The Primary Study Of Antagonistic Substance Produced By Enterococcus Faecalis ZD34 Strain

The main objective of this research was screening microbial strains that could produce antagonistic substances in foods and their raw materials.The antimicrobial activity was detected by agar diffiution assay with Micrococcus luteus,Listeria monocytogenes,Escherichia coli and Staphylococcus aureus as indicator strains.Then antimicrobial substance was confirmed elementarily as bacteriocin-like through methods as follows:the cell-free culture supernatant obtained by centrifugation of culture adjusted to pH7.0 in order to eliminate inhibitory activity of various acids and then antagonistic substance was precipitated by 60%saturation of ammumiaon sulfate.The conclusions of this study as follows:1.48 strains that could produce antagonistic substance were screened from non-sterilized raw milk.One named as strain ZD34 could secrete antimicrobial substance that had inhibitoty activity against M.luteus,L.monocytogenes and E.coli,but no inhibitory activity against S. aureus.At the same time,another strain named as ZD30 was also screened had similar culture and antimicrobial characters,but had smaller inhibitory activity compared with strain ZD34.The antagonistic substances produced by both of strain ZD30 and ZD34 could be precipitated by 60% saturation of ammumiaon sulfate.It was confirmed that both of strain ZD30 and ZD34 belong to Enterococcus faecalis on the bases of 16S rDNA sequence molecular identification.Because E. faecalis ZD34 strain had bigger antimicrobial activity and its antagonistic substance might be a new enteriocin via the advance experiments,so this research choosed strain ZD34 as main objective to research culture conditions for the growth of strain ZD34 and production of its antagonistic subatance,then optimized the best strategy of purification to obtained purified sample which was used for farther study its molecular weight and other characters.2.Through optimizing cultue conditions for growth of strain ZD34 and production of its antimicrobial substance elementarily,it was indicated that strain ZD34 could grow and produce antagonistic substance in four culture media of MRS,M17,CM and APT,and CM was the best one of them with the least non-aimed proteins or peptides were precipitated by 55%saturation of ammumiaon sulfate along with aimed antagonistic substance.Because it was benefial for the latter experiments of purification,so choosed CM broth medium as fermentation culture broth.The components of the modified CM broth medium were optimized as follows:sucrose 10.0g L^-1, tryptone 10.0 g L^-1,yeast extract 10.0 g L^-1,K₂HPO₄ 1.0 g L^-1,NaCl 2.0 g L^-1,MgSO₄·7H₂O 0.2g L^-1,Tween20 0.2%(v/v),and was adjusted to pH7.0.The improved CM broth medium was inoculated with 2%(v/v) of seed culture which was prepared in 50 mL modified CM broth medium that had been inoculated with strain ZD34 and grown at 34℃for 10 h,and then was incubated at 34℃without shaking for 28 h.The curve of growth of E.faecalis ZD34 strain and production of its antagonistic substance was drawed in modified CM broth medium inoculated with 2%(v/v) of seed culture and was incubated at 34℃,the results showed that E.faecalis ZD34 strain reached stationary phase after 26 h,and antagonistic substance was excreted along with cellular propagation.The largest yield was achieved at stationary phase correspondingly.3.The supernatant was obtained by centrifugation at 12000 rpm for 5min from modified CM broth medium inoculated with 2%(v/v) of seed culture and then was incubated at 34℃for 28h anaerobically.Then the cell-free supernatant was collected by centrifugation at 12000 rpm for 5 min and them was precipitated by 55%saturation of ammumiaon sulfate.The mixture was kept at 4℃overnight.After centrifugation at 12000 rpm for 10 min at 4℃,the deposition was solubilized in 0.2 tool L^-1 sodium acetate buffer(pH5.0) which contains 20 mmol L^-1 NaCI.This solution was the sample that was used for purification.The crude preparation was loaded in CM-52 ion-exchange chromatography and eluted with 0.2 mol L^-1 sodium acetate buffer(pH5.0) which contains 0～1.0 mol L^-1 NaCI at interval of 0.1 mol L^-1 NaCI,the active fraction was eluted by 0.2 mol L^-1 sodium acetate buffer(pH5.0) contains 0.4 mol L^-1 NaCI and it was transparent.The tubes of active fraction were mixed and could be concentrated by dialysis membrane with the molecular weight cut-off of 1000 Da or be precipitated by 80% saturation of ammumiaon sulfate overnight at 4℃,them reclaimed with sterile distilled water after centrifugation at 12000 rpm for 15 min.The reclaimed active solution re-chromatography on Sephadex G-25 gel filtration and washed by sterile distilled water.One fraction also was obtained. The active fractions were analysed by Tricine-SDS-PAGE,it was ascertained that the molecular weight of the antagonistic substance was about 3000 Da4.The active fraction from Sephadex G-25 gel filtration chromatography was further purified by C18 reverse-phase high-performance liquid chromatography(RP-HPLC).The preparation was applied to Hypersil QDS2 column(4.6mm×250 mm,elit,made in china) and washed with separation conditions as follows:mobile phases buffer A—5%(v/v) water and 0.1%(v/v) TFA in acetonitrile,buffer B—5%(v/v) acetonitrile and 0.1%(v/v) TFA in water,respectively;40min linear gradient of 40%to 100%buffer A at a flow rate of 1 ml/min.The peptide content was estimated by absorbance measuring at 280nm and the activity of each fractions were tested after vacuumized at 50℃in order to volatilize acetonitrile completely.The result indicated that antagonistic substance produced by E.faecalis ZD34 was purified by this four-step procedure,namely was precipitated by 55%saturation of ammumiaon sulfate,CM-52 ion-exchange chromatography,Sephadex G-25 gel filtration chromatography and RP-HPLC.One main fraction with retain time of 10.405 min has antimicrobial activity.Mass spectrometry analysis for the fraction from RP-HPLC with ESI-MS/MS indicated that its molecular weight was about 3434 Da.5.The characters of the antognistic substance were studied.The results showed that it was stable at pH1～13,and its antimicrobial activity almost was not influenced after was treated at 100℃for 10 min,whereas inactivated sharply after was treated at 100℃for 60 min.Therefore,this antagonistic substance was stable.The antagonistic substance was sensitive to proteinase E(37℃, pH7.0),proteinase K(37℃,pH7.0) and trypsin(30℃,pH7.0) from the fact that the antimicrobial activity was lost after treated for 6 h,which also further confirmed it was a bacteriocin.