Acidified Sodium Chlorite Antimicrobial Treatment of Broiler Carcasses

Transcription

1 1087 Journal of Food Protection, Vol. 63, No. 8, 2000, Pages Copyright, International Association for Food Protection Acidified Sodium Chlorite Antimicrobial Treatment of Broiler Carcasses G. KERE KEMP, 1 * M. L. ALDRICH, 1 AND A. L. WALDROUP 2 1 Alcide Corporation, th Avenue N.E., Redmond, Washington 98052; and 2 University of Arkansas, Department of Poultry Science, Fayetteville, Arkansas 72701, USA MS : Received 2 December 1999/Accepted 14 March 2000 ABSTRACT An acidified sodium chlorite (ASC) solution was investigated for its antimicrobial effects on broiler carcasses processed under conditions similar to those used in U.S. commercial poultry facilities. Of particular interest was the ability of the ASC solution to reduce natural bioburden in a prechill procedure. A number of parameters such as pretreatment washing of carcasses with water (no wash versus water wash), ASC concentration (500, 850, and 1,200 ppm), method of application (spray versus dip), and method of acid activation (phosphoric acid versus citric acid) were explored to evaluate disinfection conditions. ASC dip solutions (18.9 liters) were freshly prepared for groups of five prechill eviscerated carcasses per treatment (n 10 carcasses). ASC treatment was shown to be an effective method for significantly reducing naturally occurring microbial contamination on carcasses. Reductions following immersion dipping were demonstrated at all disinfectant concentrations for total aerobes (82.9 to 90.7%), Escherichia coli (99.4 to 99.6%), and total coliforms (86.1 to 98.5%). Additionally, testing showed that ASC solutions maintained stable ph and minimal chlorite ion concentration deviations throughout each treatment. The results of the parameter evaluations indicated that maximal antimicrobial activity was achieved in carcasses that were prewashed and then exposed to a 5-s dip in a solution containing phosphoric acid- or citric acid-activated ASC. At 1,200 ppm ASC, a mild but transitory whitening of the skin was noted on dipped carcasses. The results support the methods currently approved by the U.S. Department of Agriculture for the use of ASC solutions as a prechill antimicrobial intervention in U.S. poultry processing plants. The failure of decontamination techniques to eliminate naturally occurring microbes adequately during the processing of poultry concerns members of the food industry, governmental health officials, and consumers. Studies have shown that a number of bacteria including Escherichia coli, Salmonella, Pseudomonas, Enterobacteriaceae, Campylobacter, and Listeria can survive the slaughter and packaging processes. During processing, these surviving bacteria can spread from carcass to carcass and ultimately be transmitted to human consumers of the product (6, 8, 9, 18). Currently, U.S. commercial processing facilities use chlorinated water chilling (hydrocooling) to decrease product temperature and reduce the microbial load of poultry. Following evisceration, carcasses are subjected to an inside outside bird wash and then are hydrocooled at 4 C for approximately 1hincirculating water tanks containing low levels of residual chlorine (up to 50 ppm). Numerous studies have reported mixed results regarding the antimicrobial efficacy of chlorine when used as a prechill wash or spray or when used as a component of chill waters in poultry processing (3, 10, 16, 19, 24, 26). In 1971, Sanders and Blackshear (20) reported on coliform count reductions following poultry carcass washing with 0 to 230 ppm chlorine. They found that wash water chlorine concentrations of 4 to 10 ppm resulted in 0.2 to 0.4 log reductions of coliforms and that concentrations of 10 to 60 * Author for correspondence. Tel: ; Fax: ; kkemp@alcide.com. ppm resulted in 0.5 to 1.0 log reductions. However, no further reductions were achieved by raising the chlorine concentration above 60 ppm. A 1980 study by Lillard (14) reported a 0.8- to 1.0-log reduction in aerobes and a 1.4- to 1.6-log reduction in fecal coliforms in blended broiler tissue samples following chilling with 20 to 34 ppm chlorine. More recently, Waldroup et al. (27) reported a 1992 multiplant test that examined the modification of broiler processing procedures to include 20 ppm chlorine throughout most of the processing line and to provide 1 to 5 ppm free chlorine in the chill tank overflow. These combined modifications resulted in a 0.2- to 0.6-log reduction in aerobes, 0.0- to 0.3-log reductions in coliforms, and 0.0- to 0.4-log reductions in E. coli. Many researchers agree that chlorine is relatively ineffective against Salmonella spp. (10, 17, 23, 26), can act as a corrosive on plant machinery, and can combine with organic materials to generate potential mutagens (15). A recently developed disinfectant technique (Alcide Corporation, Redmond, Wash.) that uses acidified sodium chlorite (ASC or chlorous acid) can supplement chlorine hydrocooling for the bacterial decontamination of poultry carcasses. ASC is a Food and Drug Administration-approved antimicrobial for poultry, red meats, seafood, and raw agricultural commodities (fruit and vegetables) (1). Additionally, the U.S. Department of Agriculture (USDA) has granted interim approval (subject to rule-making) for ASC use as an antimicrobial intervention on poultry (4).

2 1088 KEMP ET AL. J. Food Prot., Vol. 63, No. 8 When ASC the product of NaClO 2 acidification comes into contact with organic matter, a number of oxychlorous antimicrobial intermediates are formed (7, 13). These reactive intermediates are broad-spectrum germicides that act by breaking oxidative bonds on cell membrane surfaces (13). The fundamental nonspecific oxidative mode of action of this chemistry is thought to also minimize the potential problem of acquired resistance that often arises in bacterial populations following prolonged exposure to antimicrobial procedures. A recent study performed at MicroChem Laboratory, Inc. (Fort Worth, Tex.) using a Food and Drug Administration-defined test procedure (2) showed that none of 10 test microbes developed resistance to ASC after more than 100 divisions in subinhibitory concentrations of the antiseptic (12). ASC has been successfully used as a sterilant for nonporous surfaces and devices in the medical and pharmaceutical industries, as a disinfectant in automobile air conditioning systems, and as a skin antiseptic in the dairy industry. In this study, the authors explored ASC as a prechill disinfectant treatment that could be used as an intervention early in the processing cycle to reduce bioburden and minimize the problem of cross-contamination in poultry carcasses. This manuscript reports on the evaluation of treatment parameters necessary for ASC disinfection of broiler carcasses under conditions similar to those used in U.S. commercial poultry facilities. Three concentrations of citric acid-activated ASC were compared for their antimicrobial efficacy (500, 850, and 1,200 ppm). Additionally, the effect of pretreatment washing with water (no wash versus water wash), the method of ASC application (spray versus dip), and the hypothesis that phosphoric acid and citric acid were likely equivalent activators for ASC were explored. Performance characteristics were determined by measuring reductions of aerobes, E. coli, and coliforms in carcass rinsates. MATERIALS AND METHODS The study was conducted at the University of Arkansas Poultry Science Center s pilot slaughter facility in Fayetteville, Arkansas. The carcass-handling procedures were chosen to model events that take place in poultry processing prior to slaughter and packaging. Eviscerated broiler carcasses from commercially raised 35- day-old male and female chickens were either obtained from a local slaughterhouse immediately after inside outside bird wash and before chilling, or, for some experiments, live chickens from the same source were slaughtered at the pilot facility. The carcasses were selected for standard size (1.5 to 2.0 kg) and lack of obvious defects. Collection, transport, and storage prior to treatment required a maximum of 1 h and 45 min. During this time, the carcasses were held at room temperature (approximately 20 C) to minimize any potential for the artificial reduction of carcass counts due to temperature shock from chilling. Carcass preparation. To simulate the inside outside bird wash used in commercial processing facilities, carcasses received a 5-s water wash (pretreatment wash) with a handheld hose pressurized to 60 to 80 lb/in 2 and delivering approximately 17 liters of water per min at ambient tap temperature. This wash was used to remove serum exudate or other organic matter that may have accumulated on the interior and exterior surfaces of the carcass during collection and transport. After washing, each carcass was inverted for approximately 30 s prior to antimicrobial treatment to remove any water that had accumulated within the body cavity. With the exception of experimental group T2 in experiment 1 (no prewash), all carcasses received the pretreatment wash described above. Treatment groups. Following the preparation of the carcasses, each was randomly assigned a treatment (T) group. Experiment 1: phosphoric acid-activated ASC pretreatment wash evaluation; T1 H 2 O treatment control group, no hydrocooling; T2 (n 10) experimental group immersed 5sin 1,200 ppm ASC, no pretreatment wash, no hydrocooling; T3 (n 10) experimental group immersed 5sin1,200 ppm ASC, no hydrocooling. Experiment 2: Citric acid-activated ASC concentration evaluation; T1 (n 30) H 2 O treatment control group, no hydrocooling; T2 (n 10) experimental group immersed 5sin 1,200 ppm ASC, no hydrocooling; T3 (n 10) experimental group immersed 5 s in 850 ppm ASC, no hydrocooling; T4 (n 10) experimental group immersed 5 s in 500 ppm ASC, no hydrocooling. Experiment 3: Citric acid-activated ASC application evaluation; T1 H 2 O treatment control group, simulated hydrocooling; T2 (n 10) experimental group immersed 5sin 18.9 liters, 1,200 ppm ASC followed by simulated hydrocooling; T3 (n 10) experimental group sprayed for 15 s with 5 oz of 1,200 ppm ASC followed by simulated hydrocooling. Experiment 4: Acid-activated ASC activation evaluation; T1 H 2 O treatment control group, simulated hydrocooling; T2 (n 10) experimental group immersed 5 s in 1,200 ppm phosphoric acid-activated ASC followed by simulated hydrocooling; T3 (n 10) experimental group immersed 5sin 1,200 ppm citric acid-activated ASC followed by simulated hydrocooling. ASC preparation. All ASC solutions were prepared fresh for each experimental treatment group using sodium chlorite (lot DDGH2303, 80%, technical grade; Vulcan, Birmingham, Ala.) and citric acid (lot LK0212, FCC grade; Spectrum, Savannah, Ga.) or sodium chlorite/phosphoric acid (lot 206-4, FCC grade, 85%; Rhone-Poulenc, Princeton, N.J.) and tap water to a final concentration of 500, 850, or 1,200 ppm at ambient tap water temperature (12 to 14 C). Carcasses were not exposed to additional antimicrobials. ASC solutions were monitored by Food and Drug Administration-approved procedures (1) for ph and chlorite concentrations following preparation and again after dipping each group of five carcasses. Dipping. ASC was applied by total immersion of five carcasses into a container holding 18.9 liters of ASC solution for 5 s as measured by stop-watch. An immersion time of 5 s was selected based on preliminary D-value testing (12). During immersion in the ASC solution, the carcasses were agitated manually with up and down swirling motions. The carcasses were drained (dwell time) for 30 s and were then placed in collection bags or transferred to chill-water tanks for further treatment before microbiological sampling. For each group of five carcasses treated, a fresh batch of ASC solution was prepared. This ensured that the concentration of ASC in each batch was never depleted excessively and that organic matter did not accumulate. The same procedure was followed for the control carcasses except that these carcasses were immersed in containers filled with water only.

3 J. Food Prot., Vol. 63, No. 8 ACIDIFIED SODIUM CHLORITE TREATMENT OF BROILER CARCASSES 1089 TABLE 1. Pretreatment wash with water evaluation: reductions in bacterial loads following water or ASC treatment Carcass pretreatment followed by ASC a No prewash Prewash Escherichia coli Total coliform 4.2 A, X [0.07] b 3.93 A, X [0.07] 0.28 (46.6%) c 2.5 A, X [0.21] 2.79 A, X [0.17] 0.28 (NA) 1.99 A, X [0.12] 1.69 A, X [0.13] 0.30 (50.0%) 4.15 A, X [0.09] 3.43 B, Y [0.11] 0.72 (80.9%) 3.11 A, X [0.20] 0.90 B, Y [0.28] 2.21 (99.4%) 2.15 A, X [0.10] 0.64 B, Y [0.22] 1.51 (96.9%) a Phosphoric acid-activated ASC at 1,200 ppm, 5-s dip. Within a row, different letters (A or B) indicate significance, P 0.05; by organism within a column, different letters (X or Y) indicate significance, P 0.01; NA, not applicable. Spraying. Each carcass in experiment 3, treatment group 3, was sprayed with 150 ml (5 oz) of ASC solution for 15 s followed by a 30-s dwell time. As described for dipping exposure experiments, this spray volume was derived from initial D-value testing that established volume-to-surface area ratio per unit of time on chicken skin. The sprayed carcasses were then transferred to chill-water tanks for simulated hydrocooling before being bagged for microbiological sampling. The same procedure was followed for the control carcasses except that they were subjected to a water spray only. Carcass spraying was achieved using a handheld pressurized (10 lb/in 2 ) sprayer with the exposed surfaces being sprayed in three sections: front, rear, and interior. Each section was sprayed for a total of 5 s as measured by a stop-watch. Simulated hydrocooling. Immediately after treatment and dwell time, the carcasses in experiments 3 and 4 were immersed in 60-gal tubs (12 gal chill water/carcass) of ice-chilled, 1 to 2 C water, and gently agitated for 45 min, after which the carcasses were removed individually and treated to a whole-carcass rinse procedure. The same process was followed for the control carcasses (12 gal chill water/carcass) except that immediately following water treatment the carcasses were immersed in a tank of chilled water separate from the ASC-treated carcasses. Microbiology. Post-treatment samples were collected from the control and experimental carcasses and were evaluated for microbial load. Rinse samples from experiments 1 and 2 were obtained for microbiological analysis using the whole-carcass rinse method of Cox et al. (5). Briefly, each carcass was rinsed in a plastic collection bag containing 400 ml of buffered peptone H 2 O solution with 0.1% sodium thiosulfate (lot 66H0293; Sigma, St. Louis, Mo.) (for ASC neutralization) (12). Carcasses for experiments 3 and 4 were similarly sampled after the chill-tank procedure except that samples were collected in a peptone solution lacking sodium thiosulfate. Rinse samples were transferred to sterile bottles that were placed in insulated containers containing crushed ice. The bottles remained on ice for transport to and storage at the University of Arkansas microbiology laboratory prior to analysis on the same day. All rinse samples were plated in duplicate for aerobic plate count, E. coli, and total coliform count using Petrifilm (part 6406, total aerobic plate count; part 6404, E. coli and total coliforms; 3M Center, Sait Paul, Minn.) in accordance with the manufacturer s instructions. Qualitative Salmonella analyses were also conducted using the procedures outlined in USDA LC-75 (25). Statistical design and analysis. For each experiment, separate analyses were conducted for each microbial organism. All quantitative microbiological measurements were transformed to log 10 prior to conducting an analysis of variance using the general linear model (PROCGLM) in SAS (22). A Duncan s multiple range test was performed to determine which treatments were statistically different. A P 0.05 was considered significant. RESULTS Table 1 shows the effect of carcass pretreatment washing on ASC efficacy. As expected, by removing serum exudate and other organic debris, pretreatment washing enhanced the antimicrobial activity of ASC. ASC in combination with pretreatment washing was found to reduce significantly the populations of total aerobes (F 16.39; df 3, 36; P ), E. coli (F 20.15; df 3, 36; P ), and total coliforms (F 21.12; df 3, 36; P ). Although bacterial levels were unaffected in treated carcasses that were not pretreatment washed, it should be noted that prewashing followed by ASC treatment eliminated essentially all of the bacteria from carcasses assessed for E. coli. Salmonella spp. were not found in rinse samples from either the experimental or control carcasses in any experiment in this study. Analysis of the plate counts (Table 2) showed that, when applied as 5-s dips, all three concentrations of ASC (500 ppm, 850 ppm, and 1,200 ppm) produced significant reductions in the natural populations of total aerobes (F 23.96; df 5, 54; P ), E. coli (F 63.75; df 5, 54; P ), and total coliforms (F 29.02; df 5, 54; P ). Reductions of at least 99.4% were noted for E. coli, regardless of ASC concentration. Table 3 shows that both spraying and dipping with citric acid-activated ASC, followed by simulated hydrocooling, produced statistically significant reductions in bacterial loads for total aerobes (F 23.39; df 3, 36; P

4 1090 KEMP ET AL. J. Food Prot., Vol. 63, No. 8 TABLE 2. Concentration evaluation: reductions in bacterial load following water or ASC treatment ASC a concentration 1,200 ppm 850 ppm 500 ppm E. coli Total coliform 4.25 A, X [0.13] b 3.22 A, Y [0.16] 1.03 (90.7%) c 2.52 A, X [0.14] 0.21 A, Y [0.11] 2.31 (99.4%) 2.12 A, X [0.33] 0.17 A, Y [0.10] 1.96 (98.5%) 3.85 B, X [0.07] 3.00 A, Y [0.10] 0.85 (85.6%) 2.65 A, X [0.17] 0.32 A, Y [0.18] 2.33 (99.6%) 1.70 B, X [0.16] 0.19 A, Y [0.08] 1.51 (96.0%) 3.88 B, X [0.04] 3.12 A, Y [0.09] 0.76 (82.9%) 2.58 A, X [0.20] 0.29 A, Y [0.12] 2.29 (99.5%) 2.22 A, X [0.12] 1.37 B, Y [0.10] 0.85 (86.1%) a Citric acid-activated ASC, 5-s dip. Within a row, different letter (A or B) indicate significance, P 0.05; by organism within a column, different letters (X or Y) indicate significance, P ), E. coli (F 51.06; df 3, 36; P ), and total coliforms (F 25.73; df 3, 36; P ). Again, as seen in all of the experiments, reductions in E. coli following ASC treatment were greater than those seen for aerobes or other coliforms. Dipping was shown to be a statistically more effective application procedure than spraying for all organisms tested. Table 4 presents the data from the phosphoric acid and citric acid activation comparison. As measured in this 5-s dipping experiment, both methods were equally effective activators of ASC, reducing E. coli (F 32.07; df 2,27; P ) and total coliforms (F 21.28; df 2, 27; P ). Total aerobes were not significantly affected (F 1.85; df 2, 27; P 0.177) by either activated solution in this evaluation. ASC solutions and the water controls were monitored in this study for ph and chlorite ion stability. The ph remained constant for each five-carcass batch treatment. Slight (4 to 5 ppm) reductions in chlorite concentrations were noted per five-carcass batch treatment in ASC (18.9 liters) (data not shown). A mild and transient skin whitening was observed in dipped carcasses at the very highest (1,200 ppm) dose; however, following hydrocooling this effect was completely lost. The underlying muscle and fat tissues were not visibly affected by the treatment. DISCUSSION A recently developed disinfecting technique uses ASC that has been activated by citric acid or phosphoric acid. In this study, the authors investigated a number of treatment parameters to determine conditions for the activity of ASC against naturally occurring microbials in processed poultry carcasses. The results of this study indicate that pretreatment washing of carcasses with water was particularly effective in reducing bacterial load when combined with an ASC treatment. It is hypothesized that the removal of serum exudate and other debris by prewashing prior to treatment TABLE 3. Application evaluation: reductions in bacterial load following water or ASC treatment and simulated hydrocooling ASC b application method Dip Spray E. coli Total coliform 2.80 A, X [0.07] b 2.03 A, Y [0.09] 0.77 (83.0%) c 1.52 A, X [0.08] 0.29 A, Y [0.09] 1.23 (94.3%) 0.97 A, X [0.07] 0.04 A, Y [0.02] 0.93 (88.2%) 2.83 A, X [0.06] 2.31 B, Y [0.09] 0.52 (69.8%) 1.44 A, X [0.10] 0.67 B, Y [0.07] 0.77 (82.9%) 0.82 A, X [0.14] 0.30 B, Y [0.07] 0.52 (69.7%) a Citric acid-activated ASC at 1,200 ppm. Within rows, different letters (A or B) indicate significance, P 0.05; by organism within a column, different letters (X or Y) indicate significance, P 0.01.

5 J. Food Prot., Vol. 63, No. 8 ACIDIFIED SODIUM CHLORITE TREATMENT OF BROILER CARCASSES 1091 TABLE 4. Activation evaluation: reductions in bacterial loads following water or ASC treatment and simulated hydrocooling ASC a activator Citric acid E. coli Total coliform 3.15 A [0.10] b 2.94 A [0.08] 0.21 (38.4%) c 1.54 A [0.14] 0.34 B [0.10] 1.20 (93.7%) 1.39 A [0.13] 0.36 B [0.08] 1.03 (92.0%) 3.15 A [0.10] 2.96 A [0.08] 0.20 (36.9%) 1.54 A [0.14] 0.36 B [0.13] 1.18 (93.4%) 1.39 A [0.13] 0.39 B [0.15] 1.00 (90.0%) a Activated ASC at 1,200 ppm, 5-s dip. By organism, different letters indicate significance, P most likely results in the removal of a significant amount of the extraneous oxidizable material and/or the materials that might be coating and therefore protecting the bacteria on the various exposed carcass surfaces. Therefore, it is likely that prewashing allowed better exposure of the carcass surfaces to the ASC. In separate experiments (12), the passage of carcasses through an inside outside bird washer had the effect of preconditioning the carcass surface for oxidative chemistry by removing the accumulated nonattached organic load from the skin and body cavity. The results of the experiments reported showed that E. coli was extremely susceptible at all concentrations tested; however, the higher 850 and 1,200 ppm were significantly more effective against coliforms. Dipping carcasses into the ASC solutions was demonstrably more effective than spraying. This difference most probably arises because not only does an immersion dip application bring ASC into better contact with all of the treatable surfaces, it also ensures that the active chemistry at the treatment-surface treatment-liquid interface is maintained in a more stable state for optimal performance. Both phosphoric acid and citric acid were found to be equally capable of activating ASC to kill the total aerobes, E. coli, and total coliforms. For ASC chemistry, acids primarily act as proton donors to permit the acidification of sodium chlorite. Thus, any acid that will release a hydrogen ion can be used to activate the chemistry. Secondarily, the quantity of acid used will dictate the final system ph and, therefore, the rate of dissociation of the chlorite to form the antimicrobial chlorous acid species. Recent pilot tests, however, indicate that citric acid may be a more effective activating agent than phosphoric acid for eradicating Campylobacter (12). While the exact reasons for this apparent difference have not been investigated, there is already some evidence in the literature to suggest that the additional chelating effects of citric acid may possibly be responsible. It has been shown that citric acid will act to extract divalent cations from the lipopolysaccharide region of the outer membrane, thus destabilizing the membrane structure (11, 21). Finally, significant concerns within the poultry industry today regarding the handling and remediation of phosphate-containing waste streams tends to mitigate against the routine use of phosphoric acid as an activator of ASC solutions. The antimicrobial action of ASC was found to be meaningful when investigated under conditions relevant to commercial poultry-processing plants. These data show that 5-s exposures of ASC at 1,200 ppm are capable of achieving greater log reductions than are typically seen with 1 h, or more, exposures of 20 ppm chlorinated water (27). The only notable adverse effect of ASC was a transient, mild whitening of the skin surface of those carcasses treated by dipping at the highest concentration evaluated. Subsequent tests in commercial settings have shown that this mild color change is an inconsistent finding, is subsequently lost during hydrocooling, and does not result in any organoleptic changes either in raw (postchill) or cooked poultry product. In summary, this study suggests that the ASC disinfectant technique may provide an alternative to current methods of prechill bacterial decontamination of poultry carcasses. ASC has been demonstrated to be an effective disinfectant that can be used early in the poultry-processing cycle to decrease microbial contamination. Future studies are needed to establish if utilizing ASC in combination with chlorinated chiller waters would result in greater pathogen reductions. Such a finding would support the USDA Food Safety and Inspection Service proposals for a multiple hurdle approach to pathogen control. REFERENCES 1. Anonymous Secondary direct food additives permitted in food for human consumption acidified sodium chlorite solutions. Code of Federal Regulations, 21CFR Office of the Federal Register, U.S. Government Printing Office, Washington, D.C. 2. Anonymous Testing of health-care antiseptic drug products. Code of Federal Regulations, 21 CFR (a) (1) (iii). Office of the Federal Register, U.S. Government Printing Office, Washington, D.C. 3. Barnes, E. M The effect of chlorinating chill tanks on the bacteriological condition of processed chickens, p Institute Internationale du Froid. Bulletin-Commision 4-Karlsruhe. 4. Billy, T. J Personal communication.

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