Lactobacillus casei group: identification, characterization and genetic evaluation of the stress response

Lactic acid bacteria (LAB) are an heterogeneous group of microorganisms used as starter and/or adjunctive cultures in the production of fermented foods to improve the shelf-life, the organoleptic properties and promote health. Many of these species, in fact, have been extensively characterized as probiotics several studies and clinical tests to substantiate health claims are available for several of them. During use, formulation and preservation as starter, adjunctive and/or probiotic cultures LAB are subjected to physical and chemical stresses that can influence growth, cell viability and fermentation capabilities, changing the technological fitness of the strains. This thesis focused on Lactobacillus casei, L. rhamnosus and L. paracasei with the aim to encrease the knowledge on this strains. These microbial species are involved in the fermentation of various food matrices (meat, vegetable and dairy products) and they could be used as probiotics or for the development of new functional products, as many studies reported the presence of several strains belonging to these species with probiotic features. Considering the hardness existing in the correct identification of these strains, so genetically close to each other, a wide collection of strains isolated from several sources and previously identified as belonging to L. casei group was collected (201 total strains), and a species-specific PCR, multiplex PCR, group-specific PCR and HRM analyses have been performed or developed to identify L. casei, L. rhamnosus and L. paracasei strains. Almost all the techniques resulted effective for the identification, but the group-specific PCR had to be applied as the first step of identification. Anyway, the attribution to one of the species of the group remained uncertain for some strains. Therefore, all the strains were characterized using three different assays (Rep-PCR, RAPD-PCR and Sau-PCR), in order to have a complete point of view of their genotype. As a result, a high variability was found among strains with all the three performed assays, confirming the presence of a high number of genotipes. Therafter 45 strains were selected for the development of an MLST scheme based on the analysis of internal fragments (loci) of stress related genes. The results confirmed the presence of a high variability among strains and it was not surprising to found strains with different STs. Considering that the analysis was performed on 45 strains, and the high number of STs that has been found, it would be interesting to charachterize other strains, in order to find possible correlation between the source of isolation or a specific stress response behaviour. Four-teen strains were selected for the whole genome sequencing and core-genome and pan-genome comparisons were performed, obtaining a clear distinction among L. paracasei and L. rhamnosus strains. Nonetheless, a L. paracasei and two L. casei strains grouped in L. rhamnosus cluster, confirming how hard is the discrimination among these species, because of their high level of similarity. SAPD-PCR analysis were also performed, confirming a high genomic variability among strains. The same strains where then subjected to the evaluation of their mucus binding capabilities. The binding assays were performed in vitro and the strains were tested before and after oxidative stress exposure. The behaviour of the microorganisms analysed resulted strain-dependent and analysis allowed the selection of few strains with interesting binding properties, that will be further analysed for their probiotic characteristics before to do in vivo analysis and to understand their adhesion mechanisms. At least, a strain of L. paracasei was selected for its ability to conduct malolactic fermentation under different EtOH and pH conditions studing how the expression of stress related genes was affected in the different tested conditions. The strain resulted able to performe MLF under all the tested conditions, and as expected, the lower concentration of glucose, corresponding to the lower final concentration of alcohol, demonstrated to be less stressful. Considering that the strain was able to conduct MLF also when in the presence of a concentration of glucose, corresponding to 15% v/v of ethanol, it can be considered as a potential good MLF starter culture. Another topic for the future research could be the evaluation of the stress response of this strain during MLF when adapted in a respiratory envinronment before the inoculation