| Bacillus amyloliquefaciens is considered as a safe bacterial strain by Food and Drug Administration in the USA.Accumulating evidences have reported that microorganisms and their secondary metabolites have good probiotic properties and the polysaccharide with some beneficial effects can alleviate colitis through maintaining the intestinal homeostasis.Numerous researches focus on the antimicrobial peptides and enzymes of B. amyloliquefaciens while lacking the report about the exopolysaccharides(EPS)and its role in relieving colitis.Therefore,we took B.amyloliquefaciens DMBA-K4 which obtained from Chinese Puer tea as materials and investigated its safety and identification.We then optimized its EPS yield and assessed the antioxidant activity in vitro.EPS-K4 was purified and obtained and its structure was identified by UV-Vis spectrum,FTIR,GPC and so on.We investigated the mechanism of EPS-K4 in the aspect of ameliorating colitis via gut microbiota modulation through animal trial.It provided a theoretical basis for the development of a novel immune stimulus to relieve colitis.An EPS-producing strain was isolated from Yunnan Puer tea.This strain was defined as B.amyloliquefaciens DMBA-K4 by 16 S r DNA and the physiological and biochemical properties.It was confirmed as safe and avirulent by acute oral toxicity rational test in mice.Single factor trials and response surface tests were performed to optimize its EPS yield,and its antioxidant activity in vitro was also investigated.The optimal fermentation conditions were as follow: the culture medium consisted of 4 g /L sucrose,5 g /L tryptone,10 g /L yeast,5 g /L Na Cl,p H 7.6,inoculator amount of 3.10% and fermented for 18 h.Under these conditions,the maximal yields of EPS increased by 155.7% and reached 171.31 mg/L.The vitro antioxidant results showed that EPS had strong antioxidant activity.When the concentration of EPS was 5 mg /m L,the scavenging rate of DPPH free radical and ·OH was78.60% and 75.47%,respectively.EPS-K4 was purified and obtained via anionic-exchange chromatography and gel permeation chromatography which was with an average molecular weight of 10,067 Da and consisted of mannose,glucose,fructose,and galacturonic acid with a molar ratio of 85.25∶8.40∶3.25∶2.39.It had obvious absorption peak in wavelength of 220 nm.The result of infrared spectrum showed the typical absorption peak of carbohydrate with the radical such as C-H,C=O,-OH,C-O-H and C-O-C.The main chain of EPS-K4 was→6)-β-Glcp-(1→2)-β-Glcp-(1→2)-α-Manp-(1→6)-β-Manp-(2→β-Manp-T while the branched chain was →6)-α-Manp-(1→ which was connected with →2,6)-β-D-Glcp-(1→.The SEM indicated that EPS-K4 presented a smooth flake structure with some holes in the surface.The mechanism of EPS-K4 in the aspect of relieving symptoms in colitis mice was explored from microbiota modulation,inflammatory response and intestinal barrier.The results showed that the levels of pathogenic bacteria,such as Enterococcus and Escherichia-Shigella,were significantly suppressed.In contrast,the levels of Lactobacillus and intestinal microbiota diversity were increased.EPS-K4 could stimulate mucin secretion to maintain the integrity of the gut barrier and decrease the levels of virulent substances(endotoxin,diamine oxidase,and D-lactic acid)in serum.The pro-inflammatory cytokines(TNF-α)in serum were reduced by 50.96%,conversely,anti-inflammatory cytokines(IL-10)were increased by 49.54%.In conclusion,B.amyloliquefaciens DMBA-K4 which was isolated from Yunnan Puer tea was safe and avirulent.The maximal yields of EPS from strain DMBA-K4 reached 171.31mg/L in optimal fermentation conditions and it had strong antioxidant activity.EPS-K4 which was purified and obtained mainly consisted of mannose and glucose and it had positive influence on relieving symptoms in colitis mice from microbiota modulation,inflammatory response and intestinal barrier.These findings indicated the possibility of strain DMBA-K4 and EPS-K4 in the aspect of colitis prevention.The research provided theoretical support to EPS as a chemotherapy immunostimulant and widened the utilization prospect of novel bio-polysaccharide in food and drug industry. |
