Immunoproteomics of peptidases from Lactobacillus helveticus

Abstract

Milk proteins beyond their high nutritional quality are a source of peptides with biological activity. These bioactive peptides would exert different activities in vivo, affecting the cardiovascular, endocrine, immune, digestive or nervous systems. They can be released during food processing by enzymatic hydrolysis through the action of numerous proteolytic enzymes, naturally occurring in milk or provided by exogenous supply or microbial ecosystems. However, the nature and the origin of the bacterial enzymes and among them peptidases involved in the production of such bioactive peptides are not well established. Therefore, methods of detection have to be developed to monitor these peptidases directly in cheeses during ripening. Our aim was to produce and characterise monoclonal antibodies reacting against peptidases of Lactobacillus helveticus, a lactic acid bacterium highly proteolytic, used in fermented milk products as producers of bioactive peptides. We used a strategy of pre-enrichment in intracellular peptidases to be able to produce various antibodies against peptidases from the same pool of enzymes. Two fractions enriched in peptidases, PepLHA and PepLHB were obtained from the cell free extract of Lactobacillus helveticus LH1 (FTL122, UR342 collection) and were used to immunise two sets of mice. Fusion PepLHA produced 53 hybrid clones and fusion PepLHB produced 202 hybrid clones both giving a positive reaction against fraction A and/or B. These clones were classified into 8 and 11 different families for PepLHA and PepLHB respectively. The antibodies corresponding to each family were characterised according to two methodologies: affinity columns for PepLHA, and western blot on 2D electrophoresis gels for PepLHB, and analyses by nano LC coupled on line with ESI-QTOF MS/MS. We have then produced monoclonal antibodies that react against interesting peptidases, i.e. PepN, PepC, PepX and PepQ that will be used in ELISA test