Analyss Of Extracellular Polysaccharide Synthesis Ability And Eps Gene Cluster Of Streptococcus Thermophilus Strains

Streptococcus thermophilus(S.thermophilus)is one of the most economically valuable homologous fermented lactic acid bacteria in the world and is widely used in the production of dairy products.Extracellular polysaccharide(Exopolysaccharides,EPS)produced by Streptococcus thermophilus,has a wide range of applications,since it can improve the quality of dairy products,and has been proved to have antioxidant,antimicrobial and other probiotic properties.In this study,the yield of S.thermophilus CS1~CS22 were obtained by phenol sulfuric acid method and 3,5-dinitrosalicylic acid method.According to that,differences of the EPS yield among different strains was concluded,and the highly-produced strains CS5,CS6,CS9 and CS18 were identified.The optimization of culture conditions for ropy Streptococcus thermophilus CS6,which produced a large amount of EPS,was investigated by statistically-based experimental designs in order to improve the yield of EPS.Five factors including the inoculum amount,subculture times,fermentation temperature,after-ripening time and carbon source(lactose,galactose,fructose,glucose and sucrose)were examined for their significance on the yield of EPS using Plackett–Burman factorial design.The sinificant factors were screened.The regression equation of EPS yield was set up with those siginificant factors using Central Composite Design methods.And the optimum culture conditions were obtained as followings: inoculum size 4.34%,fermentation temperature 44 ?,ripening time 24 h,ripening temperature 4 ?,subculture times 3,skim milk 10% and lactose content 1.7%.Validation experiment suggested that the yield of EPS could reached 343.75 mg/L,similar to the forecast of regression equation,and has an increased ratio of 11.64% than before.The optimization of culture conditions for ropy Streptococcus thermophilus CS6 was supplemented with carbon sources(maltose,mannose,mannitol,xylose and ribose)and nitrogen sources(peptone,soy peptone,tryptone,beef extract and whey protein concentrate)experiments.The best carbon source and nitrogen source which can help produce the highest amount of EPS were maltose and soybean peptone respectively.Gradient experimental design was carried out to obtain the optimum concentration range for the further optimization of the carbon source and nitrogen source.This offered a foundation to optimize the culture conditions of S.thermophilus CS1~CS22 whose eps gene cluster are about to be sequenced subsequently.The study on the determination of the eps gene cluster of S.thermophilus CS1~CS22: firstly,the eps gene cluster reported was analyzed and classified,and according to the conserved sequences between 5' end deo D and 3' end orf14.9,the primers were devised(8 pairs).The genomic DNA of S.thermophilus CS1~CS22were used to amplify the eps gene cluster.The PCR products were identified,purified and sequenced.And the sequence was stitching,and the whole sequence of each eps gene cluster was obtained.In this study,three complete sequences of eps gene cluster of strain CS2,CS6 and CS10,as well as most sequence fragments of the eps gene cluster in other strains were obtained.Sequence analysis showed that the eps gene cluster of CS2,CS6 and CS10 were highly homologous to that of S.thermophilus ASCC 1275(ID: CP006819.1),containing additional eps2 C and eps2 D genes associated with EPS length,which was assumed to be benificial to EPS synthesis;also containing rich glycosyltransferase genes,which were responsible for the transport of glucose,galactose,rhamnose,UDP-N-acetylglucosamine and UDP-furan galactose,Those rich glycosyltransferase genes were conducive to the formation of a unique monomer composition of the EPS.This study lays a foundation for further study of the effect of eps gene cluster on producing EPS among different strains.