人肠道微生态恒化器模拟系统建立

发布时间：2018-06-04 09:27

本文选题：人肠道菌群 + 连续培养　；参考：《华中农业大学》2008年硕士论文

【摘要】： 残留在动物可食性组织中的抗菌药物被人体摄入后,可能对人体肠道菌群产生诸多不良影响,如选择出耐药的肠道菌群或使肠道细菌耐药性增强,破坏肠道菌群的屏障作用,改变肠道菌群代谢活性等,从而对人类健康构成巨大威胁。在国际上,抗菌药残留对人体肠道菌群影响的安全性评价研究日渐成为热点。但国内由于缺乏被国际组织普遍认可的评价模型,该领域的研究工作进展缓慢。本研究利用细菌连续培养的恒化器原理,通过对稀释率和转入连续培养时间两方面条件的摸索和优化,建立了人肠道微生态恒化器模拟系统。该模拟系统是国际组织推荐的两大类模型之一,其建立将会促进国内外该领域的研究,对保障人类食品和医学安全具有极其重要的意义。本研究选用拟杆菌—胆汁—七叶苷琼脂培养基、BBL琼脂培养基、伊红美兰琼脂培养基和肠球菌选择性培养基,分别分离计数人粪便中四种优势代表菌(脆弱拟杆菌、双歧杆菌、大肠杆菌和肠球菌)。建立粪便中三种主要短链脂肪酸(SCFA,即乙酸、丙酸和丁酸)浓度测定的气相色谱方法。将50mL(20%)的新鲜粪便悬液接种于发酵罐中,厌氧条件下分批培养,定时取样,采用平板稀释计数法记录培养液中四种优势代表菌的数目,绘制其分批培养的菌体生长曲线,计算对数生长期。在此基础上,以培养液中四种优势代表菌数目和三种主要的短链脂肪酸浓度为监测指标,优化连续培养工艺,包括稀释率(0.05h~(-1)、0.07h~(-1)和0.09h~(-1))和转入连续培养的时间(第5h、第7h和第9h),建立人肠道微生态恒化器模拟系统。在优化后的培养条件下进行为期30 d的试验,进一步评价所建立的模拟系统的稳定性和可靠性。分批培养显示,四种优势菌的对数生长期不尽相同,但都包含了第3-8h。在0.07h~(-1)的条件下,系统在连续培养的前14d达到了稳态,因此0.07h~(-1)为相对较合适的稀释率。第7h转入连续培养时,系统较其他两个条件更早达到了稳态,且稳态时系统中的菌群数量和SCFA浓度也最高,因此第7h为相对较合适的转入连续培养的时间。30d试验显示,在第9-30d的系统中,四种代表菌的数量和三种SCFA的浓度变化差异均不显著,系统培养条件(如温度、搅拌速度、pH值等)的稳定性也较好。模拟系统在达到稳态后,其培养体系中的四种优势菌数量和三种SCFA浓度与接种前的粪便相比略有下降,但均在报道的健康人正常的生理参数范围内。上述试验结果表明,本研究通过对稀释率和转入连续培养时间等条件的摸索和优化,成功建立了人肠道微生态恒化器模拟系统,系统具有较好的稳定性和可靠性,能够模拟健康人的肠道微生态环境。本研究首次从菌群数量和代谢物浓度两个角度较全面地对恒化器模拟系统的稳定性、可靠性进行了评价,填补了国内研究的空白。本研究还解决了目前国内外建立同类模拟系统时存在的搅拌不均匀、液位不稳定、操作复杂而导致模拟系统相对不够稳定等问题。该恒化器模拟系统的建立可以为抗菌药残留对人肠道菌群影响的安全性评价研究构建平台,也可为新药、功能食品等的安全评价研究及药物代谢研究提供更科学的方法和途径,对保障人类食品和医学安全、促进相关学科发展具有极其重要的意义。
[Abstract]:The antimicrobial agents remaining in animal edible tissues may have many adverse effects on the intestinal flora of the human body, such as the selection of drug resistant intestinal flora or the enhancement of antimicrobial resistance of intestinal bacteria, the barrier effect of intestinal flora, and the change of intestinal microflora metabolism, so as to pose a huge threat to human health. At the international level, the research on the safety assessment of the effects of antimicrobial residues on the intestinal flora is becoming a hot topic. However, the research work in this field is slow due to the lack of an evaluation model universally recognized by international organizations. This study uses the principle of continuous culture of the chemostat of bacteria, through two parties to the dilution rate and the time to the continuous culture. The simulation system of human intestinal microecological chemostat is established. This simulation system is one of the two major models recommended by international organizations. The establishment of this simulation system will promote the research in this field at home and abroad. It is of great significance to ensure the safety of human food and medicine.
In this study, bacteriobacterium - bile - seven glucoside agar medium, BBL agar medium, ironorchid agar medium and enterococci selective medium were used to separate four dominant representative bacteria (fragile bacillus, Bifidobacterium, Escherichia coli and Enterococcus) in human feces, and three kinds of main short chain fatty acids (SCFA, b) in feces were established. A gas chromatographic method for the determination of the concentration of acid, propionic acid and butyric acid. The 50mL (20%) fresh fecal suspension was inoculated into the fermenting tank. Under the anaerobic condition, the number of four dominant bacteria in the medium was recorded by the plate dilution counting method. The growth curves of the four bacteria were plotted and the logarithmic growth period was calculated. On the basis, the optimized continuous culture process, including dilution rate (0.05h~ (-1), 0.07h~ (-1) and 0.09h~ (-1)) and the time of continuous culture (5h, 7h and 9h), was optimized by using the number of four dominant bacteria and three main short chain fatty acids in the culture medium, and the simulation system of human intestinal microecological chemostat was established. A 30 d experiment was conducted to evaluate the stability and reliability of the simulation system.
Batch culture showed that the logarithmic growth period of the four dominant bacteria was not the same, but it all contained the 3-8h. in the 0.07h~ (-1) condition, the system reached the steady state before the continuous culture, so 0.07h~ (-1) was a relatively suitable dilution rate. When the 7h was transferred to continuous culture, the system reached the steady state earlier than the other two conditions and the steady state system. The number of bacterial flora and SCFA concentration in the system is also the highest, so 7h is a relatively suitable time to transfer to continuous culture time.30d test shows that in the system of 9-30d, the difference of the number of four representative bacteria and the concentration of three kinds of SCFA is not significant, and the stability of the system culture conditions (such as temperature, stirring speed, pH value, etc.) is also better. After reaching steady state, the number of four dominant bacteria and three kinds of SCFA concentration in the culture system decreased slightly compared with the fecal phase before inoculation, but all were within the normal physiological parameters of the reported healthy people.
The experimental results show that the simulation system of human intestinal microecological chemostat has been successfully established by exploring and optimizing the dilution rate and continuous culture time. The system has good stability and reliability and can simulate the intestinal microflora of healthy people. This study is the first time from the number of bacteria group and metabolite concentration. The stability and reliability of the chemostat simulation system are evaluated by two angles, which fill the blank of the domestic research. This study also solves the problems of uneven agitation, unstable liquid level and complicated operation, which lead to the relative instability of the simulation system.
The establishment of the chemostat simulation system can provide a platform for the safety evaluation of the influence of antimicrobial residues on the intestinal flora, and also provide more scientific methods and ways for the safety evaluation and drug metabolism research of new drugs, functional foods and so on. It can ensure the safety of human food and medicine and promote the development of related disciplines. Important meaning.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R-331

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