Study On Storage Conditions Of Fecal Bacteria And Preparation Of Fecal Capsules And Analysis Of Microbial Flora

The wide variety and large numbers of intestinal flora constitute the most complex micro-ecosystem of the human body.Development of molecular biology and bio-informatics technology and big data application in biological analysis,makes people to basically understand the composition of intestinal microbial structure.With the gradual deepening of intestinal microbial research and the application of fecal microbiota transplantation(FMT)in the treatment of various diseases,a lot of stool samples were needed,and the establishment.of "stool bank" is gradually becoming a new direction of research.On the one hand,this paper analyzes the effects of fecal bacteria preparation conditions and storage conditions on the microbial community structure and flora activity of fecal bacteria,and explores the perfect and effective preparation and preservation conditions.On the other hand,we developed the fecal capsules which were more acceptable to the patients and analyzed the changes of the activity of the bacteria in the dry powder capsules after storage under different conditions.Keeping the composition of flora and the activity of flora consistent with the fresh bacteria is a vital factor in the establishment of fecal banks.This study begins with the first step of analyzing the effects of the preparation of bacterial solution,on the activity of bacteria.The collected fecal samples were prepared under both oxygen and aerobic conditions,the results of plate count of 7 kinds of culture media showed that the preparation of aerobic condition was 1?2 orders of magnitude lower than that prepared by anaerobic condition.The fecal microbial viability was analyzed using LIVE/DEAD BacLight TM Bacterial Vivacity Kits staining technique,and the results showed that under anaerobic conditions,viable bacteria are more than dead bacteria;while in the aerobic conditions,the majority is composed of dead bacteria,indicating that anaerobic conditions can be a good guarantee of the viable cells and to ensure the activity of bacteria.10%glycerol suspension and normal saline suspension were frozen immediately at-80? and stored for 1 months?2 months?6months.The number of bacteria were no significant change after 2 months and 6months storage in 10%glycerol;After storage in normal saline,bacteria number had a reduction at 2 months compared to fresh bacterial,however,there was a significant(nearly 1 log)decrease in Lactobacillus?Bacteroides and Escherichia coli at 6 months compared to fresh bacterial solution.Fecal suspension aliquots were stored at 4??-20??-80?conditions for 1?2?6 months,and culture results showed that the number of bacteria were no significant change after 1 months storage at 4??-20??-80?conditions,but with the increase of time,the difference between the storage of bacteria and the fresh bacteria was obvious at 4?.The activity of flora under the conditions of-20?and-80? showed no obvious change with time.DNA was extracted from samples of fresh suspension aliquots and stored at 4??-20??-80? conditions for 6 months suspension aliquots,and the changes of the composition of the flora were analyzed by 16S rDNA sequencing.The results showed that the relative abundance of Bacteroidetes was reduced by nearly 30%after 6 months storage at 4?compared to fresh samples,and the relative abundance of the Firmcutes was increased by about 20%,and the other Phylum were also increased,however,there was no significant difference in the composition of the flora under the conditions of-20? and-80?.The results of this study about storage conditions provide the basis for the establishment of fecal banks.In this study,we first used the vacuum freeze-drying technology to make fecal dry powder capsules.The flora activity of fecal dry powder capsules was analyzed using counting methods and LIVE/DEAD BacLight TM Bacterial Vivacity Kits.The results showed that the proportion of life and death of bacteria was not obvious at 0 days with the increase of storage time,whether it was stored at-80?,-20? or 4?.The storage conditions of the freeze-dried fecal dry powder capsules are more extensive,which provides a reference value for the commercialization of fecal powder capsules in the future.