Comparison of electrolyzed oxidizing water with other antimicrobial interventions to reduce pathogens on fresh pork

MEAT SCIENCE Meat Science 68 (2004) 463–468 www.elsevier.com/locate/meatsci

Comparison of electrolyzed oxidizing water with other antimicrobial interventions to reduce pathogens on fresh pork K.A. Fabrizio, C.N. Cutter

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Department of Food Science, 111 Borland Laboratory, The Pennsylvania State University, University Park, PA 16802, USA Received 23 January 2004; received in revised form 23 April 2004; accepted 23 April 2004

Abstract To date, the effectiveness of electrolyzed oxidizing (EO) water against bacteria associated with fresh pork has not been determined. Using a hand-held, food-grade garden sprayer, distilled water (W), chlorinated water (CL; 25 ppm), 2% lactic acid (LA), acidic EO water (EOA), or ‘‘aged’’ acidic EO water (AEOA; stored at 4 °C for 24 h) was sprayed (15 s) onto pork bellies inoculated with feces containing Listeria monocytogenes (LM), Salmonella typhimurium (ST), and Campylobacter coli (CC). Remaining bacterial populations were determined immediately following treatment, after 2 days of aerobic storage, and again after 5 days of vacuum-packaged, refrigerated storage (day 7). While LA and EOA significantly reduced ðp < 0:05Þ populations of CC at days 0 and 7, there was no significant difference ðp > 0:05Þ between antimicrobial treatments when applied to pork inoculated with ST or LM. This study demonstrates that a 15-s spray with EOA has the ability to reduce CC associated with fresh pork surfaces. However, longer contact times may be necessary to reduce other microbial contaminants. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Salmonella typhimurium; Listeria monocytogenes; Campylobacter coli; Electrolyzed oxidizing water; Pork

1. Introduction Electrolyzed oxidizing (EO) water is a relatively new disinfecting compound that has shown promise against cells suspensions of Escherichia coli (EC) O157:H7, Salmonella enteritidis, and Listeria monocytogenes (Venkitanarayanan, Ezeike, Hung, & Doyle, 1999a), E. coli O157:H7 and L. monocytogenes attached to cutting boards (Venkitanarayanan, Ezeike, Hung, & Doyle, 1999b), spoilage organisms associated with vegetables (Izumi, 1999), pathogens in solution (Fabrizio & Cutter, 2003), or pathogens attached to poultry surfaces (Fabrizio, Sharma, Demirci, & Cutter, 2002; Park, Hung, & Brackett, 2002). EO water is produced by passing a salt solution (12%) across a bipolar membrane, resulting in two solutions: an acidic solution that is characterized by a low pH, high oxidation–reduction potential (ORP), and a free chlorine concentration of approximately 50 ppm; the basic solution is composed

of a high pH and low ORP (Kim, Hung, & Brackett, 2000). Approved food grade antimicrobials such as hot or cold water, chlorine, lactic acid, or acetic acid may be applied to pork surfaces prior to and/or following evisceration through automated spray washers (large establishments) or hand-held garden-type sprayers (small establishments) to improve the microbiological safety of the carcass (Frederick, Miller, Thompson, & Ramsey, 1994; Fu, Sebranek, & Murano, 1994; Romans, Costello, Carlson, Greaser, & Jones, 2001). In the present study, the effectiveness of acidic EO water was compared with other carcass decontaminants against populations of E. coli biotype 1, coliforms, L. monocytogenes, C. coli and S. typhimurium associated with pork surfaces. 2. Materials and methods 2.1. Bacterial cultures

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Corresponding author. Tel.: +1-814-865-8862; fax: +1-814-8636132. E-mail address: [email protected] (C.N. Cutter). 0309-1740/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2004.04.013

Campylobacter coli ATCC 33559 (American Type Culture Collection, Manassas, VA), S. typhimurium

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ATCC 13311, and Listeria monocytogenes Scott A were obtained from the Food Microbiology Culture Collection at Pennsylvania State University. L. monocytogenes and S. typhimurium were grown aerobically at 35 °C for 24 h in trypticase soy broth (TSB, Difco) and stored at )76 °C in TSB containing 10% glycerol (Difco). Frozen stock cultures of C. coli were stored at )76 °C in Brucella broth (BB, Difco) containing 10% glycerol and propagated at 42 °C under microaerophilic conditions for 48 h using anaerobic jars (Becton–Dickinson, Microbiology Systems, BDMS, Cockeysville, MD) and CampyPak Plus generating envelopes (BDMS). Prior to experiments, S. typhimurium and L. monocytogenes were propagated aerobically in TSB at 35 °C for 18 h, while C. coli was propagated microaerophilically in BB at 42 °C for 48 h. Prior to inoculation in porcine feces (see below), 10 ml of each pathogen were centrifuged (Beckman Instruments Inc., model J2-21, Palo Alto, CA) at 12,000g for 15 min at 4 °C (S. typhimurium and L. monocytogenes) or at 8000g for 10 min at 4 °C (C. coli) to harvest the cells. After centrifugation, the pellets were resuspended in 10 ml of buffered peptone water (BPW) and serially diluted (1:10) in BPW to obtain approximately 5 log10 CFU/ml for all pathogens. 2.2. Porcine samples and fecal samples Pork bellies were obtained from hogs slaughtered in a local commercial establishment that used hot water as a microbiological intervention. After chilling for 24 h at 4 °C, skin-on intact surface samples were obtained from the belly section. Freshly defecated porcine feces were obtained from gestating sows housed in the facilities of the Swine Center at Pennsylvania State University. Prior to inoculation, 10 g feces was stomached (Stomacher 400, Tekmar, Cincinnati, OH) for 2 min with 90 ml BPW in a filtered stomacher bag (Spiral Biotech, Norwood, MA) containing diluted cultures of S. typhimurium, L. monocytogenes, and C. coli to obtain pathogen levels of approximately 7 log10 CFU/g of feces. Pork surfaces were inoculated as described below. 2.3. Electrolyzed oxidizing water generation EO water was generated by passing a salt (12% NaCl) solution across a charged bipolar membrane. The salt solution and deionized water were pumped into the EO water generator (ROX Water Electrolyzer, Hoshizaki America, Inc., Peachtree City, GA). According to the manufacturer, by subjecting the platinum electrodes to direct voltage (19 amperage), two types of water are generated. From the cathode side, an electrolyzed basic solution [pH 11.6, ORP of )795 mV, containing sodium hydroxide (NaOH)] and from the anode side, an electrolyzed acidic solution (pH 2.6, ORP of 1150, and ap-

proximately 50 ppm free chlorine) containing hypochloric acid (HOCl). Small amounts of oxygen and hydrogen gas were also produced during this reaction. Acidic EO water was made immediately prior to application and referred to as EOA. ‘‘Aged’’ acidic EO water (AEOA) was made 24 h prior to application and stored at 4 °C in an airtight bottle. 2.4. Spray washing experiment Each pork belly was divided into four even sections. Each section was pre-marked with edible ink using a sterile, cotton-tipped swab (Hardwood Products, Co., Guilford, MN), and a sterile 25-cm2 stainless steel template. The pork bellies were surface treated using UV light in a biological safety hood. Surfaces were evenly exposed to UV light by turning sections every 10 min for a total time of no more than 30 min (Cutter & Siragusa, 1994). UV-treated surfaces were inoculated with approximately 8 ml of porcine fecal suspension containing approximately 7 log10 CFU/ml S. typhimurium, L. monocytogenes, and C. coli using a sterile spray bottle. Bacteria on inoculated surfaces were allowed to attach for 15 min at room temperature under a biological safety hood, to obtain pathogen levels of approximately 6 log10 CFU/cm2 . Following inoculation and attachment, the pork belly sections were hung vertically on a stainless steel rack in a biological safety hood and sprayed with the antimicrobial of interest [distilled water (W; control), acidic EO water (EOA; pH 2.4–2.7, 1150 mV ORP, and 50 ppm free chlorine), ‘‘aged’’ EO water (AEOA; pH 2.3, 1150 mV ORP, and 100 ppm free chlorine), 2% lactic acid (LA), or 20 ppm sodium hypochlorite (CL)] for 15 s using a food-grade hand-held garden sprayer (Hudson, Hastings, MN; Model 67220). Once sprayed with antimicrobials, pork surfaces were transferred to a flat sterile surface and left undisturbed for 1 h at 4 °C prior to sampling. Untreated control samples also were excised prior to spray treatments. Following spray treatments, surface samples were obtained by aseptically excising a 25 cm2
0.5 cm thick piece with a sterile scalpel (Sakura, Torrence, CA) and forceps. All excised samples were diluted in 25 ml of BPW and stomached for 2 min in a filtered stomacher bag. Samples were serially diluted in BPW prior to plating onto respective media. For enumeration of mesophilic total viable counts (TVC), diluted stomachates were spiral-plated in duplicate on trypticase soy agar (TSA; Difco) using the Autoplate 4000 (Advanced Instruments, Norwood, MA) and incubated at 35 °C for 36 h. For enumeration of E. coli biotype 1 and coliforms, 1 ml of stomachates was plated in duplicate onto 3M E. coli/coliform PetrifilmTM (3M, St. Paul, MN) according to the manufacturer’s instructions and incubated for 48 h at 35 °C.

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Enumeration of S. typhimurium was performed by spiral plating in duplicate onto xylose lysine deoxycholate agar (XLD; Difco) and incubating for 24–48 h at 35 °C. L. monocytogenes was enumerated by spiral plating in duplicate onto Oxford agar (OX; Difco) and incubated for up to 48 h at 35 °C. Enumeration of C. coli was performed by spiral-plating onto modified CCDAPreston agar (mCCDA; Oxoid, Ogdensburg, NY) and incubating at 42 °C under microaerophilic conditions using anaerobic jars (BDMS) and CampyPak Plus generating envelopes (BDMS) for 48 h. All plates were enumerated manually or with the Q-count image analyzer (Advanced Instruments). The lowest level of detection of organisms was 1:30 log10 CFU/cm2 of stomachate using spiral plating procedures. In the event that no organisms were detected during spiral plating, detection was determined through enrichment procedures (see below). To ensure detection of low levels of pathogens following treatments, sample aliquots were also enriched. For S. typhimurium, 1 ml of stomached sample was added to 9 ml lactose broth and incubated for 24 h at 35 °C. Following incubation, 1 ml of pre-enrichment was added to 9 ml selenite cysteine (SC; Difco), another 1 ml was transferred to 9 ml tetrathionate broth (TT; Difco), and all tubes incubated for 24 h at 35 °C. After enrichment, samples were taken from SC and TT, streaked for isolation onto XLD agar, and incubated for 48 h at 35 °C. Typical colony morphology was identified on XLD and verified serologically using the Oxoid Salmonella latex test. For L. monocytogenes, 1 ml of stomached sample was added to 9 ml Fraser broth (Difco) and incubated for 24 h at 35 °C. Enriched samples were streaked onto OX and incubated at 35 °C for 24–48 h. Typical L. monocytogenes colonies were verified using Visual Immunoassay for L. monocytogenes (TECRA Diagnostics, Roseville, Australia) according to the manufacturer’s instructions. For C. coli, 1 ml of stomached sample was added to 9 ml of Campylobacter enrichment broth (Bolton’s formula, Oxoid) with lysed horse blood and antibiotic supplement, and incubated for 4 h at 35 °C, followed by 20 h at 42 °C under microaerophilic conditions. Enriched samples were streaked onto mCCDA agar and incubated for 24 h at 42 °C under microaerophilic conditions. Typical C. coli colonies were verified serologically using Campylobacter Agglutination Test Kit (Oxoid). Following excision of day 0 samples, the remaining portion of each belly was placed loosely in individual polypropylene bags (Seward, London, UK) to prevent cross-contamination and stored aerobically at 4 °C until sampled on day 2. On day 2, two samples were aseptically excised from each treated and untreated belly. One sample was subjected to sampling, stomaching, and enumeration procedures described above, while the other sample was individually vacuum-packaged (Koch

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Supplies) and stored at 4 °C for and additional 5 days. Following storage, the vacuum-packaged sample was enumerated as described above. 2.5. Free chlorine determination Free chlorine content of the CL and EO water treatments was measured using Hach DPD-FEAS digital titrator method (Hach Company, Loveland, CO) as described by the manufacturer. Briefly, 25 ml sample was diluted 10-fold with sterile distilled water and transferred to an Erlenmeyer flask. A DPD-Free Chlorine Powder Pillow was added to the sample and swirled to mix. The sample was titrated using 0.00564 N ferrous ethylenediammonium sulfate (FEAS) to a colorless endpoint. Free chlorine was calculated from the number obtained following titration, inclusive of the dilution factor (1:10). 2.6. Total chlorine determination Total chlorine measurements of the CL and EO water treatments were measured using Hach DPD-FEAS digital titrator method (Hach Company) as per the manufacturer. Briefly, a 10-ml sample was transferred to a 250-ml Erlenmeyer flask. One Dissolved Oxygen 3 Powder Pillow and one Potassium Iodide Powder Pillow were added and swirled to mix. One dropper (1 ml) of starch indicator was added and swirled to mix, resulting in a dark blue color. The solution was titrated using 0.113 N sodium thiosulfate to a colorless endpoint. Total chlorine content was calculated using the number obtained following titration and the digit multiplier supplied by Hach. 2.7. Determination of pH and ORP Measurements of pH were taken from the antimicrobial solutions using a Corning pH meter (Corning Inc., Corning, NY). ORP measurements of antimicrobial solutions were obtained using Corning pH meter with Orion ion electrode (Orion Research Inc., Beverly, MA). 2.8. Statistical analysis Means of bacterial populations (log10 CFU/cm2 ) from each treatment were calculated from four replications for each experiment. Data were analyzed using SPSS statistical package (SPSS Inc., Chicago, IL). General linear model with repeated measures (holding time constant) was performed to note differences ðp < 0:05Þ between means for treatments. Mean separations were performed using Tukey’s HSD multiple comparison test.

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treatments tested (Table 2). Of the compounds evaluated, LA was the most effective treatment, resulting in a 1.79, 1.46, and 1:76 log10 CFU/cm2 reduction, respectively, for days 0, 2, and 7. EOA and AEOA each afforded a 1.67 and 1:55 log10 reduction, respectively for day 0; however, by day 2, S. typhimurium was only reduced by 1.28 and 1:12 log10 CFU/cm2 . By day 7, the pathogen was reduced 1.36 and 1:1 log10 CFU/cm2 , respectively for EOA and AEOA. Of the pathogens tested, L. monocytogenes was not reduced significantly by the application of the antimicrobials (Table 2). Initially, AEOA effectively reduced the organism 1:38 log10 CFU/cm2 ; after 2 days of aerobic storage, treatments with water (W) demonstrated a 1:17 log10 CFU/cm2 reduction. By day 7, LA was the most effective antimicrobial for reducing L. monocytogenes with a 1:52 log10 CFU/cm2 reduction. When C. coli associated with pork surfaces was spray-treated, significant differences were observed with treatments of LA and EOA at day 0 (Table 2). Of these compounds, EOA was the most effective with a 1:81 log10 CFU/cm2 reduction. LA afforded the greatest reduction after 2 days of storage with a 1:74 log10 CFU/ cm2 reduction. By day 7, C. coli was only detected through selective enrichment for treatments with W, chlorine (CL), EOA, and AEOA. However, there was no significant difference between any of the treatments for day 7. When comparing the treatments with observed TVC, overall reductions diminished over 7 days, even when stored under vacuum-packaged conditions (Table 1).

3. Results and discussion 3.1. Spray washing experiment Treatments for TVC, E. coli, total coliforms (TC), L. monocytogenes, and S. typhimurium were not significantly different from each other when applied to experimentally inoculated pork bellies; however, they were significantly different from the untreated samples (Tables 1 and 2). LA and EOA water were the only treatments to significantly reduce populations of C. coli; 1.71 and 1:81 log10 CFU/cm2 , respectively (Table 2) and reductions were maintained at days 2 and 7. When populations of E. coli biotype 1 were monitored, all the treatments were significantly different from the untreated samples. However, the greatest reductions were observed with EOA and ‘‘aged’’ acidic EO water (AEOA), in which reductions of 1.13 and 1:16 log10 CFU/cm2 were observed, respectively (Table 1). After 7 days of storage, LA afforded the greatest reduction of E. coli biotype 1 (1:04 log10 CFU/cm2 ). Treatments of total coliforms afforded approximately the same reduction as for E. coli biotype 1 (Table 1). Initially, only treatments with LA, EOA, and AEOA afforded the greatest reduction of approximately 1 log10 CFU/cm2 . After day 2 and day 7 of refrigerated storage, there were no significant differences between any of the antimicrobial treatments. LA reduced coliforms 1.25 and 1:14 log10 CFU/cm2 , respectively, at days 2 and 7. At days 0, 2, and 7, populations of S. typhimurium were not significantly reduced following any of the

Table 1 Effect of spray washing distilled water, electrolyzed oxidizing water, chlorine (20 ppm), 2% lactic acid, or acidic EO water stored at 4 °C for 24 h (‘‘aged’’ EO water) on populations (log10 CFU/cm2 ) of total viable counts (TVC), Escherichia coli (EC), and total coliforms (TC) on pork bellies immediately following treatments, after 2 days aerobic storage at 4 °C and after 7 days of vacuum-packaged storage at 4 °C Treatment

Organism

Day 0

Day 2

Day 7

A

A

Untreated Distilled water Chlorine Lactic acid EO water ‘‘Aged’’ EO

TVC TVC TVC TVC TVC TVC

6.87  0.14 5.85  0.30B 5.57  0.21B 5.69  0.13B 5.64  0.68B 5.61  0.53B

6.97  0.11 6.07  0.22B 6.09  0.12B 6.93  1.75B 6.06  0.09B 6.12  0.24B

7.32  0.34B 6.89  0.13B 6.70  0.35B 6.51  0.77B 6.91  0.32B 6.86  1.01B

Untreated Distilled water Chlorine Lactic acid EO water ‘‘Aged’’ EO

EC EC EC EC EC EC

4.65  0.26A 3.71  0.24B 3.74  0.58B 3.57  0.27B 3.52  0.61B 3.49  0.69B

5.06  0.29A 3.82  0.51B 3.93  0.28B 3.77  0.39B 3.84  0.52B 3.93  0.63B

4.43  0.16A 3.80  0.51B 3.62  0.26B 3.39  0.23B 3.45  0.10B 3.47  0.39B

Untreated Distilled water Chlorine Lactic acid EO water ‘‘Aged’’ EO

TC TC TC TC TC TC

4.71  0.24A 3.75  0.23B 3.80  0.61B 3.60  0.27B 3.55  0.61B 3.52  0.71B

5.07  0.29A 3.87  0.54B 3.97  0.27B 3.82  0.34B 3.88  0.49B 3.97  0.66B

4.56  0.11A 3.87  0.50B 3.69  0.28B 3.42  0.24B 3.50  0.10B 3.56  0.33B

A;B Means within a column for a given organism on a given day sharing the same letter are not significantly different ðp > 0:05Þ. Mean square error for APC ¼ 0.04, for EC ¼ 0.11, and for TC ¼ 0.10.

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Table 2 Effect of spray washing distilled water, electrolyzed oxidizing water, chlorine (20 ppm), 2% lactic acid, or acidic EO water stored at 4 °C for 24 h (‘‘aged’’ EO water) on populations (log10 CFU/cm2 ) of Salmonella typhimurium (ST), Listeria monocytogenes (LM), and Campylobacter coli (CC) on pork bellies immediately following treatments, after 2 days aerobic storage at 4 °C and after 7 days of vacuum-packaged storage at 4 °C Treatment

Organism

Day 0

Day 2

Day 7

Untreated Distilled water Chlorine Lactic acid EO water ‘‘Aged’’ EO

ST ST ST ST ST ST

6.27  0.09A 4.91  0.36B 4.89  0.25B 4.48  0.32B 4.60  0.74B 4.72  0.77B

5.78  0.20A 4.61  0.55B 4.64  0.19B 4.32  0.32B 4.50  0.64B 4.66  0.60B

5.55  0.35A 4.73  0.48B 4.20  0.81B 3.79  0.25B 4.19  0.21B 4.45  0.42B

Untreated Distilled water Chlorine Lactic acid EO water ‘‘Aged’’ EO

LM LM LM LM LM LM

6.69  0.09A 5.68  0.20B 5.33  0.34B 5.53  0.16B 5.46  0.63B 5.31  0.55B

6.77  0.05A 5.60  0.26B 5.69  0.46B 5.86  0.32B 5.61  0.57B 5.67  0.68B

7.06  0.48A 6.23  0.51B 6.05  0.29B 5.54  0.28B 5.67  0.08B 5.64  0.17B

Untreated Distilled water Chlorine Lactic acid EO water ‘‘Aged’’ EO

CC CC CC CC CC CC

5.70  0.09A 4.37  0.34AB 4.63  0.60AB 3.99  1.04B 3.89  0.75B 3.96  0.95AB

3.98  1.13A 2.38  0.56AB 2.94  0.61AB 2.24  0.22B 2.32  0.97B 2.68  1.59AB

1.95  0.75A 1.30  0.0A 1.30  0.0A 1.38  0.15A 1.30  0.0A 1.30  0.0A

A;AB;B

Means within a column for a given organism on a given day sharing the same letter are not significantly different ðp > 0:05Þ. Mean square error for ST ¼ 0.14, for LM ¼ 0.09, for CC ¼ 0.36. * Since value of 1.30  0.0 log CFU/cm2 represents lowest detectable level by spiral plating, detection of the pathogen occurred only through enrichment.

L. monocytogenes is a psychrotrophic, facultative anaerobic bacterium. Overall numbers associated with the pathogen at day 7 were probably due to the ability of this pathogen to recover under vacuum-packaged conditions and grow at refrigeration temperatures. It has been documented that some amount of bacteria is removed physically by virtue of spray-washing (Cutter & Siragusa, 1994). Similar observations were noted in this study, as indicated by the reductions associated with water treatments. However, the lack of antimicrobial activity by some compounds, including EO or AEOA water, against some bacterial populations on pork surfaces may be due to an insufficient contact time. Several other studies also have investigated the effectiveness of EO water against food-borne pathogens under different contact times and conditions. One study compared different contact times and temperatures for reducing E. coli O157:H7 and L. monocytogenes on cutting boards (Venkitanarayanan et al., 1999b). More recently, Kim, Hung, Brackett, and Frank (2001) studied the inactivation of L. monocytogenes biofilms with EO water and demonstrated that the compound worked more effectively when cells were treated for up to 300 s. Park et al. (2002) and Fabrizio et al. (2002) examined the use of EO water to inactivate C. jejuni or Salmonella spp. on poultry. In these studies, it was determined that EO water was more effective when the contact times were increased to >10 and 40 min, respectively.

3.2. Total chlorine, free chlorine, pH, and ORP determination Previously, it was determined that storage of acidic EO water at 4 °C for 24 h (‘‘aged’’ EO water; AEOA) increased the free chlorine concentration of the solution (Fabrizio & Cutter, 2003). In this study, it does not appear that AEOA was different from EO water in pH, ORP, free, or total chlorine concentration (Table 3). When evaluated for reducing microbial populations on pork surfaces, these experiments indicated that there was no difference between either of the EO water treatments (EOA, AEOA) for any pathogenic organisms tested. Kim et al. (2000) hypothesized that the primary Table 3 Characteristics of compounds taken prior to spray washing of pork belliesa Treatmentb

pH

ORP

Free chlorine

Total chlorine

Distilled water Chlorine Lactic acid EO water ‘‘Aged’’ EO

7.81 7.82 2.33 2.79 2.94

240 626 613 1144.5 1144

N/Dc 19.9 N/D 68.25 66

N/D 20.5 N/D 74.5 73

a

Values represent the average of four replications. Treatment solutions: distilled water, chlorinated water, lactic acid (2%), electrolyzed oxidizing water (EO water), and ‘‘aged’’ EO water (stored at 4 °C for 24 h). c Values were not measured. b

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factor for inactivation of organisms with EO water was the extreme ORP value. If true, this observation may explain why both forms of EO water performed equally against the pathogens since the ORP values for the EOA and AEOA were similar. 4. Conclusion This study demonstrates that a 15-s spray with EOA water or LA has the ability to reduce some populations of undesirable bacteria (i.e., Campylobacter spp.) associated with fresh pork surfaces. However, if an increased contact time (>10 min) is needed to effectively reduce other microbial contaminants (i.e., ST, LM, EC, and TC), as has been observed in other studies, it may be necessary to determine the feasibility of incorporating such a system into pork processing. Therefore, additional experiments using EO water with extended application times are warranted. Acknowledgements This research was supported by the Pennsylvania State University, College of Agricultural Sciences Seed Grant Program, Pennsylvania Agricultural Experiment Station, and a grant from the National Pork Board. We are thankful to Drs. Beelman, Demirci, and Mills for their advice on the project. We also thank Hoshizaki Electric Co. Ltd., Sakae, Toyoake, Aichi, Japan, for providing the EO water generator used in this study. References Cutter, C. N., & Siragusa, G. R. (1994). Efficacy of organic acids against Escherichia coli O157:H7 attached to beef carcass tissue

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