Preparation Of Lactobacillus Rhamnosus Microcapsule And The Regulation Of Extracellular Polysaccharide On Intestinal Flora In Mice

Probiotics and probiotic extracellular polysaccharides have the functions of regulating the balance of the intestinal flora of the body,improving the body's immunity,lowering cholesterol,and lowering blood lipids.However,probiotics have poor tolerance to bile salts and acid environment in gastric juice,which will limit their probiotic function.The protection of probiotics by microencapsulation technology is a hot research topic at home and abroad.In this thesis,the optimal microencapsulating conditions of Lactobacillus rhamnosus were optimized,and the storage stability,tolerance of the resulting microcapsules in simulated gastric juice and release in simulated intestinal juice were studied.The extracellular polysaccharide was extracted fromL.rhamnosus by water extraction and alcohol precipitation,and its chemical composition and structure were characterized.By establishing a mouse model of acute colitis induced by dextran sodium sulfate(DSS),the effect of the extracellular polysaccharide of L.rhamnosus ZFM231 on the intestinal flora of mice was studied,and the mechanism of action was studied.The results of this thesis can provide a theoretical basis for the research and development of L.rhamnosus and its exopolysaccharides in related functional foods.The main findings are as follows:1.In this thesis,the endogenous emulsification and condensation method was used to prepare Lactobacillus rhamnosus microcapsules.The fabrication conditions of the wet microcapsules were optimized by single factor tests,followed by response surface methodology.The optimized conditions were ascertained as:whey protein concentration 7.6%,rotational speed 800 r/min,emulsifying time 2.9 h,and water oil ratio was 1:4.Under these conditions,the the protective agent was screened,and pectin was selected as the protective agent.The addition of pectin was 2:4(bacterial solution:pectin,V/V).The embedding rate of L.rhamnosus in the microcapsule lyophilized powder was determined to be 77.71±2.05%,and the particle size of microcapsule was 172±1.8?m.2.The protective effect of the lyophilized powder of microcapsules on Lactobacillus rhamnosus was evaluated by using a simulated gastric juice model in vitro.The results indicated that the number of viable bacteria in the microcapsules decreased from 8.78 log(CFU/mL)to5.82 log(CFU/mL)after 90 min treatment in simulated gastric juice,which significantly slowed down the reduction of viable bacteria compared with the non encapsulated Lactobacillus rhamnosus(the number of viable bacteria decreased from 8.89 to 3.90 log(CFU/mL)).The number of viable bacteria was 7.60 log(CFU/mL)after 90 min treatment in simulated intestinal juice,with a releasing rate of 86.56%.When the encapsulated Lactobacillus rhamnosus was stored at 4?for 28 days,the number of viable bacteria decreased from 8.78 log(CFU/mL)to7.32 log(CFU/mL),while that value for unencapsulated L.rhamnosus decreased from 8.89log(CFU/mL)to 4.50 log(CFU/mL),indicating that the storage stability of L.rhamnosus microcapsules had been greatly improved.3.The extracellular polysaccharide(EPS)of Lactobacillus rhamnosus was prepared by extracting from the fermentation liquid using water extraction method,followed by alcohol precipitation.The decolorization effect of various macroporous resins on EPS was screened.The results showed that macroporous resin D4020 was the best,and the decolorization rate reaches 75%.Analysis of the chemical components of EPS revealed thatthe contents of total sugar,protein,sulfate group and uronic acid were determined as 44.00%,3.65%,12.16%and13.00%,respectively.The monosaccharide composition of EPS was determined by ion chromatography.It was found that EPS was mainly composed of mannose,glucose,galactose,glucosamine and a small proportion of rhamnose and glucuronic acid.The UV spectrum analysis indicated that EPS might contain a small amount of protein and almost no nucleic acid and pigment.The relative molecular weight of polysaccharides was estimated as 1.32×10⁴by high performance gel permeation chromatography(HPGPC).The IR spectra analysis showed that EPS contains the characteristic absorption peaks of polysaccharides at 530 cm^-1,920 cm^-1,1060 cm^-1,1400 cm^-1,1650 cm^-1,2930 cm^-1,3400 cm^-1.¹H-NMR analysis indicated that the monosaccharides in EPS were mainly linked by?-glycosidic bonds.4.The effect of extracellular polysaccharide of Lactobacillus rhamnosus ZFM231 on intestinal microflora of mice was investigated by establishing DSS induced acute colitis model,the following results were obtained:(1)Low(100 mg/kg),medium(300 mg/kg)and high(600 mg/kg)doses of EPS were used to treat the mice with acute colitis.The macroscopical symptoms of mice with acute colitis were alleviated the addition of EPS.It was found that moderate and high doses of EPS had obvious therapeutic effect,the weight loss and colon shortening of mice were improved,DAI score was also significantly decreased,and the colon mucosal injury of mice was improved.These results indicate that EPS treatment has a good therapeutical effect on UC mice.(2)The effect of EPS on intestinal microecology of mice with acute colitis was investigated by employing 16S rDNA high-throughput sequencing and bioinformatics analysis.The results showed that compared with the normal group,the intestinal flora in DSS induced acute colitis mice changed,and the similarity of the intestinal flora between the groups reduced.The intestinal flora of mice recoverd to a certain extent after treatment with EPS,thereby improving the similarity of the intestinal flora between the groups.Compred with model group,Firmicutes/Bacteroides(F/B)ratio decreased,indicating that EPS could relieve inflammation and restore the structural imbalance of intestinal flora in mice with colitis.After treatment with EPS,Phylum Firmicum decreased significantly,but Phylum Bacteroides and Phylum Verrucae increased significantly.Compared with the model control group,the amount of acetic acid,propionic acid and butyric acid metabolized by intestinal microorganisms in EPS treated mice increased significantly.(3)After EPS treatment,the content of anti-inflammatory factor TGF-?in colonic cells of mice with acute colitis increased significantly,while the content of pro-inflammatory factor TNF-?decreased significantly.These results indicate that EPS treatment can alleviate the symptoms of UC mice to a certain extent.