Culture And Identification Of Clostridium Butyricum From Feces Of Jinhua Pigs And Its Role In Improving Fat Metabolism

There is a regulatory role for the deposition of intramuscular fat in animals.The metabolite of Clostridium butyricum(CB)is short chain fatty acids(SCFA),and its content also influences the composition of the intestinal microbiota.It has been reported that CB has been widely used in livestock industry in recent years,which mainly focuses on the regulation of fat in poultry,but little research has been done in monogastric animals,pigs.Exogenous Clostridium butyricum is not easily colonized in the intestine,and endogenous Clostridium butyricum isolated from feces can better colonize the intestine to exert its effects.Lipid metabolism affects the meat quality of animals,and intestinal flora can improve fat metabolism,including CB.Therefore,this study is aimed at discovering good CB strains,exploring the interaction between CB,intestinal microorganisms and lipid metabolism,and providing new ideas to regulate intramuscular fat deposition,improve meat quality and treat diseases caused by high-fat diet through CB de-nutrition in pigs.Part 1:Differences in butyric acid production metabolism by microorganisms in the intestinal tract of Jinhua pigs in different body conditionsFifty-five healthy Jinhua pigs of the same batch at 32 d of weaning age and 4.92±0.68 kg initial weight were reared and fed a standard maize and soybean meal diet,and slaughtered at 270 days of age.16 Jinhua pigs were selected based on the values of body weight and backfat thickness/eye muscle area after slaughter.High body fat phenotype group(H Group):backfat thickness/eye muscle area=Max(H Group)(0.119～0.350),low body fat phenotype group(L Group):backfat thickness/eye muscle area=Min(L Group)(0.080～0.103).Then the SCFA content in the intestinal surimi and the content of Clostridium perfringens in the 2 groups were measured separately.The main test results were as follows:1.Phenotypic differences in Jinhua pigs of different body conditions:there was no significant difference in body weight between the 2 groups(P>0.05),but the ratio of backfat thickness,backfat thickness and eye muscle area was highly significant higher in H Group than in L Group(P<0.0001).The above results indicated consistent with the fact that the body fat of H Group was significantly higher than that of L Group.2.The metabolism of butyric acid production by intestinal microorganisms differed among Jinhua pigs in different body conditions:the content of butyric acid was higher in the cecum and colon than in the ileum of Jinhua pigs in two groups,and the content of butyric acid was highest in the colon segment;the content of butyric acid in the cecum and colon segments of L Group was significantly higher than that of H Group.The above results indicated that the colon and cecum of Jinhua pigs were the main sites of butyric acid production,and the capacity of butyric acid production was lower in H Group than in L Group.3.Abundance of key butyric acid-producing bacteria in colonic microorganisms:analysis of the abundance of the major butyric acid-producing bacteria Clostridial cluster I,IV and XIVa in the colon contents of the 2 groups revealed that the abundance of Clostridial cluster IV in the colon of H Group was significantly lower than that of L Group(P<0.05),explaining the significantly lower abundance of butyric acid in the colon of H Group had significantly lower butyric acid content than L Group.The above results further confirmed the higher butyric acid content in the intestinal segment of low body fat Jinhua pigs.Part 2:Isolation of Clostridium butyricum of porcine originTo further investigate the effect of endogenous butyric acid-producing bacteria on lipid metabolism in low body fat phenotypic Jinhua pigs,CB was isolated from the feces of low body fat phenotypic Jinhua pigs,isolated and purified by anaerobic culture,selected suspicious colonies for 16S r RNA gene sequencing analysis,determined the SCFA concentration of colonies identified as CB,selected CB strains with high butyric acid-producing content for in vivo colonization test in mice,and investigated the effect of this CB on body fat metabolism in mice.The results of the experiment were as follows:1.CB colonies appear yellow-white or white,round or slightly irregularly round,the surface is moist and smooth.2.The morphology of 20 strains was observed based on the morphological characteristics of CB,among which 10 strains were suspected to be CB.3.10 suspected strains were sequenced with 16S r RNA gene,and 6 suspected strains were found to have more than 99%homology with the 16S r RNA gene sequence of CB by sequence BLAST comparison,and the 6 strains were identified as CB.4.The SCFA concentration of the six CB strains was measured by gas chromatography,and the strain numbered JH.CB5 was found to produce the highest concentration of butyric acid,so JH.CB5 was selected for the next step of mouse gavage test.Part 3:Effect of Clostridium butyricum on body fat metabolism in obese miceSeventeen 8-week-old male C57BL/6J mice were used for the experiment,and after1 week of adaptation,they were randomly grouped to ensure the same starting status of the experiment.The test was randomly divided into three treatment groups:the first group was the control group(Control):5 mice were given normal diet and 200μL saline by gavage;the second group was the high-fat diet group(HFD):5 mice were given high-fat diet and 200μL saline by gavage;the third group was the Clostridium butyricum group(CB):7 mice were given high-fat diet and 200μL Clostridium butyricum by gavage(10~9CFU).Gavage was performed daily at 9 a.m.for 15 weeks.During the entire study period,water was consumed normally and body weight was measured once a week.15 weeks of sampling were completed and the main results were as follows:1.The effect of CB on body fat metabolism in obese mice.body weight gain in the HFD and CB groups was significantly higher than that in the Control group,and the body weight gain in the CB group was lower than that in the HFD group;epididymal fat and subcutaneous fat in the HFD group were significantly higher than that in the CB group(P<0.05),and HE staining showed that the fat content in adipose tissue cells in the HFDgroup was higher than that in the Control and CB groups.The results suggest that gavage CB can alleviate weight gain and adiposity caused by high-fat diet.2.The effect of CB on intestinal microorganisms in mice.At the phylum level,the relative abundance of the phylum Caecalibaculum in the intestine of the HFD group decreased and that of the phylum Allobaculum increased,and at the genus level,the genus Faecalibaculum became the dominant genus;the CB group was not significantly different from the HFD group at the phylum level(P>0.05),and at the genus level,the relative abundance of Faecalibaculum decreased and that of Allobaculum increased,tending to Control group shift.The above results suggest that CB can modify the changes of intestinal microbial community induced by high-fat diet to some extent.3.The effect of CB on differential gene expression in mice.9 significantly enriched GO functions related to lipid metabolism and 2 KEGG pathways in the ileum and 2significantly enriched GO functions related to lipid metabolism in the colon in the CB group compared with the HFD group.The above results suggest that CB may affect body lipid metabolism in mice by regulating genetic pathways related to lipid metabolism in mice.In summary:1.Among the three intestinal segments of Jinhua pigs,ileum,cecum and colon,cecum and colon are the main parts of butyric acid production,and colon has the most butyric acid content.2.The content of butyric acid in the intestine of Jinhua pigs with high body fat phenotype was different from that of Jinhua pigs with low body fat phenotype,and the content of butyric acid in the intestine of pigs with high body fat phenotype was lower than that of Jinhua pigs with low body fat phenotype.3.In the colon,the butyric acid production capacity of the high body fat phenotype group was lower than that of the low body fat phenotype group,probably because the abundance of its key butyric acid producing bacteria,Clostridial cluster IV,was significantly lower than that of the low body fat phenotype group.4.CB may improve adipogenesis and slow down the trend of weight gain in obese mice by regulating gene pathways related to lipid metabolism and changing the structure of intestinal flora.