cystatins是一类天然抗菌蛋白,广泛分布于鱼类加工废弃组织。采用滤纸片扩散法和二倍稀释法,评价了鲢鱼(Hypophthalmichthys molitrix)重组cystatin C(HmCystatin C)对6株常见的水产品腐败菌的抑菌活性;并以铜绿假单胞菌和腐生葡萄球菌为典型受试菌,测定HmCystatin C与菌体及其细胞壁成分脂多糖(lipopolysaccharides,LPS)和肽聚糖(peptidoglycan,PGN)的结合情况,及对受试菌生长曲线、细胞壁、细胞质膜通透性的影响,初步探讨其抑菌机制。结果表明,HmCystatin C对除枯草芽孢杆菌外的5株腐败菌均具抑制作用,对典型受试菌的最低抑菌浓度(minimum inhibitory concentration,MIC)及最低杀菌浓度(minimal bactericide concentration,MBC)均为3.5 mg/mL;HmCystatin C能与典型受试菌结合,并且对LPS和PGN表现出亲和性。此外典型受试菌的细胞壁和细胞质膜被破坏,胞内物质渗出。因此推测HmCystatin C可能首先吸附到菌体表面并与细胞壁成分结合,进而破坏细胞完整性,导致内容物外泄后菌体死亡。该结果为阐明鱼类cystatins抑菌机制奠定实验基础,也为综合利用鱼源cystatins提供理论参考。
Cystatins, as a type of natural antibacterial protein, are widely distributed in the discarded tissues from the fish processing. The antibacterial activity of recombinant cystatin C of silver carp (Hypophthalmichthys molitrix) (HmCystatin C) against six strains of putrefied bacteria in aquatic products was evaluated by disk diffusion method and double dilution method. By measuring the binding of HmCystatin C to bacteria and their cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN), as well as the effects of HmCystatin C on the growth curve, cell wall and plasma membrane permeability, the antibacterial mechanism of HmCystatin C against typical tested bacteria (Pseudomonas aeruginosa and Staphylococcus saprophyticus) was preliminarily studied. The results showed that HmCystatin C inhibited five tested bacteria except Bacillus subtilis, and the minimum inhibitory concentration and the minimum bactericidal concentration of HmCystatin C against typical tested bacteria were 3.5 mg/mL. HmCystatin C could bind to typical tested bacteria and showed affinity to LPS and PGN. In addition, HmCystatin C damaged cell wall and plasma membrane of typical tested bacteria, resulting in leakage of cell contents. Therefore, it was speculated that HmCystatin C may first attach to the cell surface and binding to the cell wall components, thereby destroying the integrity of cells and leading to the death of bacteria after the leakage of vital cell materials. This study laid an experimental foundation for elucidating the antibacterial mechanism of fish cystatins, and provided a theoretical reference for the comprehensive utilization of fish cystatins.
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