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Poult Sci 2007. 86:123-127
© 2007 Poultry Science Association
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PROCESSING, PRODUCTS, AND FOOD SAFETY

Spoilage Microflora of Broiler Carcasses Washed with Electrolyzed Oxidizing or Chlorinated Water Using an Inside-Outside Bird Washer

A. Hinton, Jr.1, J. K. Northcutt, D. P. Smith, M. T. Musgrove and K. D. Ingram

Agricultural Research Service, Poultry Processing Research Unit, Russell Research Center, USDA, Athens, GA 30605

1 Corresponding author: ahinton{at}saa.ars.usda.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The effect of acidic, electrolyzed oxidizing (EO) water and chlorinated water on the spoilage microflora of processed broiler carcasses was examined. Carcasses were sprayed for 5 s at 80 psi with tap, chlorinated, or EO water in an inside-outside bird washer. Treated carcasses were then stored at 4°C for 0, 3, 7, or 14 d, and the microbial flora of the carcasses was sampled using the whole-carcass rinse procedure. Populations of psychrotrophic bacteria and yeasts in the carcass rinsates were enumerated. Results indicated that immediately after spraying the carcasses, significantly fewer psychrotrophic bacteria were recovered from carcasses sprayed with chlorinated or EO water than from carcasses sprayed with tap water. Furthermore, significantly fewer yeasts were recovered from carcasses sprayed with EO water than from carcasses sprayed with tap or chlorinated water. The population of psychrotrophic bacteria and yeasts increased on all carcasses during refrigerated storage. However, after 14 d of storage, significantly fewer psychrotrophic bacteria and yeasts were recovered from carcasses sprayed with EO water than from carcasses sprayed with tap or chlorinated water, and significantly fewer microorganisms were recovered from carcasses sprayed with chlorinated water than from carcasses sprayed with tap water. Pseudomonas spp. and Candida spp. were the primary microbial isolates recovered from the broiler carcasses. Findings from the present study indicate that EO water can effectively be used in inside-outside bird washers to decrease the population of spoilage bacteria and yeasts on processed broiler carcasses.

Key Words: spoilage microflora • broiler carcass • electrolyzed water • chlorinated water • inside-outside bird washer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Chlorine is widely used as a sanitizer in commercial poultry processing operations in the United States because of its low cost and its ability to kill a wide range of microorganisms on carcasses, in processing water, and on processing equipment (Lillard, 1979; Bailey et al., 1986). One method of onsite chlorination of processing water involves injecting chlorine gas into the water to produce microbicidal concentrations of hypochlorous acid in the water. Storage of large quantities of highly toxic, corrosive chlorine gas in these facilities requires the implementation of strict safety precautions to ensure employee safety (Kim et al., 2000b). Liquid or solid forms of hypochlorites are used in other facilities, but these forms of chlorine are more expensive and require more storage space than chlorine gas. The availability of safer, economical sanitizers would provide processors with alternatives to traditional forms of chlorine-based sanitizers.

Acidic and alkaline electrolyzed oxidizing (EO) water are produced in EO water generators by the electrolysis of dilute solutions of sodium chloride or other salts. The microbicidal activity of acidic EO water is due to the combination of the low pH, high oxidation-reduction potential (ORP), and elevated concentrations of hypochlorous acid (Kim et al., 2000b; Park et al., 2004) produced during electrolysis. Although alkaline EO water is not microbicidal, it can be used as a cleanser. The properties of EO water can be easily modified by altering the electrical amperage of the generator during electrolysis (Kim et al., 2000b). Salts, such as sodium chloride, are the only chemicals required during the production of EO water; therefore, the necessity of storing and handling large quantities of chlorine or other potentially dangerous chemicals is eliminated.

The microbicidal activity of acidic EO water has been successfully used in several fields to reduce microbial contamination of products. These applications include the use of EO water as a sanitizer during food preparation, as a disinfectant in medical operations, and as an antimicrobial agent to reduce contamination of agricultural crops (Wullaert, 1997). Acidic EO water has been reported to be microbicidal toward several pathogenic and indicator microorganisms associated with poultry processing (Kim et al., 2000a). Cultures of Escherichia coli O157:H7, Listeria monocytogenes (Park et al., 2004), and Salmonella enteritidis (Venkitanarayana et al., 1999a) can be completely inactivated by EO water in vitro and on cutting boards (Venkitanarayana et al., 1999b). Spraying contaminated eggs with EO water may reduce or eliminate Salmonella typhimurium, L. monocytogenes, Staphylococcus aureus, and E. coli on the surface of the eggs (Russell, 2003). Furthermore, EO water has been shown to be as effective as chlorinated water in reducing contamination of poultry meat by Campylobacter jejuni (Park et al., 2002a) and S. typhimurium. (Fabrizio et al., 2002). The purpose of the present study was to compare the ability of chlorinated and EO water to reduce the population of spoilage microorganisms on processed broiler carcasses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Carcass Treatments
Eviscerated broiler carcasses were collected from the processing line of a local commercial poultry processing facility before being sent into the final bird washer. Carcasses were placed on ice and immediately transported to a pilot plant scale poultry processing facility. For each of 3 experimental trials, carcasses were divided into 3 treatment groups of 24 carcasses each. Carcasses in each group were sprayed with tap water, a 50-ppm chlorine solution, or acidic EO water for 5 s at 80 psi in an inside-outside bird washer (IOBW). The pH of the tap water was 8.0, and the total chlorine concentration was 0.5 ppm. The 50 ppm of chlorinated water (pH 8.2) was prepared by mixing approximately 125 mL of 6.15% commercial bleach (The Clorox Co., Oakland, CA) in 150 L of tap water. Acidic EO water with a pH of 2.4, ORP of +1,180 mV, and 50 ppm of chlorine was produced with an EO water generator (Electric Aquagenics Unlimited Inc., Lindon, UT) filled with a 20% (wt/vol) solution of sodium chloride. The pH of the tap, chlorine, and EO water was measured using a handheld pH meter (model AP5, Denver Instrument, Denver, CO) before spraying the carcasses in the IOBW. The total chlorine concentration of the tap and chlorine water was also measured immediately before treating carcasses using a colorimetric reaction with N, N-diethyl-p-phenylenediamine from the CHEMetrics 2 SAM test kit (CHEMetrics Inc., Calverton, VA). The chlorine concentration of the EO water was determined using the iodometric titration method with the Hach hypochlorite test kit (model CN-HRDT, Hach Co., Loveland, CO).

Microbial Analyses
Treated carcasses were collected from the IOBW and placed in separate sterile plastic bags. Carcasses within each spray treatment group were divided into 4 smaller groups of 6 carcasses each in preparation for analysis or storage. One of the groups of 6 carcasses from each spray treatment was prepared for immediate microbial analysis, whereas the other 3 groups were placed in refrigerated storage at 4°C for 3, 7, or 14 d. The microbial flora of the carcasses was sampled using the whole carcass rinse procedure (Cox et al., 1981) by adding 100 mL of sterile neutralizing buffer (Difco Co., Detroit, MI) solution to the plastic bags containing the carcasses and shaking the carcasses on a mechanical shaker (Dickens et al., 1985) for 1 min. Carcass rinsates were decanted from the bags and serially diluted in sterile 0.1% bacto peptone solutions (Difco Co.).

The populations of psychrotrophic bacteria and yeasts in the carcass rinsates were enumerated. Psychrotrophic bacteria were enumerated on plate count agar (Difco Co.), incubated at 4°C for 10 d, and yeasts were enumerated on acidified potato dextrose agar incubated at 28°C for 3 d. Morphologically distinct colonies on each media were selected, and isolates were identified using the MIDI Sherlock Microbial Identification System (MIDI Inc., Newark, DE; Operating Manual, M. I. S., 2002; Hinton et al., 2004a).

Statistical Analysis
Data from the 3 trials were combined for statistical analyses due to insignificant trial differences or interactions. Group means of data for the number of microorganisms recovered from the carcasses were compared to determine significant differences in the size of microbial populations recovered from carcasses sprayed with tap, chlorinated, or EO water in the IOBW. Data were also analyzed to determine significant differences in the number of microorganisms recovered from the carcasses sprayed with the same solution and stored at 4°C for 0, 3, 7, 14 d. The lower limit of detection of the plating procedures used was 10 cfu/mL. For purposes of statistical analyses, a value of log10 0.99 cfu/mL was assigned to samples in which no microorganisms were recovered. Data were analyzed using GraphPad InStat version 3.05, 32 Bit for Windows 95/NT (GraphPad Software, San Diego, CA) to perform one-way ANOVA. When the AN-OVA detected significant differences in group means, the Tukey-Kramer multiple comparisons test was used to determine if treatment groups differed significantly. All significant differences were determined at P ≤ 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Psychrotrophic Bacterial Microflora
Significantly fewer psychrotrophic bacteria were recovered from carcasses after spraying with EO or chlorinated water than from carcasses sprayed with tap water (Table 1Go). Chlorination of water used to spray carcasses in IOBW has been reported to reduce the population of total bacteria on poultry carcasses (Northcutt et al., 2003). Although forcing EO water through spray nozzles may reduce the chlorine concentration of the water, the residual chlorine in the water can still reduce bacterial contamination of the carcasses (Hsu et al., 2004).


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  		Table 1. Bacterial counts (log10 cfu/mL) recovered on plate count agar incubated at 4°C for 10 d from rinsates of carcasses sprayed with tap, chlorine, or electrolyzed oxidizing (EO) water and stored at 4°C1
 
During storage, there was a significant increase in the number of psychrotrophic bacteria recovered from carcasses sprayed with tap, chlorine, or EO water then stored at 4°C. Whereas the number of bacteria recovered from carcasses sprayed with tap water or chlorinated water had increased significantly by 7 and 14 d of storage, the increase in the number of bacteria recovered from carcasses sprayed with EO water was not significantly greater until d 14. There were also significantly fewer psychrotrophs recovered on d 14 from carcasses sprayed with EO water than from carcasses sprayed with chlorinated or tap water. Significantly greater reductions in the bacterial populations of carcasses sprayed with EO water and smaller bacterial populations on these carcasses after refrigerated storage was probably due to the low pH, high ORP, and hypochlorous acid concentration of the EO water (Kim et al., 2000a). Washing poultry carcasses artificially contaminated with S. typhimurium with EO water also significantly reduced growth of the salmonellae on the carcasses during refrigerated storage (Fabrizio et al., 2002).

Although no psychrotrophs were recovered from carcasses immediately after washing with chlorinated or EO water, Pseudomonas spp. were the predominant psychrotrophs isolated from all carcasses refrigerated for 7 to 14 d (Table 2Go). Psychrotrophic bacteria generally comprise a minor portion of the microflora of fresh poultry, but some pseudomonads are capable of growing on poultry meat during refrigerated storage (McMeekin, 1977; Gallo et al., 1988; Sundheim et al., 1988). Even though commercial poultry processing operations reduce the number of pseudomonads on broiler carcasses, the population of these bacteria on carcasses increases during refrigerated storage (Hinton et al., 2004b). During changes in the size and composition of the native microflora of fresh poultry during refrigeration (Barnes and Thornley, 1966; Russell et al., 1996), the population of psychrotrophic pseudomonads increases until these bacteria become the dominant flora of spoiled, refrigerated poultry (Arnaut-Rollier et al., 1999). Pseudomonas putida was recovered from carcasses washed with chlorinated water, whereas Pseudomonas chloroaphis was isolated from carcasses washed with tap, chlorinated, or EO water.


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  		Table 2. MIDI Sherlock Microbial Identification System1 identification of selected bacterial isolates recovered on plate count agar from carcasses sprayed with tap, chlorinated, or electrolyzed oxidizing (EO) water and stored at 4°C
 
Yeast Microflora
Findings indicated that yeasts on poultry carcasses are also susceptible to the antimicrobial activity of EO and chlorinated water used in an IOBW (Table 3Go). The number of yeasts recovered from carcasses sprayed with chlorine water was significantly lower than the number of yeasts recovered from carcasses sprayed with tap water, and significantly fewer yeasts were recovered from carcasses sprayed with EO water than from carcasses sprayed with chlorine water or tap water. Between d 7 and 14 of refrigerated storage, there was a significant increase in the number of yeasts recovered from carcasses washed with tap water, whereas the number of yeasts recovered from carcasses sprayed with chlorine water increased significantly between d 3 and 14. After 14 d of refrigeration, however, there was no significant increase in the population of yeasts recovered from carcasses sprayed with EO water. Although commercial processing generally also reduces contamination of poultry carcasses by yeasts, the population of these psychrotrophic microorganisms in the microflora increases during refrigerated storage (Hinton et al., 2002). Candida spp. are among the most prevalent yeast species of isolated from commercially processed carcasses, and these yeasts were isolated from carcasses washed with tap, chlorinated, or EO water (Table 4Go).


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  		Table 3. Yeasts (log10 cfu/mL) recovered on acidified potato dextrose agar incubated at 28°C for 3 d from rinsates of carcasses sprayed with tap, chlorine, or electrolyzed oxidizing (EO) water and stored at 4°C1
 

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  		Table 4. MIDI Sherlock Microbial Identification System (MIS) identification of selected yeast isolates recovered on acidified potato dextrose agar from carcasses sprayed with tap, chlorinated, or electrolyzed oxidizing (EO) water and stored at 4°C
 
Findings from the current study indicate that EO water may be used as an alternative to chlorinated water to reduce the population of spoilage microflora of poultry carcasses. The ability of EO water to reduce the number of pseudomonad bacteria and yeasts on broiler carcasses may help to extend the shelf life of fresh, refrigerated poultry. Because processors can economically produce EO water without creating some of the safety hazards associated with water chlorination, EO water may be considered as an option to replace sanitizers currently used to reduce microbial contamination associated with poultry processing.


   ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of Jerrie Barnett, Susan Akins, Fredda J. Murray, Kathy Orr, and Alan Savage.

Received for publication December 20, 2005. Accepted for publication March 13, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Arnaut-Rollier, I., L. DeZutter, and J. Van Hoff. 1999. Identities of the Pseudomonas spp. in flora from chilled chicken. Int. J. Food Microbiol. 48:87–96.[ISI][Medline]

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Barnes, E. M., and M. J. Thornley. 1966. The spoilage flora of eviscerated chickens stored at different temperatures. J. Food Technol. 1:113–119.

Cox, N. A., J. E. Thomson, and J. S. Bailey. 1981. Sampling of broiler carcasses for Salmonella with low volume water rinse. Poult. Sci. 60:768–770.[ISI][Medline]

Dickens, J. A., N. A. Cox, J. S. Bailey, and J. E. Thomson. 1985. Automated microbiological sampling of broiler carcasses. Poult. Sci. 64:1116–1120.[ISI]

Fabrizio, K. A., R. R. Sharma, A. Demirci, and C. N. Cutter. 2002. Comparison of electrolyzed oxidizing water with various antimicrobial interventions to reduce Salmonella species on poultry. Poult. Sci. 81:1598–1605.[Abstract/Free Full Text]

Gallo, L., R. E. Schmitt, and W. Schmidt-Lorenz. 1988. Microbial spoilage of refrigerated fresh broilers. I. Bacterial flora and growth during storage. Lebensm. Wiss. Technol. 21:216–223.

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Hinton A. Jr., J. A. Cason, and K. D. Ingram. 2002. Enumeration and identification of yeasts associated with commercial poultry processing and spoilage of refrigerated broiler carcasses. J. Food Prot. 65:993–998.[ISI][Medline]

Hsu S.-Y., C. Kim, Y.-C. Hung, and S. E. Prussia. 2004. Effect of spraying on chemical properties and bactericidal efficacy of electrolysed oxidizing water. Int. J. Food Sci. Technol. 39:157–165.

Kim, C., Y.-C. Hung, and R. E. Brackett. 2000a. Efficacy of electrolyzed oxidizing (EO) and chemically modified water on different types of foodborne pathogens. Int. J. Food Microbiol. 61:199–207.[ISI][Medline]

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Lillard H. S. 1979. Levels of chlorine and chlorine dioxide of equivalent bactericidal effect in poultry processing water. J. Food Sci. 44:1594–1597.

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Park, H., Y.-C. Hung, and R. E. Brackett. 2002a. Antimicrobial effect of electrolyzed water for inactivating Campylobacter jejuni during poultry washing. Int. J. Food Microbiol. 72:77–83.[ISI][Medline]

Park, H., Y.-C. Hung, and D. Chung. 2004. Effects of chlorine and pH on efficacy of electrolyzed water for inactivating Escherichia coli O157:H7 and Listeria monocytogenes. Int. J. Food Microbiol. 91:13–18.[ISI][Medline]

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Russell, S. M., D. L. Fletcher, and N. A. Cox. 1996. Spoilage bacteria of fresh broiler chicken carcasses. Poult. Sci. 75:2041–2047.

Sundheim, G., A. Sletttn, and R. H. Dainty. 1988. Identification of pseudomonads from fresh and chill-stored chicken carcasses. Int. J. Food Microbiol. 39:185–194.

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