Separation And Purification Of Bacteriocin SE14 Screened By A Marine Derived Strain Staphylococcus Epidermidis S14

The diversity of marine ecological environment has brought the diversity of microbial resources.The results of the research for the secondary metabolites produced by marine microorganisms showed that these active substances are structurally unique and can be used as an effective drug for the treatment of cardiovascular diseases or tumors.The study is devoted to the research of secondary metabolites active substances which will provide reliable theoretical basis for marine microbial resources in medical drugs,food industry and agricultural marine preparations.The contents of this study include:?1?A total of 249 strains were isolated from the intestine and viscera of eight species marine fishes which from coastal areas of Wenzhou.Taking the food pathogens as the indicator bacteria,twenty-four strains were screened by Oxford cup method.The target strains accounted for 9.6%of the total.One of the active strains isolated from the shark intestinal tract labeled S14 was selected for follow-up study.The result of 16S rDNA sequencing for the S14 and multiple alignment with NCBI showed that the homology of the strain to Staphylococcus epidermidis was 99.0%,and named Staphylococcus epidermidis S14.Preliminary physical and chemical properties show that the supernatant of Staphylococcus epidermidis S14 was resistant acid and alkali in the different environment of acid or alkali,and the active substance can maintain stable biological bacteriostatic activity at-20?and 100?,with a good temperature tolerance.The fermentation products were treated with alkaline protease or trypsin,the activity disappeared completely and decreased after treatment with pepsin or protease K.It showed that the fermentation product of the active substance can be decomposed with proteases.?2?Optimizing the microbial fermentation processes,pre-experiment has been determined the factors affected the fermentation conditions of Staphylococcus epidermidis S14 produced antibacterial active substance were the temperature,initial pH of the culture medium,culture speed and the volume of the culture.From the single factor experiment,the optimum temperature for fermentation culture was30?,the optimum initial pH of culture medium was 6.5,the optimum culture speed was 140 rpm,and the best volume of 250 mL conical flask was 100.0 m L.Box-Benhnken test design was carried out to optimize the fermentation conditions with the single factor optimum condition as the center point.The four-factor three-level design experiment was carried out and take the inhibition zone size?mm?as the response value to test the active substance produced by Staphylococcus epidermidis S14 effect on the indicator bacteria.The response of the experimental results was analyzed by Design-Expert 8.0.6 software.The results showed that the model had the maximum response 20.0 mm when the culture temperature was 35?,and the initial medium pH was 6.4,the rotation speed was 100 rpm,and the installed volume of 250 m L conical flask was 108.3 m L.Verification of the experiment,the results show that the average value of the inhibition zone is 20.7 mm,which is consistent with the model predictive value.The model can be used to guide the fermentation process of produce active substances for the Staphylococcus epidermidis S14.?3?It has been hypothesized that protein peptides are more likely for the antibacterial active substance through the results of enzymatic hydrolysis and UV absorption detection.During the process of isolation and purification,the standard Nisin was used as the reference standard to in calculating the potency of the active substance and the purification efficiency was measured by standard protein.In the separation and purification,the experiment used ammonium sulfate precipitation of protein peptides at first,and found can not precipitate the material at the saturation of40%,when the saturation achieved 80%and 90%,the precipitated material can not inhibit the growth of the indicator bacteria,so salting out is not suitable for the rough separation of active substances in this experiment.The macroporous resin D4020was used for the crude fractionation and eluted with ethanol gradient,The collected active components were concentrated and lyophilized.The yield of the active component was 39.4%and the unit titer increased 12.2%.Then the active components of the collected were separated by ion exchange.After the cationic resin was eluted with Na₂HPO₄-citric acid buffer system,only one absorption peak appeared at 280 nm.Since the impurity protein was not removed,the cation exchange chromatography was unsuccessful for separation of the target substance.The collected active fraction was subjected to DEAE-Sepharose Fast Flow anion exchange resin and gradient eluted with 1.0 M NaCl.The yield was 37.0%,the unit titer was 46398.8 IU/mg.The active fraction was loaded onto a gel column Ultrahydrogel 250 column,and eluted with low concentration acetonitrile,The fraction was isolated and separated more than five components,two components have biological activity.The yield was 9.9%.One of the active components was separated and purified by C18 column to obtain two bioactive components.The component F1Aa is higher purity,the yield was 1.5%.The collected single component F1Aa was analyzed by LC-MS/MS,and the primary structure was analyzed and obtained.The amino acid sequence of the peptide was LLLKKPKP with a relative molecular mass of 936.3 Da.