Study On The Probiotic Properties Of Bacillus Coagulans LT3 And Its Protective Effect On LPS-induced Cecal Injury In Rats

At present,the abuse of antibiotics has caused a series of safety problems,such as some bacteria's resistance to antibiotics and the problem of drug residues,which will directly or indirectly affect human health.Therefore,more and more attention is paid to controlling the use of antibiotics in feed.Antibiotic substitutes should have no toxic effects on the animal itself and animal products,no residue or short-term residue in the body,no decomposition and denaturation in the animal intestinal environment,no induction of drug resistance,good stability in the feed,and good palatability,Can improve animal disease resistance and other probiotic effects.Probiotics not only have the above advantages,but also have the ability to balance the intestinal flora and protect the intestinal barrier.Become one of the well-deserved alternatives to antibiotics.The purpose of this study is to isolate and identify potential Bacillus coagulans with good probiotic properties,and conduct animal experiments to evaluate the protective effect and mechanism of probiotic strains on animal intestines,so as to be the application of Bacillus coagulans isolates in production practice in the future Lay the foundation.Test 1:Isolation and identification of Bacillus coagulans and research on its probiotic propertiesThe isolates were identified by colony characteristics,Gram staining,16S r RNA gene sequence sequencing and homology analysis.The probiotic properties of the isolates such as tolerance to bile salts,acids and salts and antagonism to pathogenic bacteria were systematically studied.Through SOD activity and scavenging hydroxyl free radicals,DPPH,ABTS ability and hydrogen peroxide tolerance tests to study the in vitro antioxidant capacity of the strain and the safety of antibiotic sensitivity tests.The results showed that the isolated and purified strain was Bacillus coagulans,named Bacillus coagulans LT3.Bacillus coagulans LT3 has certain acid-base salt tolerance and can grow in 0.2%bile salt environment,p H value 2.0 acid environment,8.5%salt environment;LT3 strain is resistant to Escherichia coli,Fusarium,Shigella Aspergillus flavus,Salmonella,etc.,have excellent inhibitory effects;this strain has strong scavenging ability to hydroxyl free radicals,DPPH,ABTS,respectively,83.3%,86%,93.7%;this strain also has very The high SOD activity is 46%;in the medium with the concentration of 0.4m M,0.7m M,1.0m M hydrogen peroxide,the survival rates are 86%,87%,and 84%,respectively.In addition,the strain is resistant to seven antibiotics amoxicillin(AMX),amikacin(J),tetracycline(TE),rifampicin(Li),ciprofloxacin(cyclo)and erythromycin(E)All are sensitive(S),and chloramphenicol(C30)as an intermediary(I),none of them are resistant and can be considered safe.The Bacillus coagulans LT3 we isolated and identified not only has good tolerance properties,but also has good antioxidant properties,and has potential development and utilization value in the feed and food industries.Test 2:The protective effect of Bacillus coagulans LT3 on LPS-induced cecal injury in rats32 male Wistar rats were randomly divided into 4 groups,namely CON(control)group(normal feeding),LPS(lipopolysaccharide)group(normal feeding),and HBC group(gavage1.2×10~8CFU/m L coagulation Bacillus LT3 bacterial solution),LBC group(gavage 1.2×10~3CFU/m L Bacillus coagulans LT3 bacterial solution),a total of 14 days.On the 15th day,each rat in the LPS group,HBC group,and LBC group was injected with 1 mg/kg body weight of LPS.The rats were killed by anesthesia 4h later.After the test,the kit detects the serum biochemical indicators of rats and the antioxidant indicators of rat cecum tissue;HE staining detects the damage of the rat cecum and the number of goblet cells by arizin blue staining;and Western blotting(Western Blot))Detect changes in NF?B and Nrf2 related signal pathways and TJ-related protein expression levels;use high-throughput sequencing methods to detect cecal flora.The results showed that:(1)Compared with the LPS group,the addition of LT3 strain can significantly reduce the levels of pro-inflammatory cytokines interleukin 1?(IL-1?),interleukin 6(IL-6),and tumor necrosis factor?(TNF-?)(P<0.05),increase the level of anti-inflammatory cytokine interleukin 4(IL-4).(2)LT3 strain can significantly reduce the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in the cecum tissue of rats induced by LPS(p<0.01),and make glutathione peroxidase(GSH-Px)activity and catalase(CAT)activity were significantly increased(p<0.01).(3)In the histology HE staining,it can be seen that the addition of LT3 improved the intestinal barrier damage of the cecum tissue compared with the LPS group.The results of Alixinlan staining showed that the addition of LT3 effectively enhanced the number of goblet cells in the cecum.(4)LT3 can effectively improve the body's inflammation and oxidative stress and protect the intestinal barrier from damage by regulating the TLR4/MYD88 signaling pathway,the Nrf2 signaling pathway and the expression of TJ-related proteins.(5)Through 16S sequencing,it is found that LT3 plays a positive role in the regulation of intestinal flora,increasing the abundance of beneficial bacteria.Compared with the control group,LT3 increased the relative abundance of Allobaculum,Akkermansia Muciniphila,and Verrucomicrobiae in the intestines,making Clostridia and Enterobacteriaceae)The number is greatly reduced.Conclusion:Bacillus coagulans LT3 strain can protect the cecum injury induced by LPS in rats by regulating inflammation and oxidation signal pathways and intestinal flora.