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Survival of Campylobacter spp. in poultry meat preparations subjected to freezing, refrigeration, minor salt concentration, and heat treatment
International Journal of Food Microbiology 137 (2010) 147–153
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Survival of Campylobacter spp. in poultry meat preparations subjected to freezing, refrigeration, minor salt concentration, and heat treatment Imca Sampers a,b,⁎, Ihab Habib c,d, Lieven De Zutter c, Ann Dumoulin b, Mieke Uyttendaele a a
Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium Research Group EnBiChem, Department of Industrial Engineering and Technology, University College of West-Flanders, Graaf Karel de Goedelaan 5, B-8500 Kortrijk, Belgium Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium d Division of Food Hygiene and Control, High Institute of Public Health (HIPH), Alexandria University, 165 El-Horrya Avenue, Alexandria, Egypt b c
a r t i c l e
i n f o
Article history: Received 22 April 2009 Received in revised form 9 November 2009 Accepted 22 November 2009 Keywords: Food Campylobacter spp. Chicken meat preparations Freezing Heat Water activity Salt Cooking Consumer
a b s t r a c t The survival of Campylobacter spp. under defined conditions of freezing (− 22 °C) was studied in naturally contaminated chicken skin and minced chicken meat. A decline of approximately one log10 cfu/g was observed after 1 day of freezing. No further significant reduction was achieved by prolonged storage in the freezer, although a tendency for further gradual reduction of the numbers of Campylobacter spp. present was noted. Campylobacter spp. could still be detected qualitatively (per 0.1 g) after 84 days. In a second part of this study, the survival of Campylobacter spp. in a typical minced meat preparation (minced meat supplemented with 1.5% salt (NaCl)) stored at refrigeration (4 °C) or frozen (− 22 °C) was studied. No significant reduction of the pathogen was observed if the minced chicken meat was kept at 4 °C for 14 days, opposite to approximately one log10 cfu/g reduction after 1 day when the minced meat preparation was stored in the freezer (−22 °C) for 14 days. The latter reduction is imputed to the effect of freezing as mentioned above and not due to the supplementation of NaCl to minced meat or the combination of NaCl and freezing, because similar reductions of Campylobacter spp. were noticed when minced meat (without addition of NaCl) was frozen. Finally, in a third part of the study, the survival of Campylobacter spp. subjected to a heat treatment, conform to consumer-based pan-frying, in inoculated (4.5 ± 0.2 cfu/g) as well as naturally contaminated chicken burgers (2.1 ± 0.1 cfu/g) was studied. The Campylobacter spp. numbers declined after 2 min (internal temperature reached circa 38 °C), where after 4 min (internal temperature reached circa 57.5 °C) they dropped below detectable levels (b 10 cfu/g). © 2009 Elsevier B.V. All rights reserved.
1. Introduction A myth has developed that thermophilic campylobacters are very sensitive to conditions outside the host. It is difficult to reconcile this view with the fact that these bacteria are able to infect approximately 1% of the population of Western Europe largely as a result of the consumption of contaminated food (Humphrey et al., 2007). Although there are many sources of Campylobacter spp., campylobacteriosis is predominantly believed to be associated to poultry meat, also because this food is consumed in large amounts (e.g. Vellinga and Van Loock, 2002, Wingstrand et al. 2006, Humphrey et al., 2007). A new type of fresh chicken meat, increasingly popular in Belgium are chicken meat preparations, such as chicken burgers (Uyttendaele
⁎ Corresponding author. Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure 653, B-9000 Ghent, Belgium. Tel.: +32 9 264 61 77; fax: +32 9 255 55 10. E-mail address: [email protected] (I. Sampers). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.11.013
et al., 2006). These products are stored in the supply chain as well as at the consumers, by refrigeration or freezing in order to control microbial proliferation of spoilage flora (Dooley and Roberts, 2000). However in the cold chain, the campylobacters may survive. In addition, no chemical decontamination or heat treatment is present in the processing line of (minced) chicken meat preparations. In a Belgian survey executed in 2007, a Campylobacter spp. prevalence of 48% was found in chicken meat preparations, while the average Campylobacter spp. concentration was 1.68 ± 0.64 log10 cfu/g (Habib et al., 2008b). Exposure to Campylobacter spp. of these ready-to-cook chicken meat preparations by consumption is to be restricted by thorough heat treatment (usually pan-frying), a responsibility of the food handler, the caterer or of consumers in their private homes (Uyttendaele et al., 2006). C. jejuni has been demonstrated to be extremely susceptible to a wide variety of antimicrobial treatments, food processing methods, and environmental stresses, and in addition to being difficult to culture and to maintain in the laboratory (Solomon and Hoover, 1999; Jasson et al., 2007). There is no doubt that, when challenged in broth culture, campylobacters are more sensitive to heat and acid, than for
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example salmonellas or E. coli (Humphrey et al., 2007 and Jasson et al., 2007). But next to lab data, there is a need of quantitative data on survival of Campylobacter spp. under adverse conditions from naturally contaminated foods; in the present case naturally contaminated chicken meat preparations. In the study of Sampers et al. (2008) freezing showed to have an impact on the prevalence of Campylobacter spp. in chicken meat preparations, but the reduction in Campylobacter spp. concentration in frozen samples containing skin was not significant. It is also known that Campylobacter spp. is sensitive to salt concentrations above 1.5% (ICMSF, 1996; Harrington et al., 2007). Freezing and addition of salt, are currently applied in the processing of Belgian chicken meat preparations (Sampers et al., 2008), as refrigerated or frozen storage is a common food preservation strategy of chicken meat preparations in the logistic chain or at consumer's home. Therefore the objective of this study was to determine the resistance of Campylobacter spp., by preference in naturally contaminated minced chicken meat preparations (or its raw materials) when exposed to freeze and refrigeration temperatures and/or a salt concentration of 1.5%. A heat-inactivation test by means of single-sided pan-frying of chicken burgers with flippings, to imitate the consumer, was set-up to measure the temperature during this particular heat treatment, in relation to colour change of the meat, an indication for the products' readiness for consumption and the survival of Campylobacter spp. present in chicken meat preparations.
2. Materials and methods 2.1. Survival of Campylobacter spp. upon freezing in minced meat and its raw materials Campylobacter spp. survival was determined upon freezing on naturally contaminated chicken samples. Three types of samples were considered: skin, boned upper leg with skin and minced meat (lean muscles, fatty tissues, such as chicken skin). Test portions were weighed of every sample type in a stomacher bag: 10 g skin; 12 g upper leg meat (manually skinned at the laboratory) and 13 g minced meat and put in a freezer (− 22 °C). Per sample type, the initial level of Campylobacter spp. was determined on 3 test portions. After 1, 3, 7, 10, 14, 28, 56 and 84 days, three test portions of every sample type were taken from the freezer (−22 °C) and analysed after they were thawed overnight at 4 °C. Direct enumeration and detection after enrichment of Campylobacter spp. were performed according to the guidelines of the ISO 10272:2006 method (ISO, 2006a,b). A test portion was homogenised (dilution 1:10) with Bolton enrichment broth (CM0983 plus supplement SR183, Oxoid, UK) and testing was carried out in parallel as follows. For skin, the detection at start of experiment was only performed per 1 g until counts (direct plating) dropped below the detection limit (10 cfu/g), then also detection per 10 g and per 0.1 g was conducted. (i) Direct plating (only for skin and minced meat): 1 ml of the initial homogenate (10− 1) was spread plated over four (0.3, 0.3, 0.3, and 0.1 ml) Modified Charcoal Cefoperazon Deoxycholate agar plates (mCCDA) (CM739 plus supplement SR155, Oxoid, UK). For skin also a further serial dilution (10− 2), 0.1 ml was spread plated on mCCDA. Plates were incubated microaerobically (CampyGen®) at 42 °C and enumerated after 48 h. (ii) Enrichment culture: 100 ml homogenate (detection /10 g) and 10 ml (detection /1 g) from the same sample homogenate (transferred to a sterile tube) and a further serial dilution (10− 2, detection /0.1 g) were incubated microaerobically at 42 °C for 48 h, 10 µl of each was subsequently plated onto mCCDA, and the presence of presumptive Campylobacter spp. was checked after 24 h incubation. Microscopy was used for confirmation of presumptive positive Campylobacter spp. results.
The whole experiment was repeated 4 times at independent time intervals. 2.2. Survival of Campylobacter spp. in minced meat supplemented with 1.5% NaCl Campylobacter spp. survival was determined in naturally contaminated minced chicken meat upon addition of 1.5% NaCl. Test portions (10 g) were weighed in stomacher bags after 7.5 g NaCl was added to 500 g minced meat. The initial level of Campylobacter spp., Enterobacteriaceae, Escherichia coli and total aerobic mesophilic plate count were determined on the sampling day. The rest of the test portions were divided and kept at 4 °C and −22 °C for 14 days. After 1, 3, 7 and 14 days of storage at 4 °C and − 22 °C, duplicate samples were analysed. Frozen samples were defrosted overnight at 4 °C. ISO 4833:2003 was used for the enumeration of aerobic mesophilic plate count (ISO, 2003); ISO 21528-2:2004 for the enumeration of Enterobacteriaceae (ISO, 2004); and ISO 10272:2006 for the detection and enumeration of Campylobacter spp. (conforming to the freezing experiment) (ISO, 2006a,b); the enumeration of E. coli was performed by means of a AFNOR validated (n° BRD-07/08-12/04) method (AFNOR, 2004). The salinity of the minced meat sample was determined by titration using the Mohr method (Kirk and Sawyer, 1991). Water activity was measured with a water activity kryometer, NAGY AWK-30 (NAGY Messysteme, Gäufelden, Deutschland). The whole experiment was performed twice and for each experiment the salinity and water activity were measured three times. 2.3. Survival of Campylobacter spp. upon consumer-based frying of chicken burgers The heat resistance of Campylobacter spp. was tested in inoculated and naturally contaminated chicken burgers. 2.3.1. Bacterial strains, growth conditions, and meat inoculation Campylobacter jejuni strain LFMFP 595 (poultry isolate for the collection from the Laboratory of Food Microbiology and Food Preservation (LFMFP) of Ghent University) was used for inoculation. The culture was grown in Bolton Broth at 42 °C to stationary phase. Chicken burgers (oval shaped: 11 cm–8 cm; 1.2 cm thick) were obtained from a local producer. A chicken burger (110 g) was cooled to −5 °C in order to enable to cut it easily lengthwise for inoculation. Next both inner sides of the burger were inoculated by streaking out 1 ml of the C. jejuni working culture to obtain an inoculum of approximately 106 cfu/g. Both sides of inoculated chicken burger were closed again and put in closed stomacher bags for 24 h at 4 °C. Five minutes before frying, the burger was removed from the refrigerator and placed at room temperature. The temperature of the burger was measured before frying. 2.3.2. Frying, sampling and bacterial enumeration The inoculated and naturally contaminated burgers were heated in a frying pan (newly bought in a local retail shop) of TEFAL S.A.S.® with a diameter of 24 cm on a electrical heating plate (SCHOTT instruments®, model: SLK2, 1800 W). A total of 10 g of butter (SOLO®) was heated at the highest heating state (=9) until skim disappeared. One burger per experiment was put in the pan and fried at high heat for 1.5 min (each side 45 s; maximum heat) to sear the meat, followed by frying at the lowest heating state (=1) and flipping the burger every 30 s with the intention of having a well-cooked chicken burger. Cooking times at minimum heating ranged from 0 to 8 min resulting in total cooking time including searing, between 1.5 and 9.5 min. The fried burger was lifted out of the pan with a spatula and placed on a clean paper towel to measure the temperature. Temperature was
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Fig. 1. Effect of freezing on the survival of Campylobacter spp. in naturally contaminated chicken skin (left) (standard deviations varied between 0.1 and 0.8 log10 cfu/g) or minced meat (right) (standard deviations varied between 0.1 and 0.4 log10 cfu/g) in function of incubation time (days) at − 22 °C.
taken after stabilisation (the highest temperature is noted) to get an accurate reading, because the hamburger continued to cook even though it was removed from the heat source. The temperature was measured with a thermocouple and an Agilent® 34970A Data Acquisition/Switch Unit by inserting the sensor into the geometric centre of each burger immediately after it was removed from the frying pan. Immediately after temperature measurement the burger was cut transverse, the internal colour of the burger was controlled and recorded with a digital camera (NIKON coolpix 4100). Next both halves of a burger were immediately sampled for enumeration of surviving Campylobacter spp. cells and put separately in a stomacher bag and homogenised for 1.5 min in peptone water (dilution 1/5). Enumeration of Campylobacter spp. was performed according to the guidelines of the ISO 10272:2006 method (ISO, 2006b) via direct plating as described above. 3. Results 3.1. Reduction by freezing The initial contamination level on skin (mean of 3.2 ± 0.8 log10 cfu/g) was mostly higher than for the minced meat (mean of 2.6 ± 0.5 log10 cfu/g). For the skinned boned upper legs Campylobacter spp. could not be enumerated. After 24 h storage at −22 °C a reduction of approximately one log10 cfu/g (Fig. 1) could be noticed on skin as well as on minced meat. During prolonged storage at −22 °C the number of Campylobacter spp. decreased gradually until below the detection limit (b10 cfu/g), after which only a qualitative detection was performed. The
points in Fig. 1 on the detection limit mean Campylobacter spp. were found in 0.1 g by enrichment, below the detection limit (at 0 log10 cfu/g) means that no Campylobacter spp. were detected in 0.1 g. Thus until below the detection limit, a reduction of approximately 2 log10 cfu/g for skin was found over a period of minimum 10 up to more than 28 days, while for minced meat approximately a 2 log10 cfu/g reduction was found over 10 days. Campylobacter spp. could still be detected per 0.1 g after a storage period of 84 days, as well as for the minced meat and as for the skin. During absence/presence testing Campylobacter spp. was detected per 0.1 g on the skinned, boned upper legs throughout the 84 days. 3.2. Reduction by addition of salt The salt concentration used in Belgian minced chicken meat preparations of approximately 1.5% had no impact on the number of Campylobacter spp. recovered during storage time (Table 1) since the difference in contamination level varied little and is within the range of the measurement uncertainty (± 0.325 log10) of the enumeration methods (spread plating on mCCDA), which was determined by Habib et al. (2008a). Water activity remained at 0.98 throughout the study. No significant reduction of Campylobacter spp. could be noticed at 4 °C. Again, as mentioned above, a reduction of approximately one log10 cfu/g was found when kept for 1 day at −22 °C. During both experiments a high number of E. coli (ca. 3 log10 cfu/g) was present. Freezing or refrigeration had no impact on the numbers of E. coli. The initial contamination level of Enterobacteriaceae was also approximately 3 log10 cfu/g; the mesophilic aerobic count was established as 5 to 6 log10 cfu/g. It is clear that the latter two
Table 1 Effect of 1.5% NaCl-concentration on the survival of Campylobacter spp., total mesophilic aerobic count, Enterobacteriaceae and E. coli in naturally contaminated minced meat of chicken in function of a 14 day incubation at 4 °C and − 22 °C (with standard deviation between 0.01 and 0.1 log10 cfu/g). Campylobacter (log10 cfu/g)
Total count (log10 cfu/g)
Enterobacteriaceae (log10 cfu/g)
E. coli (log10 cfu/g)
− 22 °C
4 °C
− 22 °C
4 °C
− 22 °C
4 °C
− 22 °C
Experiment 1 Aw = 0.9829 [NaCl] = 1.8 +/− 0.1% 0 2.8 1 2.5 3 2.3 7 2.1 14 2.3
2.8 1.2 1.4 1.3 1.0
5.1 5.1 5.9 N 5.0 9.8
5.1 4.8 5.1 4.9 5.1
2.9 3.1 3.6 5.7 7.0
2.9 2.8 2.8 1.6 2.5
3.1 3.0 2.9 3.0 2.6
3.1 2.9 3.0 2.8 2.9
Experiment 2 Aw = 0.9824 [NaCl] = 1.5 +/− 0.1% 0 2.1 1 1.9 3 1.8 7 1.9 14 1.4
2.1 1.1 b1 b1 b1
5.9 6.0 7.2 9.4 10.0
5.9 5.7 5.7 5.5 5.7
2.8 3.2 3.6 5.2 6.5
2.8 2.8 2.5 2.7 2.7
2.9 2.9 2.8 2.9 2.2
2.9 2.8 2.7 2.7 2.7
Incubation (days)
4 °C
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Fig. 2. Evolution of the internal temperature (°C) and colour change in the geometric centre of a chicken burger in function of the duration (s) of frying.
parameters, encompassing psychrotrophic bacteria, reached high numbers after prolonged storage (14 days) at 4 °C which was accompanied with unacceptable sensorial properties.
C. jejuni was observed once the internal temperature was raised above the maximum growth temperature of Campylobacter spp.
4. Discussion 3.3. Heat-inactivation test As shown in Fig. 2, the recorded temperatures of the geometric centre of the meat were very consistent within experiments, with standard deviations between 0.1 and 3.5 °C. After 6.5 min the burger was defined as well-cooked conforming to the internal colour. To verify the adequacy of consumer frying procedures for inactivation of Campylobacter spp. in chicken minced meat preparation, the number of surviving Campylobacter spp. cells (Fig. 3) after different time intervals was determined, this for both artificially inoculated and naturally contaminated chicken burgers. During the first 2 min no inactivation was observed. From this point on, the number of campylobacters recovered from the inoculated as well as the naturally contaminated burgers declined rapidly, and after 4 min of frying they dropped below detectable levels (b10 cfu/g) with core temperatures of 57.5 °C and 52.1 °C respectively. Thus fast killing of
Despite their importance as human pathogens little is known about how campylobacters persist in foods or non-food environments (Humphrey et al., 2007). In this study the behaviour of C. jejuni in naturally contaminated minced chicken meat preparations during storage and preparation as it might be expected to occur in the logistic chain and at the consumer's home was investigated. This study was focused on minced meat preparations because the bacteria are most likely to be uniformly spread throughout the meat by means of meat grinding. The approach selected here uses naturally contaminated chicken products to provide realistic data on the quantitative inactivation of Campylobacter spp. instead of working with inoculated samples with high numbers. It has been shown that there are differences in the behaviour of inoculated bacteria and surrogate bacteria compared to naturally found bacteria in foods and on chicken carcasses (Luber et al., 2006).
Fig. 3. Reduction of the Campylobacter concentration in a chicken burger in function of duration (s) of frying and temperature (°C). (Left: inoculated burgers; right: naturally contaminated burgers).
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Survival, inactivation of Campylobacter spp. in frozen conditions was studied for skin; skinned, deboned upper leg and minced meat to deduce the effect of any protection due to higher fat from skin on survival. Some processing plants in Belgium either freeze the skin or the whole upper legs prior to production of the meat preparations (Sampers et al., 2008). Quantitative analysis indicated that one log10 cfu/g reduction can be achieved after 1 day storage at a freezing temperature of −22 °C, from then on only a gradual reduction is achieved. No differences were obtained between naturally contaminated skin; boned, skinned upper leg and minced meat. So Campylobacter spp. are sensitive to freeze treatment and our results indicate that a long-term holding at − 22 °C can reduce the initial numbers of Campylobacter spp. below the detection limit of 10 cfu/g. However, after 84 days, Campylobacter spp. positive samples were still detected in presence/absence testing per 0.1 g. This confirms that Campylobacter spp. may persist for prolonged periods in chilled and frozen products, although reduction in the concentration and a decline in the viability were also observed during storage (Hänninen, 1981; Oosterom et al., 1983; Yogasundram and Shane, 1986; Curtis et al., 1995; Lee et al., 1998). Ritz et al. (2007) investigated the effect of freezing at − 20 °C on survival of Campylobacter spp., on the skin, below the skin and on the muscle. This study found 0.9–3.2 log decline in 2 weeks. Further freezing yielded no additional effect, in agreement with other studies (Lee et al., 1998; Zhao et al., 2003; Bhaduri and Cottrell, 2004). Most of these studies worked with inoculated samples. Data of naturally contaminated chicken meat preparations are lacking, except for a study of Georgsson et al. (2006) who worked with naturally contaminated poultry carcasses. They reported to still have positive samples after 220 days at − 20 °C. They also showed that the temperature of thawing did not have much influence on the recovery of Campylobacter spp. In our former study (Sampers et al., 2008) freezing showed to have an impact on the prevalence of Campylobacter spp. in chicken meat preparations, but the reduction in Campylobacter spp. concentration in samples containing frozen skin was not significant. Bhaduri and Cottrell (2004) also perceived that chicken skin may provide a protective microenvironment for C. jejuni. However this could not be confirmed in the present study. At present freezing of poultry meat or minced meat preparations is frequently applied in housekeeping but also in the smaller production companies for logistic reasons (Sampers et al., 2008) and not as a food processing step leading to control microbial contamination. Nevertheless freezing of chicken meat preparations (or its raw materials) could serve as an adequate product specific preventive measure that specifically reduces risk of exposing the consumer to (high) Campylobacter spp. contaminated chicken meat preparations. It is generally acknowledged by various risk assessments, the highest numbers lead to highest risks. These risk assessments all conclude that the most effective intervention measures aim at reducing the Campylobacter spp. concentration, rather than reducing the prevalence (Nauta et al., 2008). Freezing as a control measure to reduce campylobacteriosis is already suggested or introduced in countries as Sweden and Norway (Hofshagen and Kruse, 2005; Lindqvist and Lindblad, 2008), to divert Campylobacter-positive flocks to freezing. Former studies (e.g. Iceland study of Stern et al., 2003) already showed a decrease of reported cases by freezing Campylobacter spp. positive carcasses. Although during freezing Campylobacter spp. may be killed, a fraction of the C. jejuni population may survive or be sub-lethally injured (Georgsson et al., 2006 and Jasson et al., 2007). They may pose a residual risk towards the consumer. The sodium chloride concentrations usually present in chicken meat preparations (approximately 1.5%) had no influence on the survival of Campylobacter spp. on naturally contaminated minced meat, which is distinct from the findings by Harrington et al. (2007),
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who noticed increased log reductions at 1.5% NaCl, and especially 2.5% NaCl, for which reductions of at least 4 log10 units were observed using pure cultures in broth. In contrast Jasson et al. (2007) determined that 85.7% of the Campylobacter spp. population were sub-lethally damaged after 10 days exposure at 3.5% NaCl. A water activity of 0.98 was measured in minced meat, so not low enough to affect the numbers of Campylobacter spp. present. Higher NaCl concentrations were not tested as they are also not used, for sensory reasons, in poultry processing industry involved in making poultry meat preparations. Thus, freezing or chilling of minced chicken meat and addition of (minor) salt concentration alone will not contribute to a 3 to 6 log of pathogen reduction (ACMSF, 1997) proposed for enteric pathogens and cannot replace sanitary production and handling (Lee et al., 1998) to limit the pathogen's concentration in the food chain or are not a substitute for safe handling and proper cooking of poultry by the consumer. Chicken meat preparations delivered to the retail and thus as such available to the consumers, are proven to be quite highly contaminated with C. jejuni (high prevalence and occasionally high numbers) (Habib et al., 2008b). Due to insufficient rigorous or difficulties in elaboration or implementation of food safety management systems by food business operators, food that has been produced under HACCP or hygiene codes may still be unsafe (Humphrey et al., 2007; Sampers et al., 2008). So while considering a reliable risk assessment approach, it is important to stress on the importance of consumer phase in integration with production and processing phases. In relation to chicken, food borne exposure to Campylobacter spp. may take place through consumption of undercooked, internally contaminated chicken meat (such as in the case for minced chicken meat preparations) or via cross-contamination to ready-to-eat foods or hands during preparation of raw chicken (Lindqvist and Lindblad, 2008). It is generally assumed that campylobacters will not survive pasteurisation treatments or proper cooking (Nauta et al., 2008; Humphrey et al., 2007; Uyttendaele et al., 2006). Some literature indicate that the probability of cross-contamination for Campylobacter spp. via chicken meat is higher than the probability of undercooking, because the highest number of Campylobacter spp. are found on the surface of the meat, whereas internally, where the temperature is lower, the prevalence is also lower (Luber et al., 2006). This is not generally accepted. For example Bergsma et al. (2007) value the risk for infection by means of a not well performed heat treatment higher than commonly adopted. Moreover for minced meat preparations the bacteria are uniformly spread throughout the meat by means of meat grinding. Low thermal resistance of Campylobacter spp. was found during the “burger pan-frying experiment” in the present study. C. jejuni and C. coli are rather sensitive to heat and may be expressed in a D-value, but this value differs within different studies (Gill and Harris, 1982; ICMSF, 1996; Nguyen et al., 2006; Whyte et al., 2006 and Bergsma et al., 2007). Gill and Harris (1982) also noticed a high variability between strains. The thermal resistance will also depend upon the environmental conditions (food sample and its composition). With the exception of Whyte et al. (2006), who worked on naturally contaminated chicken liver; most studies established heat resistance of Campylobacter spp. with inoculated chicken samples or agar media. Majority of studies also used a boiling water immersion procedure as heat treatment. Here, the focus and set-up of the heat treatment referred to minced chicken meat preparations and on the question “how would the consumer fry his burger?”. Although single-sided cooking, used in this study, requires relatively simple cooking equipment, the flipping interval affects the cooking time. Ou and Mittal (2006) indicated that reduced cooking time is needed for higher turning-over frequency. Furthermore, single-sided pan-frying involves the uncertainty of the slowest heating spot.
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In the present study, Campylobacter spp. was already below detection limit (b10/g), before the burger was described as cookedthrough (as determined by colour change). But the consumer has to be aware that the frying time will be higher for minced meat preparations with an increased thickness. The time needed to kill the Campylobacter spp. was in this experimental set-up dependent on temperature gradient and not on the concentration present. When temperatures reached above 50 °C (core temperature), Campylobacter spp. numbers were below the detection limit (b10 cfu/g). There was no difference between inoculated and naturally contaminated meat preparations. So the consumer information given in a newsletter by the Belgian Federal Agency for the Safety of the Food Chain (FASFC, 2005) to fry their chicken meat until a internal temperature of more than 70 °C is reached, seems from the present study sufficient to be sure Campylobacter spp. are eliminated. Packages intended for supply to the final consumer containing minced meat from poultry or solipeds or meat preparations containing mechanically separated meat must bear a notice indicating that such products should be cooked before consumption (Regulation (EC) 853/2004) to make the consumer aware. However evaluation of the adequate cooking of a meat preparation based on minced poultry meat may be a problem for the consumers due to the small visual difference in colour changes between a semi- and well-cooked product in comparison to the colour changes observed during the cooking of minced beef products. Inadequate cooking of poultry may increase the risk of infections as seen in the study of Istre et al. (1984), where illness was associated with undercooked barbecued chicken meat. Worthwhile noting is that frequently during the Campylobacter spp. detection procedure a positive result could be noticed for presence/absence testing per 0.1 g, while detection per 1 and 10 g provided negative results; this was probably due to the presence of high numbers of competing ESBL E. coli (Jasson et al., 2009). 5. Conclusion Campylobacter spp. in chicken meat preparations (or their raw materials) survived during refrigerated and frozen storage or supplementation of 1.5% NaCl. Freezing could serve as an adequate product specific preventive measure that specifically reduces the chance of the (high initial) contamination of the production process, but cannot replace sanitary production and handling. Poultry contaminated with Campylobacter spp. may lead to infection if not properly handled and sufficiently cooked. Therefore, safe frying times of minced chicken meat burgers in butter were estimated, as this was one of the predominant preparation methods for this type of meat in Belgium. The heat treatment showed the difficulty to recognise a wellcooked burger. But microbiological testing in parallel showed that Campylobacter spp. were inactivated before the fried burgers looked well done. The data provided can be used in exposure assessments of Campylobacter spp. in poultry products in terms of providing both quantitative data on survival and assessing pan-frying and its ability to inactivate campylobacters. Acknowledgements This work was supported by a project fund from the Belgian Federal Public Services (FOD), Health, Food Chain Safety and Environment. We thank the local company for their cooperation and commitment. Josefien Gousseau, Ruben Froyman and Ruben Debouvere are acknowledged for their professional technical assistance in performing Campylobacter spp. analysis and helping out with experimental set-up. We also thank Leentje Braeckman and Dieter Iemants of the department of Biosystems Engineering at Ghent University for the Agilent® 34970A Data Acquisition/Switch Unit and their assistance.
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Baseline data from a belgium-wide survey of Campylobacter species contamination in chicken meat preparations and considerations for a reliable monitoring program. Appl. Environ. Microbiol. 74, 5483–5489. Hänninen, M.K., 1981. Survival of Campylobacter jejuni/coli in ground refrigerated and in ground frozen beef liver and in frozen broiler carcasses. Acta Vet. Scand. 22, 566–577. Harrington, C.S., Mordhorst, H., Gravesen, A., KnØchel, S., 2007. Survival characteristics of Campylobacter jejuni strains under different food processing stresses at chill temperatures. Zoonoses and Public Health, vol. 54 Issue s1. Congress Campylobacter, Helicobacter & related organisms, Rotterdam, the Netherlands, pp. 128–129. Hofshagen, M., Kruse, H., 2005. Reduction in flock prevalence of Campylobacter spp, in broilers in Norway after implementation of an action plan. J. Food Prot. 68, 2220–2223. Humphrey, T., O'Brien, S., Madsen, M., 2007. 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Survival and growth of Campylobacter jejuni after artificial inoculation onto chicken skin as a function of temperature and packaging conditions. J. Food Prot. 61 (6), 1609–1614. Lindqvist, R., Lindblad, M., 2008. Quantitative risk assessment of thermophilic Campylobacter spp. and cross-contamination during handling of raw broiler chickens evaluating strategies at the producer level to reduce human campylobacteriosis in Sweden. Int. J. Food Microbiol. 121, 41–52. Luber, P., Brynestad, S., Topsch, D., Scherer, K., Bartelt, E., 2006. Quantification of Campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens. Appl. Environ. Microb. 72 (1), 66–70. Nauta, M., Hill, A., Rosenquist, H., Brynestad, S., Fetsch, A., van der Logt, P., Fazil, A., Christensen, B., Katsma, E., Borck, B., Havelaar, A., 2008. A comparison of risk assessments on Campylobacter in broiler meat. Int. J. Food Microbiol. 129 (2), 107–123.
I. Sampers et al. / International Journal of Food Microbiology 137 (2010) 147–153 Nguyen, H.T.T., Corry, J.E.L., Miles, C.A., 2006. Heat resistance and mechanism of heat inactivation in thermophilic campylobacters. Appl. Environ. Microb. 72 (1), 908–913. Oosterom, J., de Wilde, G.J.A., de Boer, E., de Blaauw, L.H., Karman, H., 1983. Survival of Campylobacter jejuni during poultry processing and pig slaughtering. J. Food Prot. 46, 702–706. Ou, D., Mittal, G.S., 2006. Single-sided pan frying of frozen hamburgers with flippings for microbial safety using modelling and simulation. J. Food Eng. 80, 33–45. Regulation (EC) No 853/2004 of the European parliament and of the council of 29 April 2004 laying down specific hygiene rules for food of animal origin, annex II, section V, chapter IV. Ritz, M., Nauta, M.J., Teunis, P.F., van Leusden, F., Federighi, M., Havelaar, A.H., 2007. Modelling of Campylobacter survival in frozen chicken meat. J. Appl. Microbiol. 103, 594–600. Sampers, I., Habib, I., Berkvens, D., Dumoulin, A., De Zutter, L., Uyttendaele, M., 2008. Processing practices contributing to Campylobacter contamination in Belgian chicken meat preparations. Int. J. Food. Microbiol. 128, 297–303. Solomon, E.B., Hoover, D.G., 1999. Campylobacter jejuni: a bacterial paradox. J. Food Safety 19 (2), 121–136. Stern, N.J., Hiett, K.L., Alfredsson, G.A., Kristinsson, K.G., Reiersen, J., Hardardottir, H., Briem, H., Gunnarsson, E., Georgsson, F., Lowman, R., Berndtson, E., Lammerding, A.M., Paoli,
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G.M., Musgrove, M.T., 2003. Campylobacter spp. in Icelandic poultry operations and human disease. Epidemiol. Infect. 130, 23–32. Uyttendaele, M., Baert, K., Ghafir, Y., Daube, G., De Zutter, L., Herman, L., Dierick, K., Pierard, D., Dubois, J.J., Horion, B., Debevere, J., 2006. Quantitative risk assessment of Campylobacter spp. in poultry based meat preparations as one of the factors to support the development of risk-based microbiological criteria in Belgium. Int. J. Food Microbiol. 111, 149–163. Vellinga, A., Van Loock, F., 2002. The dioxin crisis as experiment to determine poultryrelated Campylobacter enteritis. J. Infec. Dis. 8, 19–22. Wingstrand, A., Neimann, J., Engberg, J., Nielsen, E.M., Gerner-Smidt, P., Wegener, H.C., Molbak, K., 2006. Fresh chicken as main risk factor for campylobacteriosis, Denmark. Emerg. Infect. Dis. 12, 280–285. Whyte, R., Hudson, J.A., Graham, C., 2006. Campylobacter in chicken livers and their destruction by pan frying. Lett. Appl. Microbiol. 43 (6), 591–595. Yogasundram, K., Shane, S.M., 1986. The viability of Campylobacter jejuni on refrigerated chicken drumsticks. Vet. Res. Commun. 10, 479–486. Zhao, T., Ezeike, G., Doyle, M., Hung, Y., Howell, R., 2003. Reduction of Campylobacter jejuni on poultry by low-temperature treatment. J. Food Prot. 66, 652–655.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Survival of Campylobacter spp. in poultry meat preparations subjected to freezing, refrigeration, minor salt concentration, and heat treatment Imca Sampers a,b,⁎, Ihab Habib c,d, Lieven De Zutter c, Ann Dumoulin b, Mieke Uyttendaele a a
Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium Research Group EnBiChem, Department of Industrial Engineering and Technology, University College of West-Flanders, Graaf Karel de Goedelaan 5, B-8500 Kortrijk, Belgium Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium d Division of Food Hygiene and Control, High Institute of Public Health (HIPH), Alexandria University, 165 El-Horrya Avenue, Alexandria, Egypt b c
a r t i c l e
i n f o
Article history: Received 22 April 2009 Received in revised form 9 November 2009 Accepted 22 November 2009 Keywords: Food Campylobacter spp. Chicken meat preparations Freezing Heat Water activity Salt Cooking Consumer
a b s t r a c t The survival of Campylobacter spp. under defined conditions of freezing (− 22 °C) was studied in naturally contaminated chicken skin and minced chicken meat. A decline of approximately one log10 cfu/g was observed after 1 day of freezing. No further significant reduction was achieved by prolonged storage in the freezer, although a tendency for further gradual reduction of the numbers of Campylobacter spp. present was noted. Campylobacter spp. could still be detected qualitatively (per 0.1 g) after 84 days. In a second part of this study, the survival of Campylobacter spp. in a typical minced meat preparation (minced meat supplemented with 1.5% salt (NaCl)) stored at refrigeration (4 °C) or frozen (− 22 °C) was studied. No significant reduction of the pathogen was observed if the minced chicken meat was kept at 4 °C for 14 days, opposite to approximately one log10 cfu/g reduction after 1 day when the minced meat preparation was stored in the freezer (−22 °C) for 14 days. The latter reduction is imputed to the effect of freezing as mentioned above and not due to the supplementation of NaCl to minced meat or the combination of NaCl and freezing, because similar reductions of Campylobacter spp. were noticed when minced meat (without addition of NaCl) was frozen. Finally, in a third part of the study, the survival of Campylobacter spp. subjected to a heat treatment, conform to consumer-based pan-frying, in inoculated (4.5 ± 0.2 cfu/g) as well as naturally contaminated chicken burgers (2.1 ± 0.1 cfu/g) was studied. The Campylobacter spp. numbers declined after 2 min (internal temperature reached circa 38 °C), where after 4 min (internal temperature reached circa 57.5 °C) they dropped below detectable levels (b 10 cfu/g). © 2009 Elsevier B.V. All rights reserved.
1. Introduction A myth has developed that thermophilic campylobacters are very sensitive to conditions outside the host. It is difficult to reconcile this view with the fact that these bacteria are able to infect approximately 1% of the population of Western Europe largely as a result of the consumption of contaminated food (Humphrey et al., 2007). Although there are many sources of Campylobacter spp., campylobacteriosis is predominantly believed to be associated to poultry meat, also because this food is consumed in large amounts (e.g. Vellinga and Van Loock, 2002, Wingstrand et al. 2006, Humphrey et al., 2007). A new type of fresh chicken meat, increasingly popular in Belgium are chicken meat preparations, such as chicken burgers (Uyttendaele
⁎ Corresponding author. Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure 653, B-9000 Ghent, Belgium. Tel.: +32 9 264 61 77; fax: +32 9 255 55 10. E-mail address: [email protected] (I. Sampers). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.11.013
et al., 2006). These products are stored in the supply chain as well as at the consumers, by refrigeration or freezing in order to control microbial proliferation of spoilage flora (Dooley and Roberts, 2000). However in the cold chain, the campylobacters may survive. In addition, no chemical decontamination or heat treatment is present in the processing line of (minced) chicken meat preparations. In a Belgian survey executed in 2007, a Campylobacter spp. prevalence of 48% was found in chicken meat preparations, while the average Campylobacter spp. concentration was 1.68 ± 0.64 log10 cfu/g (Habib et al., 2008b). Exposure to Campylobacter spp. of these ready-to-cook chicken meat preparations by consumption is to be restricted by thorough heat treatment (usually pan-frying), a responsibility of the food handler, the caterer or of consumers in their private homes (Uyttendaele et al., 2006). C. jejuni has been demonstrated to be extremely susceptible to a wide variety of antimicrobial treatments, food processing methods, and environmental stresses, and in addition to being difficult to culture and to maintain in the laboratory (Solomon and Hoover, 1999; Jasson et al., 2007). There is no doubt that, when challenged in broth culture, campylobacters are more sensitive to heat and acid, than for
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example salmonellas or E. coli (Humphrey et al., 2007 and Jasson et al., 2007). But next to lab data, there is a need of quantitative data on survival of Campylobacter spp. under adverse conditions from naturally contaminated foods; in the present case naturally contaminated chicken meat preparations. In the study of Sampers et al. (2008) freezing showed to have an impact on the prevalence of Campylobacter spp. in chicken meat preparations, but the reduction in Campylobacter spp. concentration in frozen samples containing skin was not significant. It is also known that Campylobacter spp. is sensitive to salt concentrations above 1.5% (ICMSF, 1996; Harrington et al., 2007). Freezing and addition of salt, are currently applied in the processing of Belgian chicken meat preparations (Sampers et al., 2008), as refrigerated or frozen storage is a common food preservation strategy of chicken meat preparations in the logistic chain or at consumer's home. Therefore the objective of this study was to determine the resistance of Campylobacter spp., by preference in naturally contaminated minced chicken meat preparations (or its raw materials) when exposed to freeze and refrigeration temperatures and/or a salt concentration of 1.5%. A heat-inactivation test by means of single-sided pan-frying of chicken burgers with flippings, to imitate the consumer, was set-up to measure the temperature during this particular heat treatment, in relation to colour change of the meat, an indication for the products' readiness for consumption and the survival of Campylobacter spp. present in chicken meat preparations.
2. Materials and methods 2.1. Survival of Campylobacter spp. upon freezing in minced meat and its raw materials Campylobacter spp. survival was determined upon freezing on naturally contaminated chicken samples. Three types of samples were considered: skin, boned upper leg with skin and minced meat (lean muscles, fatty tissues, such as chicken skin). Test portions were weighed of every sample type in a stomacher bag: 10 g skin; 12 g upper leg meat (manually skinned at the laboratory) and 13 g minced meat and put in a freezer (− 22 °C). Per sample type, the initial level of Campylobacter spp. was determined on 3 test portions. After 1, 3, 7, 10, 14, 28, 56 and 84 days, three test portions of every sample type were taken from the freezer (−22 °C) and analysed after they were thawed overnight at 4 °C. Direct enumeration and detection after enrichment of Campylobacter spp. were performed according to the guidelines of the ISO 10272:2006 method (ISO, 2006a,b). A test portion was homogenised (dilution 1:10) with Bolton enrichment broth (CM0983 plus supplement SR183, Oxoid, UK) and testing was carried out in parallel as follows. For skin, the detection at start of experiment was only performed per 1 g until counts (direct plating) dropped below the detection limit (10 cfu/g), then also detection per 10 g and per 0.1 g was conducted. (i) Direct plating (only for skin and minced meat): 1 ml of the initial homogenate (10− 1) was spread plated over four (0.3, 0.3, 0.3, and 0.1 ml) Modified Charcoal Cefoperazon Deoxycholate agar plates (mCCDA) (CM739 plus supplement SR155, Oxoid, UK). For skin also a further serial dilution (10− 2), 0.1 ml was spread plated on mCCDA. Plates were incubated microaerobically (CampyGen®) at 42 °C and enumerated after 48 h. (ii) Enrichment culture: 100 ml homogenate (detection /10 g) and 10 ml (detection /1 g) from the same sample homogenate (transferred to a sterile tube) and a further serial dilution (10− 2, detection /0.1 g) were incubated microaerobically at 42 °C for 48 h, 10 µl of each was subsequently plated onto mCCDA, and the presence of presumptive Campylobacter spp. was checked after 24 h incubation. Microscopy was used for confirmation of presumptive positive Campylobacter spp. results.
The whole experiment was repeated 4 times at independent time intervals. 2.2. Survival of Campylobacter spp. in minced meat supplemented with 1.5% NaCl Campylobacter spp. survival was determined in naturally contaminated minced chicken meat upon addition of 1.5% NaCl. Test portions (10 g) were weighed in stomacher bags after 7.5 g NaCl was added to 500 g minced meat. The initial level of Campylobacter spp., Enterobacteriaceae, Escherichia coli and total aerobic mesophilic plate count were determined on the sampling day. The rest of the test portions were divided and kept at 4 °C and −22 °C for 14 days. After 1, 3, 7 and 14 days of storage at 4 °C and − 22 °C, duplicate samples were analysed. Frozen samples were defrosted overnight at 4 °C. ISO 4833:2003 was used for the enumeration of aerobic mesophilic plate count (ISO, 2003); ISO 21528-2:2004 for the enumeration of Enterobacteriaceae (ISO, 2004); and ISO 10272:2006 for the detection and enumeration of Campylobacter spp. (conforming to the freezing experiment) (ISO, 2006a,b); the enumeration of E. coli was performed by means of a AFNOR validated (n° BRD-07/08-12/04) method (AFNOR, 2004). The salinity of the minced meat sample was determined by titration using the Mohr method (Kirk and Sawyer, 1991). Water activity was measured with a water activity kryometer, NAGY AWK-30 (NAGY Messysteme, Gäufelden, Deutschland). The whole experiment was performed twice and for each experiment the salinity and water activity were measured three times. 2.3. Survival of Campylobacter spp. upon consumer-based frying of chicken burgers The heat resistance of Campylobacter spp. was tested in inoculated and naturally contaminated chicken burgers. 2.3.1. Bacterial strains, growth conditions, and meat inoculation Campylobacter jejuni strain LFMFP 595 (poultry isolate for the collection from the Laboratory of Food Microbiology and Food Preservation (LFMFP) of Ghent University) was used for inoculation. The culture was grown in Bolton Broth at 42 °C to stationary phase. Chicken burgers (oval shaped: 11 cm–8 cm; 1.2 cm thick) were obtained from a local producer. A chicken burger (110 g) was cooled to −5 °C in order to enable to cut it easily lengthwise for inoculation. Next both inner sides of the burger were inoculated by streaking out 1 ml of the C. jejuni working culture to obtain an inoculum of approximately 106 cfu/g. Both sides of inoculated chicken burger were closed again and put in closed stomacher bags for 24 h at 4 °C. Five minutes before frying, the burger was removed from the refrigerator and placed at room temperature. The temperature of the burger was measured before frying. 2.3.2. Frying, sampling and bacterial enumeration The inoculated and naturally contaminated burgers were heated in a frying pan (newly bought in a local retail shop) of TEFAL S.A.S.® with a diameter of 24 cm on a electrical heating plate (SCHOTT instruments®, model: SLK2, 1800 W). A total of 10 g of butter (SOLO®) was heated at the highest heating state (=9) until skim disappeared. One burger per experiment was put in the pan and fried at high heat for 1.5 min (each side 45 s; maximum heat) to sear the meat, followed by frying at the lowest heating state (=1) and flipping the burger every 30 s with the intention of having a well-cooked chicken burger. Cooking times at minimum heating ranged from 0 to 8 min resulting in total cooking time including searing, between 1.5 and 9.5 min. The fried burger was lifted out of the pan with a spatula and placed on a clean paper towel to measure the temperature. Temperature was
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Fig. 1. Effect of freezing on the survival of Campylobacter spp. in naturally contaminated chicken skin (left) (standard deviations varied between 0.1 and 0.8 log10 cfu/g) or minced meat (right) (standard deviations varied between 0.1 and 0.4 log10 cfu/g) in function of incubation time (days) at − 22 °C.
taken after stabilisation (the highest temperature is noted) to get an accurate reading, because the hamburger continued to cook even though it was removed from the heat source. The temperature was measured with a thermocouple and an Agilent® 34970A Data Acquisition/Switch Unit by inserting the sensor into the geometric centre of each burger immediately after it was removed from the frying pan. Immediately after temperature measurement the burger was cut transverse, the internal colour of the burger was controlled and recorded with a digital camera (NIKON coolpix 4100). Next both halves of a burger were immediately sampled for enumeration of surviving Campylobacter spp. cells and put separately in a stomacher bag and homogenised for 1.5 min in peptone water (dilution 1/5). Enumeration of Campylobacter spp. was performed according to the guidelines of the ISO 10272:2006 method (ISO, 2006b) via direct plating as described above. 3. Results 3.1. Reduction by freezing The initial contamination level on skin (mean of 3.2 ± 0.8 log10 cfu/g) was mostly higher than for the minced meat (mean of 2.6 ± 0.5 log10 cfu/g). For the skinned boned upper legs Campylobacter spp. could not be enumerated. After 24 h storage at −22 °C a reduction of approximately one log10 cfu/g (Fig. 1) could be noticed on skin as well as on minced meat. During prolonged storage at −22 °C the number of Campylobacter spp. decreased gradually until below the detection limit (b10 cfu/g), after which only a qualitative detection was performed. The
points in Fig. 1 on the detection limit mean Campylobacter spp. were found in 0.1 g by enrichment, below the detection limit (at 0 log10 cfu/g) means that no Campylobacter spp. were detected in 0.1 g. Thus until below the detection limit, a reduction of approximately 2 log10 cfu/g for skin was found over a period of minimum 10 up to more than 28 days, while for minced meat approximately a 2 log10 cfu/g reduction was found over 10 days. Campylobacter spp. could still be detected per 0.1 g after a storage period of 84 days, as well as for the minced meat and as for the skin. During absence/presence testing Campylobacter spp. was detected per 0.1 g on the skinned, boned upper legs throughout the 84 days. 3.2. Reduction by addition of salt The salt concentration used in Belgian minced chicken meat preparations of approximately 1.5% had no impact on the number of Campylobacter spp. recovered during storage time (Table 1) since the difference in contamination level varied little and is within the range of the measurement uncertainty (± 0.325 log10) of the enumeration methods (spread plating on mCCDA), which was determined by Habib et al. (2008a). Water activity remained at 0.98 throughout the study. No significant reduction of Campylobacter spp. could be noticed at 4 °C. Again, as mentioned above, a reduction of approximately one log10 cfu/g was found when kept for 1 day at −22 °C. During both experiments a high number of E. coli (ca. 3 log10 cfu/g) was present. Freezing or refrigeration had no impact on the numbers of E. coli. The initial contamination level of Enterobacteriaceae was also approximately 3 log10 cfu/g; the mesophilic aerobic count was established as 5 to 6 log10 cfu/g. It is clear that the latter two
Table 1 Effect of 1.5% NaCl-concentration on the survival of Campylobacter spp., total mesophilic aerobic count, Enterobacteriaceae and E. coli in naturally contaminated minced meat of chicken in function of a 14 day incubation at 4 °C and − 22 °C (with standard deviation between 0.01 and 0.1 log10 cfu/g). Campylobacter (log10 cfu/g)
Total count (log10 cfu/g)
Enterobacteriaceae (log10 cfu/g)
E. coli (log10 cfu/g)
− 22 °C
4 °C
− 22 °C
4 °C
− 22 °C
4 °C
− 22 °C
Experiment 1 Aw = 0.9829 [NaCl] = 1.8 +/− 0.1% 0 2.8 1 2.5 3 2.3 7 2.1 14 2.3
2.8 1.2 1.4 1.3 1.0
5.1 5.1 5.9 N 5.0 9.8
5.1 4.8 5.1 4.9 5.1
2.9 3.1 3.6 5.7 7.0
2.9 2.8 2.8 1.6 2.5
3.1 3.0 2.9 3.0 2.6
3.1 2.9 3.0 2.8 2.9
Experiment 2 Aw = 0.9824 [NaCl] = 1.5 +/− 0.1% 0 2.1 1 1.9 3 1.8 7 1.9 14 1.4
2.1 1.1 b1 b1 b1
5.9 6.0 7.2 9.4 10.0
5.9 5.7 5.7 5.5 5.7
2.8 3.2 3.6 5.2 6.5
2.8 2.8 2.5 2.7 2.7
2.9 2.9 2.8 2.9 2.2
2.9 2.8 2.7 2.7 2.7
Incubation (days)
4 °C
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Fig. 2. Evolution of the internal temperature (°C) and colour change in the geometric centre of a chicken burger in function of the duration (s) of frying.
parameters, encompassing psychrotrophic bacteria, reached high numbers after prolonged storage (14 days) at 4 °C which was accompanied with unacceptable sensorial properties.
C. jejuni was observed once the internal temperature was raised above the maximum growth temperature of Campylobacter spp.
4. Discussion 3.3. Heat-inactivation test As shown in Fig. 2, the recorded temperatures of the geometric centre of the meat were very consistent within experiments, with standard deviations between 0.1 and 3.5 °C. After 6.5 min the burger was defined as well-cooked conforming to the internal colour. To verify the adequacy of consumer frying procedures for inactivation of Campylobacter spp. in chicken minced meat preparation, the number of surviving Campylobacter spp. cells (Fig. 3) after different time intervals was determined, this for both artificially inoculated and naturally contaminated chicken burgers. During the first 2 min no inactivation was observed. From this point on, the number of campylobacters recovered from the inoculated as well as the naturally contaminated burgers declined rapidly, and after 4 min of frying they dropped below detectable levels (b10 cfu/g) with core temperatures of 57.5 °C and 52.1 °C respectively. Thus fast killing of
Despite their importance as human pathogens little is known about how campylobacters persist in foods or non-food environments (Humphrey et al., 2007). In this study the behaviour of C. jejuni in naturally contaminated minced chicken meat preparations during storage and preparation as it might be expected to occur in the logistic chain and at the consumer's home was investigated. This study was focused on minced meat preparations because the bacteria are most likely to be uniformly spread throughout the meat by means of meat grinding. The approach selected here uses naturally contaminated chicken products to provide realistic data on the quantitative inactivation of Campylobacter spp. instead of working with inoculated samples with high numbers. It has been shown that there are differences in the behaviour of inoculated bacteria and surrogate bacteria compared to naturally found bacteria in foods and on chicken carcasses (Luber et al., 2006).
Fig. 3. Reduction of the Campylobacter concentration in a chicken burger in function of duration (s) of frying and temperature (°C). (Left: inoculated burgers; right: naturally contaminated burgers).
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Survival, inactivation of Campylobacter spp. in frozen conditions was studied for skin; skinned, deboned upper leg and minced meat to deduce the effect of any protection due to higher fat from skin on survival. Some processing plants in Belgium either freeze the skin or the whole upper legs prior to production of the meat preparations (Sampers et al., 2008). Quantitative analysis indicated that one log10 cfu/g reduction can be achieved after 1 day storage at a freezing temperature of −22 °C, from then on only a gradual reduction is achieved. No differences were obtained between naturally contaminated skin; boned, skinned upper leg and minced meat. So Campylobacter spp. are sensitive to freeze treatment and our results indicate that a long-term holding at − 22 °C can reduce the initial numbers of Campylobacter spp. below the detection limit of 10 cfu/g. However, after 84 days, Campylobacter spp. positive samples were still detected in presence/absence testing per 0.1 g. This confirms that Campylobacter spp. may persist for prolonged periods in chilled and frozen products, although reduction in the concentration and a decline in the viability were also observed during storage (Hänninen, 1981; Oosterom et al., 1983; Yogasundram and Shane, 1986; Curtis et al., 1995; Lee et al., 1998). Ritz et al. (2007) investigated the effect of freezing at − 20 °C on survival of Campylobacter spp., on the skin, below the skin and on the muscle. This study found 0.9–3.2 log decline in 2 weeks. Further freezing yielded no additional effect, in agreement with other studies (Lee et al., 1998; Zhao et al., 2003; Bhaduri and Cottrell, 2004). Most of these studies worked with inoculated samples. Data of naturally contaminated chicken meat preparations are lacking, except for a study of Georgsson et al. (2006) who worked with naturally contaminated poultry carcasses. They reported to still have positive samples after 220 days at − 20 °C. They also showed that the temperature of thawing did not have much influence on the recovery of Campylobacter spp. In our former study (Sampers et al., 2008) freezing showed to have an impact on the prevalence of Campylobacter spp. in chicken meat preparations, but the reduction in Campylobacter spp. concentration in samples containing frozen skin was not significant. Bhaduri and Cottrell (2004) also perceived that chicken skin may provide a protective microenvironment for C. jejuni. However this could not be confirmed in the present study. At present freezing of poultry meat or minced meat preparations is frequently applied in housekeeping but also in the smaller production companies for logistic reasons (Sampers et al., 2008) and not as a food processing step leading to control microbial contamination. Nevertheless freezing of chicken meat preparations (or its raw materials) could serve as an adequate product specific preventive measure that specifically reduces risk of exposing the consumer to (high) Campylobacter spp. contaminated chicken meat preparations. It is generally acknowledged by various risk assessments, the highest numbers lead to highest risks. These risk assessments all conclude that the most effective intervention measures aim at reducing the Campylobacter spp. concentration, rather than reducing the prevalence (Nauta et al., 2008). Freezing as a control measure to reduce campylobacteriosis is already suggested or introduced in countries as Sweden and Norway (Hofshagen and Kruse, 2005; Lindqvist and Lindblad, 2008), to divert Campylobacter-positive flocks to freezing. Former studies (e.g. Iceland study of Stern et al., 2003) already showed a decrease of reported cases by freezing Campylobacter spp. positive carcasses. Although during freezing Campylobacter spp. may be killed, a fraction of the C. jejuni population may survive or be sub-lethally injured (Georgsson et al., 2006 and Jasson et al., 2007). They may pose a residual risk towards the consumer. The sodium chloride concentrations usually present in chicken meat preparations (approximately 1.5%) had no influence on the survival of Campylobacter spp. on naturally contaminated minced meat, which is distinct from the findings by Harrington et al. (2007),
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who noticed increased log reductions at 1.5% NaCl, and especially 2.5% NaCl, for which reductions of at least 4 log10 units were observed using pure cultures in broth. In contrast Jasson et al. (2007) determined that 85.7% of the Campylobacter spp. population were sub-lethally damaged after 10 days exposure at 3.5% NaCl. A water activity of 0.98 was measured in minced meat, so not low enough to affect the numbers of Campylobacter spp. present. Higher NaCl concentrations were not tested as they are also not used, for sensory reasons, in poultry processing industry involved in making poultry meat preparations. Thus, freezing or chilling of minced chicken meat and addition of (minor) salt concentration alone will not contribute to a 3 to 6 log of pathogen reduction (ACMSF, 1997) proposed for enteric pathogens and cannot replace sanitary production and handling (Lee et al., 1998) to limit the pathogen's concentration in the food chain or are not a substitute for safe handling and proper cooking of poultry by the consumer. Chicken meat preparations delivered to the retail and thus as such available to the consumers, are proven to be quite highly contaminated with C. jejuni (high prevalence and occasionally high numbers) (Habib et al., 2008b). Due to insufficient rigorous or difficulties in elaboration or implementation of food safety management systems by food business operators, food that has been produced under HACCP or hygiene codes may still be unsafe (Humphrey et al., 2007; Sampers et al., 2008). So while considering a reliable risk assessment approach, it is important to stress on the importance of consumer phase in integration with production and processing phases. In relation to chicken, food borne exposure to Campylobacter spp. may take place through consumption of undercooked, internally contaminated chicken meat (such as in the case for minced chicken meat preparations) or via cross-contamination to ready-to-eat foods or hands during preparation of raw chicken (Lindqvist and Lindblad, 2008). It is generally assumed that campylobacters will not survive pasteurisation treatments or proper cooking (Nauta et al., 2008; Humphrey et al., 2007; Uyttendaele et al., 2006). Some literature indicate that the probability of cross-contamination for Campylobacter spp. via chicken meat is higher than the probability of undercooking, because the highest number of Campylobacter spp. are found on the surface of the meat, whereas internally, where the temperature is lower, the prevalence is also lower (Luber et al., 2006). This is not generally accepted. For example Bergsma et al. (2007) value the risk for infection by means of a not well performed heat treatment higher than commonly adopted. Moreover for minced meat preparations the bacteria are uniformly spread throughout the meat by means of meat grinding. Low thermal resistance of Campylobacter spp. was found during the “burger pan-frying experiment” in the present study. C. jejuni and C. coli are rather sensitive to heat and may be expressed in a D-value, but this value differs within different studies (Gill and Harris, 1982; ICMSF, 1996; Nguyen et al., 2006; Whyte et al., 2006 and Bergsma et al., 2007). Gill and Harris (1982) also noticed a high variability between strains. The thermal resistance will also depend upon the environmental conditions (food sample and its composition). With the exception of Whyte et al. (2006), who worked on naturally contaminated chicken liver; most studies established heat resistance of Campylobacter spp. with inoculated chicken samples or agar media. Majority of studies also used a boiling water immersion procedure as heat treatment. Here, the focus and set-up of the heat treatment referred to minced chicken meat preparations and on the question “how would the consumer fry his burger?”. Although single-sided cooking, used in this study, requires relatively simple cooking equipment, the flipping interval affects the cooking time. Ou and Mittal (2006) indicated that reduced cooking time is needed for higher turning-over frequency. Furthermore, single-sided pan-frying involves the uncertainty of the slowest heating spot.
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In the present study, Campylobacter spp. was already below detection limit (b10/g), before the burger was described as cookedthrough (as determined by colour change). But the consumer has to be aware that the frying time will be higher for minced meat preparations with an increased thickness. The time needed to kill the Campylobacter spp. was in this experimental set-up dependent on temperature gradient and not on the concentration present. When temperatures reached above 50 °C (core temperature), Campylobacter spp. numbers were below the detection limit (b10 cfu/g). There was no difference between inoculated and naturally contaminated meat preparations. So the consumer information given in a newsletter by the Belgian Federal Agency for the Safety of the Food Chain (FASFC, 2005) to fry their chicken meat until a internal temperature of more than 70 °C is reached, seems from the present study sufficient to be sure Campylobacter spp. are eliminated. Packages intended for supply to the final consumer containing minced meat from poultry or solipeds or meat preparations containing mechanically separated meat must bear a notice indicating that such products should be cooked before consumption (Regulation (EC) 853/2004) to make the consumer aware. However evaluation of the adequate cooking of a meat preparation based on minced poultry meat may be a problem for the consumers due to the small visual difference in colour changes between a semi- and well-cooked product in comparison to the colour changes observed during the cooking of minced beef products. Inadequate cooking of poultry may increase the risk of infections as seen in the study of Istre et al. (1984), where illness was associated with undercooked barbecued chicken meat. Worthwhile noting is that frequently during the Campylobacter spp. detection procedure a positive result could be noticed for presence/absence testing per 0.1 g, while detection per 1 and 10 g provided negative results; this was probably due to the presence of high numbers of competing ESBL E. coli (Jasson et al., 2009). 5. Conclusion Campylobacter spp. in chicken meat preparations (or their raw materials) survived during refrigerated and frozen storage or supplementation of 1.5% NaCl. Freezing could serve as an adequate product specific preventive measure that specifically reduces the chance of the (high initial) contamination of the production process, but cannot replace sanitary production and handling. Poultry contaminated with Campylobacter spp. may lead to infection if not properly handled and sufficiently cooked. Therefore, safe frying times of minced chicken meat burgers in butter were estimated, as this was one of the predominant preparation methods for this type of meat in Belgium. The heat treatment showed the difficulty to recognise a wellcooked burger. But microbiological testing in parallel showed that Campylobacter spp. were inactivated before the fried burgers looked well done. The data provided can be used in exposure assessments of Campylobacter spp. in poultry products in terms of providing both quantitative data on survival and assessing pan-frying and its ability to inactivate campylobacters. Acknowledgements This work was supported by a project fund from the Belgian Federal Public Services (FOD), Health, Food Chain Safety and Environment. We thank the local company for their cooperation and commitment. Josefien Gousseau, Ruben Froyman and Ruben Debouvere are acknowledged for their professional technical assistance in performing Campylobacter spp. analysis and helping out with experimental set-up. We also thank Leentje Braeckman and Dieter Iemants of the department of Biosystems Engineering at Ghent University for the Agilent® 34970A Data Acquisition/Switch Unit and their assistance.
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