Analysis On The Difference Of Intestinal Flora Of Centenarian Families In Luoyang Area And Study On The Separation And Identification Technology Of Bifidobacterium

The types and composition of intestinal microbes are closely related to human health.Recent studies have shown that Bifidobacterium in the intestinal tract are closely related to human health and longevity.The abundance of Bifidobacterium in the elderly is higher than that in the general population,and the relationship between Bifidobacterium and human health and longevity is gradually revealed.The study on the characteristics of intestinal flora of long-living elderly and the differences between them and their offspring will be conducive to the discovery of the genetic characteristics of intestinal flora and the analysis of specific intestinal microbial species and composition,which contributed to the theoretical and data support for the study of intestinal flora and longevity.The study on the sampling liquid selection and color culture technology in the intestinal Bifidobacterium separation technology will be helpful to the establishment of the intestinal Bifidobacterium separation and purification technology system,and help to obtain more intestinal Bifidobacterium from the elderly.Twelve stool samples were collected from three generations of four centenarian families,and the 16 S r DNA V3-V4 variable region of the sample was subjected to amplicon sequencing using the Illumina Miseq sequencing method.Furthermore,the differences and clustering of intestinal flora between centenarian families and between three generations of centenarians were compared in terms of diversity,phylum,genus and species,and the differences in the abundance of intestinal flora metabolism pathways between three generations and between families were preliminarily analyzed.Meanwhile,on the basis of comparing the effects of three fecal sampling solutions,the effects and limitations of the application of color rendering technology in the separation of intestinal Bifidobacterium were analyzed,and the correlation between the isolated intestinal Bifidobacterium and the sequencing results was preliminarily analyzed.The results show that:No significant difference was found in the Alpha diversity index of intestinal flora within and between centenarians;The relative content of Actinobacillus and Bifidobacterium in the intestinal flora of centenarians is higher than that of their offsprings;B.catulorum,B.longum and B.adolescentis are the main strains in gut microbiota of Bifidobacterium;Christensenella showed significant familial genetic characteristics and was significantly associated with BMI;Partial least squares discriminant analysis(PLS-DA)results showed that there was an obvious clustering phenomenon between the three generations,and only D and E families showed obvious clustering among the families,while the samples of A and B families could be roughly distinguished from each other despite the crossover phenomenon.The significant differences between the generation of intestinal microbiota was mainly in Verrucomicrobia comes from centenarians.The differences in intestinal microbial composition between families were mainly Firmicutes of family A and Lachnospira of family D.The differences in the abundance of metabolic pathways in the centenarian group were small,while the differences in the enrichment of metabolic pathways in the offspring and grandson groups were larger.The abundance range of D and E family was smaller than that of A and B family,and the enrichment degree of metabolic pathway was less different.Among the three fecal sample collection solutions,BS liquid medium of Bifidobacterium obtained more x-gal chromogenic colonies than the solution of 25%glycerin and normal saline.It was believed that the sample solution could better maintain the activity of Bifidobacterium in the sample and was more conducive to the separation of Bifidobacterium.The physiological and biochemical characteristics of xgal chromogenic colonies and the 16 s r DNA identification results showed that: when using x-gal as an indicator to isolate Bifidobacterium in feces,not only Bifidobacterium strains developed color.Lactobacillus salivarius,Bacillus nitratireducens,Lactobacillus gasseri,Lactobacillus vaginalis,LFHY?s,Bacillus luti and Lactobacillus fermentum were also identified from chromogenic colonies,and the chromogenic mechanism needs to be further studied.The sequencing results of intestinal Bifidobacterium of centenarians at the level of Bifidobacterium species showed certain differences from the actual Bifidobacterium species isolated,indicating that some intestinal Bifidobacterium were difficult to be isolated,and the separation and culture technology needed to be further improved.The results of this study will provide theoretical and data support for the research on intestinal probiotics and human health and longevity as well as the isolation and culture technology of intestinal Bifidobacterium.