Les protéases de paroi de <em>Lactobacillus helveticus</em> : Caractérisation moléculaire, biochimique et expression in situ dans une matrice fromagère

Lactobacillus helveticus is used in various dairy products, such as fermented milks and cheese. The higher proteolytic activity of this lactic acid bacterium contributes to the development of texture and flavour and the generation of peptides with biological or technological properties. Cell envelope proteinases (CEPs), located at the bacterial surface, are responsible for the initial hydrolysis of the major milk proteins, i.e. caseins. In contrast to other lactic acid bacteria, L. helveticus possesses two non-homologous genes of CEPs, named prtH and prtH2. However, the CEP mechanism of action, their role in the proteolysis in situ and the generation of functional properties like stretchability of emmental cheese, are still unknown. The aim of this work was to deepen knowledge on the CEPs of L. helveticus: i) their gene distribution; ii) their activity and specificity on caseins and iii) their expression in cheese matrix. The prtH2 gene was ubiquitous in the panel of 29 strains studied while the prtH gene was present in only 18/29 strains. When they are present, both genes prtH and prtH2 were expressed in different media: MRS broth culture, milk and emmental type cheese. CEP activity was determined by using synthetic substrate for 15 strains and varied by a factor 14 between strains, without significant differences between groups which possess one or two CEPs. In terms of activity and specificity on caseins, a clear distinction between strains was only found on a two regions of pure s1 casein which was not hydrolyzed by strains having only one proteinase i.e. PrtH2. Two strains, having either PrtH2 or PrtH and PrtH2, were chosen for manufacturing Emmental–type cheese. The degree of proteolysis was 1.5 times lower in cheese manufactured using the strain having one CEP (PrtH2) and a low level of autolysis. In this cheese, the kinetics of hydrolysis of s1 casein was lower from the 1st to the 20th day of ripening, in agreement with the results observed on purified s1 casein. In parallel, the stretchability of Emmental cheese was measured. Size of the strands was higher in cheese with lower proteolysis. Stretchability was statistically correlated with the presence of hydrophobic peptides. Among these peptides, a high number of peptides derived from s1 casein were identified using mass spectrometry. This study showed a biodiversity of CEPs at genetic level and at biochemical level, only on purified s1 casein. In cheese, the variability in proteolytic activity of strains led to the variability of stretchability properties of Emmental cheese. This variability in proteolysis in situ resulted from the combined action of CEPs and intracellular peptidase released. Only the use of deficient mutants in one or two CEPs would permit to determine the role of CEPs in cheese proteolysis and to use CEPs of L. helveticus as a technological lever in the generation of targeted functional properties.