| Clostridium butyricum is a kind of commensal bacteria which widely exists in the animal intestines.It has many beneficial effects,such as promoting growth,enhancing immunity,and improving intestinal health,and has been used in livestock and poultry production.However,at present,the probiotic mechanism of Clostridium butyricum is not fully understood,and the probiotic effect and physiological characteristics of different strains are significantly different.Our laboratory isolated,screened,and cultured a strain of Clostridium butyricum XKYQRL 2021(CBX 2021)from healthy pigs,which produces high butyric acid production and has good application value.In this study,CBX 2021 was used as the research object.Firstly,the overall effect of CBX2021 on the growth and health was evaluated in weaned piglets.Then,the pseudo-germ-free(PGF)mouse model was used to study the regulatory effect and mechanism of CBX 2021 on intestinal health.Finally,the regulation mechanism of CBX 2021 on intestinal health was studied and analyzed based on intestinal epithelial cells cultured in vitro.The main research contents and results are as follows:Experiment 1:This experiment systematically explored the effect of CBX 2021 on the growth and health of weaned piglets by analyzing growth performance indices,blood biochemical indices,16S rRNA microbial sequencing,and non-targeted metabolome.Forty-eight 28-day-old“Long×Large”weaned piglets were randomly divided into 2 groups with 6 replicates and 4 pigs in each replicate according to the principle of half male and half female with the same body weight.The piglets in the control(CON)group were fed with 5 mL sterile saline every day,while the piglets in the Clostridium butyricum(CB)group were fed with the same volume of CBX 2021solution(live bacteria 3×10~8 CFU/mL)for 28 days.The results were as follows:(1)Supplementing CBX 2021 significantly increased the average daily gain and significantly decreased the feed to gain ratio of piglets in the late stage(15-28 days)and the whole period(1-28 days)(P<0.05).It also significantly reduced the diarrhea index in the early stage(1-14 days)and the whole stage as well as the diarrhea frequency in each stage(P<0.05).(2)At the 28th day,compared with CON group,the levels of blood growth hormone and gastrin in the CB group were significantly increased by 6.33%and 7.74%respectively(P<0.05),and the content of ghrelin tended to increase(P=0.063);the contents of IgA,IgG,IgM and anti-inflammatory factor IL-10 in blood increased significantly by 5.90%,11.02%,7.31%and 13.05%,respectively(P<0.05),while the level of pro-inflammatory factor IL-1βdecreased by15.53%(P<0.05);the activities of D-lactic acid and diamine oxidase in blood which reflect intestinal permeability decreased significantly by 15.27%and 9.98%,respectively(P<0.05).(3)CBX 2021 obviously changed the microbial composition of piglet feces,especially increased the number of butyrate-producing beneficial bacteria such as Faecalibacterium,and decreased the abundance of pro-inflammatory bacteria such as unclassified＿Muribaculaceae and Rikenellaceae＿RC9＿gut＿group.(4)With the obvious change of microorganisms,CBX 2021 changed the metabolism of piglets,strengthened the mineral absorption and the biosynthesis of indole alkaloids and other alkaloids.In conclusion,CBX 2021 can accelerate the growth and development of weaned piglets,improve immune and intestinal barrier function,and reduce inflammation and diarrhea by optimizing intestinal microbiota structure and metabolic function.Experiment 2:In this experiment,antibiotics were used to eliminate endogenous bacteria to establish a PGF mouse model.On this basis,the effects of CBX 2021 on intestinal structure and barrier function were mainly studied,and the mechanism of CBX 2021 in regulating intestinal health was clarified.Firstly,thirty 8-week-old male Kunming mice were treated with a broad-spectrum antibiotic compound for 10 days to clear the intestinal microbiota and establish a PGF mouse model.Then,20 PGF mice were equally divided into 2 groups:CON group and CB group.They were fed with 0.2mL of normal saline and an equal volume of CBX 2021 bacterial solution(live bacteria 3×10~8 CFU/mL)for 10 days,respectively.The results were as follows:(1)Supplementing CBX 2021 can accelerate the reconstruction of intestinal microbiota in PGF mice,implant a large number of butyrate-producing bacteria,and reduce the number of cecal pro-inflammatory bacteria Enterorhabdus(P<0.05).(2)The content of butyric acid(P=0.054)and isobutyric acid(P=0.068)in the cecum of the CB group was significantly increased compared with that of CON group,along with the increase of butyrate-producing bacteria abundance.(3)Compared with the CON group,the ileal villi height and the ratio of villi height to crypt depth in the CB group were significantly increased by 41.29%and 36.61%,respectively(P<0.01),and the m RNA expression level of tight junction protein Claudin-1 was significantly increased by 56.56%(P<0.05).(4)CBX 2021 significantly reduced the content of disease-related metabolites(such as pregnenolone)in mice and promoted the digestion and absorption of carbohydrates(P<0.05).In conclusion,CBX 2021 can accelerate the production of butyric acid and isobutyric acid and positively regulate the intestinal metabolic characteristics by increasing the number of butyrate-producing beneficial bacteria,thus promoting the development of animal intestinal morphology and protecting the integrity of the physical barrier,which is beneficial to intestinal health.Experiment 3:In this experiment,CBX 2021 was co-cultured with porcine intestinal epithelial cells(IPEC-J2).The mechanism of CBX 2021 regulating intestinal health was revealed by studying its adhesion ability to intestinal epithelial cells,its effect on cell proliferation activity,inflammatory signals,and the transcriptional changes at the gene level.The results were as follows:(1)After IPEC-J2 cells were co-cultured with 1.5×10~8 CFU/mL CBX 2021 bacterial solution for 4 hours,the average adhesion number of bacteria was 50.04±2.74 CFU/cell.(2)The adhesion of CBX 2021significantly promoted the proliferation of IPEC-J2 cells after co-culture for 4-12 h(P<0.01),and the proliferation activity was the highest when co-culture for 6 hours.(3)The adhesion of CBX 2021 significantly increased the secretion of intracellular anti-inflammatory factor IL-10,while reducing the content of pro-inflammatory factor IL-1β(P<0.05).(4)The adhesion of CBX 2021 caused significant differential expression of 1892 genes in cells.KEGG enrichment analysis showed that inflammatory signaling pathways such as MAPK,PI3K-Akt and JAK-STAT were significantly affected by bacterial adhesion.Gene set enrichment analysis found that CBX 2021 significantly increased the activity of G protein-coupled receptors,while down-regulating the Notch signaling pathway and inhibiting the production of pro-inflammatory factors IL-6 and TNF.In conclusion,CBX 2021 has a good adhesion ability on IPEC-J2 cells,directly promoting the proliferation of intestinal epithelial cells,altering the expression of dozens of genes related to immunity and inflammation,and reducing the production of cellular pro-inflammatory molecules.In summary,Clostridium butyricum CBX 2021 can promote the growth and development of piglets and reduce the occurrence of intestinal diseases,mainly through optimizing the intestinal microbiota,strengthening metabolic function,improving intestinal structure and barrier function as well as improving intestinal health.Therefore,Clostridium butyricum CBX 2021 has good development and application value,and can guarantee the healthy and efficient production of livestock and poultry under the non-resistant feeding conditions. |
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