微酸性电解水对灰葡萄孢菌杀菌效果与作用机制研究
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摘要
研究了微酸性电解水对灰葡萄孢菌悬液纯培养及原位培养的杀菌效果,同时通过扫描电镜和透射电镜初步探究了其对灰葡萄孢菌的杀菌机制。研究表明,随杀菌时间和有效氯质量浓度的增加,微酸性电解水对灰葡萄孢的杀菌能力明显增强;在有效氯质量浓度为10 mg/L、杀菌处理15 min,以及有效氯质量浓度在30 mg/L、杀菌处理10 min时,对悬液中灰葡萄孢菌的杀菌率均达99. 99%。对用微酸性电解水处理后的灰葡萄孢菌落原位生长状态观察表明,微酸性电解水可以抑制菌落扩生,浓度越高抑生长作用越强,用有效氯质量浓度为30 mg/L的微酸性电解水处理菌落3 d后,菌丝逐渐变暗黄、枯萎,直至死亡,菌落停止生长,推测微酸性电解水可杀灭灰葡萄孢的新生孢子并抑制菌丝生长。通过扫描及透射电镜观察,发现微酸性电解水处理可导致灰葡萄孢孢子细胞质壁分离,细胞质溢出,细胞器溶解,细胞皱缩,这可能是引起灰葡萄孢菌无法正常生长繁殖,最终达到杀菌效果的原因。
Slightly acidic electrolyzed water is a kind of bactericidal preparation with pH value slightly lower than 7. 0 by electrolyzing dilute hydrochloric acid solution or electrolyzing low concentration dilute hydrochloric acid and electrolyte( NaCl/KCl) under non-diaphragm condition. It has a strong ability to kill pathogenic bacteria,but there is little research on the ability and mechanism of killing fungal spores by slightly acidic electrolyzed water. The killing effect of slightly acidic electrolytic water on pure culture spore suspension and in situ culture of Botrytis cinerea was studied,and the germicidal mechanism of water on Botrytis cinerea was preliminarily studied by scanning electron microscopy and transmission electron microscopy. The results showed that the bactericidal ability of slightly acidic electrolyzed water to Botrytis cinerea was increased significantly with the increase of sterilization time and available chlorine concentration( ACC), and bactericidal rate can achieve 99. 99% when the available chlorine concentration was not less than 30 mg/L and kill time was 10 min. The growth state of Botrytis cinerea colony in situ after treatment with slightly acidic electrolytic water showed that slightly acidic electrolytic water could inhibit colony expansion,and the higher the concentration was,the stronger the inhibition effect was. After 3 d of treatment with slightly acidic electrolytic water with 30 mg/L available chlorine,the mycelium gradually turned yellow and withered until death,and the colony was stopped. It was suggested that microacid electrolyzed water can kill the new spores of Botrytis cinerea and inhibit the growth of mycelia. Through microscope observation of scanning and transmission electron,it was found that treatment with acidic electrolyzed water can make separation of membrane and cell wall,cytoplasmic overflow and abnormal lysis of organelles. It was speculated that these may be the reasons which can achieve the bactericidal effect.
Slightly acidic electrolyzed water is a kind of bactericidal preparation with pH value slightly lower than 7. 0 by electrolyzing dilute hydrochloric acid solution or electrolyzing low concentration dilute hydrochloric acid and electrolyte( NaCl/KCl) under non-diaphragm condition. It has a strong ability to kill pathogenic bacteria,but there is little research on the ability and mechanism of killing fungal spores by slightly acidic electrolyzed water. The killing effect of slightly acidic electrolytic water on pure culture spore suspension and in situ culture of Botrytis cinerea was studied,and the germicidal mechanism of water on Botrytis cinerea was preliminarily studied by scanning electron microscopy and transmission electron microscopy. The results showed that the bactericidal ability of slightly acidic electrolyzed water to Botrytis cinerea was increased significantly with the increase of sterilization time and available chlorine concentration( ACC), and bactericidal rate can achieve 99. 99% when the available chlorine concentration was not less than 30 mg/L and kill time was 10 min. The growth state of Botrytis cinerea colony in situ after treatment with slightly acidic electrolytic water showed that slightly acidic electrolytic water could inhibit colony expansion,and the higher the concentration was,the stronger the inhibition effect was. After 3 d of treatment with slightly acidic electrolytic water with 30 mg/L available chlorine,the mycelium gradually turned yellow and withered until death,and the colony was stopped. It was suggested that microacid electrolyzed water can kill the new spores of Botrytis cinerea and inhibit the growth of mycelia. Through microscope observation of scanning and transmission electron,it was found that treatment with acidic electrolyzed water can make separation of membrane and cell wall,cytoplasmic overflow and abnormal lysis of organelles. It was speculated that these may be the reasons which can achieve the bactericidal effect.
引文
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[15]倪莉.微酸性电解水对蛋鸡场沙门氏菌的杀菌规律研究[D].北京:中国农业大学,2016.
[16] WILLIAMSON B,TUDZYNSKI B,TUDZYNSKI P,et al. Botrytis cinerea:the cause of grey mould disease[J]. Molecular Plant Pathology,2007,8(5):561-580.
[17] ELAD Y,WILLIAMSON B,TUDZYNSKI P,et al. Botrytis spp. and diseases they cause in agricultural systems—an introduction[J]. In Botrytis:Biology,Pathology and Control,2007:1-8.
[18]赵红霞,苟萍.灰葡萄孢菌致病机理研究进展[J].生物技术,2014,24(1):100-103.ZHAO Hongxia,GOU Ping. Advances in pathogenesis mechanism of Botrytis cinerea[J]. Biotechnology,2014,24(1):100-103.(in Chinese)
[19] YE Z,WANG S,CHEN T,et al. Inactivation mechanism of Escherichia coli induced by slightly acidic electrolyzed water[J].Scientific Reports,2017,7(1):6279.
[20]吴方丽.丙烷脒对番茄灰霉病菌的抑菌机理初探[D].杨凌:西北农林科技大学,2008.WU Fangli. Primary study on inhibiting mechanism of propamidine against Botrytis cinerea[D]. Yangling:Northwest A&F University,2008.(in Chinese)
[2] YE Z,WANG S,GAO W,et al. Inactivation mechanism and apoptotic-like changes in aeromonas hydrophila induced by slightly acidic electrolyzed water in freshwater[J]. Transactions of the ASAE,2018,61(1):305-314.
[3] KOIDE S,TAKEDA J,SHI J,et al. Disinfection efficacy of slightly acidic electrolyzed water on fresh cut cabbage[J]. Food Control,2009,20(3):294-297.
[4] BEATRICE A,HUNG Y C. Electrolyzed water and its corrosiveness on various surface materials commonly found in food processing facilities[J]. Journal of Food Process Engineering,2005,28(3):247-264.
[5]牛会平,李慧颖,吴彤娇,等.微酸性电解水与强酸性电解水消毒能力比较及分析[J].河北医药,2018(1):124-126.NIU Huiping,LI Huiying,WU Tongjiao,et al. Comparison and analysis on the disinfection efficacy of slighty acidic electrolyzed water and strongly acidic electrolyzed water[J]. Hebei Medical Journal,2018(1):124-126.(in Chinese)
[6] DEZA M A,ARAUJO M,GARRIDO M J. Inactivation of Escherichia coli O157:H7,Salmonella enteritidis and Listeria monocytogenes on the surface of tomatoes by neutral electrolyzed water[J]. Letters in Applied Microbiology,2003,37(10):482-487.
[7] ABDULSUDI I Z,YOSHINORI K,ADILI T,et al. In vitro inactivation of Escherichia coli,Staphylococcus aureus and Salmonella spp. using slightly acidic electrolyzed water[J]. Journal of Bioscience and Bioengineering,2010,110(3):308-313.
[8]叶章颖,祁凡雨,裴洛伟,等.微酸性电解水对虾仁的杀菌效果及其动力学[J].农业工程学报,2014,30(3):223-230.YE Zhangying,QI Fanyu,PEI Luowei,et al. Disinfection effect and kinetics of slightly acidic electrolyzed water for white shrimp[J]. Transactions of the CSAE,2014,30(3):223-230.(in Chinese)
[9]南松剑,王朝元,曹薇,等.微酸性电生功能水在奶牛场的消毒效果研究[J].中国奶牛,2009(10):34-37.NAN Songjian,WANG Chaoyuan,CAO Wei,et al. Study on disinfection efficacy of slightly acidic electrolyzed functional water in dairy farms[J]. Chinese Dairy Journal,2009(10):34-37.(in Chinese)
[10]赵德锟,李凌飞,谭雷姝,等.微酸性电解水对鲜切云南红梨贮藏品质影响研究[J].食品安全质量检测学报,2017,8(1):243-251.ZHAO Dekun,LI Lingfei,TAN Leishu,et al. Effects of slightly acidic electrolyzed water on storage quality of fresh cut Yunnan red pear[J]. Journal of Food Safety&Quality,2017,8(1):243-251.(in Chinese)
[11] BUCK J W,VAN M W,OETTING R D,et al. In vitro fungicidal activity of acidic electrolyzed oxidizing water[J]. Plant Disease,2002,86(3):278-281.
[12] ABBASI P A,LAZAROVITS G. Effect of acidic electrolyzed water on the viability of bacterial and fungal plant pathogens and on bacterial spot disease of tomato[J]. Canadian Journal of Microbiology,2006,52(10):915-923.
[13] KE Xiong,LIU Haijie,LIU Rong,et al. Differences in fungicidal efficiency against Aspergillus flavus for neutralized and acidic electrolyzed oxidizing waters[J]. International Journal of Food Microbiology,2010,137(1):67-75.
[14] HAMZAH M A,STEPHANIE S,ALEKSANDRA C S,et al. Bactericidal activity of neutral electrolyzed water against Bacillus cereus and Clostridium perfringens in cell suspensions and artificially inoculated onto the surface of selected fresh produce and polypropylene cutting boards[J]. Food Control,2019,96(2):212-218.
[15]倪莉.微酸性电解水对蛋鸡场沙门氏菌的杀菌规律研究[D].北京:中国农业大学,2016.
[16] WILLIAMSON B,TUDZYNSKI B,TUDZYNSKI P,et al. Botrytis cinerea:the cause of grey mould disease[J]. Molecular Plant Pathology,2007,8(5):561-580.
[17] ELAD Y,WILLIAMSON B,TUDZYNSKI P,et al. Botrytis spp. and diseases they cause in agricultural systems—an introduction[J]. In Botrytis:Biology,Pathology and Control,2007:1-8.
[18]赵红霞,苟萍.灰葡萄孢菌致病机理研究进展[J].生物技术,2014,24(1):100-103.ZHAO Hongxia,GOU Ping. Advances in pathogenesis mechanism of Botrytis cinerea[J]. Biotechnology,2014,24(1):100-103.(in Chinese)
[19] YE Z,WANG S,CHEN T,et al. Inactivation mechanism of Escherichia coli induced by slightly acidic electrolyzed water[J].Scientific Reports,2017,7(1):6279.
[20]吴方丽.丙烷脒对番茄灰霉病菌的抑菌机理初探[D].杨凌:西北农林科技大学,2008.WU Fangli. Primary study on inhibiting mechanism of propamidine against Botrytis cinerea[D]. Yangling:Northwest A&F University,2008.(in Chinese)