不同粪菌悬液处理方式对猪粪菌移植过程中细菌活性及活菌组成的影响

旨在探究不同粪菌液处理方法对其菌群活性及存活菌群物种组成的影响，为猪粪菌移植（FMT）的优化应用提供参考。本研究共设置7个试验组制备粪菌液：静置制备新鲜粪菌液组（FS）、静置制备粪菌液于-80℃冷冻1周组（FFS）、离心制备新鲜粪菌液组（FC）、离心制备粪菌液于-80℃冷冻1周组（FFC）、液氮冷冻粪便24 h静置制备粪菌液组（NS）、甘油+液氮冷冻粪便24 h静置制备粪菌液组（GNS）和液氮冷冻粪便24 h静置制备粪菌液于-80℃冷冻1周组（NFS），在37℃厌氧培养箱中平板培养48 h后，进行菌落计数。从FS、FFS、FC和FFC 4组中根据菌落形态随机挑取87个单菌落纯化后进行16S rRNA基因全长测序。结果表明，静置和离心两种方法制备新鲜粪菌液时，粪菌活性存在显著差异，离心组显著低于静置组（P<0.05）。但冷冻粪便使用静置和离心两种方法制备的菌悬液，其粪菌活性无显著差异（P>0.05）。无论是静置还是离心方法制备粪菌液，新鲜和冷冻两种不同处理方式下的粪菌活性均无显著差异（P>0.05），且对活菌的组成也无显著影响。使用静置法制备粪菌液时，FS、NS、GNS和NFS组间粪菌活性无显著差异（P>0.05）。综上表明，静置方法制备粪菌液时，超低温冷冻保存对粪菌活性及其活菌组成均无显著影响，这为大样本、长距离猪粪菌移植技术的应用提供了重要试验参考。

Effects of Different Treatments of Faecal Microbial Suspensions on the Activity and Composition of Live Bacteria in Faecal Microbiota Transplantation in Pigs

The purpose of this study is to explore the influence of different treatments on the microbial activity and live bacteria composition during the preparation of faecal bacteria suspension, so as to provide reference for optimizing the application of faecal microbiota transplantation(FMT) on pig. Seven experimental groups were set up in this study:microbial suspension from fresh feces by static settlement (FS), microbial suspension from fresh feces by static settlement and stored at -80℃ for 1 week (FFS), microbial suspension from fresh feces by centrifugation (FC), microbial suspension from fresh feces by centrifugation and stored at -80℃ for 1 week (FFC), microbial suspension from feces frozen in nitrogen by static settlement (NS), microbial suspension from feces treated with glycerin and frozen in nitrogen for 24 h by static settlement (GNS), and microbial suspension from feces frozen in nitrogen by static settlement and stored at -80℃ for 1 week (NFS). All suspensions were spread on plates and cultivated at 37℃ anaerobic incubator for 48 h. The colonies growing were counted. In addition, 87 single colonies were randomly selected from the four groups of FS, FFS, FC and FFC and purified for full-length sequencing of 16S rRNA gene. The results showed that there were significant differences in bacteria activity when preparing suspensions from fresh feces by static settlement and centrifugation methods, and the activity of faecal bacteria in the centrifugation group was significantly lower than that in the static settlement group (P<0.05). However, there was no significant influence of suspensions from frozen feces on the activity of faecal bacteria regardless of static settlement or centrifugation (P>0.05). Whether the suspensions were prepared by static settlement or centrifugation, no significant impact was found on the activity of faecal bacteria either from fresh or frozen feces (P>0.05),nor did on the live bacteria composition. When microbial suspensions were prepared by static settlement, there were no significant difference in faecal bacteria activity (P>0.05) among FS, NS, GNS and NFS groups. The above results indicate that when preparing faecal microbial suspensions by static settlement, ultra-low temperature cryopreservation has no significant effect on the activity and composition of faecal bacteria. The present research provided a useful suggestion for the application of long distance and large size faecal microbiota transplantation technology in pig production in the future.