低浓度喹诺酮对嗜热链球菌生理功能和代谢途径的影响研究

In order to explore the changes of physiology and metabolite of Streptococcus thermophilus under the stress of low concentration Quinolones (QNS), and to provide scientific evidences for comprehensive, objective evaluation of probiotic properties and application. This application based on the previous studies, screened and identified the Streptococcus thermophilus grx02 which was highly sensitive to QNS, different concentrations of QNS were used to study the influence on cell form and physiological characteristics. Cytosolic proteins were extracted from cells and separated by two-dimensional electrophoresis(2-DE), and differently expressed proteins were selected and identified by MALDI-TOF-MS, then analyze the influence of protein expression. According to the differences between protein fingerprint information , analysis the genes encoding and design primer, and real-time fluorescence quantitative RT-PCR technology was used to verify the different expression level in mRNA. Then analyze the metabolites before and after the stress to reveals the effect of mechanism and metabolic pathways of Streptococcus thermophilus under the QNS stress.

研究嗜热链球菌在低浓度喹诺酮类药物（QNS ）胁迫下细胞生理及代谢产物变化可能引起的效应，为全面、客观评价乳酸菌的益生特性和安全性提供理论依据。本项申请在前期研究基础上，筛选并鉴定对QNS高度敏感的Streptococcus thermophilus grx02，采用不同亚抑菌浓度的QNS处理，研究对细胞形态和生理特性的影响；并提取全菌总蛋白采用2-DE技术分离，对差异蛋白进行MALD-TOF-TOF-MS鉴定，分析对蛋白组表达的影响；在此基础上，根据差异蛋白指纹图谱信息，分析其编码基因并设计引物，应用实时荧光定量RT-PCR技术验证在mRNA水平上的差异表达；最后对胁迫前后的代谢产物进行分析，以揭示喹诺酮类药物胁迫下嗜热链球菌的应激机制及代谢途径的改变。

项目以本实验室前期筛选获得的专利菌株嗜热链球菌grx02为研究对象，采用不同亚抑菌浓度的喹诺酮类药物（QNS）处理，研究了S. thermophilus grx02的生长速率、微观形态、细胞膜通透性和胞内乳糖代谢关键酶活性等生理特性的变化机制；并研究了不同亚抑菌浓度的喹诺酮对S. thermophilus grx02的凝乳时间、产酸能力、产黏能力和蛋白水解能力的影响，以及对S. thermophilus grx02发酵乳的质构特性、流变特性、感官特性、活菌数和挥发性风味物质等发酵性能的影响。建立了液氮研磨一TCA/丙酮一尿素/硫脲/TBP裂解提取法制备S. thermophilus grx02全菌总蛋白的方法，优化了双向凝胶电泳技术（2-DE）的试验条件，并建立了S. thermophilus grx02菌体全蛋白和1/2MIC OFX作用下蛋白质组双向电泳表达谱，通过图谱分析软件找出表达差异显著的蛋白点。利用基质辅助光解吸/电离飞行时间质谱（MALDI-TOF-MS）对表达差异的蛋白质进行了鉴定，并对其功能进行了分类解析。结果表明低浓度氧氟沙星会对S. thermophilus grx02的生理特性、发酵性能和蛋白质表达产生影响，使许多代谢途径酶的活性受到抑制，从而降低乳酸菌的生长和代谢速度。同时诱导一些通用应激蛋白表达的上调，以提高细胞对低浓度抗生素的抵御能力。本项目的研究结果有助于进一步深入了解乳酸菌在低浓度抗生素作用下的生理状态及胁迫应答机制，为全面客观评价乳酸菌的益生特性和安全性提供理论依据。