【摘要】 本试验对黑曲霉菌丝体超临界萃取物(SE)抑制链格孢的机制进行了初步研究。采用超临界萃取从黑曲霉菌丝体中获得样品SE,使用气相色谱-质谱(GC-MS)对其萃取物进行分离鉴定,并通过细胞膜渗透性测定、核酸和总蛋白含量测定、4′,6-二脒基-2-苯基吲哚(DAPI)荧光染色法、活性氧(ROS)含量测定、呼吸代谢途径等方法对SE的抑菌机制进行研究;电导率及核酸、蛋白结果显示,SE能影响链格孢细胞膜的完整性,SE处理菌体4.5 h,电导率比阴性对照增加了37.2%,胞外核酸蛋白含量显著增加。DAPI染色结果显示,DAPI可以和链格孢内的核酸结合,经SE作用30 min,SE可引起细胞核染色质异常弥散。ROS试验结果显示,SE可以激发菌体产生过多的活性氧,SE作用链格孢40 min时ROS含量比阴性对照增加了79.67%。呼吸代谢试验结果显示,SE能抑制三羧酸循环(TCA)中的琥珀酸脱氢酶的活性,其抑制率为35.4%。SE具有较强的抑菌活性,其抑菌机制是通过影响细胞膜的正常功能、损伤细胞结构和抑制真菌的呼吸代谢等方面实现的。更多还原

【Abstract】 To investigate the antifungal mechanism of supercritical extraction(SE) from Aspergillus niger against Alternaria alternata, the chemical constituents from A.niger were extracted by supercritical extraction and analyzed by GC-MS, and the conductivity and content of macromolecules(nucleic acids and total protein), 4′6-diamidino-2-phenylindole(DAPI) staining, active oxygen(ROS) content and respiratory metabolism were studied. The results showed that SE could affect the membrane integrality, increase electrical conductivity by 37.2% with SE treated for 4.5 h, significantly increase the content of extracellular nucleic acids and total protein, respectively, compared with that of the negative control. The fluorescence microscopy and fluorescence spectrophotometer studies manifested that DAPI could integrate with A.alternata, and anomalous dispersion occurred in nuclear chromatin treated with SE for 30 min. In addition, SE stimulated the cells to produce excessive reactive oxygen, and the content of ROS increased by 79.67% with SE treated for 40 min compared with that of the negative control. The succinate dehydrogenase(SDH) activity of A.alternata was significantly inhibited by SE and the SDH activity of A.alternata was reduced by 35.4% compared to the negative control. SE had obvious antifungal activity against A.alternata. The mechanism might be that it could affect fungal membrane integrality, damage the cell structures and inhibit fungal metabolic pathways to exert its antifungal functions.更多还原