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ASSESSMENT OF SODIUM DICHLOROISOCYANURATE IN THE CONTROL OF MICROBIOLOGICAL CROSSCONTAMINATION IN BROILER CARCASS IMMERSION CHILLING SYSTEMS

PAUL WHYTE', JOHN DANIEL COLLINS and KEVINA MC GILL

Food Hygiene Laboratory Department of Large Animal Clinical Studies Faculty of Veterinary Medicine University College Dublin Ballsbridge, Dublin 4, Ireland
AND

CLAIRE MONAHAN

Dawn Farm Foods Ltd. Naas, Co. Kildare, Ireland


Accepted for Publication February 15, 2002

ABSTRACT Commercial immersion chilling systems used in poultry processing are a major source in the cross-contaminationof carcasses with pathogenic microorganisms. In the current study, the eflcacy of a chlorine based bactericidal compound, sodium dichloroisoqanurate (NADCC), to control contamination between carcasses was compared to the more traditional sodium hypochlorite solutions in a simulated carcass immersion chilling system. The addition of either sodium dichloroisocyanurate or sodium hypochlorite to the immersion chill tank water at concentrations of 50 ppm or more significantly reduced total aerobic mesophilic (37C) and psychrophilic (22C) counts when compared to corresponding untreated water samples in which dressed broiler carcasses had been immersed (P < 0.01). Both compounds significantly reduced the numbers of fecal col~orms and thennophilic campytobacters in the immersion water samples compared to corresponding control samples (P < 0.01). In contrast to the control water samples, salmonellae were not detected in immersion water samples treated with either of the chlorine based compounds.

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Journal of Food Safety 22 (2002) 55-65. All Rights Reserved. OCopyright 2002 by Food & Nutrition Press, Inc., Trumbull, Connecticut.

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P. WHYTE, J.D. COLLINS, K. MC GILL and C. MONAHAN

INTRODUCTION
The chilling of poultry carcasses in immersion tanks represents a significant hazard in terms of microbiological cross-contamination (James ef al. 1992; Geornaras et al. 1995; Fries and Graw 1999; Yang et al. 2001). The high throughputs and capacities of commercial immersion chilling systems can result in significant increases in the prevalences of pathogenic organisms such as Salmonella on carcasses exiting chill tanks. James et al. (1992) found that the prevalence of Salmonella-contaminated carcasses increased significantly following immersion chilling in potable water, with 48% and 72% of carcasses positive before and after chilling, respectively. Thornson et al. (1979) reported that 80% of uninoculated broiler carcasses became contaminated with a marker strain of S. typhimurium when unchlorinated water in the immersion tanks became positive following the processing of inoculated carcasses. Oosterom et al. (1983) found that chill tank water became heavily contaminated with C. jejuni at levels of 900 to ,1000 ch/mL; thus identifying its potential for cross-contamination. The continuous addition of chlorine to chill water has been shown to be effective in reducing or eliminating cross-contamination in these systems. James et al. (1992) found that when 25 ppm chlorinated water was used, the prevalence of Salmonella-contaminated carcasses remained almost static, with 43 % of prechill and 46 % of postchill samples reported positive. Thomson et al. (1979) reported a reduction in Salmonella contaminated carcasses from 75 % to 33% between pre- and postchill carcasses when chlorine at 20 ppm was added to the chill water. Furthermore, Mead and Thomas (1973) found that in excess of 90% of total viable counts and coli-aerogenes type bacteria were reduced from carcasses following immersion chilling. The use of elevated chlorine concentrations in chill tanks is thought to destroy microorganisms suspended in the water, thereby preventing or minimizing opportunities for cross-contamination to occur. Yang et al. (2001) reported reductions in C . jejuni and S. typhimurium counts of 3.3 and 0.7 log cfu/mL, respectively in chill water samples containing 10 ppm chlorine. However, at best, bacterial counts are reduced but not eliminated from carcasses during chilling as the crevices and feather follicles on poultry skin may protect microorganisms by making them inaccessible to the decontaminant (Mead and Thomas 1973; Mead et al. 1975; Lillard 1989). Sodium hypochlorite is frequently used in the treatment of raw water supplies and is normally added to water used in both poultry carcass washing and chilling systems. Sodium dichloroisocyanurate is currently used as a water treatment or purification aid for domestic supplies. Both compounds have been used successfully at higher concentrations as sanitizers and disinfectants

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(Griffiths et al. 1999; Aarnisalo et al. 2000; Coates 1996; Bloomfield and Miller 1989). However, to date few studies have been carried out to determine the efficacy of sodium dichloroisocyanurate (NaDCC) as a potential decontaminant within animal slaughter and processing environments. The current study was carried out in order to evaluate and compare the effectiveness of this chlorine based compound to the performance of the more traditionally used sodium hypochlorite, for the control of microbiological cross-contamination in a simulated immersion chilling system for broiler carcasses. In addition differences in the depletion rates of available chlorine between the two compound in the chill water samples was also investigated during simulated processing of the broiler carcasses.
MATERIALS AND METHODS

Broiler carcasses used in this study were obtained in a slaughter facility which processed approximately 60,000 birds per day. Live birds were electrically stunned prior to slaughter and exsanguination. Birds were mechanically defeathered following immersion for 150 s in scald tank water maintained at 52C. All carcasses were air chilled under normal processing conditions and either dispatched as whole fresh carcasses or forwarded for portioning and deboning .

Preparation of Solutions
Briefly, sodium dichloroisocyanurate solutions were prepared at 50 ppm and 100 ppm concentrations. A sodium hypochlorite solution was made up to give a final concentration of 50 ppm. Potable water was used to make up the test and control solutions. These solutions were prepared by filling four similar containers each with 20 L of water from the main supply in the broiler processing plant. One of the containers remained untreated and was designated as the control (Treatment 1). A 50 ppm NaDCC solution was prepared in the second vessel by dissolving a 1.67 g Aquatab tablet (Medentech, Wexford, Ireland) in 20 L of mains plant water (Treatment 2). Another NaDCC solution with a concentration of 100 ppm was prepared by dissolving two of the Aquatab tablets in 20 L water (Treatment 3). The sodium hypochlorite solution was made up to a concentration of 50 ppm in a suitable container by adding 10.43 mL of a 12% (w/v) stock solution to 20 L of the plants main water supply (Treatment 4 ) . All treatment solutions were allowed to stand for 30 min before the temperatures of each solution were recorded. The total chlorine concentration

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P. WHYTE, J.D. COLLINS, K. MC GILL and C. MONAHAN

of each solution in the range 5-50 ppm was determined using a Lovibond Comparator (Tintometer, Salisbury, UK). This was carried out by dissolving a 300 mg tablet of potassium iodide (BDH, Poole, UK) in 10 mL of the water sample. The concentration was then colorirnetrically Compared to a 10 mL sample of untreated water using a suitable disc in the Lovibond apparatus. Total chlorine concentrations in the 0.25-5 ppm range were measured by dissolving a DPD No. 3 tablet (Tintometer, Salisbury, UK) in 10 mL of water and comparing with an equivalent volume of untreated water in the comparator. Solutions containing chlorine concentrations exceeding 50 ppm were measured using free chlorine test strips (Medentech, Wexford, Ireland).
Immersion Treatments

Forty carcasses from within the same flock were recovered from the evisceration line immediately prior to the final carcass wash and chilling. A 150 mL sample of each test solution was obtained before any of the carcasses were immersed. To each treatment solution, 10 carcasses were immersed consecutively for 15 s. The carcasses were gently agitated in the solutions to assist in the removal of loosely bound bacterial cells. Following each sequential carcass immersion, a 150 mL sample of rinse water was recovered from each of the four vessels and placed in sterile sample bottles, The free chlorine in the rinse water samples was deactivated by adding 2.5 mL and 1.25 mL of sterile 3% (w/v) sodium thiosulphate to the 100 ppm and 50 pprn chlorine solutions, respectively. All rinse water samples were transported to the laboratory at S +4C for microbiological analysis.
Laboratory Procedures

Microbiological Analysis. All rinse water solutions were quantitatively analyzed for total viable mesophilic and psychrophilic counts, (37C and 22C), fecal coliform counts and thermophilic Campylobacrer counts. The samples were also examined for the presence of Salmonella spp. Briefly, Total Viable Counts were carried out using a WASP spiral plating device (Don Whitely Scientific, Yorkshire, UK). A volume of 50 pL from each rinse solution was spread on duplicate plates of standard plate count agar for each of the required incubation temperatures. The plates were then incubated at 37C and 22C for 48 h and 72 h, respectively. Fecal coliforms were enumerated by filtering 100 mL, 10 mL and 0.1 mL aliquots of each sample separately through 0.45 pm membrane filters. The filters were placed on membrane lauryl sulphate (Qxoid, Basingstoke, UK) agar plates and incubated for 24 h. The presence of fecal coliforms was confirmed by inoculating 10 mL and 5 mL aliquots of brilliant green bile lactose broth (BGBL) and tryptone water (TW), respectively. Both the BGBL and TW broths

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were incubated at 44C for 24 h. The presence of indole was detected in tryptone water using Kovacs reagent; this together with the formation of gas in BGBL broths was a presumptive reaction for E. coli and confirmed the presence of thermotolerant colifonns. Thermophilic campylobacters were enumerated using a three tube Most Probable Number (MPN) technique. Dilutions of each sample were prepared using 9 mL of 0.1% peptone water. For each sample, 1 mL of lo", lo-', 10' and dilutions were added to tubes containing 10 mL of Preston broth in triplicate. The tubes were incubated microaerophilically at 42C for 48 h. Following incubation, inocula from all tubes were subcultured onto charcoal cefoperazone deoxycolate agar (Oxoid, Basingstoke, UK). The plates were incubated in a microaerophilic atmosphere for 48 h at 42C. Suspected positive isolates were confirmed using Gram stains and standard biotyping techniques (MAST Diagnostics, Merseyside, UK). The number of positive tubes per dilution was recorded and a corresponding MPN referenced from tables as described by Roberts et al. (1995). The detection of Salmonella in rinse water samples was carried out by filtering 100 mL samples through 0.45 pm membrane filters. Each filter paper was then placed in 10 mL of Rappaport-Vassiliaidis (RV) broth and incubated at 42C. The RV tubes were then subcultured onto brilliant green and XLD agars after both 24 h and 48 h incubation at 37C. Suspect colonies were screened using lysine decarboxylase, urea broths and triple sugar iron slopes prior to confirming their identification with Salmnellu antisera. Statistical Analysis All microbial counts obtained in this study were converted to log,,, values prior to subsequent data analysis. Statistical significance of counts in samples a s determined using Analysis of Variance from each treatment group w (ANOVA). Statistical significance w a s defined at the P <0.01 level. Results The mean total aerobic mesophilic counts (loglo) at 37C increased significantly to 3.92 cfu/mL in the untreated control solution compared with a count of less than 1.30 prior to immersion of the carcasses ( P < 0.01). Total mesophilic counts in all of the three test solutions containing either sodium hypochlorite or sodium dichloroisocyanurate were significantly lower ( 5 1.30 cfu/mL) following immersion of the 10 carcasses compared to the corresponding count of 3.92 observed in the untreated control solution (Table 1). Baseline counts of total psychrophilic organisms were found to be higher in the untreated plant water than corresponding mesophilic counts. A moderate increase in counts was observed following immersion of the carcasses in each

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P. WHYTE, J.D. COLLINS, K. MC GILL and C . MONAHAN

of the solutions. These counts were found to increase most significantly in the unchlorinated control sample which increased from I .95 to 4.04 cfu/mL ( P < 0.01) (Table 1).

TABLE 1 . MEAN MESOPHILIC AND PSYCHROTROPHILIC COUNTS IN CONTROL AND CHLORINATED IMMERSION CHILL WATER SAMPLES

Mesophiiic Count (37 C)

Psychrophiiic Count
(22 C )

Carcass no.
0

T,

q2

V3
d.30

9 ,
4.30 4.30 4.30

T,
1.95

9 2

q 3

9 ,
4.30 4.30 1.60 1.70 1.48 1.78 1.48 1.60 1.78

~ 1 . 3 0 ' 4.30

1 2 3 4

4.30
<1.30 1.78

4.30
<1.30

4.30 4.30
4.30

2.05
2.28 2.28 2.65 3.14 3.40 3.50 3.96 3.94

4.30
4.30

2.50
2.93 3.20 3.43
4.04

4.30 ~ 1 . 3 0 c1.30

5 6
7
8

4.30 4.30 4.30


(1.30

4.30
<1.30 c1.30 4.30 (1.30 ~1.30

11.30 4.30
<1.30

1.30 1.60 1.95 2.20 2.08 1.48 1.95


2.08

4.30 4.30 1.65


2.00 1.60

2.00
2.00 1.85 1.85 2.04

4.30
4.30

9 10

3.92 3.92

4.30 4.30

1.60 1.70

4.30

4.04

2.08

1.95

2.04 2.15

'Results expressed as the total number of organisms per ml of rinse water (loglo). "qreatment groups with different superscripts are statistically significant ( p <0.01). Treatment solutions: T, = mains water (control) T2= 50ppm sodium dichloroisocyanurate T3 = lOOppm sodium dichloroisocyanurate T, = 50pprn sodium hypochlorite.

Fecal coliform counts were significantly reduced in immersion water samples containing either of the chlorine based compounds when compared with untreated control samples (P < 0.01). Coliform organisms were not detected in any of the four treatment solutions prior to carcass immersion. However, counts (loglo) increased to 5.27 cfu/mL, in the untreated control water sample after immersion of 10 carcasses compared with 2.66, 1.71 and 1.78 cfu/mL in treatments 2, 3 and 4, respectively. Interestingly, the more concentrated 100 ppm NaDCC solution did not appear to reduce coliform counts more significantly than either of the 50 ppm concentrations (Fig. 1).

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51 PW N 103 ppm N a n

50 ppm Hypochlorile

. 1

. 2

10

Carcass No.

FIG. 1. FECAL COLIFORM COUNTS IN CONTROL AND CHLORINATED IMMERSION CHILL WATER SAMPLES

Campylobacfercounts increased over 4-log fold in the untreated solution following immersion of the carcasses. In contrast, significantly lower counts of 0.84 and 1.32 cfu/mL were recovered in both of the 50 ppm solutions of NaDCC and sodium hypochlorite ( P < 0.01). Campylobacters were not detected in any of the consecutive carcass immersion water samples which contained the higher 100 ppm NaDCC concentration. Salmonella was detected in all of the unchlorinated control water samples subsequent to immersion of the fourth carcass (Table 2). No Salmonella were isolated in any of the chlorinated treatment solutions. The depletion rates of available chlorine following the immersion of broiler carcasses in the treatment solutions was similar for both of the test compounds under investigation. The initial chlorine concentration of 50 ppm in treatments 2 and 4 decreased to 25-30 ppm after ten carcasses which had been immersed in each solution.

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P. WHYTE, J.D. COLLINS, K. MC GILL and C. MONAHAN

TABLE 2. THERMOPHILIC CAMPYLOBACTER COUNTS AND SALMONELLA PREVALENCES IN CONTROL AND CHLORINATED IMMERSION CHILL WATER SAMPLES

'Salmonella Carcass no.


0

1
2 3

'TI ND 2.32
2.69

9 2

9 3

ND

ND

4 5 6 7 8 9 10

3.32 3.30

3.36 3.60 3.85 3.95


4.04 4.04

ND ND ND ND ND ND ND
0.84
0.84 0.48

ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND 0.95 1.18
1.30 1.32 1.32

T, ND ND ND
ND

+ + + + +
t

'Results expressed as the number of coliforms per lOOml of rinse water (loglo). 2Resultsexpressed as the Most Probable Number of campylobacters per ml of rinse water (Iog,J. 3Representsthe recovery of salmoneilae from 1OOml samples of rinse water. *.6Treatment groups with different superscripts are statistically significant (v <0.01). No salmonellae were isolated from samples of treatments 2,3 or 4. 'ND = not detected. + = Salmonella detected. Treatment solutions: T, = mains water (control) T2= 50ppm sodium dichloroisocyanurate T3= IOOgpm sodium dichloroisocyanurate T, = 5Oppm sodium hypochlorite.

DISCUSSION
The current study confirmed that the addition of chlorine at concentrations of 50 ppm or 100 ppm in water used for carcass chilling can significantly reduce the levels of suspended aerobic microorganisms. These findings were in agreement with previous studies where the effect of chlorine on controlling levels of microbial contamination in carcass chillers was investigated (Lillard 1980;White 1996). Interestingly, the chill water decontamination performances of sodium hypochlorite and sodium dichloroisocyanurate were not statistically different when similar concentrations were compared. The effect of chlorinating process water on fecal coliform counts in chiller tank water was highly significant. Levels of thermotolerant coliforms were

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significantly higher in the control group when compared to the other treatment groups 2 (P < 0.01). The bactericidal efficacy of both sodium hypochlorite and sodium dichloroisocyanurate were similar when used at equivalent concentrations. A similar trend was observed for Campylobucfer, with control samples containing significantly higher counts than the 3 corresponding treatment groups (P < 0.01). Campylobacters were not detected in any of the samples containing 100 ppm NaDCC which illustrated that the lethal effects of the compound were directly related to the concentration used in the chill water. These findings clearly demonstrate the benefits of using either of the chlorine compounds to prevent or reduce the occurrence of carcass cross-contamination during immersion chilling. Salmonella spp. was recovered in all of the control group water samples from the immersion of the fourth carcass onwards. However, Salmonella were not detected in any of the chlorine treated immersion water samples. These findings would suggest that both of the chlorine compounds used at appropriate concentrations were sufficient to destroy any suspended salmonellae. Yang et al. (2001) reported that S. typhimurium and C. jejuni were reduced by 3.3 and 0.7 cfu/mL (log,,,) in chill water containing 10 ppm chlorine. Furthermore, the authors failed to detect either of these organisms in chill tank water containing 30 ppm and 50 ppm chlorine concentrations. These findings are consistent with those observed in the current investigation and those reported in previous studies (Lillard 1980, 1989; James et al. 1992). The rate of depletion of chlorine in the chill water samples resulting from the immersion of broiler carcasses was substantial, with 50 ppm NaDCC and 50 ppm sodium hypochlorite solutions reduced to similar levels of 20 ppm and 25 ppm respectively, thirty minutes after dipping ten carcasses in each of the solutions. The reduction in concentrations of these compounds in immersion water systems is a result of their interactions with certain organic compounds, f al. 1979; Lillard 1980). The present in particular, organic nitrogen (Thomson e investigation demonstrates that in a commercial processing plant it would be necessary to continuously monitor chlorine levels of chill water and add chlorine as required in order to maintain an adequate concentration sufficient to destroy freely suspended pathogenic microorganisms. The routine use of elevated concentrations of chlorine in immersion chill tank water has been shown to significantly reduce the occurrence of crosscontamination (Fries et al. 1999; Yang et al. 2001). This simple intervention measure during carcass processing would result in an overall decrease in the prevalence of enteropathogen-contaminated carcasses. Furthermore, it is suggested that the use of sodium dichloroisocyanurate in broiler immersion systems is at least as effective as the more traditionally used sodium hypochlorite in reducing the counts of a range of indicator organisms and pathogens in chill

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water. This processing aid, if applied in commercial chilling systems, could significantly control carcass cross-contamination and lead, ultimately, to microbiologically safer end products. At present the Department of Agriculture and Food in Ireland does not permit the use of chemical decontaminants, including chlorine based compounds, either in carcass washing systems or in immersion chill tanks at the concentrations tested. Sufficient chlorine may be added solely for the purpose of making a factory water supply conform to a potable microbiological standard. A concentration of approximately 0.5-1 .O ppm is usually sufficient to achieve this standard. At these reduced levels, chlorine would be rapidly deactivated and cross-contamination could readily occur within water immersion tanks. The enforcement of this policy undoubtedly results in the presence of higher prevalences of pathogens on immersion-chilled carcasses and raw poultry meat products. Fortunately, the impact of this legislation is minimized as most poultry produced in Ireland is air chilled and retailed as fresh product. However, the use of these compounds in the small number of Irish plants currently using immersion chilling would seem to appear to be an appropriate intervention. This risk reduction measure could effectively be incorporated into plant specific food safety systems, including Hazard Analysis Critical Control Point (HACCP) programs. REFERENCES AARNISALO, K. et al. 2000. Bactericidal efficiencies of commercial disinfectants against Listeria monocyrogenes on surfaces. J. Food Safety 20, 237-250. BLOOMFIELD, S.F. and MILLER, E.A. 1989. A comparison of hypochlorite and phenolic disinfectants for disinfection of clean and soiled surfaces and blood spillages. J. Hosp. Infect. 13, 231-239. COATES, D. 1996. Sporicidal activity of sodium djchloroisocyanurate, peroxygen and gluteraldehyde disinfectants against Bacillus subtilis. J. Hosp. Infect. 32, 283-294. FRIES, R. and GRAW, C. 1999. Water and air in two poultry processing plants chilling facilities - a bacteriological survey. Br. Poult. Sci. 40, 52-58. GEORNARAS, I . , DE JESUS, A., VAN ZYL, E. and VON HOLY, A. 1995. Microbiological survey of a South African poultry processing plant. J. Bas. Microbiol. 35, 73-82. GRIFFITHS, P.A., BABB, J.R. and FRAISE, A.P. 1999. Mycobacterial activity of selected disinfectants using a quantitative suspension test. J. Hosp. Infect. 41, 111-121.

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JAMES, W.O., BREWER, R.L., PRUCHA, J.C., WILLIAMS, W.O. and PARHAM, D.R. 1992. Effects of chlorination of chill water on the bacteriologic profile of raw chicken carcasses and giblets. J. Am. Vet. Med. Assoc. 200, 60-63. LILLARD, H.S. 1980. Effect on broiler carcasses and water of treating chiller water with chlorine or chlorine dioxide. Poult. Sci. 59, 1761-1766. LILLARD, H.S. 1989. Factors affecting the persistence of Salmonella during the processing of poultry. J. Food Prot. 52, 829-832. MEAD, G.C., ADAMS, B.W. and PARRY, R.T. 1975. The effectiveness of in-plant chlorination in poultry processing. Br. Poult. Sci. 16, 517-526. MEAD, G.C. and THOMAS, N.L. 1973. The bacteriological condition of eviscerated chickens processed under controlled conditions in a spin chilling system and sampled by two different methods. Br. Poult. Sci. 14,413-419. OOSTEROM, J., NOTERMANS, S., KARMAN, H. and ENGELS, G.B. 1983. Origin and prevalence of Campylobacterjejuni in poultry processing. J. Food Prot. 46, 339-344. ROBERTS, D., HOOPER, W. and GREENWOOD, M. 1995. Isolation and enrichment of microorganisms. In Practical Food Microbiology, Second Ed., pp. 130-132, 142-143. Public Health Laboratory Service, London. THOMSON, J.E., BAILEY, J.S., COX, N.A., POSEY, D.A. and CARSON, M.O. 1979. Salmonella on broiler carcasses as affected by fresh water input rate and chlorination of chiller water. J. Food Prot. 42, 954-955. WHITE, P.L., BAKER, A.R. and JAMES, W.O. 1996. Strategies to control Salmonella and Campylobacter in raw poultry products. Rev. Sci. Tech. Off. Int. Epizooties 16, 525-541. YANG, H., LI, Y. and JOHNSON, M.G. 2001. Survival and death of Salmonella ryphimun'um and Campylobacterjejuni in processing water and on chicken skin during poultry scalding and chilling. J. Food Prot. 64,770776.