Impact of chilling conditions on chicken carcasses contamination by Campylobacter spp.

Transcription

1 Impact of chilling conditions on chicken carcasses contamination by Campylobacter spp. Rivoal K.*, Poezevara T., Quesne S. and Chemaly M. ANSES Laboratory of Ploufragan/Plouzané, Ploufragan, France. *Corresponding author: Campylobacter is the leading cause of bacterial gastroenteritis in many industrialized countries, with handling and consumption of raw or undercooked chicken meat the main source of infection. Risk assessment studies have indicated that campylobacteriosis associated with consumption of chicken products may be reduced 30 times by a 2 log reduction of Campylobacter concentration on carcasses (Rosenquist et al., 2003). The objective of this work is to define chilling conditions for Campylobacter reduction on poultry carcasses. For this purpose, this study was set up to investigate three major parameters in the chilling process (temperature, duration and air velocity) individually and their interaction on the behavior of Campylobacter using the Doehlert shell design. As our previous studies have shown that carcasses presenting more than 3 log cfu/g of Campylobacter would not be significantly decontaminated during the chilling process (Rivoal et al., 2014), the carcasses were artificially inoculated with 3 log cfu/g. Sixteen tests were performed using a chilling prototype: air velocity (between 1 and 3 m/s), chilling duration (between 1 and 5 hours) and temperature (between 1 and 7 C). After chilling, Campylobacter counts were conducted in accordance with the ISO standard The contamination reduction ranged from 12% to 41% of the initial concentration. Temperature had a significant effect (p=0.0045): the reduction rate decreased when the temperature increased. Interaction between temperature and air velocity had also a significant effect (p=0.007) on Campylobacter contamination. At low air velocity, the effect of temperature is very important. The reduction of the bacterial load decreased greatly when the temperature increased. At high air velocity, the effect of the increase of temperature was mitigated: bacterial reduction decreased but only slightly. The important outcome of this work is that a chilling process with low temperature can significantly reduce the bacterial load on chicken carcasses contaminated with a maximum of 3 log cfu/g. Keywords: Campylobacter; air chilling; temperature. Introduction Campylobacter is the most common cause of bacterial foodborne infection in humans in developed countries (WHO, 2012). According to the European Food Safety Authority report (EFSA, 2015), the notification rate of human campylobacteriosis in 2013 exceeded 64 per 100,000 of population in the European Union. Campylobacteriosis is characterized by mild to severe symptoms, including fever, diarrhea and abdominal pain (Butzler et al., 2004). Occasionally, infection can lead to sequelae such as reactive arthritis, meningitis, pneumonia, miscarriage and the life-threatening disease Guillain-Barré syndrome (Levin, 2007). Raw poultry meat has been identified as a major source of human infection due to crosscontamination in the kitchen to other foods eaten without further cooking, undercooking and probably direct hand-to-mouth transfer during food preparation (Cogan et al., 1999; Redmond et al., 2004). Several risk assessments concluded that reducing Campylobacter concentration on meat may have a

2 considerable impact on the number of human cases associated with broiler meat (Rosenquist et al., 2003; Nauta et al., 2007; Lake et al., 2006). In poultry processing, chilling is a crucial step that can prevent microbial growth to a level that will maximize both product safety and shelf life (Carroll and Alvarado, 2008). Generally, eviscerated carcasses are chilled by air chilling, which is more common in Europe, Brazil and Canada or by an immersion system, which is the most frequently used in the United Stated (Fluckey et al., 2003; Carciofi and Laurindo, 2007; Huezo et al., 2007; Berrang et al., 2008; Carroll and Alvarado, 2008). Lindbald et al. (2006) reported that air chilling can be more effective than immersion chilling to lower the prevalence of Campylobacter on broiler carcasses, suggesting that air chilling can injure or kill Campylobacter by desiccation. Other authors reported no change in Campylobacter numbers on carcasses treated with an air-chill procedure in a commercial processing plant (Fluckey et al., 2003; Huezo et al., 2007). In this study, the impact of different air chilling parameters individually and in interaction were studied using an experimental design built according to Doehlert matrix (Doehlert, 1970). This approach has the advantage of studying the effect of several factors alone and in combination by the means of a reasonable number of tests, with the possibility of programming the variation of all the factors in the same time, for each experiment. The objective of this work was to define air chilling conditions to reduce Campylobacter levels on poultry carcasses. Materials and methods Experimental design An experimental prototype has been designed to reproduce in our laboratory the chilling conditions found in poultry slaughterhouses. To study the impact of the different air chilling parameters individually and in interaction, the experimental design was constructed according to the Doehlert matrix (Doehlert, 1970). Three air chilling parameters were chosen: the temperature, the duration and the air flow. According to the Doelhert matrix, each parameter can vary between three and seven factors: three for the air flow (1 to 3 m/s), five for the chilling duration (1 to 5 hours) and seven for the temperature (1 C to 7 C), leading to run 16 assays by experimental design (Table 1). The levels chosen for each parameter are in accordance with the readings made in poultry slaughterhouses in our area. Microbiological analysis Chicken legs were purchased from neighbourhood supermarkets located around the city of Saint- Brieuc in France. To minimize Campylobacter contamination, chicken legs were frozen during several months. Before inoculation, chicken legs were thawed at 4 C during a night. The Campylobacter jejuni strain (Anses640) used in this study has been isolated from chicken samples and characterized by MLST as ST45. The strain was streaked onto Campylobacter blood-free selective agar plates containing charcoal-cefoperazone-deoxycholate agar (mccda) selective supplement (Oxoid, Dardilly, France) followed by incubation for 48h at 41.5 C in a microaerophilic atmosphere (85% N 2, 10% CO 2 and 5% O 2 ). After 48h of growth, a single colony was chosen and streaked onto mccda plates. After an additional 48h of growth, a single colony was transferred in Brucella broth and incubated for 24 h at 41.5 C in a microaerophilic atmosphere to obtain a bacterial concentration of about 8 log cfu / ml. The broth was diluted and streaked onto mccda for enumeration. 250µl of the 10-4 dilution were used to inoculate 25 cm 2 of the chicken leg skin corresponding to an initial concentration of about 3 log cfu/g. The chicken legs (3 by assay) were hooked in the prototype programmed with a combination of three parameters in accordance with the experimental design (Table 1). After chilling according to the experimental design, the 25 cm 2 of leg skins were cut and weighted. Campylobacter enumerations were conducted in accordance with the ISO standard Statistical analyses All bacterial counts (cfu/g) were transformed to log 10 values. Statistical analyses of the experimental design were performed using the StatGraphics software. The analysis of variance

3 (Anova) was conducted to estimate the significance of the parameters alone and in combination and the Response Surface Methodology to predict the behavior of Campylobacter as a function of the studied parameters. Results and discussion Sixteen trials combining chilling parameters were carried out and the results are shown in Table 1. The percentage of Campylobacter reduction ranged from 12.18% to 40.89% of the initial loads corresponding to a reduction of approximately 0.3 and 1.1 log cfu/g respectively. Table 1 Campylobacter concentration before and after air chilling according to the Doehlert experimental design. Assays Chilling duration (h) Chilling temperature T C Air flow (m/s) Concentration before chilling (log cfu/g) Concentration after chilling (log cfu/g) Reduction (%) The statistical results are shown in Figure 1. Only one parameter, the temperature, had a significant effect on Campylobacter reduction load (p=0.0045). The higher the temperature was, the lower the percentage of reduction was. Thus, Campylobacter reduction was higher when the chilling temperature was low. Moreover, interaction between temperature and air velocity had also a significant effect (p=0.0071) on Campylobacter contamination (Figure 1). At low air velocity, the effect of temperature was very important. The reduction of Campylobacter load decreased greatly when the temperature rose. At high air velocity, the effect of the temperature increase was mitigated: bacterial reduction decreased but only slightly. Conclusion In conclusion, this work showed that a chilling process with low temperature can significantly reduce the Campylobacter load on chicken carcasses presenting a maximum of 3 log cfu/g. Moreover, an interaction between temperature and air flow was significant. This work allowed also to determine the optimal conditions for a significant reduction of bacterial load. These conditions will be tested with other Campylobacter isolates having different STs and different characteristics concerning their virulence in order to cover the behavior of prevalent isolates in the poultry production.

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