Study On Intestinal Bacterial Diversity And Screening Of Probiotics In Tibetan Pig

Tibetan pigs are not only excellent local pig breeds in China,but also rare high-altitude small pigs in the world.They have excellent characteristics such as disease resistant,herbivorous,which are greatly owning to their own intestinal bacterial.In order to deepen the understanding of the intestinal bacteria of Tibetan pigs and discover the unique strain resources in the intestine of Tibetan pigs,and apply it to diminish the high diarrhea rate in piglets.This study cultured the intestinal bacterial and used 16 SrDNA high-throughput sequencing to study the bacterial diversity of Tibetan pig caecum.What's more,screening of probiotic strains was carried out according to short-chain fatty acid production capacity,xylanase activity,cellulase activity,acid tolerance,bile tolerance,and then the strains with excellent performance were subjected to the feeding test of the piglets the results are as follows:1.High-throughput sequencing method was used to study the bacterial diversity of Tibetan pigs.In this study,we amplified the V4-V5 region of 16 S rDNA.The original reads produced by sequencing were filtered and homogenized,and each sample contained 30,000 reads.Clustering clean reads with 97% similarity obtained 2153 OTUs in 28 samples.After species annotation with the greengene database,it was shown that the bacteria in the Tibetan cecum were divided into 16 phylum,80 families and 114 genera.Among them,Bacteroides and Firmicutes bacteria accounting for 60%-90% of the whole community were in a dominant position.However,in terms of species abundance,compared with other pig breeds,the relative abundance of Bacteroides and spirochetes was higher and the relative abundance of Firmicutes and Enterobacteriaceae was lower.This unique flora structure may be associated with the herbivorous and disease-resistant characters of Tibetan pigs.2.Culture method was used to study the bacterial diversity of Tibetan pigs.On the one hand,typical medium for intestinal bacteria like BHI,PYG,RGCA,GAM,YCFA and FAA were selected and used.On the other hand,targeting the differences in the utilization of carbon sources for different bacteria,PYG was selected as the basic medium while glucose was replaced by a variety of monosaccharides,glycans,thus PY + inulin,PY + rhamnose,PY + cellulose,PY + mucoid,PY + xylose and PY + cellose medium were designed.What's more,Marin broth 2216 E medium was selected for different tolerance to salt ions and oligotrophic 1/10 GAM medium was designed for some rapid-growing bacteria.When it came to the culture substrate,not only the agar solid medium was used,liquid medium was also used within a 96-well plate after the bacterial solution was diluted to a suitable concentration.In addition,samples were treated with 70% ethanol to selectively isolate Clostridium bacteria.A total amount of 108 strains of bacteria were isolated with the above methods.These bacteria belonged to the 4 phylum(Actinobacteria,Bacteroides,Firmicutes and Proteobacteria),26 families,36 genera(including 9 new genera),72 species(including 2 subspecies),which contained 50 known species(84 strains)and 22 potential novel species(24 strains).By consulting the relevant literature,it was found that potential probiotics accounted for 22% of the isolated species and potential pathogens accounted for 19%.3.The potential probiotics were screened according to the short-chain fatty acid production,xylanase activity,cellulase activity,acid tolerance,bile tolerance.Five probiotics strains,Bacillus velezensis Y4,Lactobacillus sp.PYG4 Bifidobacterium thermacidophilum subsp.Porcinum PYG1,Bacteroides xylanisolvens PYM1 and Prevotella sp.PYC3 were obtained.Genome analysis showed that the all of the five strains had extensive utilization ability to cellulose,hemicellulose,starch,pectins and other multiple polysaccharides.Among of them Bacillus velezensis Y4,Bacteroides xylanisolvens PYM1 and Prevotella sp..PYC3 has significantly more genes in carbohydrate metabolism.Subsequently,obtained five strains were combined into two bacterial compound agents.The combination of compound agent I contained Lactobacillus sp.PYG4,Bifidobacterium thermacidophilum subsp.Porcinum PYG1,Bacteroides xylanisolvens PYM1 and Prevotella sp.PYC3 and xylooligosaccharide had the best effect on piglets.No matter fed alone or in combination with xylooligosaccharide,it effectively reduced the diarrhea rate and improve the growth performance of piglets.