| Lactic acid bacteria exopolysaccharide(LAB EPS)is a general term for a class of carbohydrate compounds produced by lactic acid bacteria during the growth and metabolism process.,which has attracted the attention of researchers.In this project,strains with high production of exopolysaccharides and good basic probiotic properties were screened from lactic acid bacteria isolated and preserved in the laboratory in the early stage,and the biological activity of crude exopolysaccharides of the strains was studied,and a crude exopolysaccharide was selected for isolation and purification.The chemical composition,structure and biological activity of the purified components were further studied.(1)10 strains with higher exopolysaccharide production were screened from 40 strains of lactic acid bacteria by colony drawing method and phenol-sulfuric acid method;through acid tolerance(pH=2),bile salt tolerance(3.0 g/ L)test and gastrointestinal fluid tolerance test,it was found that 6 strains of lactic acid bacteria had good acid and bile salt resistance,and the survival rate of the strains in simulated gastrointestinal fluid was greater than 75%;lactic acid bacteria lysine decarboxylase activity,hemolysis The results of sex and antibiotic susceptibility experiments showed that 10 strains were preliminarily judged to be safe.Based on the above experimental results,4 strains were selected for the next stage of the experiment,and 2 strains of Lactobacillus paracasei(LZ9089,LZ9Y10),1 strain of Lactobacillus casei(LZ9183),and 1 strain of Lactobacillus brevis(LZ9285)were identified by 16 S r DNA sequencing.(2)Extract the crude exopolysaccharide of the screened strains,evaluate the bacteriostatic activity of the crude polysaccharide by the Oxford cup method,evaluate the antiallergic activity by the hyaluronidase test,and then evaluate the in vitro antioxidant activity and tumor cell activity of the crude polysaccharide.The inhibition rate was evaluated,and finally the immunomodulatory activity of crude polysaccharide was evaluated using RAW264.7 macrophages.The experimental results showed that the four exopolysaccharides had a certain inhibitory ability to Escherichia coli,and the diameter of the inhibition zone of LZ9089 ESP at 100 μg/mL was the largest,reaching17.54±2.53 mm;the inhibition rate of hyaluronic acid activity in the high-dose polysaccharide group was The hyaluronidase inhibition rate of LZ9183 EPS reached62.16±6.5% when the concentration was 500 μg/mL;the hydroxyl radical scavenging ability of polysaccharides was stronger,and the order of the hydroxyl radical scavenging rate at the concentration of 1000 μg/mL was: LZ9183 EPS> LZ9Y10 EPS> LZ9089EPS> LZ9285 EPS,and can be close to the hydroxyl radical scavenging rate of Vc;only LZ9285 EPS has weak inhibitory effect on Caco-2 cells and Hep G2 cells,but its inhibitory ability is much lower In the positive control 5-FU group;the proliferation of RAW264.7 macrophages was enhanced by the four extracellular polysaccharides.The highest proliferation rate of LZ9183 EPS at 500 μg/mL was 115.64±1.43%,and LZ9183 EPS and 285 EPS At the concentration of 1000 μg/mL,the NO concentration was significantly higher than that of the positive control LPS group.The phagocytosis rate of LZ9Y10 EPS and LZ9183 EPS was the highest at the concentration of 1000 μg/mL,reaching 137.37±0.23% and 131.86±0.84% of the blank control,respectively.Based on the basic probiotic characteristics of the strain,the production of extracellular polysaccharide and the biological activity,L.casei LZ9183 EPS was selected for isolation and purification,and its chemical composition,structure and anti-allergic activity were studied.(3)LZ9183 EPS obtained two single components LZEPS-1 and LZEPS-2 through ion exchange chromatography column and gel chromatography column.The results of chemical composition analysis and UV spectrum analysis showed that LZ9183 EPS and LZEPS-2 contained protein,the content of which was 4.94% and 1.72%,respectively.The molecular weight and monosaccharide composition of polysaccharide components were determined by HPGPC and ion chromatography,and the results showed that LZEPS-1 The molecular weights of LZEPS-2 and LZEPS-2 are 28.73 k Da and 95.58 k Da respectively.LZEPS-1 is composed of rhamnose,galactose,glucose and mannose,and the molar ratio is 4.44:4.37:86.32:4.87.LZEPS-2 is composed of glucose,galactose,It is composed of arabinose,mannose,xylose and glucuronic acid,and the molar ratio is31.67:8.32:0.64:56.72:2.33:0.32;it is found that LZEPS-1 and LZEPS-2 both have typical polysaccharide special absorption peaks by infrared spectrum analysis.It was further proved that LZEPS-1 contains pyranoside,and LZEPS-2 contains protein,mannose and uronic acid.(4)The antiallergic activities of LZ9183 EPS,LZEPS-1 and LZEPS-2 were studied by RBL-2H3 cell degranulation model.1 The inhibition rate was the highest at 500μg/mL,reaching 52.293±0.153%.The release level of HIS in each group was close to that of the normal group,the release of IL-4 was close to that of the positive control group,and the release of TNF-α was also significantly lower.In the model group,the component LZEPS-1 has a more significant effect,can effectively alleviate the allergic reaction mediated by Ig E,and has potential anti-allergic activity. |
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