黑曲霉超临界萃取物抑制链格孢作用机制的初步研究
|
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。
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.
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.
引文
[1] 张天宇. 中国真菌志[M]. 北京: 科学出版社, 2003: 1-30.
[2] 陆家云. 植物病原真菌学[M]. 北京: 中国农业出版社, 2000: 390-397.
[3] JANISIEWICZ W J, KORSTEN L. Biological control of postharvest diseases of fruits [J]. Annual Reviews of Phytopathology, 2002, 40(1): 411-441.
[4] TRIPATHI P, DUBEY N K. Exploitation of natural products as an alternative strategy to control postharvest fungal rotting of fruit and vegetables [J]. Postharvest Biology and Technology, 2004, 32(3): 235-245.
[5] ROUKAS T. Citric and gluconic acid production from fig by Aspergillus niger, using solid-state fermentation [J]. Journal of Industrial Microbiology and Biotechnology,2000,25(6):298-304.
[6] SCHUSTER E, DUNN-COLEMAN N, FRISVAD J C, et al. On the safety of Aspergillus niger,-a review [J]. Applied Microbiology and Biotechnology, 2002, 59(4/5): 426-435.
[7] 刘吴娟,陈青,王嫱,等.黑曲霉菌丝体抗菌活性及石油醚部分化学成分[J].中国实验方剂学杂志,2013,19(5):125-128.
[8] KITTURA F S, VISHU KUMAR A B, VARADARAJ M C, et al. Chitooligosaccharides-preparation with the aid of pectinase isozyme from Aspergillus niger and their antibacterial activity [J].Carbohydrate Research,2005,340(6):1239-1245.
[9] 李祝,葛永怡,陈青,等.黑曲霉发酵液抗真菌活性及稳定性研究[J].食品研究与开发,2011,32(7):141-143.
[10] 左祥,白现广,杨本寿,等.一株内生真菌黑曲霉的活性检测及鉴定[J].生物技术世界,2012,10(9):1-3.
[11] 吴新安,花日茂,岳永德,等.中草药提取物对几种植物病原菌的抗菌活性研究[J].安徽农业大学学报,2002(4):330-332.
[12] LEE H J, CHOI G J, CHO K Y. Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity[J]. Journal of Agricultural Food Chemistry, 1998, 46(2): 737-741.
[13] CHEN C Z, COOPER S L. Interactions between dendrimer biocides and bacterial membranes[J]. Biomaterials, 2002, 23(16): 3359-3368.
[14] 古力娜·达吾提,安惠霞,斯拉甫·艾白,等.地锦草有效部位对真菌琥珀酸脱氢酶活性的影响[J].中国中医药信息杂志,2010,17(11):34-38.
[15] BRADFORD M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Analytical Biochemistry, 1976, 72(12): 248-254.
[16] 傅紫琴, 王明艳, 蔡宝昌. 5-羟甲基糠醛(5-HMF)在中药中的研究现状探讨[J]. 中华中医药学刊, 2008(3): 508-510.
[17] ZEITSCH K J. The chemistry and technology of furfural and its many by-products [J]. Chemical Engineering Journal, 2001, 81(1/3): 338-339.
[18] HEITZ M, CAPEK-MENARD E, KOEBERELE P G, et al. Fractionation of Populus tremuloides at the pilot plant scale: optimization of steam pretreatment conditions using STAKE II technology [J].Bioresource Technology,1991,35(1):23-32.
[19] 王瑞芳,石蔚云.糠醛的生产及应用[J].河南化工,2008(5):14-15.
[20] 江俊芳.糠醛的生产及应用[J].化学工程与装备,2009(10):137-139.
[21] 王倩,谢明杰.木犀草素对金黄色葡萄球菌的抑菌活性及其机制[J].微生物学报,2010,50(9):1180-1184.
[22] 张小华,孙业盈,卞伟华,等.真菌细胞壁抑制剂刚果红诱导酵母细胞凋亡及其机制的研究[J].河南农业科学,2015,44(12):65-69.
[23] BOYD E F, NELSON K, WANG, F S, et al. Molecular genetic basis of allelic polymorphism in malate dehydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enterica [J]. Proceedings of the National Academy of Sciences of the United States of America,1994,91(4):1280-1284.
[24] 刘海燕,高微微,樊瑛,等.细辛挥发油抗植物病原真菌活性初步研究[J].植物病理学报,2007,37(1):95-98.
[25] ALLEN S A, CLARK W, MCCAFERY J M, et al. Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2010, 3(1): 2.
[26] HANLY T J, HENSON M A.Dynamic model-based analysis of furfural and HMF detoxification by pure and mixed batch cultures of S. cerevisiae and S. stipitis [J]. Biotechnology and Bioengineering, 2014, 111(2): 272-284.
[27] STORZ G, CHRISTMAN M F, SIES H, et al. Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium [J]. Proceedings of the National Academy of Sciences of the United States of America, 1987, 84(24): 8917-8921.
[28] ZHENG D, BAO J G, LU J M, et al. Isolation and characterization of a furfural-degrading bacterium Bacillus cereus sp. strain DS1 [J].Current Microbiology,2015,70(2):199-205.
[2] 陆家云. 植物病原真菌学[M]. 北京: 中国农业出版社, 2000: 390-397.
[3] JANISIEWICZ W J, KORSTEN L. Biological control of postharvest diseases of fruits [J]. Annual Reviews of Phytopathology, 2002, 40(1): 411-441.
[4] TRIPATHI P, DUBEY N K. Exploitation of natural products as an alternative strategy to control postharvest fungal rotting of fruit and vegetables [J]. Postharvest Biology and Technology, 2004, 32(3): 235-245.
[5] ROUKAS T. Citric and gluconic acid production from fig by Aspergillus niger, using solid-state fermentation [J]. Journal of Industrial Microbiology and Biotechnology,2000,25(6):298-304.
[6] SCHUSTER E, DUNN-COLEMAN N, FRISVAD J C, et al. On the safety of Aspergillus niger,-a review [J]. Applied Microbiology and Biotechnology, 2002, 59(4/5): 426-435.
[7] 刘吴娟,陈青,王嫱,等.黑曲霉菌丝体抗菌活性及石油醚部分化学成分[J].中国实验方剂学杂志,2013,19(5):125-128.
[8] KITTURA F S, VISHU KUMAR A B, VARADARAJ M C, et al. Chitooligosaccharides-preparation with the aid of pectinase isozyme from Aspergillus niger and their antibacterial activity [J].Carbohydrate Research,2005,340(6):1239-1245.
[9] 李祝,葛永怡,陈青,等.黑曲霉发酵液抗真菌活性及稳定性研究[J].食品研究与开发,2011,32(7):141-143.
[10] 左祥,白现广,杨本寿,等.一株内生真菌黑曲霉的活性检测及鉴定[J].生物技术世界,2012,10(9):1-3.
[11] 吴新安,花日茂,岳永德,等.中草药提取物对几种植物病原菌的抗菌活性研究[J].安徽农业大学学报,2002(4):330-332.
[12] LEE H J, CHOI G J, CHO K Y. Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity[J]. Journal of Agricultural Food Chemistry, 1998, 46(2): 737-741.
[13] CHEN C Z, COOPER S L. Interactions between dendrimer biocides and bacterial membranes[J]. Biomaterials, 2002, 23(16): 3359-3368.
[14] 古力娜·达吾提,安惠霞,斯拉甫·艾白,等.地锦草有效部位对真菌琥珀酸脱氢酶活性的影响[J].中国中医药信息杂志,2010,17(11):34-38.
[15] BRADFORD M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Analytical Biochemistry, 1976, 72(12): 248-254.
[16] 傅紫琴, 王明艳, 蔡宝昌. 5-羟甲基糠醛(5-HMF)在中药中的研究现状探讨[J]. 中华中医药学刊, 2008(3): 508-510.
[17] ZEITSCH K J. The chemistry and technology of furfural and its many by-products [J]. Chemical Engineering Journal, 2001, 81(1/3): 338-339.
[18] HEITZ M, CAPEK-MENARD E, KOEBERELE P G, et al. Fractionation of Populus tremuloides at the pilot plant scale: optimization of steam pretreatment conditions using STAKE II technology [J].Bioresource Technology,1991,35(1):23-32.
[19] 王瑞芳,石蔚云.糠醛的生产及应用[J].河南化工,2008(5):14-15.
[20] 江俊芳.糠醛的生产及应用[J].化学工程与装备,2009(10):137-139.
[21] 王倩,谢明杰.木犀草素对金黄色葡萄球菌的抑菌活性及其机制[J].微生物学报,2010,50(9):1180-1184.
[22] 张小华,孙业盈,卞伟华,等.真菌细胞壁抑制剂刚果红诱导酵母细胞凋亡及其机制的研究[J].河南农业科学,2015,44(12):65-69.
[23] BOYD E F, NELSON K, WANG, F S, et al. Molecular genetic basis of allelic polymorphism in malate dehydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enterica [J]. Proceedings of the National Academy of Sciences of the United States of America,1994,91(4):1280-1284.
[24] 刘海燕,高微微,樊瑛,等.细辛挥发油抗植物病原真菌活性初步研究[J].植物病理学报,2007,37(1):95-98.
[25] ALLEN S A, CLARK W, MCCAFERY J M, et al. Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2010, 3(1): 2.
[26] HANLY T J, HENSON M A.Dynamic model-based analysis of furfural and HMF detoxification by pure and mixed batch cultures of S. cerevisiae and S. stipitis [J]. Biotechnology and Bioengineering, 2014, 111(2): 272-284.
[27] STORZ G, CHRISTMAN M F, SIES H, et al. Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium [J]. Proceedings of the National Academy of Sciences of the United States of America, 1987, 84(24): 8917-8921.
[28] ZHENG D, BAO J G, LU J M, et al. Isolation and characterization of a furfural-degrading bacterium Bacillus cereus sp. strain DS1 [J].Current Microbiology,2015,70(2):199-205.