| Since probiotic strains are used in the food industry its security problem has been attended. Especially today, as more and more food safety problems appeared, people have deep comprehension on food security. Scientists from various countries also focus on addition of probiotic to foods, and more research is gradually increasing. In the study of probiotic safety the basic research is essential, and only in this way can the safety of probiotic food be ensured.In this study, three main lactic acid bacteria strains, as follows, Lactobacillus, Bifidobac- terium, and Streptococcus thermophilus strains the laboratory saved and the isolates of yogurt were studied, and analyzed the biological classification and identification, 16SrRNA gene PCR amplification and sequence analysis, and resistance to antibiotics, and PFGE typing, and adhesion on Hela cells in vitro in order to provide a reliable theoretical basis of tracing to the source, and monitoring, and analyzing, while consolidating all results of probiotic bacteria to establish a database to better monitor the use of lactic acid bacteria in food safety.Firstly, the laboratory storage isolates: 55 Lactobacillus strains, 46 Bifidobacterium strains, and 10 Streptococcus thermophilus strains, and 57 Lactobacillus isolates, 10 Bifidobacterium isolates, 42 Streptococcus thermophilus isolates were identified with API50CHL, and we analyzed their biochemical spectrum by clustering. The results showed that:The results of biochemical identification of the storage strains did not exactly match with the one of the given name of the manufacturers. Biochemical identification of isolates were not entirely consistent with the names marked on the bags. Detection rate of Bifidobacterium isolates of samples was lower. The typing results of strains showed that the same or similar biochemical spectrum is divided into a class of bacteria, and the biochemical spectrum of Lactobacillus strains and Bifidobacterium strains were more complex because of they had many species. The rest were relatively simple. Biological classification can not be accurately classified.Secondly, different primers of the strains were designed, and then all the strains were identified and classified by 16SrRNA gene PCR amplification and sequence analysis. The results showed that:16SrRNA identification results of the storage strains are not entirely consistent with the results of API50CHL identification. Both methods have the same results of isolates. All Streptococcus thermophilus strains have the same identification results from the two ways. From the whole, the specificity of the primers was good, and these primers were available to identify species. The identification method can significantly save the cost. 16SrRNA gene sequence typing results were not ideal.Thirdly, Lactobacillus, Bifidobacterium and Streptococcus thermophilus storage strains and yoghurt isolates were genotyped with PFGE, and the results showed that:AscI and SmaI were selected as the major restriction enzyme of Lactobacillus genus, and XbaI was the major restriction enzyme of Bifidobacterium genus, SmaI was the major restriction enzyme of Streptococcus thermophilus. The PFGE genotyping methods of these strains were established. The homology relationship of the storage strains and isolates was analyzed according to the profile of genetic relationship. Lactic acid bacteria strains were typed by PFGE molecular typing method at the gene leve, and we could determine the accuracy of other identification methods of probiotics according to the PFGE method.Comparing with the several identification methods, API50CHL biochemical identifica- tion results may cause errors which should not exist. The method of 16SrRNA gene PCR amplification was relatively stable. And this method could identify the strains at spieces level. PFGE type analysis can confirm the accuracy of identification results.Fourth, the drug resistance of the all the strains was detected by broth microdilution method according to the manual of CLSI. The results showed that:The Lactobacillus storage strains to 16 antibiotics all showed strong resistance, and most of the strains were multiple drug-resistant strains, and the resistance to isolates was slightly lower, but most of the strains also showed multiple drug resistance. The storage strains showed wider resistance range. Bifidobacterium storage strains were mostly resistant to 2-3 antibiotics, a few more strains were resistant to more than 3 antibiotics; 3 of 10 isolates showed multiple drug resistance. Resistance range of the storage strains were wider. 2 of 10 storage Streptococcus thermophilus strains showed no resistance, and the remaining were resistant to 1-6 antibiotics; Most isolates were resistant to 2-3 antibiotics. The range of their resistance showed no significant difference. Among the three species, the Lactobacillus strains showed the strongest resistance, and followed by the Streptococcus thermophilus strains, and the Bifidobacterium strains was the last one.Fifth, adhesion assay in vitro was selected, and cervical cancer Hela cells were selected as cell model, and Lactobacillus, Bifidobacterium and Streptococcus thermophilus strains was selected as beneficial bacteria, and Salmonella typhimurium AMCC50333 was selected as pathogens, then adhesion of the bacteria to Hela was analyzed, and we analyzed the effect on adhesion of these beneficial bacteria to AMCC50333. The results showed:All the strains showed adhesion properties to Hela cells, but the difference is not obvious. Strains with different species in one genus showed different adhesion. Three spieces of bacteria storage strains and isolates all could significantly inhibit the Salmonella typhimurium adhering to Hela. However, there was no significant difference. The strains which were identified as Lactococcus lactis showed strong adhesion to Hela cell, and also showed strong inhibition of Salmonella typhimurium adhering to Hela.In a whole, PFGE molecular typing method is the best typing methods, followed by 16S rRNA sequence analysis, and biochemical typing. Through a series of experiments, this study established a database of lactic acid bacteria strains Chinese food industry commonly used. |
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