Study On Exopolysaccharide Of Three Strains Of Lactic Acid Bacteria Isolated From Sour Meat Of Dong Minority

Exopolysaccharide(EPS)produced from lactic acid bacteria(LAB)can be thickener,stabilizer and moisturizer as food additives.The EPS has becoming a research hotspot because of their probiotic characteristics of intestinal immunity,antitumor and antioxidant activity.Sour meat is a traditional fermented meat product from Dong minority in Guizhou containing abundant microbial resources,which has long history and local features.In this paper,we took 3high-yield EPS LAB strains(SR2-2,SR8,SR12-1)selected from sour meat as objects to explore the feimentation,extraction,purification and antioxidant activities of EPS.The main results and conclutions are as follows:1.Adopted Single factor and orthogonal experiment to investigate the effect of medium and feimentation parameter on LAB EPS yield and optimize the fermentation conditions of 3 LAB strains.LAB SR2-2: 2% Additive volume of sucrose,tryptone:peptone=2:1,feimentation temperature was 31 ?,feimentation time was 24 h.Verify experiment measured the EPS yield under the optimized conditions was 572.06 mg/L,increased by 58.32% before optimization.LAB SR8: 3% Additive volume of sucrose,peptone:beef extract=1:1,feimentation temperature was40?,feimentation time was 24 h.Verify experiment measured the EPS yield under the optimized conditions was 627.50mg/L,increased by 36% before optimization.LAB SR12-1: 2% Additive volume of glucose,tryptone:peptone=1:1,feimentation temperature was 40?,feimentation time was 24 h.Verify experiment measured the EPS yield under the optimized conditions was436.49mg/L,increased by 21.98% before optimization.2.EPS yield of 3 LAB strains fermentation in separate was lower than multiple strains co-culture.The combination of SR8 and SR12-1 gain the most at 316.16±18.57mg/L,which was23% higher than SR8 and 2.32 times than SR12-1.3.Adopted Single factor and orthogonal experiment to investigate the effect of deproteinization,ethanol precipitation on LAB EPS yield and optimize the EPS extraction processof 3 LAB strains.LAB SR2-2:Added 80%TCA till the final concentration was 10% to remoal protein,4 times volume ethanol and precipitation for 24 h.Verify experiment measured the EPS yield under the optimized process was 806.06mg/L,increased by 53.31% before optimization.LAB SR8: Added 80%TCA till the final concentration was 10% to remoal protein,4 times volume ethanol and precipitation for 24 h.Verify experiment measured the EPS yield under the optimized process was 342.30mg/L,increased by 44.21% before optimization.LAB SR12-1: Added80%TCA till the final concentration was 4% to remoal protein,4 times volume ethanol and precipitation for 24 h.Verify experiment measured the EPS yield under the optimized process was 294.55mg/L,increased by 52.43% before optimization.4.The UV scanning showed that there were barely protein,polypeptide and nucleic acid substances in 3 LAB EPS.Three EPS fractions called E1,E2 and E3 was gained through anion exchange chromatography on DEAE-Cellulose column which eluted with deionied water,0.2M and 0.5M NaCl solution.E1 was neutral polysaccharid,E2 and E3 were acidic polysaccharid.5.Evaluation antioxidant activities in vitro of LAB SR8 EPS including DPPH free radical and hydroxyl radical scavenging activities.At the concentration of 81 ug/m L,the DPPH free radical scavenging of neutral polysaccharide E1 was significantly higher than E2,E3,CEPS(p<0.05).At the concentration of 8.0 mg/mL,the DPPH free radical scavenging of CEPS was50.41±1.40(%),lower than positive control Vc.At the concentration of 81 ug/m L,The hydroxyl radical scavenging of acidic polysaccharide E3 was significantly higher than E1,E2,CEPS(p<0.05).At the concentration of 8.0 mg/mL,the hydroxyl radical scavenging of CEPS was up to90%,close to positive control of Vc..6.Evaluation antioxidant activity in vivo of LAB SR8 EPS by establishing aging mice model.The mice's body weight of all gavage groups grew normally in which the high-dose group significantly improved liver proportion(p<0.05)as the same effect as positive control Vc.This indicated LAB SR8 CEPS was nonpoisonous,harmless and had no negative effect on mice's growth and liver health.7.Determination of antioxidant enzymes and related indicators in vivo.T-AOC: In serum,the level of dose groups were higher than model control group,of which the middle-dose and high-dose groups showed extremely significant difference compared to model control group(p<0.01).In liver,the level of all dose groups were extremely significantly higher than model controlgroup(p<0.01).CAT: In serum,activity of low-dose group showed significant difference(p<0.05)and middle-dose,high-dose groups showed extremely significant difference(p < 0.01)compared to model control group.In liver,activities of all dose groups were extremely significantly higher than model control group(p<0.01).GSH-PX: In serum,activity of low-dose group showed significant difference(p < 0.05)and middle-dose,high-dose groups showed extremely significant difference(p<0.01)compared to model control group.In liver,activities of all dose groups were extremely significantly higher than model control group(p<0.01).SOD: In serum,activities of all dose groups improved compared to model control group and high-dose group showed extremely significant difference(p<0.01).In liver,compared with blank control,the activities of low-dose,middle-dose groups significantly improved(p<0.05),and high-dose group showed extremely significant difference(p < 0.01).MDA: In serum,MDA content of all dose groups reduced compared to model control group and high-dose group showed extremely significant difference(p<0.01).In liver,MDA content of dose groups reduced compared to model control group and high-dose group showed extremely significant difference(p<0.01).MAO: In liver,activities of middle-dose and high-dose groups extremely significantly improved(p<0.01).To sum up,the study has optimized fermentation conditions and EPS extraction process of 3LAB strains,as well as the combination of co-culture to promote the EPS yield.Separated and purified all fractions of EPS of LAB SR8.It was confirmed that exopolysaccharide produced from LAB SR8 does have antioxidant activities.