Application of Highly Purified Electrolyzed Chlorine Dioxide for Tilapia Fillet Disinfection
This research aimed to develop an electrolysis method to generate high-concentration chlorine dioxide (ClO2) for tilapia fillet disinfection. The designed generator produced up to 3500 ppm of ClO2 at up to 99% purity. Tilapia fillets were soaked in a 400 ppm ClO2 solution for 5, 10, and 25 min. Results show that total plate counts of tilapia, respectively, decreased by 5.72 to 3.23, 2.10, and 1.09 log CFU/g. In addition, a 200 ppm ClO2 solution eliminated coliform bacteria and Escherichia coli in 5 min with shaking treatment. Furthermore, ClO2 and trihalomethanes (THMs) residuals on tilapia fillets were analyzed by GC/MS and were nondetectable (GC-MS detection limit was 0.12 ppb). The results conform to Taiwan’s environmental protection regulations and act governing food sanitation.
Chlorine dioxide (ClO2) is a strong oxidant widely applied for sterilization, disinfection, and waste-water treatment. It is commonly used on drinking water and environmental disinfection. It was also recommended as a commercial sanitizer to replace electrolyzed oxidizing water [1, 2], chlorine (Cl2), hypochlorous acid (HOCl), and hypochlorite (OCl−) [3–5]. Contact of chlorine dioxide with organic substances in food or water results in microbial resistance and inactivation, but it also produces four trihalomethane (THM) byproducts, that is, chloroform, bromodichloromethane, dibromochloromethane, and bromoform, which are associated with toxicity and carcinogenesis [6–9]. In Taiwan, tilapia fillets are an important economic product, and it is common practice to use sodium hypochlorite (NaClO) as a disinfecting agent for processing tilapia fillets; however, treatment of this type could lead to serious problems involving residual THMs in treated seafood [4, 10–12]. As for its application for vegetable and fruit disinfection, ClO2 gas has been successfully used to disinfect strawberries, lettuce, cabbage, and cucumbers with continuous methods [4, 13–17]. In this work, the bactericidal efficacy of ClO2 was evaluated for cleaning tilapia fillets with different cleaning methods.
Commercial ClO2 is commonly generated using chemical methods that react sodium chloride, sodium hypochlorite, or sodium chlorate with sulfuric acid or hydrochloride acid [18, 19]. The chemical method of producing ClO2 needs a strong acid (pH 2~3), inhibits Cl2 hydrolysis, and takes a long time for activation. The yield of ClO2 depends on the purity of the raw materials, the catalyst, pH, reaction time, and temperature [20–24]. Furthermore, it was discovered that electrolyzing sodium chlorite can produce highly purified ClO2 [25, 26]. Therefore, the objective of this study is to develop novel electrolysis equipment to produce highly purified, low-cost ClO2 to disinfect with water, while simultaneously monitoring trihalomethane (THM) residuals on tilapia fillets.
2. Materials and Methods
Tilapia fillets were bought from a local traditional market in Pingtung, Taiwan. The microbiological media used in this study were peptone and tryptic soy agar (TSA) purchased from Difco Laboratories (Detroit, MI, USA); these were prepared according to the manufacturer’s specifications. 3 M Coliform and E. coli Petrifilm no. 6414 were purchased from Microbiology Products 3 M Health Care (St. Paul, MN, USA).
2.2. ClO2 Electrolysis Equipment (ClO2 Generator)
The self-designed electrolysis equipment consisted of a raw material tank, an electrolyzer, an air pump, two ClO2 collecting tanks, and a cooling system (Taiwan Patent, no. 200722557) . Figure 1 shows the designed chlorine dioxide electrolysis equipment. The internal structure and reaction of the electrolyzer are shown in Figure 2. Saturated saline and sodium hypochlorite enter and mix in the electrolyzer system using a direct current (100~110 A, 7~8 V), the electrolyzed temperature was controlled to 55~65°C, and the electrolyzed material supply rate was 10 L/h. The temperature of the ClO2 collecting tank was maintained at 5~10°C by cooling water from the cooling system. NaCl was electrolyzed into NaClO2. The reaction equation is as follows:
Meanwhile, the NaClO2 was further electrolyzed, the was attracted by the cathode, and H2O was attracted by the anode to release H2 (Figure 2). The reaction equations are as follows:
The resultant ClO2 was aspirated out and collected into 5~10°C pure water in the two collecting tanks. The NaOH solution was collected separately. The oxidation/reduction potential (ORP) and pH of the ClO2 solutions were measured using an ORP/pH meter (Mettler-Toledo Seven Easy ORP/pH meter, Kaohsiung, Taiwan).
ClO2 analysis: the concentration of ClO2 was analyzed using the iodine method . The ClO2 solution at 10 mL was diluted 200 times with pure water. It was adjusted to five pH levels and then titrated with a 0.01 N sodium thiosulfite solution. The titration volumes were A, B, C, D, and E. The following calculation formulas were used to calculate the concentrations of ClO2, Cl2, , and
Here, N is the concentration of the sodium thiosulfite solution.
2.3. Cleaning Methods
Tilapia fillets were incubated at 37°C until the total plate count reached 5~6 log CFU/g. The different purities (45%~99%) of 400 mg/L ClO2 solutions were used to test the effect of tilapia disinfection. Tilapia fillets were inoculated with coliforms or E. coli at a concentration of 5~6 log CFU/g. At 99% purity, ClO2 solutions of 50, 100, and 200 ppm were used to wash the fillets by soaking or shaking treatment for 5, 15, and 25 min, and the total plate counts, coliforms, and E. coli of the fillets were determined.
2.4. Microbiological Analyses
The total plate count assay followed the China National Standard (CNS 10890 N6186)  method: 1 mL of masticated tissue liquid was serially (1 : 10) diluted in 9 mL of 0.1% sterile peptone water, and 0.1 mL portions of appropriate diluents were surface-plated on TSA. The plates were then incubated at 37°C for 48 h in duplicate. CFUs were counted and expressed per gram of sample after logarithmic conversion. The coliform and E. coli assays followed Sasithorn and Sirirat  using 1 mL of masticated tissue liquid plated on 3 M Petrifilm no. 6414 (St. Paul, MN), and the plates were incubated at 37°C for 24 h in duplicate.
2.5. Residual THMs Analyses
The analytical procedure was modified from Stack et al.’s  gas chromatographic (GC) method on an Agilent 5890 system coupled to an Agilent 5973N mass spectrometer (MS) (Palo Alto, CA). Chromatographic separation was performed using a capillary column (HP-5, 30 m × 0.32 mm, 0.25 μm phase film thickness) from Agilent Technologies. The initial temperature was 45°C for 3 min and then increased by 8°C/min to a final temperature of 220°C for 20.5 min. The injector temperature was set to 200°C. Nitrogen was used as the carrier gas at a flow rate of 38.5 mL/min.
MS was operated in the electron ionization mode at 70 eV. The mass range was scanned at 40~350 m/z and for 0.60 seconds per scan for the full-scan mode. Temperatures for the trap, manifold, and transfer line were set to 250, 50, and 280°C, respectively. All data for quantification were collected in the selected ion monitoring mode at 83 and 85 m/z for chloroform, 127 and 129 m/z for dibromochloromethane, and 173 m/z for bromoform.
2.6. Statistical Analysis
Three replicates were conducted, and each sample was assayed in duplicate. Data collected from the experiments were analyzed by an analysis of variance (ANOVA) and Duncan’s multiple range test using the SAS 8.2 program . Significant differences between tested parameters were determined based on a 95% confidence level ().
3. Results and Discussion
3.1. Effects of Different Ratios of NaClO2 for High Concentrations of ClO2
Table 1 shows that 10% NaClO2 and 20% NaCl generated 4749 ppm of total chlorine and 99.8% pure ClO2, respectively. The pH was 2.18, and ORP was 1440 mV (Table 1). When the purity of ClO2 varied from 99.5% to 99.8%, the pH increased from 2.36 to 2.18. Under an acidic condition, Cl2 easily disassociated into Cl−, and ClO2 mainly disassociated into and , with a small portion disassociating into Cl− [21, 33, 34]. A great quantity of Cl− resulted from excessive NaCl in the raw materials which contained NaCl and sodium hypochlorite. At the anode side, NaCl was converted into NaClO2 and then into NaClO3. NaClO3 was affected by the reducing reaction from the cathode side, producing Cl2, Cl− and H+. The Cl−, and H+ then formed into very small amounts of HCl. These reaction cycles generated NaOH and ClO2, producing Cl2.
3.2. Effect of ClO2 Purity on Tilapia Fillet Disinfection
Tilapia fillets were soaked in 400 ppm of 99% pure ClO2 for 5, 15, and 25 min. Results indicated that total plate counts on tilapia fillets decreased from 5.72 log CFU/g to 3.23, 2.1, and 1.09 log CFU/g, respectively (Table 2). Although ClO2 solutions contained 45%, 50%, and 60% of freely available chlorine, the bactericidal effect was not so obviously effective. One of the explanations could be that the Cl2 is not as effective as ClO2, because active oxygen molecules diminish the number of electrons on biological cell membranes and cause damage to biological enzymes on biological membranes therefore amino acid and nucleic bodies are hindered from generating proteins in biological cells [33, 35, 36]. Another reason could be that ClO2 not only reacts with electrons on biological cell membranes but also reacts with Cl2 to achieve disassociation and oxidation under an acidic condition and then forms ,, and Cl- byproducts [21, 33]. The less Cl2 there was, the higher the disinfection effect was.
3.3. Effect of Various Treatments
Three different concentrations (50, 100, and 200 ppm) of 99% ClO2 solutions were used for soaking or shaking disinfection treatment on tilapia fillets for 5, 15, and 25 min. Results are shown in Table 3. After the fish fillets were shaken in the solutions for 5 min, total plate counts were 3.49, 2.41, and 1.29 log CFU/g, respectively, and all were nondetectable after 15 and 25 min, compared to the control groups at 5.84~5.78 log CFU/g (control).
Similar results for coliforms and E. coli are shown in Tables 4 and 5. The control groups of coliform (control) were 5.23, 5.29, and 5.25 CFU/g. When tilapia fillets were treated with 50, 100, and 200 ppm of high-purity ClO2 solutions with the shaking method for 5 min, the coliform counts, respectively, decreased to 1.78, 1.07 log CFU/g, and nondetectable (Table 4). Escherichia coli also showed a > 4 log reduction after 5 min and was nondetectable after 15 and 25 min of shaking (Table 5). Both the soaking and shaking methods eliminated microbial populations; however, the results show that the soaking method was not as effective as the shaking method. Microorganisms attached to fish skin may more easily be washed out by shaking with mechanical forces . Aloisio and Francisco  claimed that ClO2 being bound to water molecules by static attraction forces under a steady state hindered the bactericidal effect.
3.4. Detection of THMs
THM residuals are a problem for the safety of chlorine-treated food materials [39, 40]. After tilapia fillets were washed by soaking or shaking in the ClO2 solution with the highest concentration (200 ppm) for 25 min, the waste solutions were analyzed for THMs using GC/MS. THMs include chloroform, dichloromethane, and methyl chloride. The results show that no THMs were detected in a used ClO2 solution after soaking (GC-MS detection limit was 0.12 ppb), as shown in Table 6. These results conform to Taiwan’s environmental protection regulations and act governing food sanitation. Furthermore, a LC-MS analysis also showed that when using a 200 ppm bactericide solution for 25 min at 25°C, residual of ClO2 solution was no detected in solution (the instrument detection limit was 0.1 ppb).
The results demonstrated the feasibility of stably producing ClO2 using electrochemical technology. The maximum concentration and purity of ClO2 were obtained when using a mixture that blended 20% NaCl and 7%~10% NaClO2 together as the electrolytes. The concentration and purity of ClO2 were 3200~4700 ppm and 99.5~99.7%, respectively. Disinfection results indicate that a 200 ppm ClO2 solution reduced the total bacterial, coliform, and E. coli counts on tilapia fillets by 3.0~4.0 log CFU/g (). The soaking wash treatment was more effective than the shaking method. A GC-MS analysis also showed that when using a 200 ppm bactericide solution for 25 min, residual THMs of the ClO2 solution were nondetectable. Bactericidal treatment with a ClO2 solution for tilapia fillets also conforms to Taiwan’s environmental protection regulations and act governing food sanitation. The ClO2 solution is indeed a safer method for treating seafood, and our novel electrolysis equipment can produce highly purified, low-cost ClO2 to disinfect with water, for immediate use for agricultural product and seafood treatment.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was financially supported by the Council of Agriculture of Taiwan under Grant no. 97AS-1.2.1-ST-A3.
R. Vaid, R. H. Linton, and M. T. Morgan, “Comparison of inactivation of Listeria monocytogenes within a biofilm matrix using chlorine dioxide gas, aqueous chlorine dioxide and sodium hypochlorite treatments,” Food Microbiology, vol. 27, no. 8, pp. 979–984, 2010.View at: Publisher Site | Google Scholar
S. D. Richardson, “Drinking water disinfection byproducts,” in The Encyclopedia of Environmental Analysis and Remediation, R. A. Meyers, Ed., pp. 1398–1421, John Wiley & Sons, New York, NY, USA, 1998.View at: Google Scholar
Ch. Rav-Acha, A. Serri, E. G. Choshen, and B. Limoni, “Disinfection of drinking water rich in bromide with chlorine and chlorine dioxide, while minimizing the formation of undesirable by-products,” Water Science and Technology, vol. 17, no. 4-5, pp. 611–621, 1985.View at: Google Scholar
J. M. Kim, T.-S. Huang, M. R. Marshall, and C.-I. Wei, “Chlorine dioxide treatment of seafoods to reduce bacterial loads,” Journal of Food Science, vol. 64, no. 6, pp. 1089–1093, 1999.View at: Google Scholar
W.-F. Lin, T.-S. Huang, J. A. Cornell, C.-M. Lin, and C.-I. Wei, “Bactericidal activity of aqueous chlorine and chlorine dioxide solutions in a fish model system,” Journal of Food Science, vol. 61, no. 5, pp. 1030–1034, 1996.View at: Google Scholar
I. H. Chen, T. S. Huang, J. Kim et al., “The bactericidal effect of chlorine dioxide against E. coli O157 : H7, Listeria monocytogenes, and Salmonella spp. inoculated on strawberries, cucumbers, and cantaloupes,” in Proceedings of the Institute of Food Technology Annual Meeting, Abstract 78F-22, Dallas, Tex, USA, 2000.View at: Google Scholar
Y. Han, T. L. Selby, K. K. Schultze, P. E. Nelson, and R. H. Linton, “Decontamination of strawberries using batch and continuous chlorine dioxide gas treatments,” Journal of Food Protection, vol. 67, no. 11, pp. 2450–2455, 2004.View at: Google Scholar
S.-Y. Lee, M. Costello, and D.-H. Kang, “Efficacy of chlorine dioxide gas as a sanitizer of lettuce leaves,” Journal of Food Protection, vol. 67, no. 7, pp. 1371–1376, 2004.View at: Google Scholar
C. C. Chung, T. C. Huang, C. H. Yu, F. Y. Shen, and H. H. Chen, “Bactericidal effects of fresh-cut vegetables and fruits after subsequent washing with chlorine dioxide,” International Proceedings of Chemical, Biological & Environmental Engineering, vol. 9, article 21, pp. 107–112, 2011.View at: Google Scholar
W. J. Masschelein, “Experience with chlorine dioxide in Brussels: generation of chlorine dioxide,” Journal American Water Works Association, vol. 76, no. 1, pp. 70–76, 1984.View at: Google Scholar
R. W. Jordon, A. J. Kosinski, and R. J. Baker, “Improved method generates more chlorine dioxide,” Water & Sewage Works, vol. 127, no. 10, pp. 44–46, 1980.View at: Google Scholar
E. M. Aieta and J. D. Berg, “A review of chlorine dioxide in drinking water treatment,” Journal American Water Works Association, vol. 78, no. 6, pp. 62–72, 1986.View at: Google Scholar
G. Gordon and B. Bubnis, “Ozone and chlorine dioxide: similar chemistry and measurement issues,” Ozone Science and Engineering, vol. 21, no. 5, pp. 447–464, 1999.View at: Google Scholar
H. H. Chen, T. C. Huang, and R. J. Tung, “Chlorine dioxide electrolysis equipment,” Inventors: National Pingtung University of Science and Technology, assignee, Taiwan Patent, no. 200722557, December 2005.View at: Google Scholar
L. Wang, J. L. Huang, and H. B. Li, “Determination of ClO2, Cl2 and in water by using continuous iodimetry,” Journal of Harbin University of Civil Engineering and Architecture, vol. 30, no. 4, pp. 70–75, 1997.View at: Google Scholar
CNS, “Method of Test for Food Microbiology-Test of Standard Plate Count,” General No 10890. Classified No N6186, 1991.View at: Google Scholar
S. A. S. Institute, “User’s Guide: Statistics,” Ver. 6.4, SAS Institute, Cary, NC, USA, 1990.View at: Google Scholar
G. L. Amy, J. Debroux, S. Sinha, P. Brandhuber, and J. Chao, “Occurrence of disinfection by-products (DBPs) precursors in source waters and DBPs in finished Waters,” in Proceedings of the 4th International Workshop on Drinking Waters Quality Management and Treatment Technology, pp. 59–70, Taipei, Taiwan, 1998.View at: Google Scholar
N. Singh, R. K. Singh, A. K. Bhunia, and R. L. Stroshine, “Efficacy of chlorine dioxide, ozone, and thyme essential oil or a sequential washing in killing Escherichia coli O157 : H7 on lettuce and baby carrots,” LWT—Food Science and Technology, vol. 35, no. 8, pp. 720–729, 2002.View at: Publisher Site | Google Scholar
L. R. Beuchat, C. A. Pettigrew, M. E. Tremblay, B. J. Roselle, and A. J. Scouten, “Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis,” Journal of Industrial Microbiology and Biotechnology, vol. 32, no. 7, pp. 301–308, 2005.View at: Publisher Site | Google Scholar
G. P. Li and H. L. Xia, “Review on the application of chlorine dioxide in food sterilization and disinfection,” Food Science and Technology, vol. 31, no. 9, pp. 21–25, 2006.View at: Google Scholar
S. Q. Hu and R. Y. Jin, “Research on sterilization of chlorine dioxide gas and its application perspective,” China Safety Science Journal, vol. 17, no. 3, pp. 153–155, 2007.View at: Google Scholar