Sterilization system using microwave and UV light

Sterilization system using microwave and UV light

Colloids and Surfaces B: Biointerfaces 25 (2002) 299– 304 www.elsevier.com/locate/colsurfb Sterilization system using microwave and UV light Shiro Iw...

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Colloids and Surfaces B: Biointerfaces 25 (2002) 299– 304 www.elsevier.com/locate/colsurfb

Sterilization system using microwave and UV light Shiro Iwaguch a, Kentaro Matsumura a, Yoshikazu Tokuoka b,*, Shiro Wakui b, Norimichi Kawashima a,b b

a Faculty of Engineering, Toin Uni6ersity of Yokohama, 1614 Kurogane-cho, Aoba-ku, Yokohama 225 -8502, Japan Biomedical Engineering Center, Toin Uni6ersity of Yokohama, 1614 Kurogane-cho, Aoba-ku, Yokohama 225 -8502, Japan

Received 15 October 2001; accepted 6 November 2001

Abstract We constructed a novel microwave–UV light sterilization system and investigated its sterilization effect. This sterilization system can emit UV light by irradiation of microwave without other power. When irradiating UV light with and/or without microwave on aqueous DMPO solution, active oxygen species such as hydroxyl radical or superoxide were generated in the solution. The amount of active oxygen species generated by irradiation of microwave and UV light was larger than that by irradiation of UV light alone. This would be due to the promotion of emission of UV light photons by microwave and UV light irradiation. Moreover, microwave– UV light sterilization was highly effective to sterilize microorganisms. The generation of active oxygen species would play an important role in sterilization of the sterilization system. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sterilization; Microwave; UV light; Active oxygen species; ESR spin trapping method

1. Introduction Nowadays putrefaction of industrial products such as foods and cosmetics by microorganisms is one of the most crucial problems. To overcome such deterioration of the products, microorganism sterilization processes have been carried out. Many sterilization technologies have meanwhile been used in various industrial fields: heat sterilization, UV light sterilization, ozone gas sterilization and so on [1–3]. In the food industry field, heat sterilization is often used. Heat sterilization is based on exposing * Corresponding author. Tel./fax: +81-45-974-5607. E-mail address: [email protected] (Y. Tokuoka).

foods to high temperature for a certain period of time. Since foods are heated from the surface by thermal conduction, only the surface of foods is sterilized in a short treatment time. In contrast, the treatment of foods at high temperature for a long time causes the quality of foods to deteriorate. Instant heat sterilization technology which can completely sterilize microorganisms is then required. Microwave is an electromagnetic wave having 300 –30 000 MHz of frequency and 1–1000 nm of wavelength. Dipolar substances, especially water, can be thoroughly heated by dielectric heating of microwave irradiation. Heating speed and thermal efficiency of dielectric heating are greater than those of common heating systems. Due to these,

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microwave has been applied to heat sterilization technology [4]. However, heat sterilization by microwave has some problems as follows: “ Temperature distribution is produced in heterogeneous materials. “ Temperature distribution is generated and thermal efficiency is varied in materials of irregular shapes. “ Temperature rise on the surface of materials is retarded by heat radiation from the surface. To obtain more effective sterilization, noble heat sterilization systems by microwave combined with other sterilization method have to be developed. UV light sterilization is also used to sterilize microorganisms in various industrial fields [2]. DNA in microorganisms absorbs UV light and is destroyed, leading to disinfection of the microorganisms. We have noted that irradiation of microwave can turn on UV lamps without other power and so constructed a novel sterilization system that can simultaneously radiate both microwave and UV light. In the present study, the sterilization effect of such microwave–UV light sterilization system was investigated.

2.2. Determination of acti6e oxygen species by ESR Active oxygen species were measured by the ESR spin trapping method [5,6]. 5,5-Dimethyl-1pyrroline-N-oxide (DMPO, SIGMA) was used as a spin-trapping agent. Five microliters of DMPO was dissolved in 5 ml of distilled water. Two microliters of the aqueous DMPO solution was poured into a quartz cell, and the cell was placed in the microwave oven. After irradiation of microwave and UV light for 10 s, ESR spectrum of the aqueous solution was measured by ESR spectroscopy. (JES-TE100, JEOL). When either hydroxyl radical or superoxide, which is a sort of active oxygen species, is generated, the characteristic ESR signal of DMPO-OH adduct can be observed. Also, ESR spectrum of aqueous DMPO solution irradiated with microwave or UV light alone was measured.

2.3. Determination of amount of UV light photon Amount of UV light photon was measured using the iron (III) oxalate method [6]. Two milliliters of aqueous iron (III) oxalate solution in a quartz cell was irradiated with UV light for 5 s. After that, 1,10-phenanthroline was added to the

2. Experimental

2.1. Microwa6e –UV light sterilization system Fig. 1 shows the microwave– UV light sterilization system constructed. A UV lamp, a 4 or 18 W low-pressure mercury lamp (NIPPO Co.), was placed in a microwave oven whose frequency was 2450 MHz. The specific wavelengths of UV light from the UV lamp were 185 and 254 nm. Every sample solution for experiment was poured into a quartz cell, and the cell was placed in the microwave oven. The distance between the UV lamp and the quartz cell was always constant. When microwave and UV light were irradiated simultaneously, the microwave turned on the UV lamp without other power. When only the UV light was irradiated, on the other hand, the UV lamp was powered by A.C. 100 V.

Fig. 1. Novel microwave – UV light sterilization system.

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aqueous solution and its absorption spectrum was measured by UV – VIS spectroscopy (V-550, JASCO Co.). Here, the absorbance of the peak at 510 nm increases with the amount of UV light photons.

2.4. Decomposition of methylene blue Aqueous methylene blue solution was placed in a quartz cell and was irradiated with microwave and UV light at the same time for 4 min. The concentration of methylene blue remaining in the aqueous solution after the irradiation was determined using UV– VIS spectroscopy (V-550, JASCO Co.). The decomposition ratio of methylene blue (R) was calculated by the equation given below: R=

C0 −C ×100, C0

where C0 and C are the initial and final concentrations of methylene blue, respectively. The decomposition ratio of methylene blue irradiated with microwave or UV light alone was also estimated.

2.5. Sterilization of Escherichia coli Escherichia coli (MN522) was suspended at 2× 104 cells per ml in aqueous 0.9 wt.% NaCl solution. The suspension was treated for 5 or 10 s with both microwave and UV light. Two hundred microliters of the treated suspension was cultured on agar medium at 37 °C for 18 h. Then, the growth of colony of E. coli was observed. The growth of E. coli in the suspension treated by microwave or UV light alone was also determined by the same method.

3. Results and discussion

3.1. Generation of acti6e oxygen species in microwa6e –UV light sterilization system It is well-known that active oxygen species are generated when 185 and 254 nm of UV light, which are used in our sterilization system, are irradiated on water or oxygen molecule. Active

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oxygen species easily react with various kinds of chemical compounds. Biomolecular substances, lipids, enzymes, proteins, etc. are attacked and destroyed by active oxygen species. In particular, polyunsaturated aliphatic acids localized into cell membranes react with active oxygen species and are converted to lipid peroxides through lipoperoxidation reactions [7]. Reactions of active oxygen species with such biomolecular substances induce cell death. Thus, it is expected that active oxygen species are effective in sterilizing microorganisms. First, we tried to confirm the generation of active oxygen species in the microwave– UV light sterilization system by the ESR spin trapping method. When either hydroxyl radical or surperoxide, which is a sort of active oxygen species, is generated in DMPO solution, we can observe the characteristic ESR spectrum with four signals of DMPO-OH adduct. Fig. 2(a) displays the ESR spectrum of aqueous DMPO solution treated at the same time with microwave and UV light. The ESR spectra of aqueous DMPO solutions irradiated with microwave or UV light alone are also shown in Fig. 2(b) and (c). The ESR signal of DMPO-OH adduct was observed after the treatment by the combination of microwave and UV light, and that by the UV light alone. In contrast, no ESR spectrum appeared for the same sample treated with microwave alone. These results suggest that active oxygen species such as hydroxyl radical or surperoxide was generated by UV light irradiation. Further, besides the signal of DMPOOH adduct, we could also find the ESR signal of DMPO-H adduct as shown in Fig. 2(a) and (b). This would be because hydrogen radical was similarly generated. In general the ESR signals of DMPO-OH adduct overlap with the 2nd, 4th, 6th and 8th ESR signals of DMPO-H adduct from low magnetic field [8]. We marked the ESR signal overlapping each other by filled square in Fig. 2(a) and (b). Fig. 3 shows the difference spectrum between Fig. 2(a) and (b). Since the ESR signal of only DMPO-H adduct marked by filled circle in Fig. 2(a) and (b) disappeared, the amount of hydrogen radical generated by the combination of microwave and UV light was equal to that by UV light alone. Moreover, the remaining signal of the

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Fig. 3. The difference spectra between ESR spectra treated by the combination of microwave and UV light and that by UV light alone.

UV light alone. These findings indicate that the amount of DMPO-OH adduct, or active oxygen species such as hydroxyl radical or surperoxide, produced by irradiation of microwave and UV light is larger than that by UV light alone. To find the reason why the amount of active oxygen species increased by microwave and UV light irradiation, we tried to measure the amount of UV light photons by the iron (III) oxalate method. Fig. 4 represents the absorption spectra of iron (III) oxalate solutions irradiated with microwave and UV light and with UV light alone. The absorbance of the peak at 510 nm increased with the amount of UV light photons. The absorbance at 510 nm after microwave and UV light irradiation was greater than that after irradiation

Fig. 2. ESR spectra of DMPO solutions. (a) Irradiation with microwave and UV light, (b) irradiation with UV light alone, and (c) irradiation with microwave alone. Filled square and filled circle show the ESR signal consisting of the signals with DMPO-OH and DMPO-H adducts, respectively.

difference spectrum resulted from the excess amount of DMPO-OH adducts. When comparison is made between the original ESR spectra, the ESR signal overlapping in treatment with microwave and UV light is greater than that with

Fig. 4. Absorption spectra of iron (III) oxalate solutions treated with microwave and UV light and with UV light alone.

S. Iwaguch et al. / Colloids and Surfaces B: Biointerfaces 25 (2002) 299–304 Table 1 Decomposition ratio of methylene blue Decomposition ratio (%) Microwave+UV light UV light alone Microwave alone

90.6 75.0 10.4

of UV light alone; indicating that emission of UV light photons is promoted by combined irradiation of microwave and UV light. Generation of excess amount of active oxygen species caused by irradiation of microwave and UV light would be due to an enhanced emission of UV light photons.

3.2. Sterilization effect of microwa6e – UV light sterilization system As a result of generation of a large amount of active oxygen species mentioned above, the microwave–UV light sterilization system is suggested to have a highly effective sterilizing action. We then tried to decompose methylene blue, typical chemical substances, with the microwave– UV light sterilization system. The results on the de-

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composition ratio of methylene blue are given in Table 1. The decomposition ratio produced by treatment with microwave and UV light was greatest, whereas irradiation of microwave alone could decompose few methylene blue. Moreover, the order of the ratio coincided with that of the amount of active oxygen species generated. This finding suggests that the microwave–UV light sterilization system has a high efficiency in decomposition of chemical substances by a large amount of active oxygen species generated. Fig. 5 shows the results of sterilization of E. coli (MN522) under various conditions. No colony was observed after irradiation of microwave and UV light for 10 s. On the other hand, it was found that lots of colonies were observed after irradiation of microwave alone, and irradiation of microwave could not produce any sterilization effect under this experimental condition. These results show that the microwave–UV light sterilization system is superior in sterilizing microorganisms to the sterilization system with microwave alone. Moreover, as seen in Fig. 5, sterilization with microwave and UV light could yield no better sterilization effect over that with UV light. When the results of decomposition of methylene blue are

Fig. 5. Sterilization of E. coli under various conditions. (a) Blank, (b) treatment with microwave alone for 5 s, (c) treatment with microwave alone for 10 s, (d) treatment with microwave and UV light for 5 s, (e) treatment with microwave and UV light for 10 s, (f) treatment with UV light alone for 5 s, and (g) treatment with UV light alone for 10 s.

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taken into consideration, provided that proper sterilization conditions for microwave are prepared, the sterilization effect of microwave– UV light sterilization would be greater than that of UV light sterilization. A series of sterilization experiments mentioned above verify that the microwave– UV light sterilization system is highly effective to sterilize microorganisms. That would be due to the amount of active oxygen species generated in the sterilization system.

4. Conclusion The sterilization effect of a novel microwave– UV light sterilization system constructed was investigated. This sterilization system was found to have a highly effective sterilizing action. Generation of active oxygen species would play an important role in sterilization with the sterilization

system. In the microwave–UV light sterilization system, moreover, irradiation of microwave could turn on the UV lamp without another power. So, this sterilization technology was found to be convenient and economical to sterilize microorganisms.

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