EFFECT OF OVERLAPPING CHICKEN PATTIES DURING AIR/STEAM IMPINGEMENT COOKING ON THERMAL INACTIVATION OF SALMONELLA SENFTENBERG AND LISTERIA INNOCUA

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1 2001 Poultry Science Association, Inc. EFFECT OF OVERLAPPING CHICKEN PATTIES DURING AIR/STEAM IMPINGEMENT COOKING ON THERMAL INACTIVATION OF SALMONELLA SENFTENBERG AND LISTERIA INNOCUA R. Y. MURPHY,*,1 L. K. DUNCAN, E. R. JOHNSON,* and M. D. DAVIS* *Department of Biological and Agricultural Engineering and Department of Mathematical Sciences, University of Arkansas, Fayetteville, AR Phone: (501) FAX: (501) Primary Audience: Food Safety Directors, Technical Service Managers, Production Managers SUMMARY Ground chicken breast patties were inoculated, throughout, to contain 10 7 cfu/g of Salmonella Senftenberg or Listeria innocua. The inoculated products were cooked in a single layer or double overlaps in a pilot-scale air/steam impingement oven following storage in an equilibrium chamber that maintained the air at dewpoint. The cooking oven was maintained at 232 C, air circulation of 13 m 3 /min, and air moisture of 60% (vol/vol). The total dwell (cooking) time was 3.5 to 5.0 min, including a dwell time of 1.0 min in the equilibrium chamber. The endpoint temperature in singlelayered patties ranged from 53 to 68 C. The temperature between the overlapping surfaces was about 50% less than that at the center of single-layered patties. After being cooked, the Salmonella or Listeria population in the products was enumerated and compared among the patties that were cooked as a single layer and as overlaps. Salmonella and Listeria in the overlapped patties were about 6 logs more than that in the products that were cooked in a single layer. This information is useful for evaluating and controlling commercial meat cooking processes. Key words: Lethality, Listeria, Salmonella, thermal processing, poultry 2001 J. Appl. Poult. Res. 10: DESCRIPTION OF PROBLEM Cooking is a primary means of eliminating pathogens from ground meat products and therefore serves to protect against foodborne disease. Salmonella is a leading cause of gastroenteritis in human [1]. Inadequate cooking is a contribut- ing factor in 67% of the Salmonella-related outbreaks [1]. In recent years, millions of pounds of cooked meat products have been recalled due to inadequate cooking [2]. Listeria monocytogenes causes listeriosis and other diseases that are deadly to immuno-compromised humans [3]. Listeria was isolated from fresh and cooked meat 1 To whom correspondence should be addressed.

2 MURPHY ET AL.: EFFECT OF OVERLAPPING CHICKEN PATTIES 405 [4]. Recent data from the Center for Disease Control (CDC) [5] indicate that about 30% of the deaths due to foodborne disease are directly caused by Listeria. In an effort to eliminate foodborne illness, the USDA [6] has implemented a performance standard that requires a 7-D (decimal reduction time) to reduce Salmonella in certain fully cooked and partially cooked poultry products. The performance standard requires that the processor provide the supporting information for the hazard analysis and decision-making documents associated with the development of Hazard Analysis Critical Control Point (HACCP) plans, critical limits, selection and frequency of monitoring, and verification procedures. The performance standards aim at ensuring product safety and also giving establishments the flexibility to adopt science-based processing procedures and controls. However, the thermal processing schedule for meeting this lethality performance standard is not specified. During commercial cooking, meat products can become overlapped on a process conveyor belt, forming double layers instead of a single layer (due to various reasons such as inadequate conveyor speed), and then remain in this position through the cooking lines. Because the dwell time for a commercial cooking process is determined basing on a single-layer product placement, overlapping products in double layers would likely result in undercooking, consequently resulting in lower pathogen destruction in these undercooked products. The objective of this study was to evaluate the effect of product overlapping on thermal inactivation of Salmonella and Listeria in chicken breast patties during air/steam impingement cooking. MATERIALS AND METHODS MEAT PRODUCT Ground chicken breast meat was obtained from a processor. The total water content was approximately 80% (wet weight basis), determined via oven drying at 110 C for 24 h. Total protein content was approximately 96% (dry weight basis), obtained using the Kjeldahl method. Total lipids content was approximately 0.2% (dry weight basis) via the Soxhlet method. Total ash content was approximately 2% (dry weight basis) by a gravimetric method and heating the sample at 550 C in a muffle furnace for 24 h. The meat samples were received frozen and were used within 3 mo. Prior to each experiment, meat samples were thawed at 4 C overnight. TESTING BACTERIA Salmonella Senftenberg and Listeria innocua were used in this study. Salmonella Senftenberg was used because it is the most heatresistant among the Salmonella species [7]. Salmonella Senftenberg ATCC was purchased [8]. For the convenience of detection, a nalidixic-acid-resistant culture was prepared as previously described by Murphy et al. [7]. A preliminary study compared the heat resistance of S. Senftenberg between the nalidixic-acidresistant and original cultures. No significant difference was found on thermal resistance of S. Senftenberg between the original and nalidixicacid-resistant cultures. Although it would be advantageous to evaluate L. monocytogenes in various thermal experiments, it is generally undesirable to risk working with this pathogen in a food-processing facility. Often, biological indicators are used in challenge studies, because they are nonpathogenic and typically more heat-resistant than pathogens, thus providing resistance information with a margin of safety [9, 10]. Therefore, we used the L. innocua strain that was developed by Fairchild and Foegeding [9] as a thermal resistance indicator for L. monocytogenes in this study. Listeria innocua was obtained from P. M. Foegeding [11]. The culture was resistant to 50 ppm rifampicin and 250 ppm streptomycin. A recent report by Walsh et al. [12] indicated that in a meat substrate, no differences were found for thermal resistance of Listeria between antibiotic-resistant and wild-type strains. The lyophilized culture was revived for 24 h at 37 C in tryptic soy broth (TSB) plus 0.6% yeast extract, 50 ppm rifampicin, and 250 ppm streptomycin. BACTERIAL INOCULATION AND PRODUCT PREPARATION According to Heddleson et al. [13], the maximum heat resistance of Salmonella occurred at stationary phase (in TSB at 35 C). In this study, stationary culture was prepared at 37 C in TSB

3 406 plus 200 ppm of nalidixic acid for S. Senfternberg and in TSB plus 0.6% yeast extract, 50 ppm of rifampicin, and 250 ppm of streptomycin for L. innocua. The counts for Salmonella and Listeria in the cultures were approximately 10 9 cfu/ml. A known weight of refrigerated ground chicken breast meat was transferred to a sterile mixer and inoculated dropwise with an equal volume of Salmonella and Listeria culture to obtain an inoculation of about 10 7 cfu/g each for Salmonella and Listeria. The inoculated meat was mixed for 4 min to ensure an even distribution of the organisms. The mixing time was determined by a preliminary experiment. After mixing, samples were weighed aseptically into 120-g portions. A sterile plastic mold was used to form patties to 127 mm diameter 12.7 mm thickness [14]. The formed patties were stored at 4 C for 2 h prior to cooking. Two of the inoculated patties that were not cooked were used to determine the initial counts (N 0 ) for Salmonella or Listeria. COOKING PRODUCTS A pilot-scale air/steam impingement oven [15] was used in this study to cook the chicken patties. The patties were randomly placed on the conveyor belt in a single layer or with double overlaps. The patties were overlapped, representing about 50% of the surface areas. Single and overlapped patties were processed together on the conveyor belt, which fed them into the equilibrium chamber where they remained 1 min and then were moved into the cooking chamber for an additional 2.5, 3.0, 3.5, or 4.0 min, respectively. During cooking, the patties were moved forward and backward inside the cooking chamber. The equilibrium chamber was maintained at a dew point temperature of 99 C by injecting 100 lb of saturated steam/h. The total dwell (cooking) time for the equilibrium chamber and oven was 3.5, 4.0, 4.5, and 5.0 min, respectively. The oven air temperature was 232 C with an air circulation of 13 m 3 /min and moisture content of 60% (vol/vol) in the oven air. During cooking, 12 thermocouple probes (type K) were placed at the patty centers or between two overlapping surfaces. The temperature was monitored every 1 s via a data acquisition unit. The lowest temperature recorded by JAPR: Research Report the probes was used. After cooking, the patties were cooled at ambient temperature (23 C). BACTERIAL ENUMERATION After each single-layer patty was cooked, it was placed in a sterile nylon meshed (the first) bag. After cooking overlapped patties, the single-layered portions were separated from the overlapped portion. Then single-layered portions were aseptically cut with a sterile knife at about 5 mm from the seams between the overlap. The single-layered portions were placed into a second bag, and the overlapped portion was placed into a third bag. The seams between the overlaps were included with the overlapped portions. All of these samples were then weighed and checked for Salmonella and Listeria. Sterile peptone solution (0.1%) was poured into each of the bags at 1:1 ratio (weight basis). The meat and peptone solutions were messaged through the bags by hand for 5 min and then blended via a stomacher for 2 min. In each case, the strained fluid was serially diluted and spreadplated over two or three plates for each dilution. Three to five serial dilutions were plated for each treatment. Salmonella was plated on TSB agar containing 200 ppm of nalidixic acid and sodium salt; Listeria was plated on TSB agar containing 0.6% yeast extract, 50 ppm of rifampicin, and 250 ppm of streptomycin. After plates were incubated at 37 C, the colonies were counted. The plates were returned to the incubator each day and recounted next day until the viable counts did not increase. The procedures were performed to ensure the recovery of injured cells. Salmonella and Listeria were verified according to Andrews et al. [16] and Hitchins [17]. DATA ANALYSIS For each trial, six patties were cooked in a single layer and six patties were cooked as overlaps. Data were averaged for each treatment of each trial. Three replicated trials were conducted. Survival rates, ln(n/n 0 ) where ln represents log e (natural logarithm), of each organism were analyzed with log-linear models. For example, with Salmonella, because ln(n/n 0 ) = ln(n) ln(n 0 ), ln(n) is modeled using ln(n 0 ) as an offset variable in SAS software procedure GENMOD,

4 MURPHY ET AL.: EFFECT OF OVERLAPPING CHICKEN PATTIES 407 N is cell counts at a certain cooking time and N 0 is initial cell counts. The counts for surviving organisms were assumed to have been sampled from negative binomial distributions [18]. Because single-layered and overlapped patties were inherently paired by design, they were specified as repeated measures (using the generalized estimating equation approach) in GENMOD. The data analysis was conducted using SAS software [19]. The following model was used to express the surviving rate, ln(n/n 0 ), of Salmonella or Listeria in the patties as a function of cooking time: ln(n/n 0 ) = τ + β(time) + γδ i2 (1) + η(time)δ i2 where i = 1 for single-layered patties, i = 2 for double-layered patties, δ i2 = 0 when i = 1, and δ i2 = 1 when i = 2. For single-patties, this model reduces to the following: ln(n/n 0 ) = τ + β(time) (2) and for double-patties, the model is as follows: ln(n/n 0 ) = (τ + γ) + (β + η)(time) (3) The parameter η represents the difference in the effect of a unit change in time ( t) on doublelayered patties versus single-layered patties. A test of whether η is significantly different from zero at α = 0.05 will indicate if there is a significant interaction between cooking time and layering or not layering. A measure of lack-offit for this model is the deviance statistic. This statistic has an associated degrees of freedom (df) and it follows an approximate chi-squared distribution. An assessment of lack-of-fit can be obtained by calculating deviance/df, where deviance/df equal to 1 would indicate a perfect fit. RESULTS AND DISCUSSION At total dwell times of 3.5, 4.0, 4.5, and 5.0 min, internal meat temperatures in the singlelayered patties were 53, 60, 65, and 68 C, respectively. The endpoint temperature in overlapped patties was about 50% lower than that FIGURE 1. Thermal profile for the center of the patty cooked in a single layer and that between two overlapped surfaces. in single-layered patties. Figure 1 illustrates a thermal profile of a single-layered patty (at center) and two overlapped patties (between the overlapping surfaces) at a dwell time of 4.5 min. Temperature elevation was delayed in the overlapped patties. The delay was observed because the rate of heat penetration is equal to -ka ( T/ X), where k is thermal conductivity of the product, A is heat transfer area, T is temperature gradient, and X is product thickness [21]. Therefore, increase of product thickness, X, decreases the rate of heat penetration. For each treatment, three sets of cell counts each were obtained for Salmonella and Listeria. The first was from the patties that were cooked in a single layer. The second was from the singlelayered portions of the overlaps, and the third was from the overlapped portions. The first set of data for each treatment (the counts from the patties cooked in a single layer) was compared with the second (the counts from the singlelayered portions). No significant (at α 0.05) differences were found for Salmonella or Listeria counts between the two sets of data, i.e., Salmonella or Listeria in the single-layered portions was not significantly different from that in the patties cooked in a single layer. For the single-layered patties, S. Senftenberg and L. innouca were not detected after the patties were cooked to 5 min (meat internal temperature 68 C). This result was consistent with previous studies by Murphy et al. [14, 22, 23]. Murphy et al. [14, 22, 23] indicated that cooking conditions affected thermal destruction of Salmonella and Listeria and found significant differences for thermal inactivation of Salmonella and Listeria between chicken patties cooked at high and low humidity. In these studies, high humidity bene-

5 408 fited thermal kill of Salmonella and Listeria [14, 22, 23]. In the present study, high humidity was obtained by injecting saturated steam into an equilibrium chamber. Because cooking conditions such as humidity can affect pathogen lethality, care should be taken when evaluating thermal destruction of pathogens when conditions (other than temperature) are unknown. At total cooking times of 3.5 to 5.0 min, more than 10 6 cfu of S. Senftenberg or L. innocua/ g were detected in the overlaps. Models were developed to correlate surviving rate, ln(n/n 0 ), of S. Senftenberg and L. innouca with cooking time. Data in the model included a constant (intercept) term, a cooking time (linear) term, and the effect of product overlap on each term. The parameter estimates for the final model are given in the Table 1. Table 1 shows that cooking time affected thermal inactivation of S. Senftenberg and L. innocua in the patties that were cooked in single layers differently from the way that it affected the inactivation in the overlapped patties (S. Senftenberg: η = , P < ; L. innocua: η = , P < ). The deviance/df value for the model was for S. Senftenberg and for L. innocua, indicating that the models fit the experimental data well. The deviance/df value can be considered a chi-squared statistic with 1 df. A perfect fit between the model and experimental data would give a deviance/df value of 1.0. When using data from Table 1 and Equations 2 and 3, the survival rate for S. Senftenberg in single-layered patties [ln(n/n 0 ) = JAPR: Research Report (time), Equation 2] and for overlapped patties [ln(n/n 0 ) = (time), Equation 3] can be modeled. Similarly, L. innocua survival rate for single-layered patties [ln(n/ N 0 ) = (time)] and for overlapped patties [ (time)] can be modeled. In this study, the thermal destruction for S. Senftenberg and L. innocua was linearly related to dwell (cooking) time. Further, we concluded that there was very little reduction of Salmonella or Listeria in the overlapped patties. Similar results have also been found [24] with similarly inoculated (7 logs) fried ground meat patties (containing beef and turkey) that were battered and breaded and then deep-fried in a single layer or as double overlaps (50% of surface areas) at 177 C for 45 or 47 s. Salmonella Senftenberg and L. innocua were not detected in the patties that were fried in a single layer; however, 5 to 6 logs of Salmonella and Listeria were detected in the patties that were fried in the overlaps. Figure 2 illustrates the thermal log 10 kill for S. Senftenberg and L. innocua in the chicken patties cooked in single layers and overlaps showing both the model predictions and experimental data. After 4.5 min, experimental values for S. Senftenberg and L. innocua approached zero. This occurrence was after 4.5 min, and the reduction of Salmonella and Listeria reached the maximum of 7 logs (the inoculation level). Therefore, data above 4.5 min were not used in the prediction equations. Further, the models from this study should only be used to predict kill rates up to 7 logs. TABLE 1. Analysis with the generalized estimating equation A model for the reduction of Salmonella Senftenberg and Listeria innocua in chicken breast patties cooked as singles or in a double layer B,C STANDARD CHI- BACTERIUM PARAMETER ESTIMATE ERROR SQUARE P Salmonella τ < Senftenberg β < γ < η < Listeria innocua τ < β < γ < η < A Generalized estimating equation is a method of analyzing longitudinal and multivariate categorical responses [25]. B Inoculated at 10 7 cfu/g. C Deviance/df = for S. Senftenberg and for L. innocua.

6 MURPHY ET AL.: EFFECT OF OVERLAPPING CHICKEN PATTIES 409 FIGURE 2. Experimental data (Symbols) and model predictions (lines) for log 10 (N 0 /N) of (a) Salmonella Senftenberg and (b) Listeria innocua. N 0 was initial cell counts, and N was the cell counts at a certain cooking time. Data above 4.5 min were not used in the model as the reduction of Salmonella and Listeria reached the maximum of 7 logs (the inoculation level) at this time. For overlapped patties that were cooked for 4.5 and 5.0 min, a study was also conducted to evaluate the thermal kill for Salmonella and Listeria on the top and bottom surfaces and along the seams of the two overlapped patties. The same procedures as described earlier were used for preparing and cooking the patties. After cooking, entire overlapped surface areas, including the top and the bottom (in contact with the conveyor belt), and the seams between the two overlapped patties were swabbed using sterile cotton tips. The tips were then spread-plated onto Salmonella or Listeria medium. From this point on, the same procedures as described earlier were followed for incubating and counting the plates. After 4.5 or 5.0 min, neither Salmonella nor Listeria was detected on the top or bottom surface of the overlapped patties (Table 2). However, Salmonella and Listeria were found along the seams between the two overlaps. These results indicated that more bacteria were killed on the patty top and bottom surfaces than on the internal portion of the patties. The difference in bacterial kill between patty top or bottom surfaces and internal areas was possibly due to the temperature gradient along the patty thickness. For chicken patties cooked in an air convection

7 410 JAPR: Research Report TABLE 2. Swabs from the top and bottom surfaces and the seams between the two overlapped patties TOTAL DWELL TIME SALMONELLA (min) SWAB LOCATION Senftenberg A LISTERIA INNOCUA A 4.5 Top surface Not detected Not detected 4.5 Bottom surface Not detected Not detected 4.5 Top seam B 2.5 ± 1.1 logs C 2.6 ± 1.9 logs C 4.5 Bottom seam B 3.3 ± 1.4 logs C 3.3 ± 1.7 logs C 5.0 Top surface Not detected Not detected 5.0 Bottom surface Not detected Not detected 5.0 Top seam B 2.3 ± 1.6 logs C 2.5 ± 1.8 logs C 5.0 Bottom seam B 3.2 ± 1.1 logs C 3.1 ± 2.1 logs C A Seven logs of S. Senftenberg or L. innocua were inoculated throughout the patties. B The length was about 150 mm. C Mean ± standard deviation. oven, a difference of more than 10 C was found between a patty surface and the center of a patty [14]. The greater survival rates of Salmonella and Listeria along the seams between the two overlapped patties indicated that the heat was transferred mainly from the patty top and bottom CONCLUSIONS AND APPLICATIONS 1. When chicken patties were cooked at an air temperature of 232 C, an air circulation rate of 13 m 3 /min, and air moisture content of 60% (vol/vol), the thermal destruction for S. Senftenberg and L. innocua was linearly related to dwell (cooking) time. 2. If the patties were overlapped during cooking, thermal kill for S. Senftenberg and L. innocua was about 6 logs lower than that in patties cooked in a single layer. Therefore, it is important to control cooking processes to avoid product overlaps. 1. Juneja, V.K., T.A. Foglia, and B.S. Marmer, Heat resistance and fatty acid composition of Listeria monocytogenes: Effect of ph, acidulant, and growth temperature. J. Food Prot. 61: USDA, Recall notification report. Food Safety and Inspection Service, United States Department of Agriculture, Washington, DC. Accessed: 3. Erkmen, O., Kinetic analysis of Listeria monocytogenes inactivation by high pressure carbon dioxide. J. Food Eng. 47: Cooksey, D.K., B.P. Klein, F.K. McKeith, and H.P. Blaschek, Reduction of Listeria monocytogenes in precooked vacuum-packaged beef using postpackaging pasteurization. J. Food Prot. 56: Salvage, B., Post-packaging interventions a must in battle against harmful organisms. Accessed April 2, USDA, Performance standards for the production of certain meat and poultry products. Government Printing Office, Washington, DC. Fed. Reg. 64: Murphy, R. Y., B.P. Marks, E.R. Johnson, and M.G. Johnson, Inactivation of Salmonella and Listeria in ground surfaces into the center of the patties. Therefore, although the seams between the two overlapped patties were exposed to the same cooking temperature, the temperature along the seams was lower than on the top and bottom surfaces. REFERENCES AND NOTES chicken breast meat during thermal processing. J. Food Prot. 62: American Type Culture Collection, Rockville, MD. 9. Fairchild, T.M., and P.M. Foegeding, A proposed nonpathogenic biological indicator for thermal inactivation of Listeria monocytogenes. Appl. Environ. Microbiol. 59: Foegeding, P.M., and N.W. Stanley, Listeria innocua transformed with an antibiotic resistance plasmid as a thermal-resistance indicator for Listeria monocytogenes. J. Food Prot. 54: Department of Food Science, North Carolina State University, Raleigh, NC. 12. Walsh, D., J.J. Sheridan, G. Duffy, I.S. Blair, D.A. Mc- Dowell, and D. Harrington, Thermal resistance of wild-type and antibiotic-resistant Listeria monocytogenes in meat and potato substrates. 13. Heddleson R.A., S. Doores, R.C. Anatheswaran, G.D. Kuhn, and M.G. Mast, Survival of Salmonella species heated by microwave energy in a liquid menstruum containing food components. J. Food Prot. 54: Murphy, R.Y., E.R. Johnson, L.K. Duncan, M.D. Davis, J.A. Marcy, and M.G. Johnson, Thermal inactivation of

8 MURPHY ET AL.: EFFECT OF OVERLAPPING CHICKEN PATTIES 411 Salmonella and Listeria in chicken breast patties cooked in a pilotscale air convection oven. J. Food Sci. 66: Stein Lab Model 102, Stein Inc., Sandusky, OH. 16. Andrews, W.H., G.A. June, P.S. Sherrod, T.S. Hammack, and R.M. Amaguana, Salmonella. Pages in: FDA Bacteriological Analytical Manual. 8th ed. Association of Agricultural Chemists, Arlington, VA. 17. Hitchins, A.D., Listeria monocytogenes. Pages in: FDA Bacteriological Analytical Manual. 8th ed. Association of Agricultural Chemists, Arlington, VA. 18. The final cell count, N, could be considered as a binomial distribution (number of surviving cells, N, out of N 0 initial bacterial cells). Due to the large initial cell counts (N 0 ) and the overdispersion among cell counts (ranging from N 0 to N), a negative binomial distribution was used. Negative binomial distribution accounts for large initial cell counts and the data overdispersion [20]. 19. SAS Institute Inc., Version 8.1. SAS Institute Inc., Cary, NC. 20. Mood, A.M., F.A. Graybill, and D.C. Boes, Special parametric families of univariate distributions. Pages in: Introduction to the Theory of Statistics. 3rd ed. D. Blackwell and H. Solomon, ed. McGraw-Hill, New York, NY. 21. Geankoplis, C.J., Principles of unsteady-state heat transfer. Pages in: Transport Processes and Unit Operations. 3rd ed. Prentice Hall, Englewood, NJ. 22. Murphy, R.Y., E.R. Johnson, J.A. Marcy, and M.G. Johnson, Survival and growth of Salmonella and Listeria in the chicken breast patties subjected to time temperature abuse under varying conditions. J. Food Prot. 64: Murphy, R.Y., E.R. Johnson, B.P. Marks, M.G. Johnson, and A.J. Marcy, Thermal inactivation of Salmonella and Listeria in ground chicken patties during convection cooking. Poult. Sci. 80: Murphy, R.Y., E.R. Johnson, and M.D. Davis, Unpublished research data from R.Y. Murphy s lab in the Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR. 25. SAS OnlineDoc, Version 8.2. SAS Institute Inc., Cary, NC. ACKNOWLEDGMENTS This study was partially supported by U.S. Poultry and Egg Association research grant. Chicken meat was kindly provided by Tyson Foods, Inc., Springdale, AR.