| Bifidobacterium animalis subsp. lactis V9 is a new potential probiotic bacteria screened out from thirty-one strains of bifidobacteria which were isolated from the stools of twelve healthy Mongolian children. For further food industrial application, the safety properties of B. animalis subsp. lactis V9, including antibiotic resistance, plasmids and antibiotic related genes were assessed, as well as its toxicological safety. Moreover, the physiological effection of B. animalis subsp. lactis V9 on the patients with diarrhoea and constipation problems were determined. Meanwhile, the whole genome sequence of B. animalis subsp. lactis V9 was sequenced to explain its probiotic properties from genetic level.The antibiotic resistance of B. animalis subsp. lactis V9 to twelve kinds of antibiotics were tested by evaluating the Minimum Inhibitory Concentrations of specific antibiotics. B. animalis subsp. lactis V9 could highly resistant to gentamicin, kanamycin and polymycin B. To nenomycin, ciprofloxacin and streptomycin, it showed a mild resistance. Moreover, it also can resistant to tetracycline and chloramphenicol. However, B. animalis subsp. lactis V9 can not tolerate to vanacomycin, penicillin G, rifampicin and erythromycin. In compared with commercial probiotic B. animalis subsp. lactis BB12, B. animalis subsp. lactis V9 showed a lower antibiotic resistance. No plasmid was detected in B. animalis subsp. lactis V9, and only rpp (tetracycline resistance related genes) and gyr A (fluoroquinolone resistance related genes) were detected out from its chromosome. Because these related genes were located in the chromosome, there is no risk for pathogenic bacteria transfer. So, B. animalis subsp. lactis V9 was safe in its antibiotic resistant properties.To evaluate the toxicological safety of probiotic B. animalis subsp. lactis V9, different dose of B. animalis subsp. lactis V9 freeze-dried powder (0.5, 5 and 15 g/kg?bw) was given to mice by gavage for fourteen days. The related indexes were measured, including weight, growing conditions, blood cell component, liver function, organ index, and the conditions of lipidmetabolism at different time (3, 7 and 14 days). Meanwhile, the liver and kidney were examined by microscope. The results showed that the sub-lethaldose (LD50) of B. animalis subsp. lactis V9 to mice was 15 g/kg?bw (about 2.25×1012 cfu/kg?bw). There was no intoxicating phenomenon and negative influence appeared after fourteen days feeding. There is no significant difference in weight change of mice in compared with control group (P>0.05), as well as the related indicators like routine analysis of blood, liver function, viscera index, serum lipids and so on. Through the gram staining microscopic examination, there is no abnormal changes in mice's liver and kidney. The results of acute toxicity analysis and related indicators showed that B. animalis subsp. lactis V9 freeze-dried powder do not have side-effects.The physiological effection of B. animalis subsp. lactis V9 to the patients with contipation and diarrhoea problems were also determined. B. animalis subsp. lactis V9 freeze-dried powder was given to one hundred and fifty-three patients with constipation, acute diarrhea and/or chronic diarrhea problems with the dose as 1.0×1010 cfu/d for three weeks. The defecation frequencies and fecal properties of patients were recorded every three days and intestinal microflora was evaluated before and after the treatment. The results showed that after treatment for three weeks, the effective rates of B. animalis subsp. lactis V9 on patients with constipation, acute diarrhea and chronic diarrhea were 95.3%, 95.4% and 89.9%, respectively. The number of bifidobacteria in facals increased significantly in compared with that of three weeks before. Meanwhile, the number of colibacillus, enterococcus, bacteroid and clostridium also changed in different degree and closed to healthy human. Clinical research showed that B. animalis subsp. lactis V9 can regulate the intestinal microbial communities, and can express remarkable effects on the patients with constipation and/or diarrhea problems.To understand the genetic basis of its probiotic properties, whole-genome sequencing of B. animalis subsp. lactis V9 was performed with a combined strategy of 454 sequencing and Solexa paired-end sequencing technology. The complete genome sequence of B. animalis subsp. lactis V9 contains a circular 1,944,050 bp chromosome with a GC content of 60.5%. Its genome contains 1,636 ORF, including 1,572 coding genes, four rRNA operons, and 52 tRNAs. When compared its whole genome with other published genomes of B. animalis subsp. lactis strains, there was a high homology which means the heredity of this subspecies is very stable, and the ratio of recombination and mutation during culture and storage is very small. Moreover, the reasons of bifidobacteria possess a higher HMOs metabolism capability and continuous growth ability under micro-anaerobic environment are explained from genetic level.
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