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Productions scientifiques Micropolluants

Unveiling the cell wall-anchored peptidase activities in Streptococcus thermophilus strains

18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France

Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Dary, A., Miclo, L.

2012

In the last few decades, increased consumers awareness about the health benefits of food products has encouraged the development of various health promoting functional foods. These foods not only contribute to the nutritional requirements but also prevent certain diseases, in order to improve the physical and mental well-being of consumers. In some cases, a bioactive peptide of food origin constitutes the active component of these products. Milk proteins are known to be a major source of bioactive peptides that may impart extra-nutritional benefits. These peptides are encrypted in the sequence of caseins and are released only after protein proteolysis in vitro or by gastric or pancreatic enzymes during the digestion of proteins in vivo or by the enzymes of lactic acid bacteria during manufacture of fermented dairy products. S. thermophilus, that possesses its own proteolytic system, is one of the most widely used lactic acid bacteria in the manufacture of fermented dairy products (yoghurt, cheese). The proteolytic system of S. thermophilus is comprised of cell surface proteinase, peptide transport system and a pool of intracellular peptidases. Thus, the question arises whether bioactive peptides added in the fermented dairy products could remain intact or not in presence of this bacterium. To answer this question, two S. thermophilus strains differing in their cell surface proteolytic activity were evaluated: strain LMD9 which possesses cell envelope proteinase activity (PrtS+) and strain CNRZ1066 without this activity (PrtS-). We studied the resistance of some milk protein derived peptides with different biological activities to S. thermophilus cell wall proteolytic system. We then incubated the peptides, namely anxiolytic [YLGYLEQ - alpha-s1-CN-(f91-97)], antihypertensive [TTMPLW - alpha-s1-CN-(f194-199), FALPQYLK - alpha-s2-CN-(f174-181)] and opioid peptide (YPFPGPI - beta-casomorphin-7, RYLGYLE - alpha-s1-CN-(f90-96)] with the non proliferating cells of LMD-9 and CNRZ1066 strains in phosphate / acetate buffer (12.5 mM, pH 6.5). Surprisingly, two different cell wall-anchored peptidase activities were highlighted. The peptides YLGYLEQ, TTMPLW, RYLGYLE, and FALPQYLK were hydrolyzed by both the strains in a similar way. Liquid chromatography coupled to mass spectrometry analysis revealed that breakdown fragments were generated after successive cleavages of amino acids from the N-terminal of these peptides which provides an evidence of an aminopeptidase activity. However, in case of beta-casomorphin-7, an X-prolyl dipeptidyl aminopeptidase activity was observed which is involved in removal of N-terminal dipeptides containing N-terminal prolyl residue. All the peptides remained stable upon incubation in cell free filtrate of these strains which showed that no intracellular peptidases were released and thus involved in the proteolysis process. Therefore, these peptidase activities may help in optimal bacterial growth which consequently increases the milk fermentation rate but at the same time may also cause degradation of bioactive peptides present or added in the fermented milk products. Hence, screening a strain of S. thermophilus having reduced cell wall proteolytic activity would be required for the production of functional fermented dairy products.

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