Screening Exopolysaccharide-producing L.helveticus And Structure,Antioxidant Activities Of Exopolysaccharides

In recent years,lactic acid bacteria exopolysaccharides have received increased attention and was widely used in medicine and food industry because of their role in modulating the rheological and physical properties to improve texture,stability and beneficial bioactivities on human health.Food-associated LAB from koumiss is granted the status of generally recognized as safe and their products,especially EPS,are regarded as suitable candidates for functional food.Koumiss as a traditional fermented dairy product is developed and known for the diversity of lactic acid bacteria and its unique physiological actions.It evaluated lactic acid bacteria from koumiss in east Inner Mongolia,not only comprehensive analyzed and identified EPS-producing lactic acid bacteria but also screened out higher EPS-producing L.helveticus.In order to improve the production of EPS,mutagenesis was used to obtain the target strains and optimized the culture condition.It explored the influence of the key enzymes in biosynthesis pathway to the structure and yield of EPS.It determined the monosaccharide,molecular weight,the primary structure and part physiological function of EPS through a variety of methods.It is a foundation for excavated nutrition and health care function of koumiss.The main results are as follows:(1)In total,72 strains were isolated from 12 koumiss samples Inner Mongolia,including 56 EPS-producing strains that were identified by phenotypic characterization,traditional method and 16S rRNA gene sequence as Enterococcus duran,Leuconosloc lactis,L.helveticus,L.casei,L.plantarum,L.delbrueckii and Leu.Mesenteroides respectively.The highest EPS-producing strain was L.helveticus and average yield to 110.39 mg/L.(2)L.helveticus SMN2-1 selected from 16 strains was mutated.UV and nitrosoguanidine respectively were used to single mutation and determined the initial mutagenesis condition.In Box-Behnken design,uv-nitrosoguanidine composite mutagenesis was under way for UV irradiation time,concentration of nitrosoguanidine and processing time as independent variables,the fatality rate for response values.The best mutation conditions was that ultraviolet irradiation time was 40.37 s,nitrosoguanidine concentration of 0.35 mg/mL,the processing time in 34.87 min,the fatality rate was 88.65%.It acquired a strain of the highest and more steadily yield of EPS named L.helveticus UN-8 by preliminary screening and secondary screening.(3)Single factor experiment of mutagenic L.helveticus UN-8 was carried out for EPS biosynthesis condition of carbon source,nitrogen source,fermentation time,fermentation temperature,initial pH and other factors.determined the the scope of each condition.Application of Plackett-Burman and Box-Behnken design,it determines the main factors and optimum condition on EPS production of L.helveticus UN-8.The amount of lactose was 21.65 g/L,fermentation time was 28.79 h,inoculation quantity was 2.01%,and yield of EPS was 408.5 mg/L that increased 2.1 times than L.helveticus SMN2-1.(4)Crude polysaccharides of L.helveticus SMN2-1and UN-8 were further purified by DEAE-52 ion exchange chromatography,gel-permeation chromatography.They molecular weight were EPS-S1:1.01×106,EPS-S2:1.5×106,EPS-U1:0.85×106,EPS-U2:1.2×106,respectively.The EPS structures were investigated by IR,HPLC,methylation,GC-MS and 1D.2D-NMR analysis,and they were composed of different proportion of glucose and galactose.(5)When lactose and glucose were substrate in the process of biosynthesis EPS of L.helveticus SMN2-1and UN-8,the relationship between enzymes activity and the EPS production was studied,and it showed that,the EPS yield,molecular weight and proportion of monosaccharide were difference,but no change in monosaccharide composition when different sugars as carbon source.A-PGM was the key enzyme to increase production of EPS.(6)The antioxidant activity of EPS-S1,EPS-U1 were evaluated with the scavenging abilities on superoxide,hydroxyl radicals and DPPH in vitro,and the results indicated that they all had good antioxidant activity,especially EPS-U1.Mice test results showed that EPS-S1 could improve a variety of antioxidant enzymes(SOD,GPx and CAT)activity and total antioxidant capacity and decreased the MDA concentration in aging-model mice’s serum,brain and liver.In addition,EPS-S1 can activate the NK and CTL cell viability and improve the level of IL-2,IL-10 and TNF-a in serum,and enhance the body immunity accordingly.