Exopolysaccharide (EPS)-producing Bifidobacterium aesculapii: screening for the presence of rfb_P gene and EPS production

Nowadays, health awareness among consumers has increased the demand for low/fat-free, but at the same time, smooth and creamy dairy products. Thus, since milk fat contributes to flavour, body and texture development of the dairy products, with its removal leading to textural and functional defects and EPSs production by food grade LAB have gained importance as biothickeners and texturizers. During the technological food process/storage, EPSs guarantee the microorganism survival/viability and better rheological properties. Furthermore, EPSs have beneficial effects on health, are involved in adhesion mechanisms, control of pathogens and induction of positive physiological response. Searching for EPSs-producing bacteria is of interest in screening of new strains as probiotic in low-fat and free-dairy foods. Several Bifidobacterium strains are able to produce EPSs, but mechanism of biosynthesis is only hypothesized; however, the priming-glycosyltransferase rfb_P seems the key enzyme in the EPS-units biosynthesis. In our work, a degenerated primer pair was designed on available rfb_P partial gene sequences of Bifidobacterium spp., which were were aligned with ClustalW. Gemi program was used for primers design. After a manual editing, PCR primers specificity was checked against the bacterial genome database using MFE-primer 2.0. Selected primers were tested in vivo: 7 Bifidobacterium aesculapii strains, MRM_3.1T, MRM_4.2, MRM_4.6, MRM_4.7, MRM_4.8, MRM_5.13, MRM_8.7, which produce a gelling exocellular matrix, were tested; Bifidobacterium longum subsp. infantis ATCC15697T and Bifidobacterium saguini DSM23967T were used as positive and negative controls, respectively. The expected amplicon products were electroforetically verified. To correlate the presence of amplicon with the metabolic production, EPSs were extracted and quantified from each strain with the method by Dubois et al. (1956). In silico and in vivo amplifications were performed to test the specificity of our designed degenerated primer pair and results, supported the reliability of our set for a rapid screening of EPS-producing Bifidobacterium spp. Furthermore, quantification analysis… have also highlighted..