Evaluation of Disinfectants Used in Pre-Chilling water Tanks of Poultry Processing Plants

I Department of Veterinary Pathology, Faculty of Agriculture and Veterinary Sciences, Universidade Estadual Paulista, Jaboticabal (FCAV/Unesp), São Paulo, Brazil. Postalcode: 14884-900 II Unit of Research and Development, Instituto Biológico, Bastos, São Paulo, Brazil III Programa de Pós-graduação em Ciência Animal, Areia, Campus II, Paraíba IV Department of Veterinary Sciences, Universidade Federal da Paraíba, Areia, Campus II, Paraíba


INTRODUCTION
During broiler carcass processing, evisceration, carcass wash, prechilling and cooling steps can favor the dissemination of microorganisms and carcass contamination (USDA, 2002).One infected broiler arriving at the processing plant may be the source of contamination of many other carcasses (Lillard, 1989;Rasschaert et al., 2008).Some microorganisms present in the broiler intestine, such as Salmonella spp., Campylobacter spp., and thermotolerant coliforms, represent a threat to public health (Scallan et al., 2011).Carcasses can be also contaminated with spoilage microorganisms that reduce the shelf life of meat products (Silva et al., 2006).
Outbreaks of foodborne diseases in Brazil remain largely uninvestigated, and consequently, few data are available.For instance, from 1999 to 2012, the Brazilian Health Surveillance Division reported 1,525 outbreaks of human foodborne salmonellosis, most of them related to food of poultry origin (Brazil, 2013).Other causes of foodborne diseases (e.g.campylobacteriosis) are rarely diagnosed in Brazil.In 2013, campylobacteriosis represented about 6.5% of all cases of human foodborne diseases in the United States of America (Crim et al., 2014) and contaminated poultry meat was the main source of this pathogen.The Brazilian legislation establishes that Salmonella spp.must be absent in broiler carcasses after processing (Brazil, 2001), but no requirements are established for the presence Campylobacter spp.
Disinfectant compounds are added to the pre-chilling and chilling water tanks with the purpose of minimizing carcass microbial

MATERIAL AND METHODS
Two separate experiments were performed to evaluate the effect of selected of disinfectants on chicken carcass microbial counts: one with meat quality indicator microorganisms, and the second with Salmonella Enteritidis.All assays were performed at 25°C.

Sampling
Seven samplings were performed.In each sampling, 18 broiler carcasses were purchased from processing plants located in the state of São Paulo, Brazil state.Carcasses were taken in isothermal containers to the Avian Pathology laboratory of the Department of Veterinary Pathology, School of Agriculture and Veterinary Sciences (FCAV/UNESP), São Paulo, Brazil.

Treatment of broilers carcasses
Six plastic buckets were filled with 30 L of cold water (14-16°C).Acidified sodium chlorite, alkyl dimethyl benzyl ammonium chloride, chlorine dioxide, peracetic acid, and sodium hypochloriteat the concentrations of50, 175, 5, 50and 5 ppm, respectively, were added to one bucket each.The sixth bucket contained only water (negative control).Three carcasses were immersed in each bucket for 25 minutes in order to simulate the pre-chilling processing step.

Microbiological analyses
Upon removal of the buckets, carcasses were placed in sterile plastic bags containing 400 mL of 0.1% Buffered Peptone Water (BPW) (Difco ™ , Detroit, Michigan, USA) and 400 mL of a mixture of neutralizers, consisting of 64.5 g of Tween 80, 20% sodium bisulfite, 7.84 g of sodium thiosulfate pentahydrate, 5 g of sodium thioglycolate, and 1.5 g of L-cysteine, all diluted in 1,000 mL of tryptone-salt solution (pH 7.2) (Espigares et al., 2003).The neutralized BPW was further processed in order to obtain the most probable number (MPN) of total and fecal coliforms and counts of viable strict and facultative aerobic mesophylls, both according to the methodology described by Downes & Ito (2001).Additionally, the presence of aerobic psychrotrophic microorganisms was determined according to Downes & Ito (2001).

Determination of the Most Probable Number (MPN)
The neutralized BPW resulting from carcass washing was serially diluted, and 1 mL of each dilution inoculated into three inverted Durham culture tubes containing10 mL of Lauryl Tryptose Broth (LTB) (Difco ™ , Detroit, Michigan, US).The tubes were incubated at 37°C for 48h.Cultured tubes presenting turbidity and gas production or effervescence by subtle shaking, were considered presumptively positive.
In order to determine the MPN of total coliforms/ mL, a sterile bacterial loop was plunged into the presumptively LTB-positive culture tubes followed by inoculation in 10 mL Brilliant Green Bile 2% (BGB) (Difco ™ , Detroit, Michigan, USA) also contained an inverted Durham tube, and incubation at 37°C for 48h.When gas production was observed on upper section of the inverted tube, the sample was considered positive.
The MPN of fecal coliforms/mL was surveyed similarly to the total coliforms, although Escherichia coli (EC) medium (Difco ™ , Detroit, Michigan, US) in an inverted Durham tube instead of BGB was used.The tubes were incubated in water bath at 45.5 ± 0.2°C for 48h and the positive samples were identified as described above, according to the methodology of Silva et al. (2007).Finally, the MPN/mL was determined based on Hoskins' table (Hoskins, 1934).

Standard count of viable strict and facultative aerobic mesophylls
This bacterial count was carried out using the pour-plate technique.Neutralized BPW obtained from carcass washing was decimally diluted in a new sterile 0.1% BPW up to 10 -6 .Then, 1 mL of each dilution was poured on disposable sterile bacteriological Petri dishes (90 x 15 mm) containing 15 mL of Plate Count Agar (PCA) (Difco ™ , Detroit, Michigan, USA) at approximately 45°C.Cultures were mixed and after agar solidification, Petri dishes were incubated at 37°C for 48h.Thereafter, colony-forming units per milliliter (CFU/mL) were enumerated.

Standard count of aerobic psychrotrophic microorganisms
A volume of 0.1 mL from the neutralized BPW and its decimal dilutions were inoculated onto PCA medium (Difco ™ , Detroit, Michigan, USA).Colonies were counted according to Downes & Ito (2001) after 10 days of incubation at 7°C.

In-vitro assessment of disinfectant activity on Salmonella Enteritidis
A spontaneous mutant of Salmonella Enteritidis resistant to nalidixic acid and to spectinomycin (SE Nal r Spc r ), stored in the bacterial culture collection of the Avian Pathology laboratory of the Department of Veterinary Pathology of FCAV/UNESP, was used in this trial.This strain was inoculated into 10 mL of Lysogeny broth (LB) (Difco ™ , Detroit, Michigan, US) and incubated at 37°C for16h under constant shaking (150 rpm).After that, the culture was centrifuged at 4,000 rpm for 20 min at 4°C and suspended in 10 mL phosphate buffered saline (PBS, pH 7.2).This wash step was repeated three times.Bacterial counts were performed in bacteriological Petri dishes (90 x 15 mm) containing Lysogeny agar (Difco ™ , Detroit, Michigan, US) with nalidixic acid (100 µg/mL) and spectinomicyn (100 µg/mL) after incubation at 37°C for 24h (Hoskins, 1934).A volume of 0.1 mL SE Nal r Spc r inoculum, containing about 4 x 10 8 colony-forming units (CFU), was added to each disinfectant solution at 14 to 16°C with or without organic matter (carcass wash solution).Each disinfectant was tested in the presence and in the absence of organic matter (carcass wash solution), as described in Table 1.Bacterial colonies were counted 5 and 25 minutes after disinfectant neutralization with 10 mL of the neutralizing solution described above.Colonyforming units (CFU) were converted to log 10 for statistical analyses.

Disinfectants
Treatments performed 5 and 25 minutes afterincubation* **A, B, C, D and E (T1) 9.9 mL of PBS + 0.1 mL of SE Nal r Spc r (T2) 9.9 mL (PBS + disinfectant) + 0.1 mL of SE Nal r Spc r (after each time point, 10 mL of neutralizer were added followed by bacterial counting) (T3) 9.9 mL of organic matter + 0.1 mL of SE Nal r Spc r (T4) 9.9 mL (disinfectant + organic matter) + 0.1 mL of SE Nal r Spc r (after each time point, 10 mL of neutralizer were added followed by bacterial counting) * T1: suspension containing the test microorganism and control PBS, with no organic matter (control or carcass wash solution); T2: suspension containing the test microorganism and the evaluated disinfectant, with no organic matter; T3: suspension containing the test microorganism andorganic matter; T4: suspension containing the test microorganism, the evaluated disinfectant solution, and organic matter.

Statistical analyses
Bacterial count results (CFU and MPN) were transformed in log 10 and means compared by Tukey's test at 5% (p<0.05)significance level.Analyses were performed using Statistical Analysis System (SAS) software version 9.1.In the second experiment, disinfectant substances were considered efficient when the SE Nal r Spc r counts decreased at least to 10 5 CFU/mL, according to the recommendations of the European Committee for Standardization (CEN, 2009).

RESULTS
The results of the effects of the tested disinfectants on the counts of meat quality indicator microorganisms in broiler carcasses are shown in Table 2. None of the disinfectant tested reduced mesophyll counts (p>0.05).Peracetic acid reduced the psychrotrophic population (4.01 CFU/mL) compared with the control solution (4.73 CFU/mL) (p<0.05).The other evaluated disinfectants did not demonstrate any activity on psychrotrophic microorganisms (p<0.05).According to the statistical analysis, acidified sodium chlorite, alkyl dimethyl benzyl ammonium chloride and peracetic acid reduced total coliform counts from 2.47 (untreated control) to 1.67 MPN/mL, 1.74 MPN/ mL, 1.75 MPN/mL and (p<0.05),respectively.These products also reduced the contamination of carcasses with thermotolerant coliforms from 2.32 MPN/mL (control) to 1.49 MPN/mL, 1.53 MPN/mL and 1.61 MPN/mL (p<0.05),respectively.Peracetic acid showed the best results in reducing meat quality indicator microorganisms, whereas sodium hypochlorite and chlorine dioxide did not affect these populations when applied at the concentration allowed by Brazilian legislation (5 ppm).
The in-vitro assessment of the action of the tested compounds on SE Nal r Spc r is presented in Table 3.
When compared with the PBS control group (T1),all tested disinfectants were efficient to reduce SE Nal r Spc r counts in the absence of organic matter(T2) (p<0.05),according to the European Committee for Standardization (CEN, 2009).Under the conditions of the present experiment, no SE Nal r Spc r was detected 5 and 25 minutes after disinfectant addition.

DISCUSSION
Microorganisms are considered good indicators of the hygienic conditions present during food manufacturing process (Hoffmann et al. 2004).For example, high coliform counts indicate post-processing contamination and/or unsuitable sanitization (Kottwitz et al. 2010).Salmonella spp., Escherichia coli and Staphylococcus aureus are examples of foodcontaminating microorganisms that cause foodborne diseases in human beings.These bacteria are frequently found in contaminated chicken meat (Jakobsen et al. 2012;Kottwitz et al. 2010;Silva et al. 2010), and therefore, should be eliminated or, at least, reduced in broiler carcasses.
The addition of disinfectants in pre-chilling tanks is an important tool to minimize the presence of microorganisms in chicken carcasses during processing.The addition of peracetic acid and many other chemical compounds in pre-chilling tanks is allowed in the United States of America (USA, 1977).On the other hand, the Brazilian legislation only allows the use of chlorinebased compounds at a maximum concentration of 5 ppm (Brazil, 1998).Therefore, studies assessing the efficacy of other disinfectants added in prechilling tanks may encourage Brazilian public health authorities to review the current legislation.According to Voidarou et al. (2007), the pre-chilling step itself without any disinfectant has little effect on reducing microorganisms in broiler carcasses.
In the present study, sodium hypochlorite and chlorine dioxide did not show any effect on the   et al. (2007), who did not observe any effect of those compounds in broiler carcass bacterial counts when added to pre-chilling tanks in processing plants.On the other hand, another study reported 1.5% and 70% reduction of coliform and Campylobacter spp.populations, respectively, using sodium hypochlorite and chlorine dioxide (Kameyama et al., 2012).However, in this study, sodium hypochlorite was added at 10 ppm and chlorine dioxide at 50 ppm (USA, 1977), which are higher than the 5 ppm currently allowed in the Brazilian legislation.Bashor et al. (2004) also reported that addition of chlorine at 25 to 35 ppm reduced Campylobacter spp.populations in broiler carcasses in 0.5 log CFU/mL of washing solution.Therefore, it is clear that the current Brazilian legislation regarding the use of sodium hypochlorite in pre-chilling tanks needs to be reviewed.Russell & Axtell (2005) reported that utilization of sodium hypochlorite at 50 ppm in pre-chilling tanks reduced carcass contamination by Salmonella spp.and Pseudomonas fluorencens, but it was not successful in reducing Escherichia coli.In an trial carried out by Mergonsi et al. (2007), the addition of chlorine to a pre-chilling tank, where organic matter was present, did not reduce the number of carcasses contaminated with Salmonella spp.Consistent results were obtained in the present experiment, where no effect of the evaluated disinfectants on Salmonella Enteritidis counts in the presence of organic matter was detected.
The results of the present study showed that peracetic acid at 50 ppm, acidified sodium chlorite at 50 ppm and alkyl dimethyl benzyl ammonium chloride at 175 ppm decreased thermotolerant coliform counts.However, Mead et al. (2000) reported that the addition of chlorine at 50 ppm during the pre-chilling stage did not prevent cross-contamination of carcasses by Escherichia coli.Buhr et al. (2005) also showed that the addition of chlorine at 20 ppm in pre-chilling tanks reduced the counts of coliforms, total aerobes, Escherichia coli and Campylobacter spp. in broiler carcasses, but did the number of carcasses positive for Salmonella spp was not decreased.
In the present study, acidified sodium chlorite decreased the population of coliforms.Oyarzabal et al. (2004) and Sexton et al. (2007), using that disinfectant in pre-chilling tanks also reported a reduction in Escherichia coli and Salmonella spp.counts in broiler carcasses.In contrast, in our study, this compound did not reduce Salmonella Enteritidis counts in the presence of organic matter.It should be noted, however, that Oyarzabal et al. (2004) and Sexton et al. (2007) applied different methods to assess disinfectant action, which could explain the divergent results.Li et al. (2002) evaluated the action of a chorine solution (50 ppm) spray on broiler carcasses and reported positive results in terms of bacterial count reduction.In the present study, acidified sodium chlorite showed better bacterial effect than other tested chlorine-based compounds (sodium hypochlorite and chlorine dioxide).The lower pH and the higher concentration (50 ppm) of sodium chlorite applied may have contributed for this result.
In agreement with the report of Bauermeister et al. (2008), in the present study, peracetic acid was more effective in reducing microbial counts than chlorinebased compounds.The weaker microbicidal effect of chlorine may be explained by the fact that it is easily neutralized by organic compounds (Northcutt & Lacy, 2000).Kich et al. (2004) observed that sodium hypochlorite (1,000 ppm), quaternary ammonium (400 ppm), and peracetic acid (53.33 ppm) were all able to reduce Salmonella Typhimurium in the absence of organic matter.These authors also reported that, in the presence of organic matter, only peracetic acid reduced the counts of this pathogen.In the present study, none of the disinfectant tested, including peracetic acid, reduced Salmonella Enteritidis counts in the presence organic matter.Differences in amounts of organic matter and disinfectant concentrations could explain the differences between our results and those described by Kich et al. (2004).
According to Sidhu et al. (2002), the inefficiency of quaternary ammonium solutions against mesophyll and psychrotrophic present in products of animal origin could be a result of resistance induced by the wide utilization of such substance as disinfectant in animal production facilities.For instance, all Pseudomonas aeruginosa strains isolated of food of animal origin were resistant to that compound (Langsrud & Sundhein, 1997).In the present study, the ineffectiveness of alkyl dimethyl benzyl ammonium chloride in reducing mesophyll and psychrotrophic microorganisms could be also a result of neutralization by organic matter.
Taking into account the antagonist effect of organic matter on disinfectant activity, as demonstrated here and previously by other authors (Kich et al. 2004;Lopes et al. 2007), it is clear that the cleaning of pre-chilling tanks is essential to enhance disinfectant efficiency against contaminating bacteria.Chlorine- based compounds, when applied at the concentration permitted by the Brazilian legislation, did not reduce the counts of meat quality indicator microorganisms nor of Salmonella Enteritidis.In the present study, acidified sodium chlorine, alkyl dimethyl benzyl ammonium chloride, and peracetic acid at 175, 50 and 50 ppm, respectively, were able to reduce total and thermotolerant coliform counts.In terms of microbial control, those three compounds presented better results than sodium hypochloriteat 5 ppm typically applied in Brazilian poultry processing plants.

Table 1 -
Experimental design of experiment 2.

Table 2 -
Average counts (log 10 )of meat quality indicator microorganisms in broiler carcasses washed with different disinfectant compounds.Means followed by the same letter in the same column do not differ by Turkey's test at significance level of 5% (p<0.05). *

Table 3 -
Average Salmonella Enteritidis (SE Nal r Spc r ) counts in the presence and absence of organic matter 5 and 25 minute after disinfectant addition.
SE Nal r Spc r = Salmonella Enteritidis resistant to nalidixic acid and to spectinomycin; *Treatments are detailed in Table1;**For each disinfectant solution, means followed by the same latter in the same column or row are not different by Turkey's test at 5% (p<0.05)significance level.Guastalli

EAL, Lopes PD, Almeida AM, Prette N, Barbosa FO, Stipp DT, Freitas Neto OC Evaluation of Disinfectants Used in Pre-Chilling water Tanks of Poultry Processing Plants evaluated
meat quality indicator microorganisms.These results are consistent with the findings of Lopes