BIOACTIVES IN MILK-DERIVED PRODUCTS: BACTERIAL PRODUCTION OF IMMUNOMODULATORY CASEIN HYDROLYSATES AND TOOLS FOR IDENTIFICATION OF IMMUNOGENIC BACTERIAL PROTEIN

During hydrolysis of bovine milk caseins, cell envelope-associated proteinases (CEPs) of lactic acid bacteria (LAB) may be able to produce hydrolysates that possess in vitro immunomodulatory activity. We studied the proteolytic activity against bovine milk caseins exerted by specific strains of LAB selected on the basis of a preliminary genomic screening for the presence of CEPs. The tested strains demonstrated diverse hydrolytic ability against different casein fractions, alphaS1- and beta-casein in particular. The 3 kDa-ultrafiltered casein hydrolysates (CHs), produced after digestion with proteinases of Lactobacillus acidophilus ATCC 4356 and Lactococcus lactis subsp. lactis GR5 demonstrated immunomodulatory activity in vitro by significantly decreasing the basal NF-kB activity in recombinant Caco-2 cell layers. Both the strains digested beta-casein mostly. However, in the case of Lactobacillus helveticus MIMLh5, the investigation proved that the immunomodulatory activity of obtained CH was comparable to the one exerted by bacteria themselves (in the absence of caseinate) and therefore, was not due to the presence of casein-derived peptides. Indeed, the surface layer (S-layer) protein of L. helveticus MIMLh5 was identified as a molecule responsible for the immunomodulation. Overall, these results emphasize the importance of this bacterial cell-derived protein while evaluating the immunological activity of CHs, and it underlines the necessity to remove such molecule in order to properly assess the bioactivity of CHs deriving from the proteolytic activity of LAB. The second part of the research was focussed on the selection of S-layer-specific single-chain variable fragment antibodies (scFvs), a new powerful tool for the study of the S-layer of L. helveticus MIMLh5, the immunologically active protein, and for its identification in milk-derived products containing L. helveticus as a starter or non starter LAB. In this study, a mix of two human synthetic phage displayed libraries (protein-directed and hapten-directed) was used to select scFvs against L. helveticus MIMLh5 S-layer protein. After three rounds of panning, four monoclonal scFv binders capable of binding to the L. helveticus MIMLh5 S-layer protein and one capable of binding not only to the mentioned protein, but also to the S-layer protein of L. helveticus ATCC 15009, which is different only in five amino acids, were obtained. All five identified novel anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their basic characterisation was performed. Anti-S-layer-scFv PolyH4 was used to develop an assay, based on Western blot analysis, for detection of the S-layer protein in Grana Padano samples. These results showed promising applications of the method for the detection of the S-layer protein in food matrices.