Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6647–6658 | Cite as

Cold plasma treatment triggers antioxidative defense system and induces changes in hyphal surface and subcellular structures of Aspergillus flavus

  • Juliana ŠimončicováEmail author
  • Barbora KaliňákováEmail author
  • Dušan Kováčik
  • Veronika Medvecká
  • Boris Lakatoš
  • Svetlana Kryštofová
  • Lucia Hoppanová
  • Veronika Palušková
  • Daniela Hudecová
  • Pavol Ďurina
  • Anna Zahoranová
Applied microbial and cell physiology


The cold atmospheric-pressure plasma (CAPP) has become one of the recent effective decontamination technologies, but CAPP interactions with biological material remain the subject of many studies. The CAPP generates numerous types of particles and radiations that synergistically affect cells and tissues differently depending on their structure. In this study, we investigated the effect of CAPP generated by diffuse coplanar surface barrier discharge on hyphae of Aspergillus flavus. Hyphae underwent massive structural changes after plasma treatment. Scanning electron microscopy showed drying hyphae that were forming creases on the hyphal surface. ATR-FTIR analysis demonstrated an increase of signal intensity for C=O and C-O stretching vibrations indicating chemical changes in molecular structures located on hyphal surface. The increase in membrane permeability was detected by the fluorescent dye, propidium iodide. Biomass dry weight determination and increase in permeability indicated leakage of cell content and subsequent death. Disintegration of nuclei and DNA degradation confirmed cell death after plasma treatment. Damage of plasma membrane was related to lipoperoxidation that was determined by higher levels of thiobarbituric acid reactive species after plasma treatment. The CAPP treatment led to rise of intracellular ROS levels detected by fluorescent microscopy using 2′,7′-dichlorodihydrofluorescein diacetate. At the same time, antioxidant enzyme activities increased, and level of reduced glutathione decreased. The results in this study indicated that the CAPP treatment in A. flavus targeted both cell surface structures, cell wall, and plasma membrane, inflicting injury on hyphal cells which led to subsequent oxidative stress and finally cell death at higher CAPP doses.


Antioxidant defense system Aspergillus flavus Cold atmospheric pressure plasma FTIR Lipid peroxidation Oxidative stress 


Funding information

This work was supported by the Slovak Research and Development Agency under the contract no. APVV-16-0216 and by a project for the building of infrastructure for the modern research of civilization diseases, ITMS 26230120006.

Compliance with ethical standard

Conflict of interest

The authors declare they have no conflict of interest.

Ethical approval

This paper does not contain any studies with human participants or animals performed by any of the authors.


  1. Alkawareek MY, Algwari QT, Laverty G, Gorman SP, Graham WG, O’Connell D, Gilmore BF (2012) Eradication of Pseudomonas aeruginosa biofilms by atmospheric pressure non-thermal plasma. PLoS One 7:13–15. CrossRefGoogle Scholar
  2. Amaike S, Keller NP (2011) Aspergillus flavus. Annu Rev Phytopathol 49:107–133. CrossRefPubMedGoogle Scholar
  3. Amare MG, Keller NP (2014) Molecular mechanisms of Aspergillus flavus secondary metabolism and development. Fungal Genet Biol 66:11–18. CrossRefPubMedGoogle Scholar
  4. Arjunan KP, Sharma VK, Ptasinska S (2015) Effects of atmospheric pressure plasmas on isolated and cellular DNA—a review. Int J Mol Sci 16:2971–3016. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bistis GN, Perkins DD, Read ND (2003) Different cell types in Neurospora crassa. Fungal Genet Rep 50:17–19. CrossRefGoogle Scholar
  6. van Bokhorst-van de Veen H, Xie H, Esveld E, Abee T, Mastwijk H, Nierop Groot M (2014) Inactivation of chemical and heat-resistant spores of Bacillus and Geobacillus by nitrogen cold atmospheric plasma evokes distinct changes in morphology and integrity of spores. Food Microbiol 45:26–33. CrossRefPubMedGoogle Scholar
  7. Bourke P, Ziuzina D, Han L, Cullen PJ, Gilmore BF (2017) Microbiological interactions with cold plasma. J Appl Microbiol 123:308–324. CrossRefPubMedGoogle Scholar
  8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. CrossRefPubMedGoogle Scholar
  9. Černák M, Ráhel’ J, Kováčik D, Šimor M, Brablec A, Slavíček P (2004) Generation of thin surface plasma layers for atmospheric-pressure surface treatments. Contrib to Plasma Phys 44:492–495. CrossRefGoogle Scholar
  10. Černák M, Černáková L, Hudec I, Kováčik D, Zahoranová A (2009) Diffuse coplanar surface barrier discharge and its applications for in-line processing of low-added-value materials. Eur Phys J Appl Phys 47:22806. CrossRefGoogle Scholar
  11. Conway GE, Casey A, Milosavljevic V, Liu Y, Howe O, Cullen PJ, Curtin JF (2016) Non-thermal atmospheric plasma induces ROS-independent cell death in U373MG glioma cells and augments the cytotoxicity of temozolomide. Br J Cancer 114:435–443. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Das D, Chakraborty A, Santra SC (2016) Effect of gamma radiation on zinc tolerance efficiency of Aspergillus terreus thorn. Curr Microbiol 72:248–258. PubMedCrossRefGoogle Scholar
  13. Dasan BG, Boyaci IH, Mutlu M (2016a) Inactivation of aflatoxigenic fungi (Aspergillus spp.) on granular food model, maize, in an atmospheric pressure fluidized bed plasma system. Food Control 70:1–8. CrossRefGoogle Scholar
  14. Dasan BG, Mutlu M, Boyaci IH (2016b) Decontamination of Aspergillus flavus and Aspergillus parasiticus spores on hazelnuts via atmospheric pressure fluidized bed plasma reactor. Int J Food Microbiol 216:50–59. CrossRefPubMedGoogle Scholar
  15. Deng X, Shi J, Kong M (2006) Physical mechanisms of inactivation of Bacillus subtilis spores using cold atmospheric plasmas. IEEE Trans Plasma Sci 34:1310–1316. CrossRefGoogle Scholar
  16. Devi Y, Thirumdas R, Sarangapani C, Deshmukh RR, Annapure US (2017) Influence of cold plasma on fungal growth and aflatoxins production on groundnuts. Food Control 77:187–191. CrossRefGoogle Scholar
  17. Ehlbeck J, Schnabel U, Polak M, Winter J, von Woedtke T, Brandenburg R, von dem Hagen T, Weltmann K (2011) Low temperature atmospheric pressure plasma sources for microbial decontamination. J Phys D Appl Phys 44:13002. CrossRefGoogle Scholar
  18. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77CrossRefPubMedGoogle Scholar
  19. Fridman A (2008) Plasma chemistry. Cambridge university pressGoogle Scholar
  20. Gaunt LF, Beggs CB, Georghiou GE (2006) Bactericidal action of the reactive species produced by gas-discharge nonthermal plasma at atmospheric pressure: a review. IEEE Trans Plasma Sci 34:1257–1269. CrossRefGoogle Scholar
  21. Graves DB (2014) Low temperature plasma biomedicine: a tutorial review. Phys Plasmas 21:80901. CrossRefGoogle Scholar
  22. Henselová M, Slováková Ľ, Martinka M, Zahoranová A (2012) Growth, anatomy and enzyme activity changes in maize roots induced by treatment of seeds with low-temperature plasma. Biologia 67:490–497. CrossRefGoogle Scholar
  23. Hojnik N, Cvelbar U, Tavčar-Kalcher G, Walsh JL, Križaj I (2017) Mycotoxin decontamination of food: cold atmospheric pressure plasma versus “classic” decontamination. Toxins (Basel) 9:1–19. CrossRefGoogle Scholar
  24. Jiang F, Zhang Y, Dusting GJ (2011) NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair. Pharmacol Rev 63:218–242. CrossRefPubMedGoogle Scholar
  25. Joshi SG, Cooper M, Yost A, Paff M, Ercan UK, Fridman G, Friedman G, Fridman A, Brooks AD (2011) Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli. Antimicrob Agents Chemother 55:1053–1062. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kaminskyj S, Jilkine K, Szeghalmi A, Gough K (2008) High spatial resolution analysis of fungal cell biochemistry—bridging the analytical gap using synchrotron FTIR spectromicroscopy. FEMS Microbiol Lett 284:1–8. CrossRefPubMedGoogle Scholar
  27. Kiššová I, Deffieu M, Samokhvalov V, Velours G, Bessoule JJ, Manon S, Camougrand N (2006) Lipid oxidation and autophagy in yeast. Free Radic Biol Med 41:1655–1661. CrossRefPubMedGoogle Scholar
  28. Kogelschatz U (2003) Dielectric-barrier discharges: their history, discharge physics and industrial applications. Plasma Chem Plasma Process 23:1–46. CrossRefGoogle Scholar
  29. Laroussi M (2005) Low temperature plasma-based sterilization: overview and state-of-the-art. Plasma Process Polym 2:391–400. CrossRefGoogle Scholar
  30. Laroussi M (2014) From killing bacteria to destroying cancer cells: 20 years of plasma medicine. Plasma Process Polym 11:1138–1141. CrossRefGoogle Scholar
  31. Laroussi M, Leipold F (2004) Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. Int J Mass Spectrom 233:81–86. CrossRefGoogle Scholar
  32. Lazovic S, Puac N, Radic N, Hoder T, Malovic G, Ráhel’ J, Černák M, Petrovic ZL (2008) Mass spectrometry of diffuse coplanar surface barrier discharge. Publ l’Observatoire Astron Beogr 84:401–404Google Scholar
  33. Lu X, Naidis GV, Laroussi M, Reuter S, Graves DB, Ostrikov K (2016) Reactive species in non-equilibrium atmospheric-pressure plasmas: generation, transport, and biological effects. Phys Rep 630:1–84. CrossRefGoogle Scholar
  34. Mai-Prochnow A, Clauson M, Hong J, Murphy AB (2016) Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma. Sci Rep 6:1–11. CrossRefGoogle Scholar
  35. Mcintyre M, Müller C, Dynesen J, Nielsen J (2001) Metabolic engineering. Adv Biochem Eng Biotechnol 73:103–128. PubMedCrossRefGoogle Scholar
  36. Mir SA, Shah MA, Mir MM (2016) Understanding the role of plasma technology in food industry. Food Bioprocess Technol 9:734–750. CrossRefGoogle Scholar
  37. Misra NN, Tiwari BK, Raghavarao KSMS, Cullen PJ (2011) Nonthermal plasma inactivation of food-borne pathogens. Food Eng Rev 3:159–170. CrossRefGoogle Scholar
  38. Mohamad R, Mohamed MS, Suhaili N, Salleh MM, Ariff A (2010) Kojic acid: applications and development of fermentation process for production. Biotechnol Mol Biol Rev 5:24–37Google Scholar
  39. Mošovská S, Medvecká V, Halászová N, Ďurina P, Valík Ľ, Mikulajová A, Zahoranová A (2018) Cold atmospheric pressure ambient air plasma inhibition of pathogenic bacteria on the surface of black pepper. Food Res Int 106:862–869. CrossRefPubMedGoogle Scholar
  40. ten Bosch L, Pfohl K, Avramidis G, Wieneke S, Viöl W, Karlovsky P (2017) Plasma-based degradation of mycotoxins produced by Fusarium, Aspergillus and Alternaria species. Toxins (Basel) 9:1–12. CrossRefGoogle Scholar
  41. Panngom K, Baik KY, Nam MK, Han JH, Rhim H, Choi EH (2013) Preferential killing of human lung cancer cell lines with mitochondrial dysfunction by nonthermal dielectric barrier discharge plasma. Cell Death Dis 4:e642–e648. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Park BJ, Takatori K, Sugita-Konishi Y, Kim IH, Lee MH, Han DW, Chung KH, Hyun SO, Park JC (2007) Degradation of mycotoxins using microwave-induced argon plasma at atmospheric pressure. Surf Coatings Technol 201:5733–5737. CrossRefGoogle Scholar
  43. Puač N, Živković S, Selaković N, Milutinović M, Boljević J, Malović G, Petrović ZL (2014) Long and short term effects of plasma treatment on meristematic plant cells. Appl Phys Lett 104:214106. CrossRefGoogle Scholar
  44. Roth S, Feichtinger J, Hertel C (2010) Characterization of Bacillus subtilis spore inactivation in low-pressure, low-temperature gas plasma sterilization processes. J Appl Microbiol 108:521–531. CrossRefPubMedGoogle Scholar
  45. Ryu YH, Kim YH, Lee JY, Shim GB, Uhm HS, Park G, Choi EH (2013) Effects of background fluid on the efficiency of inactivating yeast with non-thermal atmospheric pressure plasma. PLoS One 8:1–9. CrossRefGoogle Scholar
  46. Sakudo A, Toyokawa Y, Misawa T, Imanishi Y (2017) Degradation and detoxification of aflatoxin B1 using nitrogen gas plasma generated by a static induction thyristor as a pulsed power supply. Food Control 73:619–626. CrossRefGoogle Scholar
  47. Selcuk M, Oksuz L, Basaran P (2008) Decontamination of grains and legumes infected with Aspergillus spp. and Penicillum spp. by cold plasma treatment. Bioresour Technol 99:5104–5109. CrossRefPubMedGoogle Scholar
  48. Šimončicová J, Kaliňáková B, Kryštofová S (2017) Aflatoxins: biosynthesis, prevention and eradication. Acta Chim Slovaca 10:123–131. CrossRefGoogle Scholar
  49. Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47:389–394. CrossRefPubMedGoogle Scholar
  50. Su X, Tian Y, Zhou H, Li Y, Zhang Z, Jiang B, Yang B, Zhang J, Fang J (2018) Inactivation efficacy of non-thermal plasma activated solutions against Newcastle disease virus. Appl Environ Microbiol 81:996–1002. CrossRefGoogle Scholar
  51. Suhem K, Matan NN, Nisoa M, Matan NN (2013) Inhibition of Aspergillus flavus on agar media and brown rice cereal bars using cold atmospheric plasma treatment. Int J Food Microbiol 161:107–111. CrossRefPubMedGoogle Scholar
  52. Thirumdas R, Sarangapani C, Annapure US (2014) Cold plasma: a novel non-thermal technology for food processing. Food Biophys 10:1–11. CrossRefGoogle Scholar
  53. Weiss M, Gümbel D, Hanschmann EM, Mandelkow R, Gelbrich N, Zimmermann U, Walther R, Ekkernkamp A, Sckell A, Kramer A, Burchardt M, Lillig CH, Stope MB (2015) Cold atmospheric plasma treatment induces anti-proliferative effects in prostate cancer cells by redox and apoptotic signaling pathways. PLoS One 10:e0130350. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Yoshino K, Matsumoto H, Iwasaki T, Kinoshita S, Noda K, Iwamori S (2013) Monitoring of sterilization in an oxygen plasma apparatus, employing a quartz crystal microbalance (QCM) method. Vacuum 93:84–89. CrossRefGoogle Scholar
  55. Zahoranová A, Henselová M, Hudecová D, Kaliňáková B, Kováčik D, Medvecká V, Černák M (2016) Effect of cold atmospheric pressure plasma on the wheat seedlings vigor and on the inactivation of microorganisms on the seeds surface. Plasma Chem Plasma Process 36:397–414. CrossRefGoogle Scholar
  56. Zhou R, Zhou R, Zhang X, Zhuang J, Yang S, Bazaka K, Ostrikov K (2016) Effects of atmospheric-pressure N2, He, air, and O2 microplasmas on mung bean seed germination and seedling growth. Sci Rep 6:32603. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Juliana Šimončicová
    • 1
    Email author
  • Barbora Kaliňáková
    • 1
    Email author
  • Dušan Kováčik
    • 2
  • Veronika Medvecká
    • 2
  • Boris Lakatoš
    • 1
  • Svetlana Kryštofová
    • 1
  • Lucia Hoppanová
    • 1
  • Veronika Palušková
    • 1
  • Daniela Hudecová
    • 1
  • Pavol Ďurina
    • 2
  • Anna Zahoranová
    • 2
  1. 1.Institute of Biochemistry and Microbiology, Faculty of Chemical and Food TechnologySlovak University of TechnologyBratislavaSlovakia
  2. 2.Department of Experimental Physics, Faculty of Mathematics, Physics, and InformaticsComenius UniversityBratislavaSlovakia

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