In Vitro Studies

The effects of hardness and pH of water used to prepare electrolyzed oxidizing (EO) water and bleach solutions on the bactericidal activity of sanitizer prepared from the water were examined. EO water and bleach solutions were prepared with hard water of 0, 50, 100, and 200 mg/l as CaCO3 at pH 5, 6, 7, and 8. Increased water hardness tended to increase free chlorine and oxidation-reduction potential (ORP) and decrease pH of EO water. Chlorine levels also increased with water pH. Water hardness and pH only had minor effect on the pH of bleach solutions. Increasing hardness to 50 mg/l increased antimicrobial effect of EO water against Escherichia coli O157:H7, but reduced when water hardness further increased to 100 mg/l or higher. Water pH had no effect on EO water produced against E. coli O157:H7. Water hardness had no significant effect on bactericidal activity of EO water against Listeria monocytogenes but elevated water pH decreased bactericidal activity of EO water produced against L. monocytogenes. Bleach solution prepared using hard water at 200 mg/l or at pH 7 or higher had significant lower efficacy in inactivating E. coli O157:H7, but had no effect on the inactivation of L. monocytogenes. Results indicate that increasing the hardness or pH of water used to prepare EO water or bleach solutions will decrease the bactericidal activity of sanitizers prepared from the water.

The food industry has recognized electrolyzed oxidizing water (EOW) as a promising alternative decontamination technique. However, there is not a consensus about the sanitizing mechanism of EOW. In this study, we evaluated the disinfection efficacy of different types of EOW on Escherichia coli. Based on the hypothesis of hydroxyl radicals existing in EOW, in the present study, the hydroxyl radicals existed in slightly acidic electrolyzed water (SAEW) and acidic electrolyzed water (AEW) diluted to different levels were detected quantitatively. An ultraviolet (UV) spectrophotometer was used to scan EOW with different pH values. Accounting for the results of UV scanning to EOW with different pH value and the disinfection efficacy of different types of EOW, it can be concluded that considering the lower chlorine concentration of EOW compared with traditional chlorine disinfectants, the existing form of chlorine compounds rather than the hydroxyl radicals played important role in the disinfection efficacy of EOW.

In the current study, the effectiveness of slightly acidic electrolyzed water (SAEW) on an in vitro inactivation of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Salmonella spp. was evaluated and compared with other sanitizers. SAEW (pH 5.6, 23 mg/l available chlorine concentration; ACC; and 940 mV oxidation reduction potential; ORP) was generated by electrolysis of dilute solution of HCl (2%) in a chamber of a non-membrane electrolytic cell. One milliliter of bacteria suspension (ca. 10-11 log10CFU/ml) was mixed with 9 ml of SAEW, strong acidic electrolyzed water (StAEW; ca. 50 mg/l ACC), sodium hypochlorite solution (NaOCl; ca.120 mg/l ACC) and distilled water (DW) as control and treated for 60 s. SAEW effectively reduced the population of E. coli, S. aureus and Salmonella spp. by 5.1, 4.8, and 5.2 log10CFU/ml. Although, ACC of SAEW was more than 5 times lower than that of NaOCl solution, they showed no significant bactericidal difference (p > 0.05). However, the bactericidal effect of StAEW was significantly higher (p < 0.05) than SAEW and NaOCl solution in all cases. When tested with each individual test solution, E. coli, S. aureus and Salmonella spp. reductions were not significantly different (p > 0.05). These findings indicate that SAEW with low available chlorine concentration can equally inactivate E. coli, S. aureus and Salmonella spp. as NaOCl solution and therefore SAEW shows a high potential of application in agriculture and food industry as an environmentally friendly disinfection agent.

Neutral (NEW) and acidic (AEW) electrolyzed water were stored in open or closed glass bottles under light or dark conditions at 20 C for 30 days. The pH, oxidation reduction potential (ORP), electrical conductivity (EC), available chlorine concentration (ACC), dissolved oxygen (DO), and bactericidal efficiency of NEW and AEW were determined during storage or before and after storage, respectively. The pH and EC of NEW and AEW remained unchanged in storage. The ORP, ACC and DO of AEW decreased 22%, 100% and 52% under open storage conditions, respectively. Light had no significant effects on the physicochemical properties of NEW (P > 0.05). Bactericidal efficiency was not markedly affected by storage conditions for NEW, but decreased significantly for AEW under open storage conditions. Electrolyzed water should be stored in closed containers or used immediately to prevent the loss of available chlorine that is one of the main contributing factors for antimicrobial activity.

Electrolyzed oxidizing (EO) water has proved to be effective against foodborne pathogens attached to cutting boards and poultry surfaces and against spoilage organisms on vegetables; however, its levels of effectiveness against Listeria monocytogenes and Salmonella Typhimurium in cell suspensions have not been compared with those of other treatments. In this study, the oxidation reduction potentials (ORPs), chlorine concentrations, and pHs of acidic and basic EO water were monitored for 3 days at 4 and 25 C after generation. There were no differences between the pHs or ORPs of acidic and basic EO waters stored at 4 or 25 C. However, the free chlorine concentration in acidic EO water stored at 4 C increased after 24 h. In contrast, the free chlorine concentration in acidic EO water stored at 25 C decreased after one day. Cell suspensions of Salmonella Typhimurium and L. monocytogenes were treated with distilled water, chlorinated water (20 ppm), acidified chlorinated water (20 ppm, 4.5 pH), acidic EO water (EOA), basic EO water (EOB), or acidic EO water that was aged at 4 C for 24 h (AEOA) for up to 15 min at either 4 or 25 C. The largest reductions observed were those following treatments carried out at 25 C. EOA and AEOA treatments at both temperatures significantly reduced Salmonella Typhimurium populations by >8 log10 CFU/ml. EOA and AEOA treatments effectively reduced L. monocytogenes populations by >8 log10 CFU/ml at 25 C. These results demonstrate the stability of EO water under different conditions and that EO water effectively reduced Salmonella Typhimurium and L. monocytogenes populations in cell suspensions.

To identify the primary component responsible in electrolyzed oxidizing (EO) water for inactivation, this study determined the concentrations of hypochlorous acid (HOCl) and hypochlorite ions (OCl-) and related those concentrations to the microbicidal activity of the water. The ultraviolet absorption spectra were used to determine the concentrations of HOCl and OCl- in EO water and the chemical equilibrium of these species with change in pH and amperage. EO water generated at higher amperage contained a higher chlorine concentration. The maximum concentration of HOCl was observed around pH 4 where the maximum log reduction (2.3 log10 CFU/ml) of Bacillus cereus F4431/73 vegetative cells also occurred. The high correlation (r = 0.95) between HOCl concentrations and bactericidal effectiveness of EO water supports HOCl s role as the primary inactivation agent. Caution should be taken with standard titrimetric methods for measurement of chlorine as they cannot differentiate the levels of HOCl present in EO water of varying pHs.

This study was undertaken to evaluate the efficacy of electrolyzed oxidizing (EO) and chemically modified water with properties similar to the EO water for inactivation of different types of foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Bacillus cereus). A five-strain cocktail of each microorganism was exposed to deionized water (control), EO water and chemically modified water. To evaluate the effect of individual properties (pH, oxidation-reduction potential (ORP) and residual chlorine) of treatment solutions on microbial inactivation, iron was added to reduce ORP readings and neutralizing buffer was added to neutralize chlorine. Inactivation of E. coli O157:H7 occurred within 30 s after application of JAW EO water with 10 mg/l residual chlorine and chemically modified solutions containing 13 mg/l residual chlorine. Inactivation of Gram-positive and -negative microorganisms occurred within 10 s after application of ROX EO water with 56 mg/l residual chlorine and chemically modified solutions containing 60 mg/l residual chlorine. B. cereus was more resistant to the treatments than E. coli O157:H7 and L. monocytogenes and only 3 log10 reductions were achieved after 10 s of ROX EO water treatment. B. cereus spores were the most resistant pathogen. However, more than 3 log10 reductions were achieved with 120-s EO water treatment.

This study investigates the properties of electrolyzed oxidizing (EO) water for the inactivation of pathogen and to evaluate the chemically modified solutions possessing properties similar to EO water in killing Escherichia coli O157:H7. A five-strain cocktail (1010 CFU/ml) of E. coli O157:H7 was subjected to deionized water (control), EO water with 10 mg/liter residual chlorine (J.A.W-EO water), EO water with 56 mg/liter residual chlorine (ROX-EO water), and chemically modified solutions. Inactivation (8.88 log10 CFU/ml reduction) of E. coli O157:H7 occurred within 30 s after application of EO water and chemically modified solutions containing chlorine and 1% bromine. Iron was added to EO or chemically modified solutions to reduce oxidation reduction potential (ORP) readings and neutralizing buffer was added to neutralize chlorine. J.A.W-EO water with 100 mg/liter iron, acetic acid solution, and chemically modified solutions containing neutralizing buffer or 100 mg/liter iron were ineffective in reducing the bacteria population. ROX-EO water with 100 mg/liter iron was the only solution still effective in inactivation of E. coli O157:H7 and having high ORP readings regardless of residual chlorine. These results suggest that it is possible to simulate EO water by chemically modifying deionized water and ORP of the solution may be the primary factor affecting microbial inactivation.

Acidic electrolyzed water (acidic EW), which is prepared by the electrolysis of an aqueous NaCl solution, has recently become of great importance for disinfection in a variety of fields, including medicine, the food industry and agriculture. In a previous paper we showed that: 1) acidic EW is a mixture of hypocholorite ion, hypochlorous acid and chlorine, depending upon the pH; 2) hypochlorous acid is primarily responsible for disinfection in the case of Escherichia coli K12 and Bacillus subtilis PCI219, both in clean culture media. In practice, however, the use of acidic EW is in many cases severely hampered due to the presence of a variety of non-selective reducing agents. In view of the salient nature of acidic EW, it is therefore strongly urged to establish an optimum way to use acidic EW in a variety of systems. The present paper is the first report on our attempt along this line in order to characterize the nature of the chemical changes that the bactericidal activity of the acidic EW deteriorates in the presence of organic materials, which include amino acids and proteins.

Hypochlorous acid (HOCl) is probably the most widely used disinfectant worldwide and has an important role in inflammatory reaction and in human resistance to infection. However, the nature and mechanisms of its bactericidal activity are still poorly understood. Bacteria challenged aerobically with HOCl concentrations ranging from 9.5 to 76 M exhibit higher ability to form colonies anaerobically than aerobically. Conversely, aerobic plating greatly increased lethality after an anaerobic HOCl challenge, although anaerobic survival did not depend on whether HOCl exposure was aerobic or anaerobic. Even a short transient exposure to air after anaerobic HOCl challenge reduced anaerobic survival, indicative of immediate deleterious effects of oxygen. Exposure to HOCl can cause lethal DNA damage as judged by the fact that recA sensitivity to HOCl was oxygen dependent. Antioxidant defenses such as reduced glutathione and glucose-6-phosphate dehydrogenase were depleted or inactivated at 10 M HOCl, while other activities, such as superoxide dismutase, dropped only above 57 M HOCl. Cumulative deficiencies in superoxide dismutase and glucose-6-phosphate dehydrogenase rendered strains hypersensitive to HOCl. This indicates that part of HOCl toxicity on Escherichia coli is mediated by reactive oxygen species during recovery.


Scroll to Top