辣木籽抗菌肽对金黄色葡萄球菌生物膜的影响

Effect of Moringa oleifera Seeds Antimicrobial Peptide on Staphylococcus aureus Biofilm

辣木籽抗菌肽MOp2、MOp3对金黄色葡萄球菌具有良好的抑菌效果,但是其对生物膜是否产生影响尚未清楚。采用结晶紫半定量法、荧光显微镜观察、酶解实验和分子对接技术等探讨从辣木籽蛋白水解物中分离鉴定到的新型抗菌肽MOp2、MOp3对金黄色葡萄球菌生物膜的影响。结果表明:MOp2、MOp3的最小抑制生物膜质量浓度(MBIC)和最小清除生物膜质量浓度均为4 mg/mL和8 mg/mL;1×MBIC下MOp2与MOp3对金黄色葡萄球菌生物膜清除率分别为63.28%和67.90%;荧光显微镜发现,MOp2、MOp3处理后的生物膜黏附减少,细菌数量减少;细菌初期黏附率在1×MBIC的MOp2、MOp3处理后分别降低44.19%和50.77%,且表面疏水性降低;酶解实验表明,金黄色葡萄球菌生物膜胞外聚合物中含量最多的为胞外蛋白(质量分数49.01%),其次为胞外多糖和胞外DNA,且MOp2、MOp3会抑制胞外聚合物中这3种成分的分泌合成;分子对接结果显示,MOp2、MOp3均可以与生物膜形成关键蛋白AgrA、CshA、LuxS和SarA结合,这可能对金黄色葡萄球菌群体感应系统造成一定影响,从而抑制生物膜形成。研究旨在为辣木籽抗菌肽在食品工业中的防腐保鲜应用提供理论依据。

Moringa oleifera seed antimicrobial peptides MOp2 and MOp3 had good antibacterial effects on Staphylococcus aureus(S.aureus),but their effects on biofilm were still unclear.Therefore,crystal violet semi-quantitative method,fluorescence microscopy,enzymatic digestion experiment and molecular docking technology were used to study the effects of the newly identified antimicrobial peptides MOp2 and MOp3,which were isolated from hydrolysates of Moringa oleifera seeds proteins,on S.aureus biofilm.The results showed that the minimum biofilm inhibitory concentration(MBIC)and minimum biofilm eradication concentration of MOp2 and MOp3 were both 4 mg/mL and 8 mg/mL,respectively.At 1×MBIC concentration,both MOp2 and MOp3 exhibited biofilm clearance rates of 63.28%and 67.90%,respectively.It was found by fluorescence microscopy that after treatment with MOp2 and MOp3,biofilm adhesion decreased and bacteria numbers reduced.The initial bacterial adhesion rates decreased by 44.19%and 50.77%after treatment with 1×MBIC of MOp2 and MOp3,respectively,and the surface hydrophobicity also decreased.Enzymatic digestion experiments showed that the extracellular proteins(mass fraction 49.01%)were the most abundant component in S.aureus biofilm extracellular polymeric substances,followed by extracellular polysaccharides and extracellular DNA.MOp2 and MOp3 could inhibit the secretion and synthesis of the three components in extracellular polymeric substances.The results of molecular docking showed that MOp2 and MOp3 could both bind to key biofilm formation proteins AgrA,CshA,LuxS and SarA in S.aureus,which might have certain effects on the quorum sensing system of S.aureus and thus inhibit biofilm formation.The study aimed to provide a theoretical basis for the application of Moringa oleifera seed antimicrobial peptides in preservation of food industry.