Development Of A Chemostat System Simulating Human Intestinal Ecology

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.