冷等离子体对单核增生李斯特菌的杀菌机理

doi:10.3864/j.issn.0578-1752.2020.24.013

摘要/Abstract

摘要：

【目的】作为一种新兴的非热灭菌技术,冷等离子在食品行业中得到了广泛的应用。通过探讨冷等离子体对细菌细胞膜的破坏效果,阐述其抗菌机制,为冷等离子体在食品行业的应用提供参考。【方法】本研究以单核增生李斯特菌（Listeria monocytogenes,LM）为试验菌株,研究冷等离子体处理对LM形态和胞内物质的影响,阐述LM细胞膜完整性的变化;冷等离子体处理后,通过测定细胞膜脂肪酸含量和类型的变化,比较8-苯胺-1-萘磺酸荧光强度的改变,反映冷等离子体对LM细胞膜流动性的影响。通过测定碘化丙啶荧光、电导率和β-半乳糖苷酶活性的变化,观察冷等离子体对LM细胞膜通透性的改变。最后,通过检测胞内活性氧和活性氧相关基因表达量的变化,阐明冷等离子体对LM细胞膜造成的氧化损伤。【结果】冷等离子体处理后,LM细胞膜表面观察到破损变形的结构,胞内蛋白质和DNA分别下降了68 mg?mL^-1和14 μg?mL ^-1,证明冷等离子体破坏了细胞膜的完整性。冷等离子体处理使LM细胞膜中不饱和脂肪酸的含量从40.17%上升至53.91%,饱和脂肪酸的含量从53.68%下降到41.57%,8-苯胺-1-萘磺酸荧光强度从8.99下降到3.73,说明冷等离子体处理后细胞膜的流动性增加。此外,冷等离子体处理后,碘化丙啶可以透过细胞膜,与胞内遗传物质结合发出红色荧光,电导率由0.15 mS?cm^-1上升至0.33 mS?cm^-1,β-半乳糖苷酶活性也发生了明显的变化,OD_420nm从0.274提高至0.683,说明LM细胞膜通透性提高。胞内活性氧（reactive oxygen species,ROS）荧光和荧光强度的变化说明,冷等离子体刺激细胞膜上ROS的产生,因此对细胞膜造成了氧化损伤。qRT-PCR结果显示冷等离子体处理下调了perR和recA的相对表达量,分别下降43.29%和52.71%,而sigB的相对表达量上调了89.42%,揭示冷等离子体处理条件下,微生物在基因层面的氧化应激和调控机制。【结论】冷等离子体的活性基团通过对细胞膜的作用,破坏LM的细胞活性,起到了抑制效果。

关键词: 冷等离子体, 单核增生李斯特菌, 细胞膜, 流动性, 氧化损伤

Abstract:

【Objective】 As a novel non-thermal sterilization technology, the cold plasma has been widely applied in food industry. The aim of this study was to explore the antibacteral mechanism of cold plasma against bacteria at cytomembrane level, thus providing a foundation for the application of the cold plasma in food industry. 【Method】 In this study, the Listeria monocytogenes (LM) was selected as the test strain. After cold plasma treatment, the changes of morphology and intracellular materials were detected to reveal the integrity of LM cytomembranes. The changes of cell membrane fatty acid content and type, as well as the 8-aniline-1-naphthalene sulfonic acid (ANS) fluorescence intensity were also measured to detect the fluidity change of cytomembranes after cold plasma treatment. The changes of cytomembrane permeabilization were detected by propidium iodide (PI) fluorescence, conductivity and β-galactosidase activity. Finally, in order to demonstrate the oxidative damage of LM cytomembrane caused by cold plasma treatment, the changes of reactive oxygen species (ROS) and reactive oxygen species related genes were detected as well. 【Result】After cold plasma treatment, damaged and deformed structures were observed on the surface of the LM cytomembrane, the contents of proteins and DNA were decreased by 68 mg?mL ^-1 and 14 μg?mL ^-1, respectively, which showed that cold plasma destroyed the integrity of cytomembrane. After cold plasma treatment, the content of unsaturated fatty acids in LM cytomembrane increased from 40.17% to 53.91%, along with the decrease of saturated fatty acids from 53.68% to 41.57%. The ANS fluorescence intensity decreased from 8.99 to 3.73, indicating the increase of cytomembrane fluidity caused by cold plasma action. In addition, cold plasma treatment resulted in the penetration of PI and reacted with DNA to emit red fluorescence. By analyzing the changes of β-galactosidase activity and conductivity (increased from 0.15 mS?cm ^-1 to 0.33 mS?cm^-1), it was concluded that the cold plasma enhanced the permeabilization of LM cytomembrane. The changes of ROS fluorescence intensity indicated that cold plasma stimulated the generation of ROS on cytomembrane, thus exerting oxidative damage to cytomembrane. Finally, the results of qRT-PCR showed that cold plasma down-regulated the expression of perR and recA genes by 43.29% and 52.71%, while up-regulated the expression of sigB gene by 89.42%, which disclosed the regulatory mechanism of oxidative stress in microorganism at genic level.【Conclusion】Through the action on cell membrane of the active groups of cold plasma, the viability of LM was inhibited and eventually died.

Key words: cold plasma, Listeria monocytogenes, cytomembrane, fluidity, oxidative damages

窦勇,姚妙爱,闾怀中,胡佩红,董静. 冷等离子体对单核增生李斯特菌的杀菌机理[J]. 中国农业科学, 2020, 53(24): 5104-5114.

DOU Yong,YAO MiaoAi,LÜ HuaiZhong,HU PeiHong,DONG Jing. Antibacterial Mechanism of Cold Plasma Against Listeria monocytogenes[J]. Scientia Agricultura Sinica, 2020, 53(24): 5104-5114.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.24.013

https://www.chinaagrisci.com/CN/Y2020/V53/I24/5104

图/表 9

图1

表1

图2

图3

表2

图4

图5

图6

图7

参考文献 32

[1]	刘海泉, 赵强, 孙晓红, 吴启华, 潘迎婕, 赵勇. 多重PCR快速检测食品中单核细胞增生性李斯特菌. 中国农业科学, 2010,43(23):4893-4900.
	LIU H Q, ZHAO Q, SUN X H, WU Q H, PAN Y J, ZHAO Y. Rapid detection of Listeria monocytogenes in food by multiple PCR. Scientia Agricultura Sinica, 2010,43(23):4893-4900. (in Chinese)
[2]	PARK S H, BALASUBRAMANIAM V M, SASTRY S K. Quality of shelf-stable low-acid vegetables processed using pressure-ohmic- thermal sterilization. LWT - Food Science and Technology, 2014,57(1):243-252. doi: 10.1016/j.lwt.2013.12.036
[3]	ZHU Y L, LI C Z, CUI H Y, LIN L. Antimicrobial mechanism of pulsed light for the control of Escherichia coli O157:H7 and its application in carrot juice. Food Control, 2019,106:106751. doi: 10.1016/j.foodcont.2019.106751
[4]	杨天歌, 邓红, 李涵, 孟永宏, 雷佳蕾, 马婧, 郭玉蓉. 超高压杀菌处理冷破碎猕猴桃果浆的条件优化及其贮藏期杀菌效果. 中国农业科学, 2018,51(7):1368-1377. doi: 10.3864/j.issn.0578-1752.2018.07.014
	YANG T G, DENG H, LI H, MENG Y H, LEI J L, MA J, GUO Y R. Optimization of ultra-high pressure sterilization conditions on the kiwi fruit pulp produced by cold crushing method and its sterilization effect during storage period. Scientia Agricultura Sinica, 2018,51(7):1368-1377. (in Chinese) doi: 10.3864/j.issn.0578-1752.2018.07.014
[5]	陶瑞, 史智佳, 贡慧, 杨震, 刘梦. 超声协同诱导剂对枯草芽孢杆菌芽孢致死的作用. 食品科学, 2018,39(11):95-100.
	TAO R, SHI Z J, GONG H, YANG Z, LIU M. Inactivation of Bacillus subtilis spores by ultrasonic treatment combined with inducer. Food Science, 2018,39(11):95-100. (in Chinese)
[6]	乔维维, 黄明明, 王佳媚, 严文静, 章建浩, 杨龙平. 低温等离子体对生鲜牛肉杀菌效果及色泽的影响. 食品科学, 2017,38(23):237-242.
	QIAO W W, HUANG M M, WANG J M, YAN W J, ZHANG J H, YANG L P. Effect of cold plasma on sterilization and color of fresh beef. Food Science, 2017,38(23):237-242. (in Chinese)
[7]	KIM H J, YONG H I, PARK S, CHOE W, JO C. Effects of dielectric barrier discharge plasma on pathogen inactivation and the physicochemical and sensory characteristics of pork loin. Current Applied Physics, 2013,13(7):1420-1425. doi: 10.1016/j.cap.2013.04.021
[8]	李兆杰, 杨丽君, 刘小菁, 王静, 刘玉敏, 李春喜. 辉光放电低温等离子体技术对微生物的杀菌动力学和杀菌机制. 食品科学, 2015,36(11):167-171. doi: 10.7506/spkx1002-6630-201511032
	LI Z J, YANG L J, LIU X J, WANG J, LIU Y M, LI C X. Bactericidal kinetics and mechanisms of low temperature glow discharge plasma. Food Science, 2015,36(11):167-171. (in Chinese) doi: 10.7506/spkx1002-6630-201511032
[9]	LIN L, LIAO X, LI C Z, ABDEL-SAMIE M A, CUI H Y. Inhibitory effect of cold nitrogen plasma on Salmonella Typhimurium biofilm and its application on poultry egg preservation. LWT-Food Science and Technology, 2020,126:109340. doi: 10.1016/j.lwt.2020.109340
[10]	张爱静, 李琳琼, 王鹏杰, 高瑀珑. 热胁迫对大肠杆菌细胞膜和膜蛋白的影响. 中国农业科学, 2020,53(5):1046-1057. doi: 10.3864/j.issn.0578-1752.2020.05.015
	ZHANG A J, LI L Q, WANG P J, GAO Y L. Effects of heat stress on cell membrane and membrane protein of Escherichia coli. Scientia Agricultura Sinica, 2020,53(5):1046-1057. (in Chinese) doi: 10.3864/j.issn.0578-1752.2020.05.015
[11]	SANTOS A L, GOMES N C M, HENRIQUES I, ALMEIDA A, CORREIA A, CUNHA A. Contribution of reactive oxygen species to UV-B-induced damage in bacteria. Journal of Photochemistry & Photobiology B Biology, 2012,117:40-46.
[12]	吴学友, 朱悦, 陈正行, 鞠兴荣. 乳酸菌细菌素Durancin GL对单增李斯特菌的抗菌活性及机制. 食品科学, 2019,40(23):73-78.
	WU X Y, ZHU Y, CHEN Z H, JU X R. Antibacterial activity and mechanism of Durancin GL against Listeria monocytogenes. Food Science, 2019,40(23):73-78. (in Chinese)
[13]	ZHAO L, ZHANG H, HAO T Y, LI S R. In vitro antibacterial activities and mechanism of sugar fatty acid esters against five food-related bacteria. Food Chemistry, 2015,187(15):370-377. doi: 10.1016/j.foodchem.2015.04.108
[14]	李长城, 白伟娟, 贾真, 方婷, 陈锦权. 温度和盐含量对三文鱼中单增李斯特菌热失活的影响. 食品工业, 2018,39(12):50-53.
	LI C C, BAI W J, JIA Z, FANG T, CHEN J Q. Effect of temperature and salt on thermal inactivation of Listeria monocytogenes in salmon. Food Industry, 2018,39(12):50-53. (in Chinese)
[15]	王婷婷, 李春, 李佳栋, 杜鹏, 刘丽波, 徐占文, 杨雨泽. 乙醇胁迫对植物乳杆菌膜生理及粘附性的影响. 食品科学, 2019,40(18):63-69.
	WANG T T, LI C, LI J D, DU P, LIU L B, XU Z W, YANG L Z. Effect of ethanol stress on membrane and adhesion of lactobacillus plantarum. Food Science, 2019,40(18):63-69. (in Chinese)
[16]	LI S G, HUANG Y, AN F P, HUANG Q, GENG F, MA M H. Hydroxyl radical-induced early stage oxidation improves the foaming and emulsifying properties of ovalbumin. Poultry Science, 2019,98(2):1047-1054. doi: 10.3382/ps/pey370 pmid: 30101285
[17]	CAI X T, WANG X, CHEN Y C, WANG Y D, SONG D F, GU Q. A natural biopreservative: Antibacterial action and mechanisms of Chinese Litsea mollis Hemsl. extract against Escherichia coli DH5α and Salmonella spp. Journal of Dairy Science, 2019,102(11):9663-9673.
[18]	WASTON A L, CHIU N H L. Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells- Lactobacillus helveticus. Journal of Microbiological Methods, 2016,128(8):58-60. doi: 10.1016/j.mimet.2016.06.030
[19]	ESCUDERO R, SEGURA J, VELASCO R, VALHONDO M, DE AVILA M, GARCIA-GARCIA A B, CAMBERO M I. Electron spin resonance (ESR) spectroscopy study of cheese treated with accelerated electrons. Food Chemistry, 2019,276:315-321. doi: 10.1016/j.foodchem.2018.09.101 pmid: 30409600
[20]	李凤珠, 张玉礼杨吉霞. 运用实时荧光定量PCR法研究榨菜腌制过程中细菌和真菌的数量变化. 食品与发酵工业, 2019,50(18):58-64.
	LI F Z, ZHANG Y L, YANG J X. Evaluation of quantity dynamics of bacteria and fungi during curing process of Zhacai by quantitative PCR. Food and Fermentation Industries, 2019,50(18):58-64. (in Chinese)
[21]	ZHAO W, YANG R J, GU Y J, LI C Y. Effects of pulsed electric fields on cytomembrane lipids and intracellular nucleic acids of Saccharomyces cerevisiae. Food Control, 2014,39:204-213. doi: 10.1016/j.foodcont.2013.11.015
[22]	LIU Z W, ZENG X A, NGADI M, HAN Z. Effect of cell membrane fatty acid composition of Escherichia coli on the resistance to pulsed electric field (PEF) treatment. LWT- Food Science and Technology, 2017,76:18-25. doi: 10.1016/j.lwt.2016.10.019
[23]	GREBOWSKI J, KROKOSZ A, PUCHALA M. Membrane fluidity and activity of membrane ATPases in human erythrocytes under the influence of polyhydroxylated fullerene. Biochimica et Biophysica Acta, 2013,1828(2):241-248. doi: 10.1016/s0005-2736(98)00020-0 pmid: 9630653
[24]	ANUVA S, BIJAN K P, NIKHIL G. Photophysics of DNA staining dye Propidium Iodide encapsulated in bio-mimetic micelle and genomic fish sperm DNA. Journal of Photochemistry and Photobiology B: Biology, 2012,192(2):58-67.
[25]	LIU P, XIE J X, LIU J H, JIA O Y. A novel thermostable β- galactosidase from Bacillus coagulans with excellent hydrolysis ability for lactose in whey. Journal of Dairy Science, 2019,102(11):9740-9748. doi: 10.3168/jds.2019-16654 pmid: 31477300
[26]	XIE Y F, JIANG S H, LI M, GUO Y H, CHENG Y L, QIAN H, YAO W R. Evaluation on the formation of lipid free radicals in the oxidation process of peanut oil. LWT-Food Science and Technology, 2019,104:24-29. doi: 10.1016/j.lwt.2019.01.016
[27]	OLATUNDE O O, BENJAKUL S, VONGKAMJAN K. Dielectric barrier discharge cold atmospheric plasma: Bacterial inactivation mechanism. Journal of Food Safety, 2019,39:1-10.
[28]	MOMTAHAN N, CROSBY C O, ZOLDAN J. The role of reactive oxygen species in i n vitro cardiac maturation. Trends in Molecular Medicine, 2019,25(6):482-493. doi: 10.1016/j.molmed.2019.04.005 pmid: 31080142
[29]	REN X Y, AN P P, ZHAI X, WANG S, KONG Q J. The antibacterial mechanism of pterostilbene derived from xinjiang wine grape: A novel apoptosis inducer in Staphyloccocus aureus and Escherichia coli. LWT-Food Science and Technology, 2019,101:100-106.
[30]	RUHLAND B R, RENIERE M L. Sense and sensor ability: Redox-responsive regulators in Listeria monocytogenes. Current Opinion in Microbiology, 2019,47:20-25. doi: 10.1016/j.mib.2018.10.006 pmid: 30412828
[31]	VAN DER VEEN S, VAN SCHALKWIJK S, MOLENAAR D, DE VOS W M, ABEE T, WELLS-BENNIK M H J. The SOS response of Listeria monocytogenes is involved in stress resistance and mutagenesis. Microbiology, 2010,156(2):374-384. doi: 10.1099/mic.0.035196-0
[32]	FERREIRA A, O'BYRNE C P, BOOR K J. Role of sigma (B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes. Applied and Environmental Microbiology, 2001,67(10):4454-4457. doi: 10.1128/aem.67.10.4454-4457.2001 pmid: 11571142

基因名称 Gene name	前引物（5'-3'） Forward primer (5'-3')	后引物（5'-3'） Reverse primer (5'-3')
16S RNA	ACCGTCAAGGGACAAGCA	GGGAGGCAGCAGTAGGGA
perR	CAACTGTATATAATAATTTACGCGT	TGCTGCGAAATGTTCTACTT
recA	CAAGCACAAGGCGGAACAG	CAACGGAGTCAATTACTAGCATAT
sigB	TATTTGGATTGCCGCTTACC	TTTCGGACCTAACTCTTTGATT

脂肪酸成分 Fatty acid composition	样品Sample
脂肪酸成分 Fatty acid composition	试验组 Test group	对照组 Control group
C12:0 (%)	2.12±0.03a	1.43±0.02b
C14:0 (%)	6.01±0.13a	5.45±0.07b
C16:0 (%)	16.45±0.75a	19.48±1.03b
C16:1 (%)	27.27±1.25a	21.26±0.77b
C17:0 (%)	2.73±0.05a	2.84±0.02a
C18:0 (%)	14.26±0.93a	24.48±1.02b
C18:1 (%)	26.64±0.16a	18.91±0.84b
不饱和脂肪酸 Unsaturated fatty acid (%)	53.91±1.76a	40.17±0.78b
饱和脂肪酸 Saturated fatty acid (%)	41.57±0.82a	53.68±1.64b
短链脂肪酸 Short chain fatty acid (%)	8.13±0.09a	6.88±0.15b