Study On Optimization And Evaluation Of In Vitro Human Intestinal Microorganism Simulation System

The human intestinal flora is diverse and numerous,which can be called the "microbial organ" of the human body.Intestinal flora not only has the function of microbial digestion,but also has a close relationship with host health.At present,there is no scientific and effective method for the regulation of intestinal flora.Cobalt-porphyrins are the general name of all compounds containing the cooing ring and contain various forms of vitamin B12 and its analogues.Cobalt-porphyrins are used by more than 76%of intestinal microbes,but less than 25%have the genes to synthesize them.Therefore,there is a complex competition and communication mechanism between intestinal bacteria for cobalt-porphyrins.However,we do not know what effect the different cobalt-porphyrins have on intestinal flora and how they change intestinal flora.In order to solve the above problems,first of all we need a reasonable study model of intestinal flora,because the animal intestinal flora have a big difference with human intestinal flora.Besides,animal experiments have long cycles and poor repeatability,so this experiment abandoned the traditional animal model,we adopted the in vitro human intestinal simulation model.However,the model still has disadvantages such as lack of uniform medium standards,cumbersome and expensive detection methods and long data return time.In view of the above situation,we firstly optimized the in vitro intestinal simulation system,including the optimization of medium,gas environment,immobilized fermentation technology and detection method.Secondly,by using 16S rRNA sequencing,ITS sequencing,gene pathway prediction,typical association analysis and other detection and analysis methods,the influences of several cobalt-porphyrins on intestinal flora structure and metabolism were investigated by in vitro intestinal simulation system.Lastly,a type of model bacteria was selected to study its mechanism at the surface level gradually deep to transcriptome level and ribosomal switch level.The main conclusions of this paper were summarized as following:(1)Established a short-term culture medium system for fecal bacteria,obtained the short-term culture formula which is closer to the human fecal flora and the short-term culture formula with the fastest growth of bacteria density;A rapid detection method(gas chromatogram technology)of intestinal flora for in vitro simulation system was developed.Through the evaluation,it was found that it had certain ability to distinguish and classify samples.The quantitative detection accuracy of the bacteria was about 70%;The microflora of intestinal mucosa was simulated by gel pellet fermentation.(2)Both cobalt-porphyrins(mecobalamin and cyanocobalamin)decreased the alpha diversity of intestinal flora.Meanwhile,cyanocobalamin significantly increased the relative abundance of acinetobacter.Both cobalt-porphyrins inhibited amylase activity of toxigenic bacteria and had no significant effect on cellulase activity:Both cobalt porphyrins promoted lipid metabolism,Inhibition of transcription,nitrogen metabolism,starch and sucrose metabolism;The relative abundance of Megasphaera spp.was significantly increased by the addition of Me-Cbl-C-10-Br in the in vitro intestinal simulation.Long-term supplementation of high concentrations of cobalt-porphyrins(methcobalamin and methbromide)can lead to intestinal fungal disorders.(3)The treatment of mecobalamin and cyanocobalamin mainly resulted in differences in the transcription of phosphate metabolism,carbon metabolism,ABC transporter and other related genes of Escherichia coli.The disconnection effect of cyanocobalamin on ribosome switch was stronger than that of cobalamin.In the condition of high concentration of cyanocobalamin,E.coli will turn off the ribosome switch and reduce the translation and synthesis of ButB gene.In the condition of mecobalamin,E.coli can also turn on the relevant ribosomal switch to increase the expression of PocR,a cell-cycle regulatory protein,so as to achieve rapid growth and reproduction.This paper studied the influence of gas environment on in vitro simulation system and developed a method for rapid detection of intestinal flora in vitro simulated fermentation broth.The flora of intestinal mucous layer was simulated by gel-pellet fermentation.This study can provide technical support for intestinal in vitro simulation for the convenience of future studies on intestinal flora;The effect of cobalt-porphyrins on intestinal flora and metabolism was investigated by using the in vitro colon model,providing guidance for vitamin B12 supplementation and dietary intake of cobalt-rich porphyrins for special population.It was found that long-term intake of high concentration of cobalt-porphyrin would cause intestinal fungus disorder,which may change the clinical medication of cobalt-methylamine.This paper found that different cobalamin can affect the growth of intestinal bacteria by regulating different ribosome switches,which provides a basis for the application of cobalamin in regulating intestinal flora in the future.