Research Note. Institute for Food Quality and Food Safety, University of Veterinary Medicine, Hannover, Bischofsholer Damm 15, Hannover, Germany

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1 845 Journal of Food Protection, Vol. 71, No. 4, 2008, Pages Copyright, International Association for Food Protection Research Note Effect of Different Concentrations of Carbon Dioxide and Oxygen on the Growth of Pathogenic Yersinia enterocolitica 4/O:3 in Ground Pork Packaged under Modified Atmospheres C. STROTMANN, T. VON MUEFFLING, G. KLEIN,* AND B. NOWAK Institute for Food Quality and Food Safety, University of Veterinary Medicine, Hannover, Bischofsholer Damm 15, Hannover, Germany MS : Received 3 July 2007/Accepted 15 December 2007 ABSTRACT The influence on Yersinia enterocolitica counts of a gradual increase of carbon dioxide concentrations (percentage by volume in air) during packaging and storage of ground pork meat artificially contaminated with this pathogen was evaluated. Ground meat was packaged under customary conditions using modified atmospheres with various carbon dioxide percentages (0, 30, 50, 70, and 100% CO 2 by volume; for atmospheres of less than 100% CO 2, the rest of the gas was O 2 ). The packs were stored at 2 C for 12 days. During the entire storage time, counts of Y. enterocolitica were determined by the spread plate method for direct plate counts (DPCs). Microbiological shelf life of the stored ground pork also was assessed by total mesophilic aerobic bacterial plate counts (APCs). Y. enterocolitica counts were not significantly different (P 0.05) in the ground pork packaged under the various CO 2 -enriched atmospheres. The growth of Y. enterocolitica was nearly entirely inhibited in all tested modified atmospheres containing the protective CO 2. However, in ground pork packaged with 100% oxygen, there was a significant decrease (P 0.05) in the DPC for Y. enterocolitica from 4.30 log CFU/g (day 0) to 3.09 log CFU/g at the end of the storage time (day 12). The decrease was presumably due to the marked increase in APC seen only in those packages stored under 100% O 2. Packaging with high CO 2 concentrations had significant inhibitory effect (P 0.05) on the growth of mesophilic aerobic bacteria. In Germany, yersiniosis is the third most common cause of human bacterial enteric infections, after salmonellosis and campylobacteriosis (25). Infections in Europe are mainly attributed to the virulent strains of the serovars O:3, O:5,27, and O:9. Pigs are considered the most important reservoir for human pathogenic strains of Yersinia enterocolitica (7). In these animals, the pathogen is most often isolated from the intestine and the tonsils. Contamination of meat with Y. enterocolitica may take place during slaughtering and subsequent processing of the carcass (9). Therefore, consumption of ground pork (including also insufficiently heated meat) represents a risk factor for the consumer. The psychrotrophic properties of Y. enterocolitica add the risk, because the pathogen can grow in fresh meat during storage at cool temperatures. Because extended shelf life of fresh meat products is preferred by retail markets, their production of these products has become increasingly centralized, and cooling, packaging, and storage often is conducted under modified atmospheres (MAs) (21). MA packaging (MAP) may be defined as the enclosure of food products in gas-barrier materials, in which the gaseous environment has been * Author for correspondence. Tel: ; Fax: ; guenter.klein@tiho-hannover.de. changed (30). Normal air is replaced by different gas mixtures (14) to extend the microbiological shelf life and to maintain the desired red color of the meat. In MAP, carbon dioxide mainly is used because of its bacteriostatic effects (6). In some applications required in red meat packaging, the atmospheres also are enriched with oxygen (60 to 80% O 2 by volume in air) to prevent decoloration by metmyoglobin (2). Concerns have been expressed that increasing the shelf life of MAP meat may allow growth of human pathogens to an unsafe level while the sensorial quality seems unaltered (8, 15). Rönner (26) considered Y. enterocolitica more resistant to carbon dioxide than other pathogens, thus representing a greater health risk for products stored in MAs. Several studies have been published on the growth of Y. enterocolitica in MAP meat (17, 19, 23). However, there is little information on the growth of this pathogen especially in ground pork packaged with various carbon dioxide concentrations. The aim of the present study was to evaluate the effects of a gradual increase of carbon dioxide in MAs on the growth and survival of pathogenic Y. enterocolitica. MATERIALS AND METHODS Bacterial strain and preparation of inoculum. Y. enterocolitica subsp. enterocolitica DSM (German Collection of

2 846 STROTMANN ET AL. J. Food Prot., Vol. 71, No. 4 Microorganisms and Cell Cultures, Braunschweig, Germany) was used for the inoculation studies. The bioserotype of the Y. enterocolitica strain was 4/O:3 (isolated from a clinical specimen). A pure culture of the Y. enterocolitica strain was grown in brain heart infusion broth (Merck, Darmstadt, Germany) at 30 C for 24 h to stationary phase. At stationary phase, Y. enterocolitica reached a density of approximately 10 8 CFU ml 1 in the broth. The incubated broth was diluted with physiological NaCl peptone water (0.75%), and the dilution was used immediately for the inoculation of the ground pork samples with a target inoculum of 10 4 CFU g 1. The Y. enterocolitica level was tested for 10 packages before packaging under MAs to ensure the desired inoculation level. Preparation of meat samples. Five hind quarters from pork carcasses were selected 1 day postmortem at a commercial German abattoir. Low-fat meat from the semimembranosus and semitendinosus muscles and the quadriceps femoris muscle was used for ground pork preparation. To obtain a standardized fat content of the ground meat, pork backfat was added before grinding. Muscles and backfat were cut into approximately 5- to 8-cm pieces and then sequentially ground in a meat grinder (Seydelmann KG, Aalen, Germany) through plates with orifices 8 mm in diameter. The ground meat was collected in a sterile plastic container, and portions (n 6 per packaging treatment and sampling time) weighing g were placed on polypropylene trays (187 by 137 by 50 mm; ES-Plastik GmbH & Co. KG, Hutturm, Germany). The surface of each sample was inoculated with 1 ml of the inoculum applied by drops, and the inoculum was mixed into the meat with a sterile spatula. Meat packaging. The inoculated ground meat was packaged under practical (industrial) conditions using MAs with various percentages of carbon dioxide (MA 1,MA 2,MA 3,MA 4, and MA 5 were 0, 30, 50, 70, and 100% CO 2 by volume; for atmospheres of less than 100% CO 2, the rest of the gas was O 2 ). MA 2 (30% CO 2 and 70% O 2 ) was considered the customary MA for red meat. For creating different variations of the MAs, the gases BIOGON C (carbon dioxide) and BIOGON O (oxygen) were used (Linde AG, Höllriegelskreuth, Germany). Meat was packaged with a semiautomatic tray sealer (Sealpac GmbH, Oldenburg, Germany). To obtain defined fractions of carbon dioxide and oxygen in the MAs, a proportional gas mixer (PBI Dansensor Deutschland GmbH, Neuwied, Germany) was connected upstream from the tray sealer. Air was evacuated from the package once, one gas flush was applied, and the package was sealed. To ensure exact gas concentrations, the insertion was tested before packaging until the exact amount of gas was reached inside preliminary packages. The initial gas volume to meat weight ratio was 1.6:1. Uninoculated controls were similarly packaged and stored for each batch. The packaging film used was a multilayered polyethylene terephthalate film with a polyvinylidene chloride oxygen barrier and antifog properties: O 2 transmission rate of 8 cm 3 /m 2 day bar at 23 C and 50% relative humidity, and CO 2 transmission rate of 20 cm 3 /m 2 day bar at 23 C and 50% relative humidity (Wipak GmbH & Co. KG, Walsrode, Germany). Storage and sampling of meat. Six samples were collected from the ground meat before packaging for ph measurements and microbiological analysis. The packaged meat was stored in a cooling chamber (Viessman GmbH & Co. KG, Allendorf, Germany) at 2 C 0.5 C for 12 days. Six packs per packaging variation were analyzed on storage days 1, 4, 8, and 12. Gas analysis and surface ph. The different packs were analyzed for CO 2 and O 2 immediately after packaging and at each sampling date. Gas composition was determined using a Check Mate 9900 head space gas analyzer (PBI Dansensor Deutschland) for O 2 and CO 2. After a sample was removed aseptically for microbiological analyses, the surface ph of the meat was measured. Values were obtained by using a standardized device with a combination of a glass electrode (4.0 and 7.0 buffers) and a ph meter (Knick GmbH, Berlin, Germany). Microbiological analysis. At each sampling time, 250 g of the ground pork in each package was removed and homogenized separately for 2 min in a sterile stomacher bag. From these thoroughly mixed ground pork samples, subsamples for further microbiological investigations were taken. Meat subsamples of 10 g were removed aseptically and homogenized in 90 ml of 0.1% peptone water for 2 min with a Stomacher 400 Lab Blender (Seward, Norfolk, UK). Cell counts were determined by plating serial dilutions of the homogenate. Total aerobic plate counts (APCs) were made on plate count agar (Merck) plates that had been incubated aerobically at 37 C for 72 2h. For enumeration of Y. enterocolitica DSM with the direct plate count (DPC) method, decimal dilutions were spread on duplicate plates of cefsulodin-irgasan-novobiocin (CIN) agar (Merck) containing an antibiotic supplement and incubated at 25 C for 24 2 h. From each second sample, representative presumptive colonies were picked from the plate, and the identities of the isolates were confirmed by further tests. Colonies on CIN agar that were small to medium with a dark red center ( bull s eye ) surrounded by a small transparent border were presumed to be Y. enterocolitica. Colonies with the characteristic appearance were tested by Gram staining followed by biochemical identification on triple sugar iron agar (Merck) and urea agar. Identification and confirmation of Y. enterocolitica was made by a PCRbased method (target gene was the chromosomal ail gene) described previously (29). Serotyping was carried out using commercial O:3 antiserum (SIFIN GmbH, Berlin, Germany). Statistical analyses. Data were analyzed by analysis of variance with the general linear model procedure of the SAS statistical package, version 6.12 (SAS Institute Inc., Cary, N.C.). Different MAs and storage times were compared on the basis of log CFU per gram and ph. Significance was set at P RESULTS AND DISCUSSION Gas composition. The gas conditions in the head space of the different MAP trials during storage are shown in Table 1. On day 0, no standard deviation is given because the exact gas concentration was tested in preliminary packages before packaging of samples. Initial oxygen concentrations in packs with 100% CO 2 (MA 5 ) were all below 0.1% immediately after packaging. From day 0 of storage to day 1, significant differences for the gases were detected. On all following days, no significant changes were seen. However, the influence of significant differences of up to about 20% CO 2 (e.g., MA 4, 70% on day 0 to 51% on day 1) on growth of Y. enterocolitica is still questionable. In other investigations, these differences in carbon dioxide concentration did not have any effect on the growth rate of Y. enterocolitica. For example, Pin et al. (24) reported that an increase of 26% CO 2 resulted in a twofold increase in the lag phase of Y. enterocolitica but not in an increase of the growth rate of this pathogen because of the lack of proportionality between lag phase and specific growth rate. Therefore, in our study we used higher differences in con-

3 J. Food Prot., Vol. 71, No. 4 Y. ENTEROCOLITICA IN GROUND PORK PACKAGED UNDER MODIFIED ATMOSPHERES 847 TABLE 1. Evolution of carbon dioxide and oxygen measured in the headspace of the gas-packed trays and ph values measured directly on meat surface during storage Mean SD measurements a Gas Storage time (days) MA 1 MA 2 MA 3 MA 4 MA 5 CO 2 (%) b b b b O 2 (%) b b b ph b a Values are the mean of six samples per analytical group (n 6). Modified atmospheres: MA 1,0%CO 2 and 100% O 2 ;MA 2, 30% CO 2 and 70% O 2 ;MA 3, 50% CO 2 and 50% O 2 ;MA 4, 70% CO 2 and 30% O 2 ;MA 5, 100% CO 2 and 0% O 2. b Significant changes between storage days (P 0.05). centrations (0, 30, 50, 70, to 100% CO 2 ) in MAP to evaluate the effects. ph measurements. Initial ph of the ground pork was 5.7 and generally did not vary (Table 1) with MAP over the entire storage period. No significant differences occurred in ph between MA 1 and MA 4 (P 0.05). Changes in ph on ground pork packaged with 100% CO 2 (MA 5 ) had a slight but significant decrease from an initial ph of 5.7 to 5.5 on day 12 (P 0.05). However, such a slight change did not have any reported impact on the growth rate of Y. enterocolitica in ground meat, which is in accordance with results reported by other authors (3, 5) indicating that Y. enterocolitica can grow under a wide range of ph conditions. FIGURE 1. Mean ( standard deviation) total aerobic plate counts (log CFU per gram, n 6) in the different MAPs (MA 1, 0% CO 2 ;MA 2, 30% CO 2 ;MA 3, 50% CO 2 ;MA 4, 70% CO 2 ;MA 5, 100% CO 2 ; in atmospheres of less than 100% CO 2, the rest of the gas was O 2 ) at different sampling dates during storage (days 0, 1, 4, 8, and 12). Total APC. The growth data for total aerobic bacteria in the ground pork stored under various atmospheres are presented in Figure 1. In fresh ground pork, APCs were around 5.8 log CFU/g. This count was slightly higher than the process criterion (m) of 5.7 log CFU/g for ground meat at the end of production according to Regulation (EC) 2073/2005 (1) but below the limit (M) of 6.7 log CFU/g. These data suggest that modifying the atmosphere to contain carbon dioxide levels above 50 % (MA 3 through MA 5 ) has no effect on APCs. Nearly constant APCs were found throughout the storage time (12 days at 2 C) in these MAPs (no significant differences, P 0.05). The APCs in these packaging treatments did not differ significantly (P 0.05) and were detected at a plateau close to the initial level of approximately 5.8 log CFU/g. In agreement with Viana et al. (28), treatment MA 1 with 100% O 2 had the shortest shelf life. Already after 4 days of storage in MA 1, the APC increased significantly (P 0.05) by 0.9 log CFU/g, reaching a maximum of 8.78 log CFU/g at the end of the storage period (day 12). Changes in the range of 10 8 log CFU/g are associated with meat spoilage by off-odor and possible slime development (22, 16). In this study, the background microflora of the fresh ground pork in the high-oxygen treatment was presumably dominated by psychrotrophic bacteria, e.g., Pseudomonas and Acinetobacter. These microorganisms will not influence the ph of the ground pork as much as will Lacobacillus spp., which would dominate under vacuum or high carbon dioxide concentrations. This difference in flora may explain the lack of effect of the total APC increase on ph of the ground pork in this packaging treatment. In accordance with Gill and Tan (13), Devlieghere et al. (6), Farber (8), Labadie (18), and Martínez et al. (20), the total APC in this study was strongly influenced by the concentration

4 848 STROTMANN ET AL. J. Food Prot., Vol. 71, No. 4 TABLE 2. Effect of different modified atmospheres on the survival of Y. enterocolitica DSM in artificially contaminated ground pork meat stored at C Mean SD direct plate counts (log CFU/g) a Storage time (days) MA 1 MA 2 MA 3 MA 4 MA A AB B B B A B B B B A A A A A A B B B B a Values are the mean of six samples per analytical group (n 6; n 10 on day 0). Modified atmospheres: MA 1,0%CO 2 and 100% O 2 ;MA 2, 30% CO 2 and 70% O 2 ;MA 3, 50% CO 2 and 50% O 2 ;MA 4, 70% CO 2 and 30% O 2 ;MA 5, 100% CO 2 and 0% O 2. Within the same row, means followed by different letters are significantly different (P 0.05). of carbon dioxide; inhibition increased with higher carbon dioxide concentrations. Effect of the different MAPs on growth of Y. enterocolitica. The minced meat was examined before inoculation and was free of Y. enterocolitica. No Y. enterocolitica isolates were recovered from control samples during storage time, as determined by spread plating of initial dilutions onto selective CIN agar. Growth of Y. enterocolitica is presented as a function of storage time at 2 C in Table 2. Despite a slight decrease in Y. enterocolitica numbers, none of the investigated MAs containing carbon dioxide (MA 2 through MA 5 ) resulted in a marked reduction of the initial 4.3 log CFU/g Y. enterocolitica numbers. At no time did the decline exceeded 1 log CFU. Similar effects on meat packaged in carbon dioxide enriched atmospheres have been reported previously. In pork with a ph below 5.8 that was packaged in 100% CO 2 and stored at 4 C, the growth of Y. enterocolitica was suppressed. However, in pork packaged under the same conditions but with an initial ph of 6.0, growth of Y. enterocolitica was not inhibited (4). Manu-Tawiah et al. (19) also observed rapid growth of this pathogen in fresh pork chops (ph 6.0) stored in carbon dioxide enriched atmospheres of 40% CO 2 plus 0% O 2 plus 60% N 2 and 40% CO 2 plus 10% O 2 plus 50% N 2 and stored at 4 C. Gill and Reichel (12) reported Y. enterocolitica growth in an atmosphere of 100% CO 2 at 5 C in beef (ph 6.0) but not at 2 or 0 C. Thus, in addition to temperature effects, the lack of sensitivity of Y. enterocolitica to carbon dioxide enriched atmospheres could be caused by high initial ph values of the meat. Packaging the ground pork under high-oxygen atmospheres (MA 1 ) resulted in a slight but significant (P 0.05) reduction in Y. enterocolitica by the end of storage time (Table 2). The mean Y. enterocolitica numbers measured at day 12 by DPC in the ground pork packaged under 100% O 2 (MA 1 ) were significantly lower (P 0.05) than those for any of the carbon dioxide enriched atmospheres (MA 2 through MA 5 ). The effect of high-oxygen atmospheres on Y. enterocolitica growth has not been described conclusively, but there is information indicating that oxygen has an additional inhibitory effect on the growth of Y. enterocolitica in packaged meat (11, 24). In agreement with the findings of Fukushima and Gomyoda (10) and Kleinlein and Untermann (17), in our study the Y. enterocolitica count reduction observed in MA 1 (100% O 2 ) probably primarily was due to the marked increase in total APC. This is supported by the fact that the decrease took place at very late in the storage period (days 8 through 12) when total APCs reached high numbers (7.98 to 8.71 log CFU/g). Some authors have reported inhibition of Y. enterocolitica by the natural microflora in mixed cultures in raw pork (10, 27). These reports support the hypothesis that the inability of Y. enterocolitica to multiply appeared to be associated with the presence of competitive microflora. Thus, inhibition was not the consequence of a depletion in essential nutrients or an unfavorable change in ph. Similar mechanisms can explain the slight decrease of Y. enterocolitica in the high-oxygen packaging (MA 1 ). Although the present study was only a simulation of contamination at the processing level, the data indicate that contaminated ground pork stored under customary MAs (30% CO 2 and 70% O 2 ) may result in consumer exposure to Y. enterocolitica. Because a reduction of Y. enterocolitica counts in artificially contaminated ground pork in this study could not be achieved, the risk for consumers seems to be dependent mainly on the initial level of this pathogen and the way the product is treated before consumption (raw or heated). Therefore, a prevention strategy along the food chain is necessary. According to Fredriksson-Ahomaa et al. (9), improvements in the slaughtering process may reduce the Y. enterocolitica incidence in pork meat. 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