菲律宾蛤仔（Ruditapes philippinarum）抑菌酶解物的制备与作用机理研究

【作者】 万慧一；

【导师】 魏玉西；

【作者基本信息】 青岛大学 ， 微生物学， 2012， 硕士

【摘要】 本文以菲律宾蛤仔为研究对象，筛选有抑菌活性酶解物并优化其酶解工艺：对酶解物进行初步的分级纯化,并研究抑菌活性影响因素、探讨其作用机理。研究结果如下：1、菲律宾蛤仔胃蛋白酶解产物对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酿酒酵母和副溶血弧菌均有一定的抑菌活性,其中对金黄色葡萄球菌和枯草芽孢杆菌的抑菌活性最强，对四联微球菌则无明显的抑菌活性。最优酶解工艺条件为：酶解时间2h,温度35℃,pH2,加酶量3000U,料水比1：4。2、菲律宾蛤仔抑菌活性酶解物相分了质量主要分布在5000D以下,相对分子质量小于1000D的组分占27.31%,相对分子质量在1000～3000D之间的组分占42.19%,相对分子质量在3000～5000D之间的组分占28.49%,相对分子质量大于5000D的组分仅占1.91%。菲律宾蛤仔抑菌活性酶解物经超滤杯分级后得到3个组分：组分Ⅰ(3KD<Mr<5KD),组分Ⅱ(1KD<Mr<3KD),组分Ⅲ(Mr <1KD);其中,组分Ⅱ对金黄色匍萄球菌的抑制作用最强,其次是组分Ⅲ(Mr <1KD),组分Ⅰ对金黄色葡萄球菌的抑菌活性最弱。高温、储存时间过长和强碱性环境均会降低组分Ⅱ对金黄色葡萄球菌生长的抑制能力；而紫外线处理对组分Ⅱ的抑菌活性没有影响。3、菲律宾蛤仔抑菌酶解物组分Ⅱ使金黄色葡萄菌和枯草芽孢杆菌的菌落形态发生变化,在对数生长期抑制2种菌的的菌体分裂。研究发现2种菌培养液中蛋白、可溶性糖和紫外吸收物质的含量增加,液电导率升高,还可以检测到碱性磷酸酶、β—半乳糖苷酶的活性,且2种细菌的磷代谢均受到严重阻碍。扫描电镜结果显示：随着菲律宾蛤仔抑菌酶解物组分Ⅱ作用时间的延长,金黄色葡萄球菌菌体细胞间发生结团现象,形状变得不规则,大量细胞粘连在一起,随后大量胞内物质外溢,细胞间的界限完全丧失,几乎融合为一个整体；枯草芽孢杆菌菌体细胞表面变得粗糙,细胞开始粘连,胞间界限变得模糊不清,随后出现破损现象,处理9h后大量胞内物质外泄,仅能从贱存菌体碎片辨别出小部分菌体大概形态,大部分菌体已裂解死亡。透射电镜结果显示：随着菲律宾蛤仔抑菌酶解物组分Ⅱ作用时间的延长,金黄色葡萄球菌菌体表面变得粗糙,细胞质分布不均匀,固缩较严重,中间有颜色较深的团状物质出现,随后大量胞内物质外泄,处理9h后大量菌体裂解死亡；枯草芽孢杆菌菌体细胞开始皱缩,细胞质不均匀分布,开始出现质壁分离现象,随后细胞壁破裂明显,大量胞内物质外泄,仅可以看到残留的模糊核质区,细胞裂解死亡。SDS-PAGE图谱显示,金黄色葡萄球菌和枯草芽孢杆菌80KD和25KD这两个分子量段的蛋白谱带随着菲律宾蛤仔抑菌酶解物组分Ⅱ作用时间的延长而逐渐变浅,而其它分子量段的蛋白谱带逐渐消失。因此,可以推测菲律宾蛤仔抑菌酶解物组分Ⅱ的作用机理可能是破坏了菌体细胞壁和细胞膜,改变了菌体细胞的通透性,导致大量胞内物质外泄,并进入菌体细胞内部影响其正常的生理代谢，从而引起菌体裂解死亡更多还原

【Abstract】 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.更多还原