我国中高纬度地区冷水鱼肠道乳酸菌多样性、系统发育及优势种群Enterococcus的遗传多态性研究

Bacteriocins are ribosomally-synthesized peptides or proteins with antimicrobial activity, produced by different groups of bacteria. Many lactic acid bacteria (LAB) produce bacteriocins with rather broad spectra of inhibition. Several LAB bacteriocins offer potential applications in food preservation, and the use of bacteriocins in the food industry can help to reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods which are more naturally preserved and richer in organoleptic and nutritional properties. The main purpose of this work is to select cold-adapted lactic acid bacteria (LAB) from the intestinal tract of cold-water fishes from middle and high latitude regions, china，including the Eerqisi river, Xinjiang and Helongjiang river basin.Taxonomic identity and genetic diversity of strains isolated are determined by using universal 16S rRNA gene sequences and phenotypic characteristics. Accurate identification to the species level of the LAB population is obtained by means of using species-specific primers PCR and genomic fingerprintings (PFGE, rep-PCR, AFLP). In addition, we will use barcoded pyrosequencing to comprehensively survey bacterial community composition and the diversity of lactic acid bacteria (LAB) in intestinal contents of various cold-water fishes. We recently showed that that members of the genus Enterococcus are among the dominant lactic acid bacteria (LAB) in culture collections from the intestinal tract of cold-water fishes. In this work, we will apply rapid MLSA （Multilocus sequence analysis） genotyping method to determine the genetic heterogeneity of enterococci in the intestinal tract of cold-water fishes. By PCR amplification of structural genes for enterocins, antibiotic resistance and virulence genes, we will detect antimicrobial activity and virulence traits of Enterococcus isolates and compare their occurrence in clonal clusters generated by MLST and genotypic fingerprints(PFGE, rep-PCR).

产细菌素、安全的耐低温乳酸菌在食品、药物等领域具有很大的应用潜力。本项目拟通过广泛收集新疆北部地区、黑龙江流域分布的冷水鱼样本，采用16S rRNA基因高通量测序技术，揭示不同冷水鱼肠道微生物群落结构组成及乳酸菌多样性；优化培养基方案，从冷水鱼肠道中分离筛选各种乳酸菌，阐明可培养乳酸菌菌株的分类学地位、系统发育及生理生化、代谢特征；采用MLSA、PFGE方法，以及毒力基因、细菌素基因的多重PCR，揭示冷水鱼肠道可培养的优势乳酸菌种群Enterococcus spp.的遗传多态性，并阐明不同来源的菌株形成的特殊遗传谱系是否具有寄主(或者环境)特异性。项目的开展将为揭示肠道细菌的遗传多态性与栖息环境、来源寄主、生理代谢的相关性提供极好的模式研究，有助于丰富微生物种群遗传学、生态地理学理论。同时为进一步开发乳酸菌及其代谢产物作为天然的生物保鲜剂、人类药物和动物饲料益生菌制剂奠定理论基础。

1．利用5 种培养基对冷水鱼肠道中的低温乳酸菌进行分离鉴定，共分离得到纯培养物670 株。16S rRNA基因测序结果显示，670株乳酸菌主要分属于9个属，包括Lactobacillus、Carnobacterium、Enterococcus、Lactococcus、Streptococcus、Leuconostoc、Weissella、Brochothrix、Vagococcus，其中以Lactobacillus、 Carnobacterium、Enterococcus占优势：Lactobacillus包括L. casei、L. plantarum、L. sakei、L. paracasei、L. paraplantarum、L. pentosus，其中前三者为优势种；肠球菌(Enterococcus)包括E. faecium、E. faecalis、E. durans、E. hermaniensis、E. rivorum、E. thailandicus，肉杆菌(Carnobacterium)包括C. divergens、C. maltaromaticum两个种。其生长温度范围在4~37 oC之间，最适生长温度为15~24 oC，属于低温菌范畴。有9 株菌株的最适生长温度在20 oC以下并且当温度超过24 oC时停止生长，属于专性嗜冷菌。.2．以金黄色葡萄球菌（Staphylococcus aureus）CICC21600、李斯特氏菌（Listeria monocytogenes）、枯草芽孢杆菌（Bacillus subtilis）及革兰氏阴性菌大肠杆菌（Escherichia coli）作为指示菌，采用agar spot test法对优势种群Enterococcus的抑菌活性的筛选，再用肠球细菌素基因的引物（AS-48、entA、entB、entP、L50A and L50B、bacA-31）对已知产细菌素的Enterococcus进行PCR扩增，结果显示，有10株菌含有细菌素基因，他们分别是BC-M-5、GC-C-2、LH-M-3a、LB-L-7-C、GB-E-4、LD-M-4、GB-C-3、GB-R-8、GC-M-3和GD-M-2-B。其中菌株GB-E-4含有AS-48基因和entP基因，菌株LH-M-3a、GC-M-3和GD-M-2-B含有 entA基因，