Lactobacilli have a worldwide industrial use as starters in the manufacturing of fermented dairy products. Moreover, some Lactobacillus strains have probiotic characteristics leading to increasing the use of lactobacilli in fermented food products. Increasing of probiotic lactobacilli in food products and nutritional supplements underscores the need to evaluate and correctly identify these useful bacteria. The aims of this study were to evaluate putative probiotic lactobacilli isolated from different sources as fermented milk products from Egypt and faeces from two new born children (twins) for establishment of probiotic strains, to clone and transform a bile salt hydrolase gene for genetic improvement of Lactobacillus strains to act as probiotics. Chapter I of the present study deals with for identification of LAB using molecular methods, like e.g. ARDRA, PFGE, and sequencing of the 16S rRNA gene, to achieve accurate identification for selection of probiotics. The objectives of chapter II were evaluation and characterization of 26 Lactobacillus strains through probiotic selection criteria (acid tolerance, bile salts resistance and antimicrobial activity). Also, a safety assessment of some Lactobacillus isolates was done. We report a survey of susceptibility of 26 Lactobacillus against 13 antibiotics including inhibitors of cell wall synthesis, protein and nucleic acid synthesis. Resistance to bile salts is one of the important criteria for selection and establishment of probiotic strains with good health benefits. Most of Lactobacillus strains showed some resistance to bile salts. In chapter III, we describe that conjugated bile salt hydrolase gene (cbh) is responsible for de-conjugation of bile acids. The cbh-gene and its regulatory sequences (promoter, ribosomal-binding site and terminator) from highly bile salts resistance Lactobacillus plantarum BfEL 92122 strain were amplified by PCR, cloned into E. coli vectors. The cbh was so strongly expressed in E. coli that transformants could be identified directly on indicator plates containing bile salts as a substrate, indicating that the cbh gene might be generally useable as a food-grade indicator gene. Cbh was recloned into several E. coli/Lactobacillus shuttle vectors. Electrotransformation of L. gasseri, L. casei and L. rahmnosus strains with these recombinant plasmids and expression of cbh gene significantly improved resistance against ox-gall, glycodeoxycholic- and taurodeoxycholic acid. For probiotic bacteria to survive in the product and after consumption in the consumer’s gastrointestinal tract, inhibitory effect of bacteriophages and autolysis during industrial fermentation should be also considered. Therefore, chapter IV focused on lysogeny and autolysis of lactobacilli caused by bacteriophages or their enzymes and the characterization of a bacteriophage, phi Lbgas1.from L. gasseri. The establishment of a prophage cured derivative of this strain is reported. Finally, we purpose for completion of characterization prospective in vivo studies on survival of Lactobacillus strain in animal models (like e.g. Gottingen minipigs). These studies may then form the basis for clinical evaluation the potential of the selected strains. health properties |
