Activity of Electrolyzed Oxidizing Water Against Penicilium expansum in Suspension and on Wounded Apples

JFS: Food Microbiology and Safety Vol. 69, Nr. 1, 2004
D.O. OKULL AND L.F. LABORDE

ABSTRACT: Spores of Penicillium expansum, the primary organism responsible for the occurrence of patulin in apple juice, were exposed to electrolyzed oxidizing (EO) water in an aqueous suspension and on wounded apples. Full-strength and 50% EO water decreased viable spore populations by greater than 4 and 2 log units, respectively. Although EO water did not prevent lesion formation on fruit previously inoculated with P. expansum, cross-contamination of wounded apples from decayed fruit or by direct addition of spores to a simulated dump tank was substantially reduced. EO water, therefore, has potential as an alternative to chlorine disinfectants for controlling infection of apples by P. expansum during handling and processing operations.

Keywords: Penicillium expansum, electrolyzed oxidizing water, apples, patulin

Introduction

Penicillium expansum is the primary cause of postharvest decay of apples (Paster and others 1995). Besides causing economic losses, fruit contaminated with P. expansum can accumulate high levels of the mycotoxin patulin (Taniwaki and others 1992). Recently, the U.S. Food and Drug Administration (USFDA) issued a final ruling requiring juice processors to implement Hazard Analysis Critical Control Point plans to control food safety hazards in their operations (Federal Register 2001). The USFDA has issued guidelines for minimizing patulin in apple juice, which include purchasing only sound, tree-picked fruit and culling or trimming after storage to eliminate moldy, damaged, or rotten apples (USFDA 2002b). Of particular concern are small processing operations in which poor-quality apples may be used to make cider (Brackett and Marth 1979). According to current USFDA estimates (USFDA 2002a), if 1 rotten apple containing 10000 parts per billion (ppb) patulin is used along with 200 sound apples to make juice, the resulting patulin level in the juice could exceed USFDA’s action level of 50 µg/kg. However, culling may not be completely successful in eliminating patulin from apples because lesions may occur within the interior of the fruit that are not apparent upon visual inspection (Paterson and others 2000). Minimizing inoculum levels and preventing crosscontamination from occurring during handling and storage is, therefore, an important strategy for preventing the occurrence of apple decay and resulting presence of patulin in juice products (Rosenberger 1999).

Chlorine, at levels of 100 to 200 ppm is currently recommended for control of postharvest pathogen spores in dump tanks and other recirculating water systems (Willet and others 1989). However, the use of chlorine has disadvantages, such as the corrosion of metal equipment, reliance on manual monitoring of chlorine concentrations,
sensitivity to organic load, effectiveness within a narrow pH range, and the formation of harmful chlorinated byproducts (Roberts and Reymond 1994). Alternative measures for controlling postharvest decay include treating apples with chlorine dioxide (Roberts and Reymond 1994), hydrogen peroxide (Baldry 1983), acetic acid vapor (Sholberg 2000), ozone (Spotts and Cervantes 1992), and calcium salts (Conway and others 1999).

Electrolyzed oxidizing (EO) water is a novel disinfectant that has been shown to effectively reduce pathogenic bacteria in cell suspensions (Venkitanarayanan and others 1999a) on plastic cutting boards (Venkitanarayanan and others 1999b) and on the surface of vegetables (Izumi 1999; Bari and others 2003). Suzuki and others (2002) demonstrated inhibitory activity of EO water on the growth of Aspergillus parasiticus and production of aflatoxin. EO water is produced by electrolysis of aqueous sodium chloride to produce an electrolyzed basic aqueous solution containing dilute sodium hydroxide at the cathode and an electrolyzed acidic solution at the anode (Kim and others 2000a).

The activity of EO water against P. expansum has not previously been demonstrated. Therefore, the objectives of this study are to determine (1) the activity of EO water against an aqueous suspension of P. expansum spores, (2) the efficacy of EO water in preventing decay from occurring on inoculated wounded apples, and (3) the efficacy of EO water in preventing cross-contamination of P. expansum introduced into process solutions.

Materials and Methods

Preparation of inoculum

Penicillium expansum (PP497A), an isolate from infected pear fruit, was obtained from the Penn State Fusarium Research Center (Univ. Park, Pa., U.S.A.) and adapted to apple fruit as follows: A 30-mm-wide strip of skin was removed around the equators of 3 fresh apples with a vegetable peeler; 500 µL of a 10⁶ colony-forming units
(CFU)/mL spore suspension of the isolate was then evenly applied to the exposed flesh, and the inoculated apple incubated at 25 °C for 6 d, during which more than 50% of the peeled strip showed visible decay with spore formation. The procedure was repeated once before inocula from the decayed apples were plated onto potato
dextrose agar (PDA) for use in all experiments.

Cultures of the organism were maintained on PDA (Difco, Sparks, Md., U.S.A.) slants. Inoculum was produced by growing the organism on PDA slants for approximately 8 d at 25 °C, at the end of which the entire slant surface was covered with spores of the mold. Five milliliters of sterile 0.01% Tween 80 (polyoxyethylene sorbitan monooleate, VWR, Chester, Pa., U.S.A.) were then added to each of the slants, and the tubes were shaken gently to disperse
the spores. The suspension was then filtered through 8 layers of cheesecloth to remove mycelial debris. The number of spores was determined using a Bright-LineTM hemacytometer (Hausser Scientific, Horsham, Pa., U.S.A.) and confirmed by PDA plate counts. All reagents and equipment were sterilized by autoclaving at 121 °C for 21 min.

EO water

An ROXâ„¢ Water Electrolyzer (Hozishaki America, Inc., Peachtree City, Ga., U.S.A.) was used to generate EO water. A 0.1% sodium chloride solution was electrolyzed in a cell containing inert positively charged and negatively charged platinum electrodes separated by a bipolar membrane composed of vinylidine polyflouride. By subjecting the electrodes to direct current voltage at 19.0 amps, 2 types of water were generated: an electrolyzed basic aqueous solution containing dilute sodium hydroxide (NaOH) produced from the cathode and an electrolyzed acidic solution. Experiments were conducted using the acidic portion.

Determination of free chlorine, pH, and oxidation reduction potential

Free chlorine content of acidic EO water was measured using a Hach DPD-FEAS digital titrator (Hach Co., Loveland, Colo., U.S.A.) calibrated to measure up to 25 ppm chlorine in a 25-mL solution according to the manufacturer’s directions. Briefly, 25-mL samples diluted 10-fold with sterile deionized water were transferred into
50-mL beakers. A DPD-free CL powder pillow was added to each sample and stirred to mix. These were then titrated using 0.00564 N ferrous ethylenediammonium sulfate to a colorless endpoint. Free chlorine was calculated from the number obtained following titration, inclusive of the dilution factor. Values for pH and oxidation-reduction potential (ORP) were determined using an Accumet pH meter (model AR25, Fisher Scientific, Pittsburgh, Pa., U.S.A.) with Orion Ion and ORP electrodes (Orion Research, Beverly, Mass., U.S.A.).

Inactivation of spores in suspension

One milliliter of the stock spore suspension containing approximately 10⁷ CFU/mL of inoculum was deposited on an individual sterile 0.22 µm cellulose acetate filter fitted onto a 47-mm vacuum filter holder (VWR). Ten milliliters of acidic undiluted EO water or EO water diluted with sterile distilled water to 75%, 50%, or 25% (v/v) of the original concentration was dispensed onto the filter. Sodium hypochlorite (chlorine) solutions containing 100 or 200 ppm chlorine with or without adjustment to pH 7 were included for comparison. Sterile distilled water served as a control. The filter holder was shaken gently to suspend the spores in the treatment solution. Following treatment times of between 15 s and 300 s, the solution was vacuum filtered through the disc, and the remaining spores were washed once with 10 mL of sterile distilled water. The filter was transferred to a 250-mL Erlenmeyer flask containing 50 mL of 0.01% Tween 80 solution, and the spores were dislodged by a 2 min ultrasound treatment in an AquasonicTM ultrasonicator (VWR) operating at a frequency of 60 Hz. Serial dilutions from the flask were then spread onto pre-poured PDA plates and incubated at 25 °C for 48 h to determine the amount of viable spores. Each experiment consisted of duplicate treatments that were repeated 3 times.

Inoculated apple study

Macintosh apples obtained from a local grocery store were held at 4 °C until ready for use. Before each experiment, 19 apples were washed with mild detergent, rinsed in distilled water, wiped dry with clean paper towels, and allowed to equilibrate to room temperature. An 8-mm circular wound was made to a depth of 3 mm at 5 equidistant locations on each apple using a sterile cork borer and scalpel. Each wounded apple was inoculated by dipping in 500 mL of distilled water containing 10⁶ CFU/mL of P. expansum spores for 10 s and then air-dried for 1 h. Inoculated apples were then added to a 30-L water bath filled with 20 L of full strength EO water (100% EO), EO water diluted with an equal volume of tap water (50% EO), or 200 ppm chlorine at pH 9.3 or 6.9. Inoculated apples were also held in distilled water as a control.

Conditions in a commercial dump tank were simulated by continuously agitating the apples using a mechanical stirrer and recirculating pump. Following a 5-min treatment, the apples were removed and allowed to dry in air at 25 °C for 1 h. Apples were stored in ventilated plastic bags at 25 °C for 6 d. These experiments were repeated once, and the activity of treatment solutions was expressed as the percent number of wounds showing decay after the storage period.

Cross-contamination study

Nineteen uninoculated, wounded apples were added to the water bath containing EO or chlorine solutions and exposing them to either a single decayed apple or a known concentration of spores. Decayed apples were prepared as described before for the adaptation of P.expansum PP497A on fresh apples. For experiments in which spores were added directly to the water bath, the concentration of the stock solution was calculated to achieve approximately the same level of spores released by a decayed apple held for 5 min in the water bath containing distilled water. Following 5-min treatments in EO water or chlorine as previously described, the
apples were stored at 25 °C for 6 d, and the percent number of wounds showing decay was determined. All experiments were replicated 2 times.

Statistical analyses

Mean CFU for in vitro treatments and the mean number of infected wounds in apple experiments were compared using 1-way ANOVA procedures. Tukey’s HSD test was used to determine significant differences between treatments, and all analyses were performed with Minitab statistical software (Minitab Inc., State College,
Pa., U.S.A.).

Results and Discussion

Inactivation of spores in aqueous suspension

The physicochemical properties of EO water or chlorine solutions and reductions in viable P. expansum spores after treatment for up to 300 s are shown in Table 1. The pH of undiluted acidic EO water increased from 3.1 to 6.5 after diluting with 3 equal volumes of distilled water. ORP and free chlorine values within the same dilution range decreased from 1133 to 851 mV and 59.6 to 10.1 ppm, respectively.

Table 1—Inactivation of Penicillium expansum spores by electrolyzed oxidized (EO) water and sodium hypochlorite solutions

^a Numbers within each column with different letters indicate significant (P  0.05) differences between treatments. ORP = oxidation-reduction potential.
^b pH was adjusted using 5 N HCl.

Processors would benefit by using diluted EO water because it would reduce the volume that must be generated while minimizing the potential for equipment corrosion to occur at excessively low pH levels. The ORP value for unacidified 100 ppm chlorine was less than all EO water solutions. When the pH of each chlorine solution was lowered with HCl, free chlorine remained unchanged while ORP values in each case increased. This can be attributed to the acid induced conversion of hypochlorite ion (OCl–) to the more reactive hypochlorous
acid form (HOCl) (Kim and others 2000b, Eifert and Sangley 2002). ORP values measured at each treatment interval did not significantly (P ≤ 0.05) change with time (data not shown).

Inactivation of P. expansum spores by EO water or chlorine was significantly affected (P ≤ 0.05) by dilution level or concentration and exposure time (Table 1). Decreases in viable spores were initially rapid with over a 4.6 log reduction observed after 300 s of exposure to undiluted EO water. Unacidified chlorine was initially less effective than EO water, although a greater than 4 log reduction was achieved with a 200-ppm solution after 300 s. Consistent with the positive effect of pH adjustment on ORP values, neutralization of chlorine solutions increased the rate of spore inactivation.

Figure 1 confirms the relationship between ORP of EO water or chlorine solutions and inactivation of P. expansum spores. A strong correlation (R2 = 0.89) was observed between ORP and inactivation of spores following exposure to each sanitizer for 60 s irrespective of the treatment used. Kim and others (2000b) demonstrated the relationship
between sanitizer effectiveness and ORP and proposed that ORP is a suitable indicator of antibacterial activity. Our findings support a similar conclusion for inactivation of P. expansum spores.

Figure 1— Relationship between oxidation-reduction potential (ORP) of 25%, 50%, 75%, and 100% EO water and chlorine with or without pH adjustment and log10 reduction of Penicillium expansum spores treated for 60 s.

A tailing effect observed after 15 s of treatment indicates that prolonged exposure to the sanitizers may not necessarily increase spore inactivation. Further treatment of spores by vacuum filtering the solutions followed by addition of fresh solution, or successive treatment with the alkaline and acid EO fractions did not enhance inactivation (data not shown). This effect is consistent with a study by Kirsten and Nielsen (1995), who found varied resistance to disinfectant treatments among related fungal species. They speculated that fungal resistance to disinfectant agents may be related to the hydrophobic nature of their spore surfaces. The spore suspensions in this experiment were prepared in a solution of Tween 80 to increase their dispersion in the treatment solution. Inouye and others (2001) found that the presence of Tween 80 in liquid media reduced the effectiveness of antimicrobial compounds because of the formation of micelles, which could prevent the interaction of the compounds with spores.

Inactivation of spores on inoculated apple wounds

Table 2 shows the properties of treatment solutions used in water-bath experiments with wounded, inoculated apples. Because 200 ppm chlorine was more effective than 100 ppm in inactivating P. expansum spores, this concentration was used for apple treatments. The properties of each solution mirrored those obtained in the previous experiment, in which ORP, for example, was highest for 100% EO water and lowest for the control.

Table 2—Frequency of decay (%) on inoculated^a apple wounds treated with electrolyzed oxidizing (EO) water or
sodium hypochlorite in a simulated dump tank and stored at 25 °C for 6 d

^a Apple wounds were inoculated by dipping in a 106 colony-forming units/mL spore suspension for 10 s. ORP = oxidation-reduction potential.
^b Wounded apples were exposed to treatment solutions for 5 min and stored at 25 °C for 6 d.
^c pH was adjusted using 5 N HCl.

None of the treatments prevented decay lesions from developing on the apples. Mari and others (1999) were similarly unable to prevent decay of nectarines and plums from Monilinia laxa using postharvest applications of chlorine dioxide and peracetic acid. In addition, Koffmann and Penrose (1987) failed to prevent P. expansum decay on inoculated apples and pears using various fungicides. It is possible that some spores may be embedded within the
apple tissue and are thus protected from contact with sanitizers. Interactions between the sanitizer and organic matter on the apple surface may also contribute to this lack of activity.

Prevention of cross-contamination

Addition of a single decayed apple to the water bath resulted in a P. expansum spore population of approximately 10⁶ CFU/mL. It has previously been shown that P. expansum spores can reach 10³ CFU/mL or higher in apple dump tanks (Spotts and Cervantes 1986). The level used in this experiment would thus simulate severe contamination.

When apples were held in the water bath containing distilled water for 5 min (control), all of the wounds became infected. However, both EO water and 200 ppm chlorine significantly (P ≤ 0.05) reduced the rate of infection and decay (Table 3). Undiluted EO was more effective than all other treatments, whereas 50% EO water and acidified chlorine had an approximately equal effect. Dilution of EO water with tap water resulted in approximately twice the rate of infection compared with undiluted EO water. When the pH of chlorine solutions was lowered from 10.2 to 7.1, the number of infected wounds decreased by 50%, from 34% to 16.7%.

Table 3—Penicillium expansum cross-contamination of uninoculated wounded apples in a simulated dump tank containing electrolyzed oxidized water (EO water) or sodium hypochlorite

^a Numbers within each column with different letters indicate significant (P ≤ 0.05) differences between treatments. ORP = oxidation-reduction potential.
^b Apples were exposed to treatment solutions for 5 min and stored at 25 °C for 6 d.
^c Single decayed apple resulted in a P. expansum spore population of approximately 106 colony-forming units (CFU)/mL in dump tank.
^d Spore concentration in stock suspension was calculated to achieve 10⁶ CFU/mL in the dump tank.
^e pH was adjusted using 5 N HCl.

Despite similar inoculum levels, direct addition of spores into the water bath resulted in higher infection rates compared with addition of a single decayed apple. Because the 10⁶ CFU/mL level was determined after a decayed apple was held in the water bath for 5 min, fewer numbers of spores may initially be present as they gradually detach from conidiophores and surrounding mycelial structures. Thus, direct addition of spores to the water bath results in higher numbers of spores that are immediately accessible to the apple wounds.

Conclusions

EO water is a promising alternative to chlorine sanitizers for minimizing postharvest infection of apples in dump tanks and other recirculating water systems. However, the results from this study demonstrate the difficulty in inactivating spores on wounded apples once they have become contaminated. Nevertheless, the use of EO water or other sanitizers in process solutions is warranted because it reduces the potential for cross-contamination to occur in apple handling systems. A possible limitation to the large-scale use of EO water is the cost associated with equipment and electrical consumption. More economical uses that do not require large volumes of water, such as spray applications, may offer savings in cost.

Because treatments to prevent cross-contamination are only partially effective, it is essential to minimize inoculum levels and thus reduce the incidence of patulin in juice by harvesting only tree-picked apples, handling them carefully to avoid wounding, and following a regular cleaning and sanitizing program in handling and storage facilities.

Acknowledgment

This research was supported by the Pennsylvania State Agricultural Experiment station and by funding from the Pennsylvania Dept. of Agriculture. The authors thank Dr. Ali Demirci of the Penn State Dept. of Agricultural and Biological Engineering for providing the electrolyzed oxidizing water.

References

Baldry MGC. 1983. The bacterial, fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid. J Appl Bact 54:417–23.

Bari ML, Sabina Y, Isobe S, Uemura T, Isshiki K. 2003. Effectiveness of electrolyzed oxidized water in killing Escherichia coli O157:H7, Salmonella Enterit-idis, and Listeria monocytogenes on the surfaces of tomatoes. J Food Prot 66:542–8.

Brackett RE, Marth EH. 1979. Patulin in apple juice from roadside stands in Wisconsin.J Food Prot 42:862–3.

Conway WS, Janisiewiscz WJ, Klein JD, Sams CE. 1999. Strategy for combining heat treatment, calcium infiltration, and biological control to reduce postharvest decay of ‘Gala’ apples. Hortscience 34:700–4.

Eifert JD, Sanglay GC. 2002. Chemistry of chlorine sanitizers. Dairy Food Environ Sanit 22:534–8.

[USFDA] U.S. Food and Drug Administration. 2002a. Guidance for industry. Juice HACCP hazards and controls guidance. 1st ed. (Draft Guidance). Washington, D.C.: USFDA Center for Food Safety and Applied Nutrition. Released for comment on 12 Sept 2002. Available at: http://www.fda.gov/OHRMS/DOCKETS/ 98fr/02d-0333-gdl0001.doc. Accessed 12 Feb 2003.

[USFDA] U.S. Food and Drug Administration. 2002b. Juice HACCP regulator training.Washington, D.C.: USFDA Center for Food Safety and Applied Nutrition. Office of Field Programs. Sept 2002. Available at: http://www.cfsan.fda.gov/~comm/juiceman.html. Accessed 13 Feb 2003.

Federal Register. 2001. Hazard analysis and critical control point (HAACP); procedures for the safe and sanitary processing and importing of juice; final rule. Federal Register 66(13):6137–202.

Inouye S, Tsuruoka T, Uchida K, Yamaguchi H. 2001. Effect of sealing and Tween 80 on the antifungal susceptibility testing of essential oils. Microbiol Immunol 45:201–8.

Izumi H. 1999. Electrolyzed water as a disinfectant for fresh-cut vegetables. J Food Sci 64:536–9.

Kim C, Hung YC, Brackett RE. 2000a. Efficacy of electrolyzed oxidizing (EO) water and chemically modified water on different types of foodborne pathogens. Int J Food Microbiol 61:199–207.

Kim C, Hung YC, Brackett RE. 2000b. Roles of oxidation-reduction-potential in electrolyzed oxidizing and chemically modified water for the inactivation of food-related pathogens. J Food Prot 63:19–24.

Kirsten BN, Nielsen PV. 1995. Fungicidal effect of 15 disinfectants against 25 fungal contaminants commonly found in bread and cheese manufacturing. J Food Prot 59:268–75.

Koffmann W, Penrose LJ. 1987. Fungicides for the control of blue mold (Penicillium spp.) in pome fruits. Sci Hortic 31:225–32.

Mari M, Cembali T, Beraldi E, Casalini L. 1999. Peracetic acid and chlorine dioxide for postharvest control of Monilinia laxa in stone fruits. Plant Dis 83:773– 6.

Paster N, Huppert D, Barkai-Golan R. 1995. Production of patulin by different strains of Penicillium expansum in pear and apple cultivars stored at different temperatures and modified atmospheres. Food Addit Contam 12:51–8.

Paterson RRM, Archer S, Kozakiewicz Z, Lea A, Locke T, O’Grady E. 2000. A gene probe for the patulin metabolic pathway with potential for use in patulin and novel disease control. Biocontrol Sci Tech 10:509–12.

Roberts RG, Reymond ST. 1994. Chlorine dioxide for reduction of postharvest pathogen inoculum during handling of tree fruits. Appl Environ Microbiol 60:2864–8.

Rosenberger DA. 1999. Postharvest decays: research results and future directions. In: CA storage: meeting the market requirements. Proc. from the CA Storage Workshop; Ithaca, N.Y.; 18 Aug 1999. NRAES Publ. 136. Ithaca, N.Y.: NaturalResource Agric. Eng. Serv., Cornell Univ. p 49–62.

Sholberg P, Haag P, Hocking R, Bedford K. 2000. The use of vinegar vapor to reduce postharvest decay of harvested fruit. Hortscience 35:898–903.

Spotts RA, Cervantes LA. 1986. Populations, pathogenicity, and benomyl resistance of Bortrytis spp., Penicillium spp., and Mucor Piriformis in packinghouses. Plant Dis 70:106–8.

Spotts RA, Cervantes LA. 1992. Effect of ozonated water on postharvest pathogens of pear in laboratory and packinghouse tests. Plant Dis 76:256–9.

Suzuki T, Noro T, Kawamura Y, Fukunaga K, Watanabe M, Ohta M, Sugiue H, Sato Y, Kohno M, Hotta K. 2002. Decontamination of aflatoxin-forming fungus and elimination of aflatoxin mutagenicity with electrolyzed NaCl anode solution. J Agric Food Chem 50:633–41.

Taniwaki HM, Hoenderboom CJM, Vitali AA, Eiroa MNU. 1992. Migration of patulin in apples. J Food Prot 55:902–4.

Venkitanarayanan K, Ezeike GOI, Hung Y-C, Doyle MP. 1999a. Efficacy of electrolyzed oxidizing water for inactivating Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes. Appl Environ Microbiol 65:4276–9.

Venkitanarayanan K, Ezeike GOI, Hung Y-C, Doyle MP. 1999b. Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes on plastic kitchen cutting boards by electrolyzed oxidizing water. J Food Prot 62:857–60.

Willet M, Kupferman G, Roberts R, Spotts R, Sugar D, Apel G, Ewart H, Bryant B. 1989. Integrated management of postharvest diseases and disorders of apples, pears, and cherries. Post-Harvest Pomol Newsl 7:1–4.