R.Y. MURPHY, B.P. MARKS, E.R. JOHNSON, AND M.G. JOHNSON ABSTRACT:

Transcription

1 JFS: Thermal Inactivation Kinetics of Salmonella and Listeria in Ground Chicken Breast Meat and Liquid Medium R.Y. MURPHY, B.P. MARKS, E.R. JOHNSON, AND M.G. JOHNSON ABSTRACT: Thermal inactivation of Listeria innocua and 6 Salmonella serotypes in ground chicken breast meat was compared to that in peptone (0.1%) - agar (0.1%) solution. Inoculated samples were packed in a thin-wall metal tube and submerged in a water bath at temperatures ranging from 55.0 to 70.0 C. For Salmonella and Listeria, the D values in ground chicken breast meat at 55 to 70 C were higher (p ) than those in peptone-agar solution; however, the z values were not significantly different. Complete first-order inactivation models, with Arrhenius temperature dependency, were developed for each inoculum and medium. Key Words: bacteria, poultry, cooking, lethality, modeling Introduction THE U.S. DEPARTMENT OF AGRICULTURE FOOD SAFETY AND Inspection Service (USDA-FSIS) has published a final rule intended to improve the safety of meat and poultry products. In the background statement, the agency states: performance standards spell out the objective level of performance establishments must meet during their operations in order to produce safe and nonadulterated products, but allow the use of plantspecific processing procedures, other than those prescribed in the current regulation. (FSIS 1999). Quantitative information on lethality of pathogens is required for any processing schedule, to show that it meets the lethality performance standard. The knowledge of thermal inactivation (death) kinetics of potential pathogens in commercial products is essential for proper thermal process design and operation. The heat resistance of pathogens in meat is influenced by meat species, muscle type, ph, fat content, and other environmental factors (Ghazala and others 1995; Veeramuthu and others 1998). Thermal resistances of some bacteria were higher in cooked meat broth than in conventional 0.05 M phosphate buffer at ph 7.0 (Bell and Delacy 1984). D-values ranging from 1.99 to min were cited by Ghazala and others (1995) for Streptococcus faecium heated under pasteurization conditions in different heating media, such as brain-heart infusion broth, luncheon meat emulsion, ham broth, normal saline, nonfat milk, infant food, and buffalo milk. However, much published information on the inactivation kinetics of Salmonella and Listeria is based on limited experimental studies conducted with nutrient medium broth (Smith 1995), meat slurries (Abdul-Raouf and others 1993), or via external inoculation (on the surface) of meat (Manu-Tawiah and others 1993; Nychas and Tassou 1996; Fu and others 1995a, 1995b; Kim and others 1994). Most thermal inactivation kinetic data for Salmonella and Listeria were developed with liquid media (Cole and others 1993; Stephens and others 1994; Pruitt and Kamau 1993; Membré and others 1997). One consequence is that they have little direct application to real poultry products in commercial processes. The kinetic values for thermal inactivation of Salmonella and Listeria have not been reported in ground chicken breast meat products, although a z value of 6.4 C for Salmonella Senftenberg in chicken à la king was reported (Angelotti and others 1960). In turkey thigh meat, Veeramuthu and others (1998) obtained D values of , 13.24, and 3.43 min at 55, 60, and 65 C, respectively, and a z value of 5.4 C for the same organism. Goodfellow and Brown (1978) reported a z value of 5.6 C for six Salmonella serotypes in heated ground beef. Orta-Ramirez and others (1997) reported a z value of 6.3 C for S. Senftenberg in ground beef. Due to the highly complicated structure and composition in meat, thermal inactivation kinetics of microorganism within the meat could differ from those in liquid media or on external surfaces (Rodriguez-Estrada and others 1997). Additionally, Liu and others (1997) stated that the total process lethality, the cumulative effect of a given heat treatment on biological factors, could be affected by meat composition. The kinetic studies into microbial inactivation in real products, as opposed to traditional liquid culture, should provide a model system more representative of the food environment. The objectives of this study were to evaluate the thermal inactivation of Salmonella and Listeria in ground chicken breast meat and to compare the thermal lethality kinetics of these pathogens in ground chicken breast meat with those in liquid medium. Results and Discussion Raw product composition The total water content of the ground breast meat was about 78% (w/w, wet basis), using an oven drying method at 110 C for 24 h. The total protein content was about 96% (w/w, dry basis), using the Kjeldahl method. The total lipids content was about 0.12% (w/w, dry basis), using the Soxhlet method. The total ash content was about 2.1% (w/w, dry basis), using a gravimetric method and heating the sample at 550 C in a muffle furnace for 24 h. Thermal history Thermal history was monitored during treatments (Fig. 1). The heating lag time, required for the samples to reach within 0.5 C of the bath temperature, was taken into consideration in the calculations of the kinetic parameters, per Eq. 1 and 2. Conversely, the cooling lag time was not considered, because the center temperature of each sample dropped to 23 C within just a few seconds. 706 JOURNAL OF FOOD SCIENCE Vol. 65, No. 4, Institute of Food Technologists

2 Survival of Salmonella and Listeria in chicken breast meat Analysis of uninoculated controls revealed no Salmonella or Listeria initially present in the meat samples used in this study. The initial inoculum range of 7.0 to 7.5 log 10 (CFU/g) was attained in all trials. Survivor curves were constructed by plotting recovered CFU/ g of sample versus heating time. As expected, as heating temperature increased from 55 to 70 C, survival of Salmonella and Listeria decreased (Fig. 2 and 3). In general, semilogarithmic survivor curves showed a linear decline in population over heating time. However, some of the survival curves exhibited possible shouldering. Kotrola and Conner (1997) reported that shouldering could be caused by the poor heat transfer through the heating medium. Nonetheless, the curves for the most part illustrated a logarithmic destruction of cells, which allowed for reliable calculation of mean D values. Thermal Inactivation Kinetics in Chicken Breast Meat Salmonella. The average D values for Salmonella in chicken meat (Table 1) were calculated from the survival curves (r ). Kinetic rate constants (approximately 2.303/D) of to 9.68 min -1 were obtained for Salmonella at a temperature range of 55 to 70 C. The z value was 6.53 C, with a correlation coefficient of (Fig. 4). The activation energy (E) and the Table 1 Mean D values for Salmonella and Listeria in chicken breast meat and peptone (0.1%) - agar (0.1%) at 55 to 70 C. Culture Temperature Chicken Breast Meat Peptone-Agar Solution ( C) D (min) r 2 D (min) r 2 Salmonella Listeria Arrhenius constant (a) were kj/mol and min -1, respectively (Fig. 5). In comparing our results with the limited previous literature, Veeramuthu and others (1998) tested S. Senftenberg on ground turkey thigh meat and obtained D values of min at 55 C, min at 60 C, and 3.08 min at 65 C, on phenol red sorbitol agar plate. They reported a z value of 5.4 C. Orta-Ramirez and others (1997) reported that D values of S. Senftenberg in ground beef ranged from 53.0 to 0.22 min at temperatures of 53 to 68 C, Fig. 1 Center temperatures of small (8.5 g) chicken meat samples at various bath temperatures. Fig. 3 Survival of Listeria in chicken meat at different bath temperatures Fig. 2 Survival of Salmonella in chicken meat at different bath temperatures Fig. 4 Logarithm of D values for Salmonella in chicken meat and peptone-agar Vol. 65, No. 4, 2000 JOURNAL OF FOOD SCIENCE 707

3 Salmonella and Listeria in Chicken... with a z value of 6.25 C. Goodfellow and Brown (1978) calculated D values in ground beef inoculated with 6 Salmonella serotypes (without S. Senftenberg) and obtained D values of 61 to 62, 3.8 to 4.2, and 0.6 to 0.7 min at 51.6, 57.2, and 62.7 C, respectively, with a z value of 5.56 C. Because of the difference in testing material and strains, it was expected that D and z values in this study would vary from the previous published data. It was previously reported that S. Senftenberg was more heat resistant than the other five types of Salmonella commonly used in thermal destruction tests (Murphy and others 1999). This greater resistance is reflected in the larger D values in the literature for S. Senftenberg, as compared to the cocktail in this study. These differences emphasize the importance of test material, pathogen serotype, and environment in determining parameters for kinetic models used in food safety programs. Listeria. The average D values for Listeria in chicken meat (Table 1) were calculated from the survival curves, with correlation coefficients ranging from 0.87 to The kinetic rate constants (approximately 2.303/D) were to min 1 at 55 to 70 C. The z value was 6.29 C, with a correlation coefficient of (Fig. 6). The activation energy (E) and the Arrhenius constant (a) were kj/mol and min 1, respectively (Fig. 7). In previous studies with L. innocua, Moody and others (1998) estimated thermal inactivation at 70 C for a liquid thermal process and obtained D values of and min for bath and continuous processes, respectively. In their study, paired equivalent isothermal exposures (PEIE) and equivalent point (EP) methods were used to estimate the Arrhenius kinetic parameters from the data obtained by continuous flow process. D values of and min were obtained respectively from PEIE and EP calculations. The z values from their study, 4.5 to 5.1 C, were lower than our value (6.29 C). With L. monocytogenes, Fain and others (1991) reported a D value of 1.2 min at 63 C. In their study, less than one logarithm reduction was obtained for L. monocytogenes in ground beef at 52 C for 100 min (Fain and others 1991). D values of L. innocua were reported to be times greater than those of L. monocytogenes (Foegeding and Stanley 1991). The z values of 6.8 to 6.9 C were obtained, respectively, for two L. innocua cultures in buffer solution at a temperature range of 56 to 66 C (Foegeding and Stanley 1991). A z value of 5.3 C was obtained for L. innocua PFEI in skim milk heated at 56 to 66 C. The z values in chicken meat from our study were approximately 8% lower than those determined in buffer solution by Foegeding and Stanley and 16% higher than those in skim milk. Our activation energy for L. innocua in ground chicken meat of kj/mol is in the range of to kj/mol reported for L. innocua in phosphate buffer and skim milk, respectively (Foegeding and Stanley 1991). Although our results are in general agreement with others, the differences between D and z values obtained in this study and those from previous literature may reflect the impact that different food materials can have on thermal inactivation kinetics. Comparison of chicken meat with liquid medium The D values of Salmonella and Listeria in chicken meat were higher (p ) than those in peptone-agar solution at each respective temperature (Table 1). However the z values in peptone-agar solution (6.26 and 6.36 C for Salmonella and Listeria, respectively) were not significantly different from those in chicken meat (Fig. 4 and 6). A protective effect of medium composition on thermal lethality, as reflected in the D values, was also reported in other foods. Fain and others (1991) reported that D values of L. monocytogenes for fatty beef were double those for lean beef at 57 and 63 C and that different z values of L. monocytogenes were obtained between fatty (6.3 C) and lean ground beef (5.2 C). Line and others (1991) determined D values of E. coli 0157:H7 in ground beef and concluded that D values in fatty ground beef exceeded those for lean ground beef at 52, 57, and Fig. 6 Logarithm of D values of Listeria in chicken meat and peptone-agar Fig. 5. Natural logarithm of k (min -1 ) for Salmonella in chicken meat and peptone-agar solution at 55 to 70 C Fig. 7 Natural logarithm of k (min -1 ) for Listeria in chicken meat and peptone-agar solution at 55 to 70 C 708 JOURNAL OF FOOD SCIENCE Vol. 65, No. 4, 2000

4 63 C. Ababouch and others (1995) found that B. subtilis was more heat resistant in oil than in buffer. Additives (sodium chloride, sodium lactate, polyphosphate, and fat) also may enhance thermal resistance of pathogens. Kotrola and Conner (1997) observed this with E. coli O157:H7 in turkey meat at 52 to 60 C and believed that this increase was due to the reduction in water activity caused by the additives binding water in the heating medium. Thermal inactivation of S. Senftenberg in milk and milk by-products was reported to decrease in the presence of low-molecular-weight solutes (Kornacki and Marth 1993). This protective effect could be due to the influence of low-molecular-weight solutes (lactose and salts) on osmolality, which in turn offers protection from thermal inactivation (Kornacki and Marth 1993). Consistent isothermal heating temperatures were achieved Sample preparation According to Heddleson and others (1991), the maximum heat resistance of Salmonella occurs at 24 h (in stationary phase). Therefore, for each trial, a 24 h culture was prepared individually for each serotype in TSB (plus 200 ppm of nalidixin this study. Therefore, the practical implications derived from our results concern the chemical nature of the food constituents, which play critical roles in affecting the thermal inactivation of pathogenic bacteria. The composition of the heating medium is known to have a strong influence on thermal resistance in bacteria, which was confirmed in this study. The rate of pathogen thermal inactivation increases with decreasing solid content in liquid foods (Annous and Kozempel 1998). Therefore, the prediction of bacterial thermal inactivation in a certain food currently cannot be reliably derived from data obtained from another food or model system. Also, there may be variable influence of heating medium on microbial inactivation, depending on the heating temperatures and materials used. The resulting kinetic parameter should be useful in designing and estimating thermal processes for the poultry industry. Materials and Methods Raw material Ground and formed chicken breast patties (114.3 mm dia 14.8 mm thick, avg 53 g) were obtained from a commercial processor. Six original patties were used to determine moisture, protein, fat, and ash contents according to AOAC (1990). Prior to the thermal treatments, the patty samples were screened, per FDA methods (Andrews and others 1995; Hitchins 1995), for the presence of naturally-occurring Salmonella and Listeria and were found free of both. The same batch of meat (approximately 10 kg) was used throughout the study. Prior to experiments, the patties were thawed for 16 h at 4 C and deformed using a sterilized spatula in a beaker. During chicken meat handling, care was taken in order to avoid contamination, and the meat was kept at 0 to 4 C prior to inoculation. Test organisms Inoculation with several similar or dissimilar organisms could give insight into possible microbial interaction; therefore, both Salmonella and Listeria were used in this study. Six Salmonella serotypes (S. Senftenberg, S. Typhimurium, S. Heidelberg, S. Mission, S. Montevideo, and S. California) were chosen as the target microorganisms because of their potential to occur in chicken products. S. Senftenberg (ATCC 43845) was purchased from American Type Culture Collection (Rockville, Md., U.S.A.). The other 5 Salmonella cultures were obtained from Amy Waldroup (Department of Poultry Science, University of Arkansas, Fayetteville, Ark., U.S.A.). A nalidixic acid resistant culture of each serotype was prepared as previously described (Murphy and others 1999). Although, the acid resistance of the Salmonella cultures might have affected the heat resistance, this effect was not quantified. Subcultures for use as inocula were prepared from the stock culture as required. Listeria innocua M1 was be used in this study as a model for L. monocytogenes. Listeria innocua M1 was obtained from P.M. Foegeding (Department of Food Science, North Carolina State University, Raleigh, N.C., U.S.A. ). The Listeria culture was resistant to 50 ppm of rifampicin and 250 ppm of streptomycin. The lyophilized culture was revived in tryptic soy broth (Difco, Detroit, Mich., U.S.A.) for 24 h at 37 C before use. ic acid for the Salmonella) at 37 C. The count for each Salmonella serotype and the Listeria in those 24 h cultures was determined to be approximately 10 9 CFU/ml. Just prior to the thermal treatment, an equal-volume mixture (1.0 ml of each culture) was blended dropwise into ground chicken breast meat (100 g, ph about 5.9), or a sterile agar (0.1%) peptone (0.1%) aqueous solution (ph about 5.7), in a sterilized beaker to obtain uniform inoculation levels of 10 7 to 10 8 CFU/g. Diluted agar solution was used in the liquid medium in order to increase the viscosity and minimize convection effects within the aqueous solution. Four parallel tests were conducted to ensure the consistency of the mixing procedure in the meat. Thermal treatments After mixing with the culture, samples (8.5 g of meat) were loaded into thin-wall (0.7 mm) metal containers (8.23 mm dia mm length) and immersed in a heated circulation water bath at 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, or 70.0 ( 0.1) C. After thermal treatment, the samples were immediately removed and placed in an ice-water cooling bath until the sample center reached 23 C (in a few seconds). The samples were kept on ice less than 10 min before plating. The thermal history was recorded (every 1 s) by a data acquisition system, via thermocouples (40-gauge, type E) placed at the sample center, sample surface, and in the heating and cooling baths. Enumeration Enumeration of Salmonella and Listeria was based on FDA procedures (Andrews and others 1995; Hitchins 1995). Prior to thermal treatment, 25 g of inoculated chicken meat (that is, untreated control) was combined with 225 ml of sterile peptone solution (0.1%) in a sterile nylon-mesh-lined polyethylene bag and blended in a Stomacher (Lab Blender 400, Tekmar Co., Cincinnati, Ohio) for 2 min. For each thermally-treated sample, the entire sample (8.5 g) was combined with 0.1% peptone solution (76.5 ml) and blended by the Stomacher for 2 min. In each case, the wash fluid was serially diluted, and 0.1 ml (for 10 CFU/g) or 1 ml (for 10 CFU/g) was spread-plated over 2 or 3 plates, respectively, in duplicate for each dilution. A total of 3 to 5 serial dilutions were plated for each treatment. Salmonella was plated on TSB-N agar (containing 200 ppm of nalidixic acid, sodium salt). Listeria was plated on TSB-YE-R-S agar (containing 0.6% yeast extract, 50 ppm of rifampicin, and 250 ppm of streptomycin). The plates were incubated at 37 C for 72 h for Salmonella and for 96 to 144 h for Listeria, and the colonies were then counted. The plates were returned to the incubator and re- Vol. 65, No. 4, 2000 JOURNAL OF FOOD SCIENCE 709

5 Salmonella and Listeria in Chicken... counted until viable counts did not increase further. It should be noted that, for the heated chicken meat samples, up to 144 h was required for the heat-injured Listeria to recover. Survivor curves and primary models Survivor curves were generated by plotting the logarithm of survivor colony counts versus the heating time at each tested temperature. The following linear primary model of Buchanan and others (1997) was used to model the thermal destruction of Salmonella and Listeria and to determine the decimal reduction times. X t X 0 [t t L ] (1) X t X 0 s (t t L ) [t t L ] (2) where X t log 10 (cfu/g) at time t, X 0 log 10 (cfu/g) at time t 0, s slope of the survivor curve, t time (min), and t L duration of lag period (min). First-order kinetics and an Arrhenius equation were used to estimate the kinetic parameters as follows: N N 0 e kt (3) k a e E/RT (4) where N/N 0 is the fraction of survivors, k is the destruction rate (min 1 ), a is the Arrhenius constant (min 1 ), E is the activation energy (J/mol); R is the universal gas constant (8.314 J/mol/K), and T is the product temperature (K). Calculation of D, z, k, and E values Triplicate thermal inactivation trials were performed at seven temperatures from 55 to 70 C. The D (decimal reduction) values for Salmonella and Listeria at each trial temperature were calculated by taking the negative inverse of the relevant s value (Ghazala and others 1995). The z values (temperature change to change the D values 10-fold) were determined as the negative inverse slope of the log 10 D in contrast to heating temperature plot. The k, rate of inactivation at a tested temperature, was the negative slope of the ln(n/n 0 ) in contrast to heating time plot at each tested temperature and could be estimated as 2.303/D. The activation energy of thermal inactivation (E) was calculated from the negative slope of the ln(k) in contrast to inverse temperature (1/T) plot. The Arrhenius constant (a) was calculated from the intercept of the ln(k) in contrast to 1/T regression. Slopes and r 2 values were determined using least squares linear regression. Mean parameter values from experiments were compared via paired t-tests ( = 0.05). References Ababouch LH, Grimit L, Eddafry R, Busta FF Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils. J. Appl. Bacteriol. 78: Andrews WH, June GA, Sherrod PS, Hammack TS, Amaguana, R.M Salmonella. In: FDA Bacteriological Analytical Manual 8 th edition. Arlington, Va.:Association of Agricultural Chemists, Chapter 5. Angelotti R, Foter MJ, Lewis KH Time-temperature effects on salmonellae in foods. II. Behavior at warm holding temperatures. Thermal-death-time studies. U.S. Dept. of Health, Education, and Welfare, Public Health Service, Report F60-5, Cincinnati, OH. Cited in Veeramuthu GJ, Price JF, Davis CE, Booren AM, Smith DM Thermal inactivation of Escherichia coli O157:H7, Salmonella senftenberg, and enzymes with potential as time-temperature indicators in ground turkey thigh meat. J. Food Protect. 61: Annous BA., Kozempel MF Influence of growth medium on thermal resistance of Pediococcus sp. NRRL B-2354 (Formerly Micrococcus freudenreichii) in liquid foods. J. Food Protect. 61: Bell RG, DeLacy KM Heat injury and recovery of Streptococcus faecium associated with the souring of club-packed lunchen meat. J. Appl. Bacteriol. 57: Brown WL Designing Listeria monocytogenes thermal inactivation studies for extended shelf-life refrigerated foods. 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Manu-Tawiah W, Myers DJ, Olson DG, Molins RA Survival and growth of Listeria monocytogenese and Yersinia enterocolitica in pork chops packaged under modified gas atmospheres. J. Food Sci. 58: Membré JM, Thurette J, Catteau M Modeling the growth, survival, and death of Listeria monocytogenes. J. Appl. Microbiol. 82: Moody V, Smerage G, Teixeil A, Foegeding P, Lindsay J Estimating thermal inactivation kinetics of microorganisms from a continuous dynamic thermal treatment process. Presentation IFT Annual Meeting Book of Abstracts; 1998 June 20-24, Chicago, Ill. Institute of Food Technologists. p 176. Abstract nr Murphy RY, Marks BP, Johnson ER, Johnson MG Inactivation of Salmonella and Listeria in ground chicken breast meat during thermal processing. J. Food Protect. 62: Nychas GJE, Tassou CC Growth/survival of Salmonella enteritidis on fresh poultry and fish stored under vacuum or modified atomosphere. Lett. Appl. Microbiol. 23: Orta-Ramirez A, Price JF, Hsu YC, Veeramuthu GJ, Cherry-Merritt JS, Smith D M Thermal inactivation of Escherichia coli O157:H7, Salmonella senftenberg, and Enzymes with potential as time-temperature indicators in ground beef. J. Food Protect. 60: Pruitt KM, Kamau DN Mathematical models of bacterial growth, inhibition and death under combined stress conditions. J. Ind. Microbiol. 12: Rodriguez-Estrada MT, Penazzi G, Caboni MF, Bertacco G, Lercker G Effect of different cooking methods on some lipid and protein components of hamburgers. Meat Sci. 45: Smith MG Survival of E. coli and Salmonella after chilling and freezing in liquid media. J Food Sci. 60: Stephens PJ, Cole MB, Jones MV Effect of heating on the thermal inactivation of Listeria monocytogenes. J. Appl. Bacteriol. 77: Veeramuthu GJ, Price JF, Davis CE, Booren AM, Smith DM Thermal inactivation of Escherichia coli O157:H7, Salmonella senftenberg, and enzymes with potential as time-temperature indicators in ground turkey thigh meat. J. Food Protect. 61: MS received 6/11/99; revised 3/8/00; accepted 4/10/00. This project was partially supported by a grant from the U.S. Poultry & Egg Association. Authors Murphy and E.R. Johnson are with the Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR Author M.G. Johnson is with the Dept. of Food Science, University of Arkansas. Author Marks is with Department of Agricultural Engineering, A.W. Farrall Hall, Michigan State University, East Lansing, MI Direct inquiries to Marks ( marksbp@msu.edu). 710 JOURNAL OF FOOD SCIENCE Vol. 65, No. 4, 2000