| Endophyte refers to live in healthy plant tissues and organs and causes no obvious symptoms. The species of endophyte is rich and distributes widely. So endophyte is an important part of the microbic system. In this paper, the endophyte DNN6 isolated from Laminaria japonica Aresch was researched. By optimization of culture conditions of strain DNN6, and its protein and polysaccharide in fermentation brothwere separated, purified and analyzed. Finally purified protein and polysaccharide were applied to preservatize Pseudosciaena polyactis. The results were as follows:1. The optimization of fermentation conditions of strain DNN6 was investigated by the orthogonal design method. The optimal fermentation conditions of strain DNN6 could be recognized as follows:50g/L sucrose,0.2g/L (NH4)H2PO4,5g/L NaCl,0.2g/LMgSO4.7H2O, 1g/L K2HPO4, cultured at 40℃ for 2 days. The antibacterial activity of the optimization of fermentation conditions of strain DNN6 was better than the original culture conditions of strain DNN6, and the antibacterial activity of supernatant was better than the cell.2. Using ammonium sulfate to separatethe protein of strain DNN6, the saturation of ammonium sulfate was 85%. Purication conditions of strain DNN6 protein by anion exchange chromatography column:the 0.05mol/L pH 8.7 Tris-HCl was selected as the initial bufferand protein of strain DNN6 could be eluted completely by 0.75mol/L NaCl. The protein of strain DNN6 was separated to four components by DEAE-52 anion exchange chromatography. The protein component F4 had the best bacteriostatic activity. Scavenging rates of 1000μg/mL of protein component F4 on the superoxide anion radical and hydroxyl radical were 75.68% and 4.8%. The protein component F4 was detected by SDS-PAGE electrophoresis, and the results showed that the protein component F4 had nine bands with molecular mass 170,55,41,39, 37,34,20,14,8 kDa, which could be watched obviously. To analysis molecular weightunder 10 kDa, the result of MALDI-TOF/TOF-MS mass spectrometry accurate molecular weight was 8770.6 Da.3. Using alcohol to separate the polysaccharide of strain DNN6, and the volume of anhydrous alcohol was 2.5 times of strain DNN6 supernatant liquid. Purication conditions of polysaccharide of strain DNN6:using Sephadex G-100 molecular sieve chromatography, the eluent was double distilled water.The polysaccharide was separated to two kinds of components EPS1 and EPS2. EPS1 had the best bacteriostatic activity. Scavenging ratesof 250μg/mL of polysaccharide component EPS1 on th esuperoxide anion radicalwas76.14%, and scavenging rates of 125μg/mL of polysaccharide component EPS1 on hydroxylradical was 23%. The results of gas chromatography and infrared spectrum analysis showed that The monosaccharide composition of EPS1 was glucose. The characteristic absorption peak indicated-OH,-C≡C-,-C=C-, etc.4. Preservatize Pseudosciaena polyactis by proteincomponent F4 and polysaccharide component EPS1.The results suggested that the concentration of proteincomponent F4 was 800μg/mL, which had better preservative activity than other treatment groups. The shelf life was four days. On the fourth day of storage, Pseudosciaena polyact was still hadgood smell, the suface gloosy, muscle elasticity and texture closely, eyes sunken slightly and scales slightly off. The total number of bacteria was 4.961gcfu/g. pH value was 7.03.TVB-N value was 12.7mg/100g. TBA value was 0.89mg/kg. It conformed to the national secondary standard. The concentration ofpolysaccharidecomponent EPS1 was 100μg/mL. It had better preservation activity than other treatment groups. The shelf life was four days. On the fourth day of storage, Pseudosciaena polyactissmelt fresh, the suface gloosy, eyes almost level, muscle elasticity and texture closely, the scalesuneasy to fall off. The total number of bacteria was 4.981gcfu/g. pH value was 7.0.TVB-N value was 12.9mg/100g. TBA value was 0.87mg/kg. It conformed to the national secondary standard. |
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