| Exopolysaccharide(EPS)of Streptococcus thermophilus is a natural high molecular polymer that is produced and secreted into the cell during growth and metabolism of the bacterium.As a new type of natural food additive,it has anti-tumor,anti-oxidation and other benefits and application potential.In this study,EPS-producing conditions of S.thermophilus strain CS6 were optimized;EPSs that were produced in skim milk supplemented with 2% lactose were separated and purified to determine their functional groups and molecular weights,and the primary analysis of the antioxidant activity of EPSs was carried out.The PCR method was used to analyze the eps gene clusters.The aim was to predict the monomer composition of EPS,and combined with the study of the antioxidant activity of EPS,provided a theoretical basis for the study of the structure-activity relationship of EPSs of S.thermophilus.In the previous study of our research group,a S.thermophilus CS6 with outstanding EPS production was obtained.In this study,response surface analysis was used to determine the optimal EPS fermentation conditions for CS6 strain.First,the PB experiment was conducted through a single-factor initial p H value of the medium experiment,combined with the optimal level range of the remaining six single factors(subculture times,inoculum amount,fermentation temperature,after-ripening time,maltose content,and soybean peptone content)determined in previous study.Screening the key factors in the single factor;using response surface center composite experiment method(CCD)to opt imize the EPS extraction process,the optimal culture conditions were as follows: soybean peptone content was 5.86%,subculture times was 3.32,and inoculum amount was 3.83%,initial p H was 6.5,the fermentation temperature was 42°C,the after-ripening time was 24 h,and the maltose content was 2.5%.Through verification experiments,the crude EPS content was 683.24 ±2.51 mg/L,which was close to the predicted value of 681.88 mg/L.It was proved that this model can simulate and predict the optimal fermentation conditions of EPS produced by S.thermophilus CS6.In this study,EPS produced by the addition of 2% lactose in skim milk were separated and purified by DEAE-52 column to obtain two elution fractions.FT-IR spectral analysis revealed that the above two fractions all had polysaccharide-specific functional groups and were polysaccharides,which were named neutral polysaccharide EPS-1 and acid polysaccharide EPS-2,respectively.HPLC analysis showed that the liquid chromatogram of EPS-1,a polysaccharide component,had a small peak next to a single peak,and was then further purified by Sephadex column for subsequent studies.The liquid chromatogram of EPS-2 was presented a relatively single and symmetrical single peak indicates that EPS-2 was purified on a DEAE-52 column to obtain relatively uniform components with relatively high purity.The retention time was substituted into the dextran standard curve,and the average molecular weights of EPS-1 and EPS-2 were 6914 Da and 20295 Da,respectively.In vitro antioxidative activity analysis of EPS indicated that both neutral component EPS-1 and acidic component EPS-2 had a certain amount of free radical scavenging capacity,and its free radical scavenging rate increased with the increase of polysaccharide concentration,and The free radical scavenging capacity of the acidic component EPS-2 is higher than that of neutral components.In the previous study of our research group,we analyzed the partial eps gene clusters of 22 strains of Streptococcus thermophilus.Based on this study,primers were designed based on the eps gene cluster information of the existing strains,and the eps gene clusters of the 22 strains were finished.Sequencing analysis obtained the complete eps gene cluster sequence information of CS12,CS13 and CS15 strains of Streptococcus thermophilus,and the sequence information of most eps gene cluster fragments of other strains.Bioinformatics analysis revealed that the eps gene clusters of the CS12,CS13,and CS15 strains were as high as 99% homologous to the ASCC 1275 strain.The homology to the eps gene cluster of Sfi39,Sfi6 and EU20 strains can reach 96%.The CS12,CS13,and CS15 strains contain additional EPS length genes(eps2C and eps2D)that are related to EPS length,presumably involved in the regulation of EPS molecular weight.At the same time,the above three strains all have eps E~eps J genes and are responsible for encoding different glycosyltransferases,so that the extracellular polysaccharides produced by different strains have differ ent monomer components... |
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