Preparation,Structure Identification And Application Of Extracellular Polysaccharide From Lactic Acid Bacteria

Lactic acid bacteria(LAB)is the main source of probiotics,which can adhere to the mucosal surface and has probiotic properties such as regulating intestinal flora balance,lowering cholesterol and resisting oxidation.Extracellular polysaccharide(EPS)is a kind of carbohydrate secreted into the medium during the growth and metabolism of lactic acid bacteria.It has unique rheological properties and can be used as a stabilizer,gel and thickener in food,especially for fermented dairy products.In addition,EPS also has the functions of scavenging oxygen free radicals,increasing antioxidant enzyme activity and reducing lipid peroxidation.At present,the EPS yield is used as an indicator without considering the antioxidant activity of the polysaccharide to screened the major strains of EPS.Based on this,this study first screened 66 strains of lactic acid bacteria with high EPS yield and high antioxidant capacity and then identified the strains.The most suitable EPS conditions was obtained through the main factor screening and response surface methodology,and the crude polysaccharide was purified by ion exchange chromatography.The molecular weight and monosaccharide composition of the polysaccharide were characterized by gel permeation chromatography,infrared spectroscopy and liquid chromatography.Finally,the EPS strain and EPS were used for fermentation in sheep milk and its texture were studied by TA.XT plus physical property tester.1.The 66 strains of lactic acid bacteria were screened by the yield of extracellular polysaccharides.The results showed that all of the strains selected in the experiment could produce EPS with the different EPS yield.The ranged of EPS yield from 13.47~237.14 mg/L.There were 23 strains had a less than 50 mg/L yield,23 strains of 50~100 mg/L and 9 strains between 100~130 mg/L.Only 11strains higher than 130 mg/L,that 11 strains were rescreened in the next experience.2.11 strains of lactic acid bacteria were screened by the antioxidant activity of extracellular polysaccharides.The DPPH radical scavenging activity ranged from 26%to 38%and the ferrous ion chelation rate ranged from 14%to 40%.The highest yield of EPS strain was B62,its DPPH free radical scavenging activity was33.22%and the ferrous ion chelation rate was 29.36%.Five strains were identified by 16S rDNA.The results showed that strains B55 and B62 were both Lactobacillus fermentum,strains 7830,B30 and K2 were Lactobacillus plantarum,Lactobacillus acidophilus and Lactobacillus helveticus,respectively.3.The EPS-producing medium of lactic acid bacteria B62 was optimized by PB and response surface test.The optimal medium for producing extracellular polysaccharide was 5%sucrose,3.5%peptone and 0.45%potassium dihydrogen phosphate according to the optimal culture conditions.The results of the validation test showed EPS:217.98±13.24mg/L,DPPH radical scavenging activity:65.12±2.75%,ferrous ion chelation rate:41.20±3.68%,which increased 49.8%,96%and 40.3%than before optimization,respectively.4.The EPS purification components EPS1a,EPS2b,EPS3c,EPS4d and EPS5e were obtained by ion exchange chromatography from LAB B62,7830,K2,B55 and B30.The molecular weights were 2.41×10~4Da,1.62×10~4Da,6.42×10~3Da,6.45×10~3Da and 1.26×10~4Da,which determined by gel permeation chromatography.Infrared spectroscopy results show that these polysaccharides contain characteristic absorption peaks,in which the terminal carbons of EPS1a,EPS2b and EPS3c were consisted with interaction of?-and?-,while the terminal carbons of EPS4d and EPS5e were?-type.The analysis of monosaccharide composition showed that EPS1a composed of guluronic acid,rhamnose and galactose with a molar ratio of 2.7:1:2.4,EPS2b and EPS3c were composed of guluronic acid,EPS4d was composed of Gluuronic acid,glucose,galactose and the molar ratio was 1:1.1:1.2,EPS5e consists of glucose and galactose and the molar ratio was 1.6:1,which indicates that the EPS structure produced by different strains is quite different.5.The application results of EPS-producing strains in goat milk fermentation showed that the addition of EPS-producing strains could promote acid production and improve the texture of sour goat milk,acidity,hardness,consistency,viscosity and viscosity index compared with the control group.The B62 strain and EPS were added to the fermented goat milk in different proportions.It can be found that the pH of the fermented goat milk with strain B62 and EPS added at 4 h when the end of the fermentation assay was lower than 4.3 and the acidity was higher than 90°T,which was significantly higher than control group;DPPH radical scavenging activity and viscosity index of 3%B62 fermented goat milk were 69.03%and14.59gsec respectively,the ferrous ion chelation rate of 2%B62 fermented goat milk was 57.03%and the hardness of 1%B62 fermented goat milk adding was12.79g,which were 128%,61.6%,153%and 34.3%higher than the control group,respectively.The consistency and viscosity of the fermented goat milk with 1.5%EPS were 295.07gsec and 341.25mPa·s,which were 16.6%and 31.4%higher than the control group,respectively.These indicating that the addition of Lactobacillus fermentum B62 or EPS can improve the texture of fermented goat milk and improve oxidation resistance effectively.