation leading to change in accessibility to the CEP, or a change in CEP selectivity. Moreover, bacterial cells exhibited different rate of lysis according to the NaCl content. High salt concentrations above or equal to 3% led to an earlier lysis, regardless of pH values, in particular for the strains which are constitutively prone to lysis. This study suggests that among the various equipment in CEPs of L. helveticus strains, those harboring at least PrtH3 have higher proteolytic activity on both s1-and -caseins. These strains have CEP activity that decreases only at high level of salt and that is distinct between both caseins. The consequences of these different capabilities to degrade caseins and to lyse will be assessed in Swiss-type cheeses under three levels of NaCl concentrations.