Purification Of Exopolysaccharides Of Lactobacillus Rhamnosus And Their Hypolipidemic And Intestinal Flora-regulating Activities

Studies have shown that Lactobacillus rhamnosus extracellular polysaccharide?EPS?has many biological activities,such as anti-oxidation,lowering serum cholesterol,lowering blood fat,improving intestinal micro-ecological balance,and enhancing immunity.A polysaccharide-producing bacterial strain,Lactobacillus rhamnosus ZFM231 was screened in our lab.This thesis aims to provide a theoretical basis for the development of Lactobacillus rhamnosus related foods.In order to improve the yield of EPS,fermentation conditions of Lactobacillus rhamnosus ZFM231 were optimized.The EPS was obtained by water extraction and alcohol precipitation,and followed by the separation and purification.Chemical composition of the purified fractions was analyzed,their structures were characterized.The biological activities of EPS and its purified fractions,including hypolipidemic activities and intestinal flora regulatory were investigated.The main findings are as follows1.The optimal fermentation conditions of Lactobacillus rhamnosus for the production of EPS were ascertained as:the optimal incubation time 24h,and the optimal carbon source was glucose.Under these optimal conditions,the yield of EPS was 220±0.4 mg/L.2.The chemical composition analysis showed that the total sugar,protein,sulfate and uronic acid contents of EPS were 61.65%,1.34%,16.81%and 10.53%,respectively.The UV spectrum showed that the crude EPS contains a small amount of protein and is basically free of nucleic acids and pigments.The molecular weight of EPS measured by high performance gel permeation chromatography?HPGPC?was 2.76×10⁴Da.The infrared spectrum showed that EPS has the characteristic absorption peaks of polysaccharides,being at 3400 cm^-1,2930cm^-1,1650 cm^-1,815 cm^-1,and 590 cm^-1.3.The EPS was separated by DEAE-52 column,obtaining four fractions EPS-0,EPS-0.1,EPS-0.3 and EPS-0.5,with a yield of 21.25%,25%,18.75%and 20%.These fractions underwent further purification on a Sephadex G-100 chromatography column,providing four purified components,which were labeled as EPS-0M,EPS-0.1M,EPS-0.3M and EPS-0.5M,with a recovery of 88%,90%,89%and 88%,respectively.Their molecular weights were measured as 1.40×10⁴,1.01×10⁴,1.42×10⁴,and 2.47×10⁴ Da,respectively.;their total sugar contents were determined to be 70.28%,73.83%,61.71%,53.96%;their sulfate contents were5.73%,8.75%,15.81%,18.84%;and their uronic acid contents were 9.81%,8.38%,10.24%and 21.22%,respectively.EPS and the purified fractions are mainly composed of mannose,glucose,galactose,together with a small amount of rhamnose,xylose,fucose and arabinose.The UV spectrum showed that the purified fractions were basically free of nucleic acids,proteins and pigments,with the exception of EPS-0.3M,which contained a small amount of protein that is difficult to remove,indicating an effectiveness of the purification.The purified fractions were characterized by IR spectra,¹H and ¹³C NMR spectra.4.The in vitro antioxidant capacity of EPS and the four purified components were determined.The results showed that the reducing power,free radical?DPPH,hydroxyl radical,superoxide anion radical?scavenging ability and iron ion chelation ability of the purified fractions were basically better than those of the EPS.The cholesterol-lowering and triglyceride-lowering activities of the four purified fractions were evaluated by the high cholesterol and high triglyceride model of Hep G2.EPS-0.5M was found to possess the strongest hypolipidemic activity among the four fractions.EPS fractions exhibited an appreciable inhibition on lipase,suggesting that inhibition on lipase is possibly one of the pathways of EPS to reduce the blood lipid.5.The effects of the purified EPS fractions on healthy human intestinal microflora were studied by constructing an in vitro fermentation model of intestinal microbes.The results of thin layer chromatography?TLC?showed that after 24 h of fermentation,the degradation rate of polysaccharides in intestinal microorganisms reached a maximum of 84.33%.After 24 h of fermentation,EPS was decomposed by the intestinal flora to produce short-chain fatty acids such as acetic acid,propionic acid,and butyric acid.Among them,the concentration of acetic acid in EPS-0.5M group is the highest;the concentration of propionic acid in EPS group is almost higher than that in Glc group;the concentration of butyric acid in EPS-0.1M group and EPS-0.5M group is much higher than that in EPS-0M,EPS-0.3M and Glc group.6.16S rDNA high-throughput sequencing was used to investigate the effect of EPS on intestinal microbial diversity and flora structure.The results showed that the species diversity of each sample in the EPS-0M group and EPS-0.5M group was richer.The results of PCo A analysis showed that the source of the difference in the flora composition between the Glc group and the EPS group was the carbon source.The correlation between the Genus classification level of bacteria and SCFAs was evaluated by environmental factor association analysis.The results showed that the correlation coefficients between acetic acid,propionic acid,butyric acid,valeric acid and species composition were large.The main strains affecting SCFAs production are Phascolarctobacterium,Eggerthella,Parabacteroides,Bacteroides,Faecalibacterium,Alistipes,Collinsella,etc.The correlation coefficient between isobutyric acid,isovaleric acid and species composition is small,and there is almost no significant difference.