The Structure Of Exopolysaccharide From Bacillus Amyloliquefaciens And Its Probiotic Mechanism

Bacillus and its exopolysaccharide?EPS?can improve intestinal flora balance,and stimulate intestinal immune response,especially in preventing and treating acute diarrhea caused by enterotoxigenic Escherichia coli?ETEC?.However,due to the unthorough elucidation of its probiotic mechanism,and the neglect of the probiotic function of the main metabolite EPS,the precise application of Bacillus EPS in animal production was limited,unstable and untargeted.The present study isolated an individual Bacillus strain produces EPS with anti-hemagglutination activity against ETEC.And then the study elucidated the structure of its EPS and analyzed the inhibitory effect of EPS against ETEC,as well as the regulation impact of EPS towards lactic acid bacteria.The study finally identified the protection effect of EPS to intestinal cells facing oxidative damage induced by Pb²⁺.This study may shed light on the application of Bacillus EPS in preventing and treating animal stress and improving food safety,which is a difficult issue in the animal farming.It may also provide a new idea for utilizing EPS as a new feed additive to realize its precise control in the animal farming.The main conclusions are as follows:?1?A potential probiotic yielding a large amount of EPS with high anti-hemagglutination activity against ETEC was isolated from the feces of healthy piglets.The strain was identified as Bacillus amyloliquefaciens JN4 by 16S rRNA sequencing and routine physiological and biochemical tests.EPS from B.amyloliquefaciens JN4 was purified by DEAE-Sepharose FF and Sepharose CL-6B,with the recovery rate of 90.2%.The pure EPS renamed as EPS-JN4could inhibit the hemagglutination of ETEC at the titer of 4 g·L^-1.The molecular weight of EPS-JN4 was 8 kDa and the degree of polymerization was approximately 50.EPS-JN4 was consisted of two main monosaccharide,glucose and fructose,with a molar ratio of 1:46.1,indicating EPS-JN4 as a fructan with a small molecular weight.Furthermore,by Fourier infrared spectroscopy,methylation analysis and nuclear magnetic resonance analysis,EPS-JN4 was presumed to be a levan-type fructan with branched chains,containing 7 repeat units,each of which contained five residues of?2?6?-?-fructosyl and one residue of?1,2?6?-?-fructosyl as branch points,and the side chain was linked to only one 2-?-fructosyl residue.Compared with other fructan,EPS-JN4 had higher activity in inhibiting ETEC hemagglutination.?2?EPS-JN4 acts as a receptor analogue when binding to ETEC,thereby inhibits the adhesion of ETEC to intestinal cells.EPS-JN4 showed synergistic effect with galactooligosaccharide in inhibition of ETEC towards intestinal cells.High titers of EPS-JN4could inhibit the growth of ETEC and postpone its logarithmic growth phase,but it cannot kill ETEC completely.Studies have shown that these effects were carried out through the inhibition of the respiratory metabolism of ETEC by cell surface adhesion.EPS-JN4 also showed positive impact on the inhibition expression of pro-inflammatory genes in intestinal cells that were mediated by ETEC,and also reduced the inhibitory effect of ETEC on the expression of anti-inflammatory genes,both of which lead to the slowing down of ETEC mediated inflammatory response.?3?EPS-JN4 was stable enough to resist the degradation of?-amylase and gastric acid in the digestive system of animals.EPS-JN4 exhibited significantly positive effect on the proliferation of Lactobacillus in faecal flora,especially on the species Lactobacillus reuteri.The growth rate of L.reuteri JN101 exposed to EPS-JN4 was slower than that cultured with glucose.Although the final biomass was not significantly different,the main metabolites,such as lactic acid and acetic acid,were 10.5%lower and 23.9%higher in the culture exposed to EPS-JN4 than that with glucose,respectively.By contrast,when exposed to EPS-JN4,the surface hydrophobicity and intestinal adhesion of L.reuteri JN101,were significantly raised by 73.5%and 2.6 times respectively.The contents of cell membrane proteins and lipids were also increased by 40.2%and 104.8%,among which,the content of unsaturated C18 fatty acids increased by 86.8%.The higher surface hydrophobicity and more abundant S-layer protein and elongation factor Tu in L.reuteri JN101 cells made them easier adhesion to intestinal cells.?4?Metabolomics analysis showed that the metabolites between L.reuteri JN101 dosed with EPS-JN4 and glucose were significantly different.A total of 13 metabolites were up-regulated,among which N-acetyl-arginine,glucoside and 3-phosphoglyceride increased by112.36,84.51 and 19.62 times,respectively.These different metabolites could be originated in various metabolic pathways,including the arginine synthesis and metabolic pathway which may be the most important caused by different carbon sources,the metabolic pathways of spannolipids and phosphoglycerates leading to different cell membrane composition,and the metabolic pathways of glycolic acid and dibasic acid that caused differences in extracellular lactic acid and short-chain fatty acid contents.?5?EPS-JN4 can scavenge oxygen and hydroxyl free radicals and harbors lipid peroxidation inhibition activity,suggesting it can acts as an antioxidant that can inhibit DNA breakage caused by various free radicals.EPS-JN4 could capture free radicals generated by Pb²⁺induced cell metabolism,improve the activity of antioxidant enzyme system,prevent peroxidation damage of cell membrane lipid,and therefore improve the survival rate of cells.At the same time,Pb²⁺could be absorbed directly by EPS-JN4,the intestinal cells therefore avoided oxidative damage induced by Pb²⁺.Isothermal adsorption thermodynamic analysis showed that the adsorption process was consistent with Langmuir model,and Q_max was 53.2mg·g^-1.According to adsorption kinetics analysis,the adsorption process is fast and efficient,which conforms to the pseudo-second-order kinetics equation.The equilibrium adsorption value had reached 93.1%at 100 min,and then reached the equilibrium value at about 240 min.Fourier-transform infrared spectral analysis showed that Pb²⁺was absorbed by EPS-JN4through hydroxyl and carboxyl groups.