Cold Plasma in Food Technology
Possible Applications for Surface Sterilization
- Fig. 1: The four states of matter.
- Fig. 2: The plasma-tube system (schematic).
- Fig. 3: Inactivation of Methicillin-resistant Staphylococcus aureus. In the upper row are the untreated control samples, in the middle row the samples after 20 seconds of treatment and in the lower row the samples after 30 seconds of treatment. The previously prepared dilution series, are from left to right, with decreasing bacterial cell concentration (10-1 to 10-8 cfu/mL). (private photo taken by Dr. B. Ahlfeld).
- Fig. 4: Swabbing of cold plasma-treated norovirus samples. (private photo taken by Dr. A. Binder).
Non-thermal atmospheric pressure plasma, also called cold plasma, has gained increasing importance as a new sterilization method in recent years. Various studies have shown a microbial reduction effect against a variety of microorganisms on the surface of different matrices. This report focuses on two semi-direct plasmas, working with ambient air. The effectiveness of these plasma systems was investigated against a variety of food-borne pathogens.
Each matter undergoes the physical states from solid to liquid to gaseous after energy supply. If more energy is supplied to this third gaseous state, the gas particles are excited or ionised. Plasma, the fourth state of matter, is formed (fig. 1).
Natural plasmas are thermal (‘hot’) plasmas. They represent 99% of the visible matter of the universe and have gas temperatures of several million Kelvin (K). A typical characteristic of hot plasmas is that all particles (electrons, ions and neutral molecules) are in a thermodynamic equilibrium. Hot plasmas are, for example, terrestrial and astrophysical plasmas, which include lightning, the northern lights and solar winds. Furthermore, thermal plasmas only appear as artificially generated plasmas on earth. These plasmas are used in industry applications and in medicine for cauterisation . However, the high temperatures limit the range of applications of hot plasmas.
Therefore, the focus of research shifted to the development and establishment of new technologies for generating cold plasmas. The electric field applied for generating cold plasmas is lower than the one used to generate thermal plasmas, which results in ionisation of only one electron or ion per billion neutral atoms or molecules. Electrons at very high temperatures are produced in the electric field. These electrons only transfer a fraction of their energy to the neutral atoms and molecules, resulting in only a slight heating of them. In case of that, cold plasmas have a clear temperature difference between electrons and the other gas particles. Hence, cold plasmas are described as non-equilibrium plasmas due to the temperature divergence of the various plasma gas particles.
Cold Plasma Technologies
In this studies, two semi-direct plasma systems, so-called hybrid plasmas, were used (first model FlatPlaSter 2.0.
and successor system the plasma-tube, terraplasma) (fig. 2). The generated ‘plasma cloud’ consists of different plasma species, e.g., UV radiation, ROS and RNS.
In contrast to the plasma tube system, the first model can sterilize surfaces with a maximum size of a 96-well plate. This demonstrates that the development of expandable plasma technologies, like the plasma-tube enables the treatment of larges surfaces. This is the reason why an integration of this efficient plasma system is possible in industrial processes, e.g., the food-production chain, application in conveyor belts or in packaging machines. Additional advantages are the saving of resources due to the use of inexpensive ambient air and the particularly environmentally friendly character.
Basics of the Bactericidal Effect of Cold Atmospheric Pressure Plasmas
As mentioned above, radical plasma species have a bactericidal effect. Besides the concentration of reactive plasma species, their chemical composition and the applied voltage also influences the level of reduction. A high voltage produces a cold plasma with high concentration of more bactericidal radical nitrogen species.
The effect of radical plasma species against pathogenic microorganism, e.g., viruses, bacteria, fungi, depends also on various factors. Gram-negative bacterial species are only protected by a thin cell wall with extracellular lipopolysaccharides, while gram-positive bacterial species have a thicker cell membrane due to more murein layers. The thicker outer cell membrane results in a higher resistance against many exogenous factors, e.g., radical plasma species .
Exogenous factors can influence the inactivation of bacterial cells, too. The matrix on which the bacterial cells are located can protect the bacterial cells against radical plasma species, e.g., by cavities in the surface. Furthermore, proteins and lipids react with plasma species, too. This reaction is known as the quencher-effect and results in a decrease of the bactericidal effect of radical plasma species . Defence mechanisms of the pathogens include the active migration of bacteria into deeper matrix layers and the exhibition of secondary pathogenicity factors in the cell membrane like a natural antioxidant, e.g., carotenoids, which protect the bacterial cells against radical plasma species .
Applying Cold Plasma on Food Products
Using cold plasma for treating different food surfaces entails the challenge of an effective reduction of microorganisms while maintaining the product quality.
First in vitro investigations into the reduction of food pathogens were carried out with the first model. For this experimental run, various pathogenic or spoilage bacterial species (e.g., B. cereus, S. aureus, E. faecium, P. aeruginosa, E. coli, MRSA, ESBL-forming E. coli) were selected. All pathogens were reduced by up to 4 lg steps after 30-second plasma treatment (fig. 3).
In order to test the efficacy of cold plasma against viruses, a human pathogen norovirus strain (GII.4) was chosen. The strain was isolated during a disease outbreak. For the experiment, norovirus suspension, obtained from a human stool sample, was transferred to petri dishes, dried (fig. 4) and then plasma treated for 30 seconds up to 15 minutes . The study showed that the plasma treatment resulted in a time-dependent significant reduction of the norovirus.
The development of the plasma-tube system opens new possibilities for the in vivo reduction of food-borne pathogens on food. In order to test the effectiveness of the new plasma-tube system on food pre-packaged rolled fillet of ham was selected. Rolled fillet of ham is not heat-treated during production or by consumers before consumption. During production, however, the ubiquitously present gram-positive human pathogenic bacterial species Listeria monocytogenes or the gram-negative Salmonella enterica Serovar Typhimurium can be transferred to the product, e.g., by sick employees.
This study showed a significant higher germ reduction of L. monocytogenes as well as S. Typhimurium after applying higher voltage and generating more bactericidal radical nitrogen species . A low-voltage setting resulted in the generation of an ozone-based ‘plasma cloud’ with a decreased inactivation of both bacterial species. In contrast to nitrogen species, radical oxygen species, especially ozone, dissociate with increasing humidity level of the ambient air due to the so-called ‘quencher-effect’ and have no bactericidal potential against bacterial species.
Probably initial sublethally damaged bacterial cells were lethally damaged during storage. This underlines the suitability of plasma treatment in combination with retail packaging, the distribution logistics and storage under cool storage conditions.
Chemical and physical investigations conducted with a near-infrared spectrometer on the plasma-treated ham resulted in no relevant changes.
All in all, plasma treatment seems to lead to a significant inactivation of pathogenic microorganisms without any loss of food quality.
The application of plasma treatment to reduce microorganisms on different food surfaces is an innovative technology for providing microbiological safety of food while maintaining food quality.
Further investigations are necessary to establish plasma-technologies, such as the plasma-tube system, on other food and environmental materials with other bacterial species.
Karolina A. Lis1, Corinna Kehrenberg1, Birte Ahlfeld1
1 Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Germany
Karolina A. Lis
Institute for Food Quality and Food Safety
University of Veterinary Medicine Hannover
 Fridman, A., A. Chirokov, and A. Gutsol, Non-thermal atmospheric pressure discharges. Journal of Physics D: Applied Physics, 2005. 38(2): DOI:10.1088/0022-3727/38/2/R01
 Raiser, J. and M. Zenker, Argon plasma coagulation for open surgical and endoscopic applications: state of the art. Journal of Physics D: Applied Physics, 2006. 39(16): p. 3520-3523. DOI:10.1088/0022-3727/39/16/S10
3. terraplasma-GmbH, Oberflächenmikroentladungstechnologie.
4. Laroussi, M., et al., Plasma Medicine - Applications of Low-Temperature Gas Plasmas in Medicine and Biology. Cambridge, 2012. 1. DOI:10.1017/CBO9780511902598
5. Ehlbeck, J., et al., Low temperature atmospheric pressure plasma sources for microbial decontamination. Journal of Physics D: Applied Physics, 2010. 44(1): p. 013002. DOI:10.1088/0022-3727/44/1/013002
6. Heinlin, J., et al., Plasma medicine: possible applications in dermatology. J Dtsch Dermatol Ges, 2010. 8(12): p. 968-76. DOI:10.1111/j.1610-0387.2010.07495.x
7. Shimizu, T., et al., Characterization of microwave plasma torch for decontamination. Plasma Processes and Polymers, 2008. 5(6): p. 577-582. DOI:10.1002/ppap.200800021
8. Shimizu, T., J. Zimmermann, and G. Morfill, The bactericidal effect of surface micro-discharge plasma under different ambient conditions. New Journal of Physics, 2011. 13(2): p. 023026. DOI:10.1088/1367-2630/13/2/023026
9. Hirschberg, J., Grundlegende Untersuchungen zur Wirkung kalter Plasmen auf kutane Lipidsysteme. Technische Universität Clausthal, Dissertation, 2017: p. 151. DOI:10.21268/20170831-142144
10. Laroussi, M., D. Mendis, and M. Rosenberg, Plasma interaction with microbes. New Journal of Physics, 2003. 5. DOI:10.1088/ 1367-2630/5/1/341
11. Joshi, S.G., et al., Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli. Antimicrob Agents Chemother, 2011. 55(3): p. 1053-62. DOI:10.1128/AAC.01002-10
12. Korachi, M. and N. Aslan, The Effect of Atmospheric Pressure Plasma Corona Discharge on pH, Lipid Content and DNA of Bacterial Cells. Plasma Science and Technology, 2011. 13(1). DOI:10.1088/1009-0630/13/1/20
13. Lu, H., et al., Bacterial inactivation by high-voltage atmospheric cold plasma: influence of process parameters and effects on cell leakage and DNA. J Appl Microbiol, 2013. 116(4): p. 784-94. DOI:10.1111/jam.12426
14. Laroussi, M. and F. Leipold, Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. International Journal of Mass Spectrometry, 2003. 233(1–3): p. 81-86. DOI:10.1016/j.ijms.2003.11.016
15. Critzer, F., et al., Atmospheric plasma inactivation of foodborne pathogens on fresh produce surfaces. Journal of Food Protection®, 2007. 70(10): p. 2290-2296. DOI:10.4315/0362-028X-70.10.2290
16. Niemira, B.A. and J. Sites, Cold plasma inactivates Salmonella Stanley and Escherichia coli O157: H7 inoculated on golden delicious apples. Journal of Food Protection®, 2008. 71(7): p. 1357-1365.
17. Ragni, L., et al., Non-thermal atmospheric gas plasma device for surface decontamination of shell eggs. Journal of Food Engineering, 2010. 100(1): p. 125-132. DOI :10.1016/j.jfoodeng.2010.03.036
18. Surowsky, B., S. Bußler, and O.K. Schlüter, Chapter 7 - Cold Plasma Interactions With Food Constituents in Liquid and Solid Food Matrices, in Cold Plasma in Food and Agriculture. 2016, Academic Press: San Diego. p. 179-203.
19. Fernandez, A., et al., Effect of microbial loading on the efficiency of cold atmospheric gas plasma inactivation of Salmonella enterica serovar Typhimurium. Int J Food Microbiol, 2012. 152(3): p. 175-80. DOI:10.1016/j.ijfoodmicro.2011.02.038
20. Hury, S., et al., A parametric study of the destruction efficiency of Bacillus spores in low pressure oxygen-based plasmas. Letters in Applied Microbiology, 1998. 26(6): p. 417-421. DOI:10.1046/j.1472-765X.1998.00365.x
21. Bermúdez-Aguirre, D., et al., Effect of atmospheric pressure cold plasma (APCP) on the inactivation of Escherichia coli in fresh produce. Food Control, 2013. 34(1): p. 149-157. DOI:10.1016/j.foodcont.2013.04.022
22. Clauditz, A., et al., Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infect Immun, 2006. 74(8): p. 4950-3. DOI:10.1128/IAI.00204-06
23. Lee, H., et al., Inactivation of Listeria monocytogenes on agar and processed meat surfaces by atmospheric pressure plasma jets. Food microbiology, 2011. 28(8): p. 1468-1471. DOI:10.1016/j.fm.2011.08.002
24. Surowsky, B., et al., Impact of cold plasma on Citrobacter freundii in apple juice: inactivation kinetics and mechanisms. Int J Food Microbiol, 2014. 174: p. 63-71. DOI:10.1016/j.ijfoodmicro.2013.12.031
25. Perni, S., G. Shama, and M. Kong, Cold atmospheric plasma disinfection of cut fruit surfaces contaminated with migrating microorganisms. Journal of Food Protection®, 2008. 71(8): p. 1619-1625. DOI:10.4315/0362-028X-71.8.1619
26. Ahlfeld, B., et al., Inactivation of a foodborne norovirus outbreak strain with nonthermal atmospheric pressure plasma. MBio, 2015. 6(1). DOI:10.1128/mBio.02300-14
27. Anonymous, Robert Koch Institute: Infection epidemiological yearbook of notifiable diseases for 2016. Robert Koch-Institute, 2016.
28. Lis, K.A., et al., Inactivation of Salmonella Typhimurium and Listeria monocytogenes on ham with nonthermal atmospheric pressure plasma. PLoS One, 2018. 13(5): p. e0197773. DOI:10.1371/journal.pone.0197773
29. Lee, K., et al., Sterilization of bacteria, yeast, and bacterial endospores by atmospheric-pressure cold plasma using helium and oxygen. JOURNAL OF MICROBIOLOGY-SEOUL-, 2006. 44(3): p. 269.
30. Ali, S., et al., Comparison of Two Whole-Room UV-Irradiation Systems for Enhanced Disinfection of Patient Rooms Contaminated with MRSA, carbapenemase-producing Klebsiella pneumoniae and Clostridium difficile spores. J Hosp Infect, 2017. DOI:10.1016/j.jhin.2017.08.011