| In this article,fresh intestinal tract of the Apostichopus japonicas was used as raw material,the crude extract yield of polypeptide and antibacterial activity were taken as indicators,and using two extraction methods by the methanol and acetic acid extracts peptides from the intestinal tract of Apostichopus japonicas.Single factor and orthogonal experiment were used to optimize the extraction parameters.Four components with different molecular weight ranges were prepared by the ultrafiltration.The antimicrobial activities against Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Bacillus cereus,Vibrio anguillarum,Vibrio Parahaemolyticus and Vibrio Harveyi were tested.Sephadex G-25 column chromatography was used for separation and purification,and the antibacterial activities of the obtained components were detected.Finally,the polypeptides with antibacterial activities were analyzed by high performance liquid chromatography(HPLC),the results are as follows:1.Optimization of extraction methods and conditions of antimicrobial peptide from the intestinal tract of the Apostichopus japonicasWith the yield of crude extract as the index,single factor and orthogonal experiment were used to optimize the two extraction methods.The optimum conditions of the two extraction methods were obtained.The methanol extraction method was as follows: the ratio of material to liquid was 0.3 mg/m L,the concentration of the extract was 80%,the extraction time was 24 hours,and the centrif ?gal speed was 8000 r/min.The maximum yield of the two extraction methods was 8.33%.The acetic acid extraction method is as follows: the ratio of material to liquid is 0.3 mg/m L,the concentration of the extract is80%,the extraction time is 24 hours,and the centrif ?gal speed is 8000 r/min.The highest yield obtained by orthogonal design was 7.41%.The content of polypeptide in the crude extracts of the two groups was determined.The results showed that the concentration of polypeptide in methanol crude extract was 3.26 mg/m L and that in acetic acid crude extract was 2.62 mg/m L.2.Ultrafiltration analysis and determination of antimicrobial activityThe crude extracts of methanol and acetic acid were separated into four molecular weight polypeptide groups by tangential flow ultrafiltration system,which were <1k D,1~3k D,3~5k D and 5~10k D respectively.It was found that the groups above 3k D needed four times of ultrafiltration to separate different molecular weight polypeptides,while those below 3k D needed at least five times.The antimicrobial activity of polypeptides from different molecular weight groups of methanol and acetic acid against seven kinds of bacteria was tested by turbidimetry.The growth curve of each bacteria for 24 hours was drawn and the antimicrobial activity of each group of polypeptides was obtained.The results showed that the polypeptide of methanol ultrafiltration group < 1k D had the strongest antimicrobial activity in the molecular weight ultrafiltration groups of methanol and acetic acid.3.Sephadex G-25 purification and determination of antibacterial activitySephadex G-25 column was used to purify the < 1kD polypeptide group after ultrafiltration.After elution,three elution components were obtained,which were named P1,P2 and P3.The antimicrobial activity of the three elution components was determined by the method of inhibition circle and turbidimetry.The results showed that P2 had antimicrobial activity against Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Vibrio parahaemolyticus and Vibrio anguillae.The circle diameter was 16.5 mm,15.7mm,14.6 mm,13.9 mm and 15.1 mm.The minimum inhibitory concentration(MIC)was1000 ?g/m L,1000 ?g/m L,2000 ?g/m L,3000 ?g/m L and 2000 ?g/m L.At the same time,the effects of heating time,heating temperature and freeze-thaw times on the antimicrobial activity of P2 were analyzed.The results showed that heating time and temperature had no significant effect on P2,while freeze-thaw times had significant effect on P2.Tricine-SDS-PAGE electrophoresis showed that P2 was a single band on the separating gel with a molecular weight of 4.5k D.Finally,the composition of P2 was analyzed by HPLC,and P2 was composed of 5 substances. |
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