ФИЗИКА ПЛАЗМЫ, 2013, том 39, № 9, с. 857-861
МЕТОДИЧЕСКИЕ ЗАМЕТКИ
УДК 533.9
INACTIVATION OF POSSIBLE MICROMYCETE FOOD CONTAMINANTS USING THE LOW-TEMPERATURE PLASMA AND HYDROGEN PEROXIDE
© 2013 г. M. Cerovsky*, J. Khun**, K. Rusova*, V. Scholtz**, H. Sou skova***
*Department of Food Preservation, Faculty of Food and Biochemical Technology, Institute of Chemical Technology in Prague **Department of Physics and Measurements, Faculty of Chemical Engineering, Institute of Chemical Technology in Prague ***Department of Computing and Control Engineering, Faculty of Chemical Engineering, Institute of Chemical Technology in Prague e-mail: scholtz@aldebaran.cz Поступила в редакцию 22.01.2013 г. Окончательный вариант получен 14.03.2013 г.
The inhibition effect of hydrogen peroxide aerosol, low-temperature plasma and their combinations has been studied on several micromycetes spores. The low-temperature plasma was generated in corona discharges in the open air apparatus with hydrogen peroxide aerosol. Micromycete spores were inoculated on the surface of agar plates, exposed solely to the hydrogen peroxide aerosol, corona discharge or their combination. After incubation the diameter of inhibition zone was measured. The solely positive corona discharge exhibits no inactivation effect, the solely negative corona discharge and solely hydrogen peroxide aerosol exhibit the in-activation effect, however their combinations exhibit to be much more effective. Low-temperature plasma and hydrogen peroxide aerosol present a possible alternative method of microbial decontamination of food, food packages or other thermolabile materials.
DOI: 10.7868/S0367292113090023
INTRODUCTION
The fungal and other microbial contaminants may present a serious problem in medicine, food processing and other areas and those elimination is often desired in many area. One common method is the inac-tivation of microorganisms on surfaces by hydrogen peroxide, however due to preoxide high oxidative potential this method may be limited by the material applied on, ecological problems, safety manipulations with the chemicals or other problems (e.g. economical viewpoint). On the other side there are numerous of works describing the microbicidal effects of low-temperature plasma. The presented work studies the possibility of the use of combination of low-temperature plasma with the hydrogen peroxide for the inactivation of micromycetes (fungal) spores on surfaces what may help to reduce the adverse side effects. The biological effects of low temperature plasma, devoted mainly to the killing of prokaryotic bacteria may be found in reviews [1—4], various applications in human medicine in the article [5] and decontamination of the medical products may be found in [6]. The fungicidal effect of low-temperature plasma are presented less often and was mentioned e.g. in [7, 8] or in our recent work [9], where we have studied the inactivation of micromycet-es spores in positive corona discharge. This work follows up our previously presented insights and describes the fungicidal effect of the synergy combination of low-temperature plasma generated in corona discharge and hydrogen peroxide aerosol. This combi-
nation leads to the decrease of the operation time up to several seconds and may be applicable for the treatment of food surface or food packages, preventing the mould overgrow and food spoilage, or for the decontamination of other thermolabile materials.
MATERIALS AND METHODS
The low temperature plasma was generated using the modification of current apparatus previously described in [10]. The modification allowed the addition of hydrogen peroxide aerosol into the atmosphere of applied discharge. The discharge burned between the point and grid electrodes. The distance between them is 6 mm. The point electrode was represented by the tip of a syringe needle situated vertically to the grid electrode. The grid consisted of stainless steel wire of diameter 0.25 mm forming the net with a mesh size of 0.8 cm. The discharge was stabilized by the connection of a serial resistance of 20 Mfi into the circuit. The discharge, similar to the pin-to-ring geometry, burns on the tip of the needle; on the closes parts of the wire burn secondary parasite discharges of inverse polarity but of lower intensity as it was observed by naked eye. Despite it, in this work, we call the discharge as positive corona if the polarity of the point electrode is positive and of the grid electrode is negative. Contrary, we call the discharge as negative corona if the polarity of the point electrode is negative and of the grid electrode is positive. For more details about the corona dis-
Fig. 1. Schematic experimental arrangement and the picture of the apparatus.
charge stabilisation and its characteristic see e.g. papers [11, 12]. The aerosol of hydrogen peroxide was generated by the ultrasonic nebulizer (Sun-up S.A. Model 3019) and driven into the discharge. The distance between the surface applied on and the grid electrode was set to 12 mm. The schematic experimental arrangement and the picture of the apparatus are shown in Fig. 1.
In experiments the spores were prepared by common microbiological method by taking the loopful of spores from the surface of grown culture and diluting in sterile physiological saline to obtain appropriate concentration. The number of spores in the suspension significantly exceeds the number of other mycelium cells. This initial suspension was diluted to obtain the concentration of approximately 100 cfu/plate or 100 cfu/cm2 for the experiments with low or high concentration, respectively. The clear YGC agar plates (9 cm in diameter) were used for inoculation. Consequently, the plates were exposed to the solely discharge, solely peroxide aerosol or the discharge burning in the atmosphere of hydrogen peroxide aerosol. After the exposition the exposed plates were incubated at 25°C for 5 days. After this time the survival spores begin to grow and form colonies, the inhibition effect was observed as clear inhibition zones bounded by colonies grown from surviving spores. Due to the size of particular colony the error of the inhibition zone diameter was estimated as 5 mm.
The parameters of discharge and peroxide aerosol varied to test the influence of the discharge polarity and several peroxide concentration. In case of solely discharge, the positive corona discharge burns at the voltage of 3.5 kV, the negative corona discharge burns at the voltage of 2.7 kV. Both voltages correspond to the current of 500 ^A. In case of the combination of discharge with the aerosol, the higher atmosphere hu-
midity influences the discharge and to obtain the some current of 500 ^A, the voltage was decreased to 3.3 kV and 2.6 kV for the positive and negative discharge, respectively. The influence of the hydrogen peroxide concentration in the aerosol was negligible. Concentrations of hydrogen peroxide water solutions were 0, 3, 10 and 30%. Nebulized hydrogen peroxide was mixed with the air and this aerosol flowed to the discharge area. The flow of the aerosol was adjusted to the values of 2.0 ± 0.1 L/min and the volume concentration of nebulized hydrogen peroxide solution in the mixture was 0.40 ± 0.03 mL/L.
In the first experiment, the characteristic of the spores inactivation of one fungal species Talaromyces striatus was measured for both discharge polarities, various hydrogen peroxide concentration and low spores concentration of 100 cfu/plate. The relatively low concentration was used due to assumption that for some parameters the inactivation may have no or low effect only and in the case of high concentration it may not be visible. T striatus was selected as known thermoresistant species and frequent contaminant in food processing. Used wild strain also origin as a contaminant from food factory. In the second experiment, due to the results of first experiment one discharge polarity and one peroxide concentration were chosen and the characteristic of spores inactivation was extended to other common fungal species. In this case the inacti-vation was relatively effective so the high spores concentration of 100 cfu/cm2 was used to approve the inactivation effect in the conditions of high contamination.
RESULTS AND DISCUSSION
In the first experiment with low concentration of inoculated spores of 100 cfu/plate, the characteristic
of the inactivation of Talaromyces striatus spores for both discharge polarities, various hydrogen peroxide concentrations and exposition times was studied. Results for solely discharge and solely hydrogen peroxide aerosol are shown in Tables 1 and 2, respectively. It is visible that the solely positive corona discharge has no inhibition effect neither for low spores concentration. Contrary to the positive corona discharge, the solely negative corona discharge or solely peroxide aerosol has the microbicidal effect on spores. Results for the combination of hydrogen peroxide aerosol with negative and positive discharges are shown in Tables 3 and 4, respectively and it is visible that the combination of the discharge and peroxide aerosol has synergy effects for the microbicidal efficiency. The combination of the hydrogen peroxide aerosol with the negative corona has higher efficiency than the combination with the positive one. For better visibility, the graph comparing the inhibition zones diameter on the exposition time for solely negative corona discharge, solely aerosol and their combination is shown in the Fig. 2.
Second experiment was focused to the mutual comparison of the inactivation effect on several mi-cromycetes species and has the aim to approve the in-activation in high spores concentration of 100 cfu/cm2. The apparatus was set to negative corona discharge (2.6 kV, 500 ^A) and hydrogen peroxide concentration of 10%, because in this concentration peroxide does not present so high oxidative reagent and in the comparison with 30% concentration
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