Purification And Characterization Of Bacteriocin From A Pediococcus Acidilactici

Bacteriocins were metabolited products of food-grade microorganisms, and some of which were used as food preservative to impove the food safety. A bacteriocin producing strain Pediococcus acidilactici was isolated from fermented vegetable food in the previous study. The cultive conditions were optimized, the purification technologies and the activity determination method were studied, and the characteristics of the bacteriocin were also explained.The antibacterial substance secreted by Pediococcus acidilactici was treated with acidities and some proteinases including hydrogen pemxidase, pepsin, trypsin, papain and proteinase K. The results suggested that the antibacteriocin substance was not H2O2 and acid metabolization, and it was sensitive to some proteinaceous. So the study showed it was bacteriocin, and it was called Pediocin. Inoculated in MRS culture medium, the cultive conditions of maximum production of bacteriocin were optimized. The beginning pH was 6.5, the cultivate temperature was 37℃and the cultivate time was 16 hours.The bacteriocin was extracted from the culture and purified by three steps. The bacteriocin was adsorped to the cell at pH6.0, and desorped at pH2.0, based on that, the bacteriocin was collected by the method of adjusting the culture pH and centrifuging. After dialysing and freezing-dry, it was further purified through SP Sepharose Fast Flow ion-exchange chromatography and Sephadex G-50 gel filtration. The protein concentration was detemind by coomassie brilliant blue pigmentation method, and activity was determind by the method of two-fold dilution. The yield of bacteriocin was 0.19%, and the specific activity was 4.526×104AU/mg. The specific activity was increased by 34.54 fold. Tricine-SDS-PAGE indicated its molecular weight was about 4600Da.The characteristics of Pediocin were completed reseached. Heat treatment under 20℃-100℃for 20min did not destroy its activity. It was found to be stable over a wide pH range between 1 and 13, activity was lost at pH 14 and above after 3 hours at 20℃. Treatment with organic solvents did not cause any loss of activity. And it still had activity after freezing and deepfreezing. The bacteriocin secreted by Pediococcus acidilactici was safe and high efficiency, so it had a favorable potential application in food preservation. The purification technology and characteristics studies in this work provided theoretic basis and technical parameters for the bacteriocin's industrialization.