The Philippines Clam (ruditapes Philippinarum) Antibacterial Mechanism Of Enzyme Preparation And Research

The antibacterial activity of hydrolysates from Ruditapcs philippinarum was screened and the optimized hydrolysis condition was investigated. Furthermore, the preliminary purification, the factors affecting its antibacterial activity and antimicrobial mechanism were also studied. The results were as follows:1.The hydrolysates with pepsin have certain antibacterial activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Saccharomvces cerevisiae and Vibrio parahaemolyticus. The strongest antibacterial activity against Staphylo-coccus aureus and Bacillus subtilis. and no significant antibacterial activity against Bacillus subtilis. The optimal enzymatic hydrolysis conditions were time2h, temperature35℃, pH2, the enzyme dosage3000U, the ratio of material to water1:4.2.The molecular weight(Mr) of most hydrolysate with pepsin was below5000D, component with Mr<1000D accounted for27.31%, component with Mr between1000D and3000D accounted for42.19%, component with Mr between3000D and5000D accounted for28.49%and component with Mr>10000D only accounted for1.91%. The antibacterial hydrolysates of Ruditapes philippinarum was classificated into three components by ultrafiltration cup:component Ⅰ (3KD<Mr<5KD), component Ⅱ (1KD<Mr<3KD), component Ⅲ (Mr<1KD). The components Ⅱ had the strongest antibacterial activity against Staphylococcus aureus, followed by component Ⅲ (Mr<1KD).The antibacterial activity of components Ⅰ (3KD<Mr<5KD) was the weakest. High temperature, long storage time and strong alkaline environment will reduce the antibacterial activity of hydrolysate with pepsin against Staphylococcus aureus; UV treatment has no effect on the antibacterial activity.3.The antibacterial hydrolysate of Ruditapes philippinarum changed the colony morphology of Staphylococcus aureus and Bacillus subtilis and inhibited the growth of two kinds of bacteria in the logarithmic phase. The results showed that the content of protein, soluble sugar and UV-absorbing compounds and the conductivity in the culture medium of two kinds of bacteria increased. It could be also detected the activity of alkaline phosphatase and beta-galactosidase in the culture solution and phosphorus metabolism of the two kinds of bacteria were seriously hampered. The results of scanning electron microscope revealed that it emerged the phenomenon of agglomeration with the extension of response time in the culture solution of Staphylococcus aureus and the cell shape became irregular, a large number of cells sticked together, afterwards intracellular material spilled, the boundaries between cells completely lost and cells almost mixed as a whole. The cell surface of Bacillus subtilis becomes rough, the cells began to adhere and intercellular boundaries became blurred, and subsequently cells broke, intracellular material leaked when treating nine hours, probably form of Bacillus subtilis could be identified from the remnants of the bacterial debris, most of the bacteria had been completely cracked and died. Transmission electron microscope results showed that with the extension of treated time the surface of Staphylococcus aureus becomes rough, uneven distribution of cytoplasm, serious condensation, darker substances appeared in the middle, followed by large numbers of intracellular material leakage, the massive cells became dead when treated for nine hours. Bacillus subtilis cells began to shrink, ocytoplasm distributed uneven, began to showed the phenomenon of plasmolysis, followed by the rupture of cell wall significantly, intracellular material leaked, only could see the residual and ambiguity nucleoplasm area. Therefore cells were killed. SDS-PAGE results showed that protein bands whose molecular weight were80KD and25KD faded with extension of duration of action in Staphylococcus aureus and Bacillus subtilis, while other protein bands gradually disappeared. Therefore, it was speculated that the hydrolysates of Ruditapes philippinarum could destroy cell wall and membrane of the two kinds of bacteria, change the permeability of cells, result in a large number of intracellular substances leakage and enter within the bacterial cells to affect their normal physiological metabolism which led to death.