Factors affecting the antibacterial action of acidified sodium chlorite (ASC), a widely used disinfectant, have not been determined. This study investigated the significant factors suggesting efficient production method to maximize bactericidal action of ASC.
Methods and Results
The effects of (i) preparation procedures (total three methods); (ii) initial concentrations of reactants: sodium chlorite (SC) and citric acid (CTA) (up to maximum solubility of each reactant) and (iii) final pH values (3·0 and 2·5) to the bactericidal action of ASC were investigated with a fixed final concentration of SC (10 ppm) using various foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aureus). The antimicrobial compounds produced and the bactericidal effects depended on the preparation procedure and the initial concentrations of the reactants. The ASC prepared by premixing highly concentrated reactants (in particular > 40%) followed by dilution (dilution after reaction, DAR) was more effective in inactivating foodborne pathogens, and it produced higher antimicrobial compound (Cl2 and ClO2) yields than the other procedures. A 5-min treatment with ASC, produced using the other procedures, resulted in a reduction of < 3·5 log CFU ml−1 (Gram positive = 0·18–0·78; Gram negative = 0·03–3·49 log CFU ml−1), whereas ASC produced with the DAR procedure using the saturated reactants completely inactivated all of the test pathogens within 5 min without recovery (initial concentration = 6·94–7·08 log CFU ml−1).
The ASC production with the DAR procedure using the saturated reactants maximizes both the antimicrobial compound yields and bactericidal effects of the ASC solutions.
Significance and Impact of the Study
This study will contribute to increase the efficiency of ASC treatments for disinfections reducing the effective SC concentrations for industrial use.
Foodborne illnesses have been frequent throughout the world in recent decades, and their negative impacts have been recorded in many previous studies (Wilcock et al. 2004). Thus, the importance of microbiological safety is growing in the food industry, and various intervention methods have been developed, the antimicrobial effectiveness of which have been evaluated using food matrices and related environments (Sofos and Geornaras 2010; Kwak et al. 2011; Goodburn and Wallace 2012). However, modern consumers tend to be very concerned about the presence of potential chemical risks in the food chain, which can be caused by the persistence of residual toxic compounds in processed foods (Kher et al. 2013). Thus, researchers, food manufacturers, food managers and consumers are interested in increasing the effectiveness of antimicrobials while reducing their level of usage (Valero and Frances 2006; Pei et al. 2009; Kim and Rhee 2013; Ye et al. 2013).
Acidified sodium chlorite (ASC) was recently evaluated as an effective and efficient alternative disinfectant to chlorine (Ruiz Cruz et al. 2006; Liao 2009). In various studies, ASC has been applied widely to a variety of food products, including fresh carrot, cabbage, meat and poultry (Gill and Badoni 2004; Inatsu et al. 2005; Ruiz-Cruz et al. 2007; Sexton et al. 2007; Keeton and Eddy 2008; Stopforth et al. 2008). The use of ASC at < 1200 ppm sodium chlorite (SC) (pH 2·3–2·9) has been approved for the decontamination of food products (e.g. finfish, poultry, processed fruits and vegetables, and red meat) in several countries, including Canada, the EU and New Zealand (CFIA 2001; FSANZ 2003; EFSA 2006; FDA 2011).
Acidified sodium chlorite solutions are prepared by adding various types of acids, which are mostly food-grade [e.g. citric acid (CTA), lactic acid and phosphoric acid], to acidify a SC solution to pH < 3·0 or by simply mixing equal volumes of aqueous SC solution with an acid solution immediately prior to use (Rao 2007). This generates chlorous acid (HClO2), which gradually decomposes to form chlorate ion (MClO3), chloride ion (Cl−), chlorine (Cl2) and chlorine dioxide (ClO2; USDA 2008). The hypothetical antimicrobial action of ASC involves uncharged chlorous acid penetrating the bacterial cell envelope to disrupt protein synthesis by reacting with sulfhydryl, sulphide and disulphide in nucleotides (Warf and Kemp 2001).
Acidified sodium chlorite is widely used in the food industry, but there is a lack of information on the major factors that affect the bactericidal action of ASC. Most of the previous investigations of the antimicrobial activity of ASC in various foods have purchased commercial ASC products, which comprise SC and acid bases, and the solutions have been prepared according to the manufacturer's instructions (Gonzalez et al. 2004; Inatsu et al. 2005; Ruiz Cruz et al. 2006; Ruiz-Cruz et al. 2007; Stopforth et al. 2008; Keskinen et al. 2009). The procedures used to prepare ASC have been described briefly in some research publications, where organic acid (OA) was added slowly to the SC solution until it reached the required pH (Allende et al. 2009; Fan et al. 2009).
Figure 1 shows the three different procedures that can be used to prepare ASC solutions, as follows: (i) mixing equal volumes of OA and SC solution at the final concentrations (conventional procedure, C); (ii) the gradual addition of a highly concentrated acid solution to SC solution up to the final concentration (acidification procedure, A) and (iii) reaction using high-concentration reactants, followed by dilution to the final concentration (dilution after reaction procedure, DAR). The bactericidal action of ASC is derived from the reaction of SC and OAs. The preparation procedure, initial concentrations of reactants and pH of ASC solutions are important factors that determine the antimicrobial activity of ASC, but variations in the bactericidal effects of ASC have not been tested by exploring each factor.
Thus, to promote the use of ASC in the food industry and to improve the treatment efficiency, it is necessary to evaluate the bactericidal action of ASC by testing each factor to develop an optimized procedure and to maximize the bactericidal action of ASC. The objective of this study was to investigate the bactericidal action of ASC solutions, which were prepared using three different procedures (C, A and DAR procedures), with different initial concentrations of reactants and various pH levels, thereby determining the most efficient preparation procedure for the practical application of ASC to bacterial inactivation.
Materials and methods
Preparation of ASC solutions
Sodium chlorite (CAS No. 7758-19-2, purity = 80%) and CTA (CAS No. 77-92-9, purity > 99·5%) were purchased from Sigma-Aldrich (St Louis, MO). All of the chemical solutions were prepared immediately before the experiments. Initially, the saturated solutions of SC (40%, w/v) (water solubility at 17°C = 39 g per 100 ml) (INCHEM 2012) and CTA (70%, w/v) (water solubility at 20°C = 57·6–77·1 g per 100 ml; INCHEM 2005) were prepared by dissolving each material in sterile distilled water (SDW), before diluting the saturated solutions to the appropriate level for use (SC = 20, 10 and 5%, and 20 and 10 ppm; CTA = 40, 20, 10 and 5%, and 10 000, 1000, 5000 and 500 ppm). The ASC solutions were prepared using three different procedures, as described below (Table 1).
Table 1. Composition of the acidified sodium chlorite solutions (SC, 10 ppm) used in this study
(A) Preparation procedure
(B) Final status of ASC solution
SC concentration (w/v)
Reactant volume (a)
CTA concentration (w/v)
Reactant volume (b)
SC final concentration
CTA final concentration
SC, sodium chlorite; CTA, citric acid.
For the DAR preparations, 30 – (a + b) ml of sterile distilled water was added to adjust the final volume to 30 ml.
The 20 ppm SC solution (15 ml) and 1000 or 10 000 ppm CTA solutions (15 ml) were placed in a water bath (VS-1205SW1; Vision Scientific Co. Ltd, Daejoen, Korea) at 22°C and then mixed gently for 1 min in a 50-ml conical tube (Becton Dickinson, Franklin Lakes, NJ). The total volumes of the final ASC solutions were 30 ml, so each reactant was diluted twofold (final concentrations: SC = 10 ppm; CTA = 500 and 5000 ppm).
Acidification procedure (procedure A)
The 10 ppm SC solution (30 – xn ml) and concentrated CTA solutions (70, 40, 20, 10 and 5%) were placed in a water bath at 22°C. Aliquots (xn) of the CTA solutions were added to the 10 ppm SC solution and mixed gently for 1 min in a 50-ml conical tube (total volume = 30 ml).
Dilution after reaction procedure (DAR procedure)
The concentrated SC (40, 20, 10 and 5%) and CTA (70, 40, 20, 10, and 5%) solutions, and SDW were placed in a water bath at 22°C. Aliquots (a or b) of each reactant were mixed gently for 1 min, before dilution to the final concentration (10 ppm SC; 500 and 5000 ppm CTA) with SDW [30 – (a + b) ml] (total volume = 30 ml).
pH, chlorine and chlorine dioxide concentration measurements
The pH values of the prepared ASC solutions were measured using a pH meter (MP 200; Mettler-Toledo Inc., Columbus, OH) at 22°C. To determine the concentrations of the active bactericidal compounds in the ASC solutions, the concentrations of Cl2 and ClO2 were measured with a colorimeter (DR/800 Colorimeter; HACH, Loveland, CO) using the N,N-diethyl-p-phenyldiamine (DPD) colorimetric method, according to the manufacturer's instructions (method numbers 8021, 10069, and 10126).
Three strains each of E. coli O157:H7 (ATCC 35150, 43889 and 43890), Salmonella Typhimurium (ATCC 19585, 43174 and DT 104 killercow), Staph. aureus (ATCC 23235, 25923 and NRS 111) and Listeria monocytogenes (ATCC 19111, 19115 and 19117) were used in this study. All of the test strains were obtained from the Food Microbiology Culture Collection at Korea University (Seoul, Korea) and were stored at –80°C in tryptic soy broth (TSB; Difco, Detroit, MI) supplemented with glycerol (20%). The stock microorganisms were subcultured monthly on tryptic soy agar (TSA, Difco).
Each strain was incubated separately in TSB at 37°C (E. coli O157:H7, Salm. Typhimurium and Staph. aureus) or at 30°C (L. monocytogenes) for 24 h before use in the experiments. Three strains of each bacterium were combined in a plastic 50-ml conical tube. The bacterial cells were harvested by centrifugation at 3000 g (Centra-CL2; Needham Heights, MA) for 15 min and washed twice using 0·85% sterile NaCl solution. The final pellets were resuspended in 0·85% sterile NaCl solution.
The bacterial cell suspensions were inoculated into ASC solutions, which were prepared with different procedures and with various reactant concentrations, to yield approximately 7 log CFU ml−1 of the test microorganisms. Aliquots (1 ml) of samples treated with ASC were withdrawn from each treatment group at 1, 3 and 5 min. To stop the residual bactericidal action of ASC, 0·1% sodium thiosulphate buffer (Sigma-Aldrich) was added to each sample dilution, according to a previous validation study (Kemp and Schneider 2000). All of the experiments were performed in triplicate.
The aliquots removed from each treatment group at specific times were spread-plated onto TSA in duplicate. To obtain a lower detection limit (1 CFU ml−1), 1-ml aliquots were spread onto five TSA plates. After incubation at 37°C (E. coli O157:H7, Salm. Typhimurium and Staph. aureus) or 30°C (L. monocytogenes) for 24 h, typical colonies were counted and the cell counts were expressed as log CFU ml−1 values. For treatments where the test microorganisms were totally inactivated to the ‘not detected’ level, injured cell recovery was confirmed after further enrichment. The aliquot (1 ml) removed from each treatment group at a specific time was inoculated into 10 ml of TSB and incubated at 37°C (E. coli O157:H7, Salm. Typhimurium and Staph. aureus) or 30°C (L. monocytogenes) for 24 h. The enriched samples were streaked onto sorbitol MacConkey agar [(SMAC), Difco, E. coli O157:H7], xylose lysine desoxycholate agar [(XLD), Difco, Salm.Typhimurium], Oxford agar (L. monocytogenes) and Baird-Parker (BP) medium (Difco) supplemented with 5% egg yolk tellurite emulsion. The results were recorded as positive (typical colonies) or negative (atypical colony or none) after incubation (SMAC and XLD: 37°C, 24 h; Oxford agar: 30°C, 24 h; BP medium: 35°C, 48 h). The experiments were performed in triplicate.
The effects of the preparation procedure and the reactants concentrations on the production of active bactericidal compounds were evaluated by analysis of variance (anova) using sas (sas version 9·3; SAS Institute Inc., Cary, NC). The least significant differences were analysed among the treatment conditions (P <0·05) using Tukey's studentized range test.
Production of antimicrobial compounds in ASC prepared with three different procedures
Figure 2 shows the concentrations of the antimicrobial compounds (chlorine and chlorine dioxide) in ASC produced using three different procedures (C, A and DAR). The final pH level of the ASC solution seldom affected the yield of Cl2 and ClO2. The final concentration of SC (10 ppm) was the same in all of the ASC solutions, but the amounts of Cl2 and ClO2 produced differed greatly, depending on the preparation procedure and the concentrations of the reactants. The Cl2 and ClO2 concentrations in ASC prepared using procedure C and A were 0·03–0·27 (ASC-C) and 0·01–0·07 ppm (ASC-A), respectively, whereas ASC prepared using the DAR procedure (ASC-DAR) had higher levels of Cl2 (0·16–5·05 ppm) and ClO2 (0·06–1·94 ppm). In ASC-DAR produced using >40% reactants, the yields of Cl2 and ClO2 were significantly higher than those prepared with the other procedures (P <0·05). In general, the Cl2 and ClO2 concentrations increased with the concentrations of the reactants. The Cl2 and ClO2 concentrations in ASC-DAR prepared using 40% SC + 70% CTA were approximately 5·0 and 1·9 ppm, respectively.
Bactericidal action of ASCs prepared using the three different procedures
Figure 3 shows the bactericidal action of ASCs prepared using the C, A and DAR procedures at pH 3·0. Overall, ASC-DAR had significantly higher microbial inactivation effects (0·05–7·08 log CFU ml−1 reduction after 5 min treatment) than ASC-C (0·01–0·17 log CFU ml−1) and ASC-A (0·01–0·40 log CFU ml−1) at pH 3·0. In particular, ASC-DAR prepared using >40% reactants has an extremely high antimicrobial activity (6·94–7·08 log CFU ml−1 reductions) compared with the other formulations. No reductions (0·06–0·40 log CFU ml−1) were observed after treatment with ASC-A even when > 40% reactants were used in the preparation. Reductions of 6·92–7·09 log CFU ml−1 were obtained with ASC-DAR using 40% SC + 40% CTA and 40% SC + 70% CTA. Salm. Typhimurium was the most sensitive (total inactivation within 1 min by ASC-DAR using 40% SC + 70% CTA) among the foodborne pathogens tested (log reductions with same treatment = 2·89–4·51 log CFU ml−1).
At pH 2·5, the bactericidal actions of ASCs prepared using the three different procedures are shown in Fig. 4. Similar to the pH 3·0-treated group, ASC-DAR had significantly higher bactericidal effects (0·18–7·08 log CFU ml−1 reductions after 5 min treatment) than ASC-C (0·08–1·47 log CFU ml−1) and ASC-A (0·13–3·56 log CFU ml−1) at pH 2·5. The responses of Gram-positive foodborne pathogens (Fig. 4b,d) to ASC solutions at pH 2·5 shared the same trend as the pH 3·0 treatment group, while the log reductions of Gram-negative foodborne pathogens (Fig. 4a,b) increased significantly as the pH decreased.
All of the 5-min treatment groups are shown separately in Fig. 5 to demonstrate the significant differences between the effects of the ASCs at pH 3·0 and 2·5. The bactericidal action of ASC against Gram-negative foodborne pathogens (E. coli O157:H7 and Salm. Typhimurium) was enhanced greatly by reducing the pH level. At pH 2·5, ASC-DAR and ASC-A had stronger bactericidal effects on Gram-negative foodborne pathogens when the reactant concentrations were increased (2·58–3·56 log CFU ml−1 reduction after 5 min treatment) compared with the pH 3·0 treatment group (0·01–0·40 log CFU ml−1). For the ASC-DAR treatment group, reductions of >4 log CFU ml−1 were obtained even when <40% reactants were used in the preparation (reductions with ASC-DAR using 20% SC + 20% CTA for 5 min: E. coli O157:H7 = 4·67 log CFU ml−1; Salm. Typhimurium = 6·92 log CFU ml−1).
Table 2 shows the results of the survival test for the most effective treatment (ASC-DAR prepared using > 40% reactants). All of the test foodborne pathogens were inactivated completely within 5 min using 10 ppm ASC-DAR, and there was no recovery when the ASC-DAR was prepared with 40% SC + 70% CTA.
Table 2. Survival of the test foodborne pathogens after treatment with 10 ppm ASC-DAR, which was prepared using > 40% reactants
Acidified sodium chlorite solution is prepared by mixing solutions that contain the chlorite ion () and hydrogen ion (H+), as described earlier. In general, ClO2 is known to be one of the active antimicrobial compounds produced by the acidification of SC, so some researchers have called ASC an ‘aqueous ClO2’ (Lin et al. 1996; Fu et al. 2007; Bang et al. 2011). The concentrations of reactants used in previous studies have varied greatly (chlorite solutions = 0·1–25%, acid solutions = 0·6–50%) for the ASC solutions and aqueous ClO2 (Lin et al. 1996; Yuk et al. 2005, 2006; Fu et al. 2007; USDA 2008; Allende et al. 2009; Inatsu et al. 2010; Bang et al. 2011). Most previous comparisons of the antimicrobial efficiency of various food product sanitizers have employed commercial products (Sanova®) manufactured by the Ecolab Inc. (St. Paul, MN), which comprise 25% SC (Sanova base) and 50% CTA solutions (Activator base; Yuk et al. 2005, 2006; Ruiz Cruz et al. 2006; Ruiz-Cruz et al. 2007; Stopforth et al. 2008).
To optimize the preparation guidelines for ASC, the present study analysed variations in the bactericidal actions of ASC solutions, which depended on the preparation procedure (C, A, and DAR), the concentration gradients of the reactants (SC and CTA) and the final pH of ASC solutions. The ASC solution prepared using procedure C, which is generally known as the conventional procedure, had a much lower bactericidal effect on pathogenic bacteria than the ASC solution prepared using the DAR procedure. In addition, the yields of Cl2 and ClO2 and the bactericidal action were higher when the concentrations of the reactants were higher. With the ASC-DAR solution prepared using the maximum level of reactants (40% SC + 70% CTA), all of the pathogenic bacteria tested were inactivated completely within 5 min and there was no recovery of injured cells. Thus, the present study suggests that the DAR procedure improves the bactericidal action of ASC.
From a toxicological perspective, however, the DAR procedure has obvious adverse effects because it increases the Cl2 and ClO2 production levels, which are known to be toxic compounds. The established tolerable daily intakes of Cl2 and ClO2 are 5 mg l−1 (5 ppm) and 0·03 mg kg−1 body weight in drinking water, respectively (WHO 2005). For food applications, however, the approved SC concentrations allowed in ASC solutions are 50–150 ppm with a pH of 2·8–3·2 or 500–1200 ppm with a pH of 2·5–2·9 (JECFA 2007). The final concentration of SC used in this study was only 10 ppm. All of the foodborne pathogens tested (initial concentration: approximately 7–8 log CFU ml−1) were inactivated completely within 5 min using ASC-DAR with <40% reactants, whereas ASC-C and ASC-A prepared with the same SC concentration had insignificant antimicrobial activity. Thus, food manufacturers or researchers could prepare an ASC solution with a much higher bactericidal activity using the DAR procedure, with smaller amounts of reactants in the final ASC solution. Therefore, the DAR procedure with saturated reactants could be used to maximize the bactericidal action of ASC solution in the food industry.
To the best of our knowledge, this is the first study to demonstrate an increased bactericidal action for ASC based on the optimized combination of reactants. Deshwal et al. (2004) reported that the decomposition reaction of acidic SC was of the first order with respect to both the chlorite and acid concentration (pH), and it was concluded that the reaction rate increased with decreasing pH, increasing chlorite concentration and increasing temperature (15–35°C). The present study confirmed that the bactericidal actions of ASC solutions and the yields of antimicrobial compounds (Cl2 and ClO2) increased with the concentrations of the reactants. However, this study had the following limitations: (i) the effect of temperature gradients on the bactericidal action of ASC prepared using various procedures and (ii) the ‘critical point’ where the minimum concentration of reactants used in the DAR procedure increases the bactericidal efficiency of ASC solutions to a significant level remains unknown. Therefore, further investigations are required to identify the major factors that affect the antimicrobial activity of ASC and to optimize the ASC preparation procedure for food products or food manufacturing-associated instrument surfaces with respect to the characteristics of each food product, quality deterioration (appearance, flavour and taste) and the toxicological dose in humans.
This work was supported by a Korea University grant. The authors thank the Institute of Biomedical Science and Food Safety and Korea University Food Safety Hall for providing their equipment and facilities.