The Scientific World Journal

Volume 2014 (2014), Article ID 619038, 7 pages

http://dx.doi.org/10.1155/2014/619038

Research Article

Application of Highly Purified Electrolyzed Chlorine Dioxide for Tilapia Fillet Disinfection

Chen-Hsing Yu,¹ Tzou-Chi Huang,² Chao-Chin Chung,¹ Hao-Hsun Huang,¹ and Ho-Hsien Chen¹

¹Department of Food Science, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan

²Department of Biological Science and Technology, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan

Received 4 December 2013; Accepted 23 December 2013; Published 17 February 2014

Academic Editors: I. Ortiz and R.-F. Yu

Copyright © 2014 Chen-Hsing Yu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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.

1. Introduction

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

2.1. Materials

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 (ClO 2 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) [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 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:

Figure 1: Designed chlorine dioxide electrolysis equipment: (a) material, (b) electrolyzer, (c) electronic control system, (d) air pump, (e) collecting tank, and (f) cooling system.

Figure 2: Internal structure and reaction of the electrolysis system.

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 [28]. 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) [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 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 [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 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 [32]. Significant differences between tested parameters were determined based on a 95% confidence level ().

3. Results and Discussion

3.1. Effects of Different Ratios of NaClO 2 for High Concentrations of ClO 2

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.

Table 1: Constituents of chlorine dioxide aqueous solution on different constituent ratios of electrolytes.

Electrolyte constituent ratio
20% NaCl

7% NaClO₂
8% NaClO₂
9% NaClO₂
10% NaClO₂

Constituents of chlorine dioxide aqueous solution

    ClO2 (ppm)
3193.76d
3612.31c
4232.49b
4738.36a

     (ppm)
16.02a
ND
ND
ND

     (ppm)
ND
ND
ND
ND

    Cl₂ (ppm)
ND
9.75b
10.64a
10.64a

    Total chlorine* (ppm)
3209.78d
3622.06c
4243.12b
4749.00a

    ClO₂ purity** (%)
99.50a
99.73a
99.75a
99.78a

    pH (unit)
2.36a
2.34a
2.38a
2.18b

    Oxidation reduction potential (millivolt)
1.32c
1.38b
1.44a
1.44a

Means in the same column followed by different superscripts are significantly different at (Duncan’s multiple range test).

*Total chlorine (ppm) = ClO₂ + + + Cl₂.

**ClO₂ purity = [ClO₂/(ClO₂ + + + Cl₂)] × 100%.

3.2. Effect of ClO 2 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.

Table 2: Total bacterial counts of tilapia fillets for various purities in 400 ppm chlorine dioxide solutions.

Treatment time (min)
ClO₂ purity (%)

45%
50%
60%
70%
80%
90%
99%

Total bacteria count log (CFU/g)*

5
5.60a
5.51a
4.53b
4.38c
4.13d
3.96e
3.23f

15
5.44a
5.43a
4.3b
4.09c
3.98d
3.29e
2.1f

25
5.33a
4.3b
3.13c
2.91b
2.74e
2.54f
1.09g

Means in the same column followed by different superscripts are significantly different at (Duncan’s multiple range test).

*Original microbial load: 5.72 log (CFU/g).

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).

Table 3: Total bacterial counts for tilapia fillets disinfected by soaking or shaking treatments at different chlorine dioxide concentrations.

Washing method
Concentration (ppm)
Cleaning time (min)

5
15
25

Total bacteria count

log (CFU/g)

Soaking
Control
5.84ax
5.80ax
5.78ax

50
5.73abx
5.62abxy
5.53aby

100
5.43bx
5.01aby
4.63bz

200
4.73cx
4.23by
3.64cz

Shaking
Control
5.83ax
5.74ax
5.85ax

50
3.49dx
ND
ND

100
2.41ex
ND
ND

200
1.29fx
ND
ND

Means in the same column followed by different superscripts are significantly different at (Duncan’s multiple range test).

Means in the same row followed by different superscripts are significantly different at (Duncan’s multiple range test).

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 [37]. Aloisio and Francisco [38] claimed that ClO₂ being bound to water molecules by static attraction forces under a steady state hindered the bactericidal effect.

Table 4: Coliform reduction of tilapia fillets disinfected by soaking or shaking treatments at different chlorine dioxide concentrations.

Wash method
Concentration (ppm)
Cleaning time (min)

5
15
25

Coliforms

log (CFU/g)

Soaking
Control
5.23ax
5.33ax
5.21ax

50
4.80bx
3.22bz
4.06by

100
4.17cx
2.33cy
ND

200
3.23dx
1.23dy
ND

Shaking
Control
5.23ax
5.29ax
5.25ax

50
1.78ex
ND
ND

100
1.07fx
ND
ND

200
ND
ND
ND

Means in the same column followed by different superscripts are significantly different at (Duncan’s multiple range test).

Means in the same row followed by different superscripts are significantly different at (Duncan’s multiple range test).

Table 5: Escherichia coli reduction of tilapia fillets disinfected by soaking or shaking treatments at different chlorine dioxide concentrations.

Wash method
Concentration (ppm)
Cleaning time (min)

5
15
25

E. coli  log (CFU/g)

Soaking
Control
5.23ax
5.33ax
5.22ax

50
3.08bx
2.21by
1.1cz

100
2.25cx
1.14cy
ND

200
1.13dx
ND
ND

Shaking
Control
5.56ax
5.51ax
5.58ax

50
1.1dx
ND
ND

100
ND
ND
ND

 200
ND
ND
ND

Means in the same column followed by different superscripts are significantly different at (Duncan’s multiple range test).

Means in the same row followed by different superscripts are significantly different at (Duncan’s multiple range test).

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).

Table 6: Total trihalomethane (THM) residuals by GC/MS.

Item
Total trihalomethanea (ppb)

Methyl chloride
ND

Dichloromethane
ND

Chloroform
ND

The instrument detection limit was 0.12 ppb.

4. Conclusions

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.

Acknowledgment

This work was financially supported by the Council of Agriculture of Taiwan under Grant no. 97AS-1.2.1-ST-A3.

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