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.


Introduction
Chlorine dioxide (ClO 2 ) 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 (Cl 2 ), hypochlorous acid (HOCl), and hypochlorite (OCl − ) [3][4][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][7][8][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][11][12]. As for its application for vegetable and fruit disinfection, ClO 2 gas has been successfully used to disinfect strawberries, lettuce, cabbage, and cucumbers with continuous methods [4,[13][14][15][16][17]. In this work, the bactericidal efficacy of ClO 2 was evaluated for cleaning tilapia fillets with different cleaning methods.
Commercial ClO 2 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 ClO 2 needs a strong acid (pH 2∼3), inhibits Cl 2 hydrolysis, and takes a long time for activation. The yield of ClO 2 depends on the purity of the raw materials, the catalyst, pH, reaction time, and temperature [20][21][22][23][24]. Furthermore, it was discovered that electrolyzing sodium chlorite can produce highly purified ClO 2 [25,26]. Therefore, the objective of this study is to develop novel electrolysis equipment to produce highly purified, low-cost ClO 2 to disinfect with water, while simultaneously monitoring trihalomethane (THM) residuals on tilapia fillets.   [27]. 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 ClO 2 collecting tank was maintained at 5∼10 ∘ C by cooling water from the cooling system. NaCl was electrolyzed into NaClO 2 . The reaction equation is as follows:

Materials and Methods
Meanwhile, the NaClO 2 was further electrolyzed, the ClO 2 − was attracted by the cathode, and H 2 O was attracted by the anode to release H 2 ( Figure 2). The reaction equations are as follows: cathode : overall : The resultant ClO 2 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 ClO 2 solutions were measured using an ORP/pH meter (Mettler-Toledo Seven Easy ORP/pH meter, Kaohsiung, Taiwan). ClO 2 analysis: the concentration of ClO 2 was analyzed using the iodine method [28]. The ClO 2 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 ClO 2 , Cl 2 , ClO 2 − , and ClO 3 − : The Scientific World Journal  Figure 2: Internal structure and reaction of the electrolysis system.
Here, N is the concentration of the sodium thiosulfite solution.

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 ClO 2 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, ClO 2 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.

Microbiological Analyses.
The total plate count assay followed the China National Standard (CNS 10890 N6186) [29] 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 [30] using 1 mL of 4 The Scientific World Journal   [31] 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 m 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.

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 [32]. Significant differences between tested parameters were determined based on a 95% confidence level ( < 0.05). Table 1 shows that 10% NaClO 2 and 20% NaCl generated 4749 ppm of total chlorine and 99.8% pure ClO 2 , respectively. The pH was 2.18, and ORP was 1440 mV (Table 1). When the purity of ClO 2 varied from 99.5% to 99.8%, the pH increased from 2.36 to 2.18. Under an acidic condition, Cl 2 easily disassociated into Cl − , and ClO 2 mainly disassociated into ClO 2 − and ClO 3 − , 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 NaClO 2 and then into NaClO 3 . NaClO 3 was affected by the reducing reaction from the cathode side, producing Cl 2 , Cl − and H + . The Cl − , and H + then formed into very small amounts of HCl. These reaction cycles generated NaOH and ClO 2 , producing Cl 2 .

Effect of ClO 2 Purity on Tilapia Fillet Disinfection.
Tilapia fillets were soaked in 400 ppm of 99% pure ClO 2 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 ClO 2 solutions contained 45%, 50%, and 60% of freely available chlorine, the bactericidal effect was not so obviously effective. One of the The Scientific World Journal 5  explanations could be that the Cl 2 is not as effective as ClO 2 , 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 ClO 2 not only reacts with electrons on biological cell membranes but also reacts with Cl 2 to achieve disassociation and oxidation under an acidic condition and then forms ClO 2 − , ClO 3 − , and Cl − byproducts [21,33]. The less Cl 2 there was, the higher the disinfection effect was.

Effect of Various Treatments.
Three different concentrations (50, 100, and 200 ppm) of 99% ClO 2 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 ClO 2 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 [37]. Aloisio and Francisco [38] claimed that ClO 2 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 ClO 2 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 ClO 2 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 6 The Scientific World Journal  also showed that when using a 200 ppm bactericide solution for 25 min at 25 ∘ C, residual of ClO 2 solution was no detected in solution (the instrument detection limit was 0.1 ppb).

Conclusions
The results demonstrated the feasibility of stably producing ClO 2 using electrochemical technology. The maximum concentration and purity of ClO 2 were obtained when using a mixture that blended 20% NaCl and 7%∼10% NaClO 2 together as the electrolytes. The concentration and purity of ClO 2 were 3200∼4700 ppm and 99.5∼99.7%, respectively. Disinfection results indicate that a 200 ppm ClO 2 solution reduced the total bacterial, coliform, and E. coli counts on tilapia fillets by 3.0∼4.0 log CFU/g ( < 0.05). 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 ClO 2 solution were nondetectable. Bactericidal treatment with a ClO 2 solution for tilapia fillets also conforms to Taiwan's environmental protection regulations and act governing food sanitation. The ClO 2 solution is indeed a safer method for treating seafood, and our novel electrolysis equipment can produce highly purified, low-cost ClO 2 to disinfect with water, for immediate use for agricultural product and seafood treatment.