【作者】 魏亚静；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2008， 硕士

【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。更多还原

【Abstract】 The antibiotic residues in food-producing animals may enter the food and be consumed by humans.This would result in a potential risk for human intestinal flora, such as increase of the population of resistant enteric bacteria,disruption of the colonization barrier,alteration of the metabolic activity of intestinal flora,and threaten human health.Nowadays,safety evaluation of residues of veterinary,antibiotics in human food is focusing on influence of human intestinal flora,and many people are interested in the studies gradually.But in China,this research has little progression because of lacking models approved by international organization.The dilution ratio and the time of transferring to continuous cultivation were optimized,and a chemostat system which could simulate human intestinal microecology was established.This system is one of the two models which are recommended by international organization.Development of this system will promote the research mentioned above and be significant to protect human health.In this study,bacillus-bile-esculin agar culture medium,BBL agar culture medium, eosin methylthioninium chloride agar culture medium and enterococcus element were chosen as the selective medium for Bacteroides fragilis.Bacillus bifidus,Escherichia coli and Enterococci.A gas chromatography method was established to determine the concentrations of three kinds of main short chain fatty acids（SCFA,including ethanoic acid,propionic acid and butyric acid）.50ml（20%） fresh excrement suspension was inoculated into fermentor and cultured under anaerobic condition.The viable number of four species of predominant bacterium in fixed time was counted by pour plate method. Then their growth curve in vitro batch culture was drawn and the exponential phase was calculated.On the base of the number of four species of predominant bacteria and the concentration of three kinds of main SCFA in broth culture,the dilution rate of continuous culture(0.05h^-1,0.07h^-1 and 0.09h^-1) and the time of shifting into continuous culture（5h, 7h and 9h） were optimized,and a chemostat system which could simulate human intestinal microecology was established.The long-term test of 30 days was carried out under the optimal condition,and the stability and reliability of this system were appraised through statistics analysis to the data.The batch culture indicated that exponential phase of four species of predominant bacteria were different,but they were in the period of 3-8h.The 0.07h^-1 was the relative appropriate dilution ratio,because it was the suitable condition which could make system reach a steady-state in 14d continuous cultivation.The 7h was the relative appropriate the time of shifting into continuous culture,because system could reach a steady-state earlier than other two conditions,and the flora quantity and the SCFA concentration of system were also highest in the stable state.In the 30 days test,there were no significance on the changes of four species bacterium quantity and three kinds of SCFA concentration in the 9th-30th days system.The stability of culture conditions of system（such as temperature, mixing speed,pH value and so on） is high.When the system achieved the stable state,the numbers of four species of predominant bacterium and the concentration of three kinds of main SCFA in culture solution were slightly decreased comparing with the fresh feces,but within the reported healthy person’s normal physiological parameter area.According to the experiment data,a chemostat system which could simulate human intestinal microecology has been established,by groping and optimizing the continuous cultivation dilution ratio and the time of shifting in continuous cultivation.The system has good stability and the reliability.It can simulate the intestinal tract environment of healthy human.The research evaluate the stability and reliability of the chemostat system in the flora quantity and metabolite concentration,filling a domestic gap in this field.This study solves the unstable problem of system induced by uneven stirring,unstable liquid-level and complex operation in the similar system at home and abroad.The development of this system could provide a platform for safety assessment research of the antibiotics residues on human intestinal microflora and propose a scientific approach to assess the safety of new drugs and functional foods and research of drug metabolism.It will promote the research mentioned above and be significant to protect human health.更多还原