Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (24): 5104-5114.doi: 10.3864/j.issn.0578-1752.2020.24.013

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Antibacterial Mechanism of Cold Plasma Against Listeria monocytogenes

DOU Yong1(),YAO MiaoAi1,LÜ HuaiZhong1,HU PeiHong2,DONG Jing1   

  1. 1Department of Grain Engineering and Food & Drug, Jiangsu Vocational College of Finance & Economics, Huai’an 223003, Jiangsu
    2Huai’an Zhengchang Feed Co., Ltd, Huai’an 223003, Jiangsu
  • Received:2020-04-17 Accepted:2020-06-10 Online:2020-12-16 Published:2020-12-28
  • Contact: Yong DOU E-mail:douyong1979@163.com

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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

Fig. 1

Graphical abstract of mechanism of cold plasma on cytomembrane"

Table 1

Primer sequence of different genes"

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

Fig. 2

Response surface of the effects of power, treatment time and flow rate on the inhibitory effect of cold plasma"

Fig. 3

Effect of cold plasma treatment on cell membrane integrity A: TEM results of control; B: TEM results of CP treatment; C: SDS-PAGE results; D: Protein concentration; E: DNA concentration. * means significant difference (P<0.05). The same as below"

Table 2

Compositions and contents of fatty acids in different samples"

脂肪酸成分
Fatty acid composition		 样品Sample
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

Fig. 4

ANS fluorescence intensity of different samples"

Fig. 5

Effect of cold plasma treatment on cell membrane permeability A: PI fluorescent images of control; B: PI fluorescent images of CP treatment; C: Changes of conductivity and β-galactosidase"

Fig. 6

Effect of cold plasma treatment on cell fluorescence intensity A: ROS fluorescent images of control; B: Fluorescent images of CP treatment for 20 min; C: Fluorescent images of CP treatment for 40 min; D: ROS fluorescence intensity"

Fig. 7

ESR spectrogram of different samples (A), Changes of expression of perR, recA and sigB genes (B)"

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