Survival and growth of Listeria monocytogenes on sausage formulated with inoculated and stored rework product

Food Control 17 (2006) 981–986 www.elsevier.com/locate/foodcont

Survival and growth of Listeria monocytogenes on sausage formulated with inoculated and stored rework product Hristo Daskalov a

a,¤

, Joe Momfre b, John N. Sofos

b

Department of Hygiene, Technology and Control of Food and FoodstuVs, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria b Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80523-1171, USA Received 3 February 2005; received in revised form 14 July 2005; accepted 15 July 2005

Abstract To assess the eVect of including contaminated rework on survival and growth of Listeria monocytogenes, two sausage formulations (one American, Bologna sausage; and one Bulgarian, Stranja sausage) were inoculated with the pathogen and stored for 4 days at 10 °C plus 15 h at 30 °C. After storage, both rework types were included (at 20% and 40%) in corresponding fresh sausage emulsions and heated to 68, 70 and 71.7 °C; fresh Bologna and Stranja emulsions served as controls and were inoculated with 24 h broth cultures of the same 10-strain mixture of L. monocytogenes and thermally treated to the same temperatures. The results showed that heating to 68 and 70 °C inactivated 3–4 log CFU/g of the initial concentration of L. monocytogenes cells (>7 log CFU/g), while heat treatment to 71.7 °C in the center of experimental samples reduced counts by 6 log CFU/g. Survival of L. monocytogenes in samples heated to 68 and 70 °C was higher in controls. Control samples of Stranja emulsion heated to 71.7 °C allowed higher growth (P < 0.05) during storage (5 days at 10 °C) as compared to other control and experimental rework samples. The Stranja emulsion had a higher fat content (20.2%) compared to the Bologna emulsion (11%). This study provides evidence about the possible danger when potentially contaminated rework is stored and then introduced into fresh product formulations. © 2005 Elsevier Ltd. All rights reserved. Keywords: Listeria monocytogenes; Sausage; Rework

1. Introduction Listeria monocytogenes has been associated with human illness for more than 70 years, but only during the last two decades has it been recognized as an important foodborne pathogen. Disease caused by L. monocytogenes is mostly associated with consumption of ready-to-eat products. According to data of the United States Centers for Disease Control and Prevention (CDC) (Mead et al., 1999), listeriosis illnesses and deaths in the USA are estimated at 2500 and 500, respectively. The US Food and Drug Administration (FDA) and the

*

Corresponding author. Tel.: +359 42 699537; fax: +359 42 670624. E-mail address: [email protected] (H. Daskalov).

0956-7135/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.07.007

US Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) have established a “zero tolerance” policy under which ready-to-eat foods contaminated with detectable levels of L. monocytogenes are deemed adulterated. Thus, L. monocytogenes is considered as an “adulterant” and any ready-to-eat food that contains this microorganism (in a 25 g sample) is subject to a Class I recall and/or seizure in the USA (Harris, 2002). Tompkin (2002) noted that despite eVorts to eradicate the pathogen from ready-to-eat foods, L. monocytogenes contamination continues to occur. Levine, Rose, Green, Ransom, and Hill (2001) reported up to 5% contamination of some ready-to-eat foods with L. monocytogenes. L. monocytogenes was also the primary cause of food recalls due to biological reasons (35 of 39 recalls of

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cooked meats and 35 of 60 recalls for any bacterial adulteration) requested by the USDA/FSIS (http:// www. fsis.usda.gov/OA/recalls/rec.intr.htm) in 2000. Ready-to-eat foods such as frankfurters, Bologna sausage and others may pose a greater risk for listeriosis in the event of postprocess surface contamination. In certain instances, sausages not sold before the expiration date, or sausages with some defects, may be used as rework in newly formulated batches of products. It is not known how this practice may inXuence survival, destruction or growth of L. monocytogenes, if present in the rework. The purpose of this study was to estimate survival and growth of L. monocytogenes in rework sausage and its potential for survival during heat processing and storage of fresh sausage formulated with rework.

2. Materials and methods

parts of cooked sausages, were cut into very small pieces and assayed. The measurement of pH was done with a US-10 (Denver Instruments, Ultra Basic, Denver, Colorado, USA) pH meter. Ten samples of 10 g each from both types of sausages were placed into 18-oz NASCO Sterile Whirl Pack® Filter bags (NASCO, Fort Atkinson, Wisconsin) containing 40 ml of sterilized maximal recovery diluent (MRD; 1.0 Bacto™ Peptone (Difco, Becton Dickinson, Sparks, MD, USA) and 8.5 g sodium chloride (Fisher) in 1:1 distilled water) and homogenized (Masticator, IUL Instruments, Barcelona, Spain) for 2 min. Samples from diVerent parts of cooked sausages were taken for the evaluation of protein and lipid content. Percentage of protein was determined with a LECO CHN-1000 machine (St. Joseph, MI, USA). Nitrogen content (%) was multiplied with the coeYcient 6.23 to calculate protein (% on a wet basis). Lipid content was determined by the Soxhlet method in the dry matter of product.

2.1. Preparation of sausage formulations Two replicate experiments were conducted for all sausage formulations. The two sausage formulations (one American, Bologna sausage; and one Bulgarian, Stranja sausage) were prepared at the Colorado State University Meat Science Laboratory (Fort Collins, Colorado, USA). The ingredients of the sausages were as follows: Bologna formulation (kg); pork trimmings (30% fat) 12.3, NaCl 0.3, dextrose 0.3, dry mustard 0.135, corn syrup solids 0.3, polyphosphate 0.06 (sodium tripolyphosphate + sodium hexametaphosphate), sodium nitrite 0.0024, sodium erythorbate 0.0075, paprika 0.038, onion powder 0.0075, garlic powder 0.0075, coriander 0.0075, white pepper 0.0075, and ice 1.45 (10%). The Bulgarian/Stranga sausage formulation consisted (kg) of pork trimmings (20% fat) 4.5, pork trimming (50% fat) 10.5, soybean protein 0.45, sodium caseinate 0.3, starch 0.15, NaCl 0.33, sodium nitrite 0.0015, sodium tripolyphosphate 0.05, ascorbate/ vitamin C 0.006, black pepper 0.060, nutmeg 0.060, and ice 4. After preparation of the emulsions of both kinds of sausages (Bedie et al., 2001; Samelis et al., 2001, 2002), 40% of the batches were stuVed in 50 mm diameter cellulose casings and cooked in a smoke house. The other 60% of the emulsions were placed in sterile plastic bags and stored at (¡12 °C). 2.2. Physical and chemical analyses Immediately after cooking and chilling, sausage samples were analyzed for water activity (aw), pH, protein and lipid content. Water activity was determined with an AquaLab (Model Series 3/Decadon Devises, Ltd, Pullman, Washington, USA) water activity meter. Ten samples of approximately 2 g each were taken from diVerent

2.3. Listeria monocytogenes strains, inoculum preparation and inoculation for rework preparation A 10 strain L. monocytogenes (NA19; 103M; ScottA; NA3; 101M; PVM1; PVM2; PVM3; PVM4; 558) mixture was used for inoculation of the sausage (Bedie et al., 2001; Samelis et al., 2001, 2002). Each strain was propagated (30 °C, 24 h) and maintained on tryptic soy yeast extract agar (TSAYE, Difco) slants at 4 °C. Strains were subcultured monthly. The strains were activated by transferring a loopful of one colony of each strain into 9 ml of tryptic soy broth yeast extract (TSBYE, Difco) and incubating at 30 °C for 24 h. One day (24 h) before inoculation, all activated strains were inoculated again into 9 ml of TSBYE (Difco). After 24 h of incubation the contents of all tubes with L. monocytogenes TSBYE cultures were mixed in a sterile bottle (200 ml volume) with a Vortex-2 Genie. Five serial dilutions from the mixed strain L. monocytogenes composite culture were made by transferring 10 ml into bottles of 90 ml buVered peptone water (BPW, Difco). The average cell concentration in the last dilution was 4.6 log CFU/ml. Cooked sausages were sliced (0.5 cm thickness) with sterile knives and 12 slices (total of 150 g) were placed into individual 18-oz NASCO Sterile Whirl-Pack® Filter bags (NASCO, Fort Atkinson, WI, USA). The inoculation (Bedie et al., 2001; Samelis et al., 2001, 2002) of slices was done with 1.5 ml of composite culture (4.6 log CFU/ml). After introduction of the inoculum in the bags samples were massaged by hand for 2 min. All inoculated samples for both sausage formulations were stored in the bags for 4 days at 10 °C followed by 15 h at 30 °C. Samples (Wve bags) of each sausage were used to determine the contamination level (mean log CFU/g),

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immediately after inoculation. The same quantity of sterile BPW as the weight of the sample was added into the sterile Whirl-Pack® Wlter bags containing the samples. Samples were homogenized (Masticator, IUL Instruments, Barcelona, Spain) for 2 min. From the homogenized sample, 1 ml portions were serially-diluted in 9 ml of sterile 0.1% BPW and appropriate dilutions were plated onto TSAYE for determination of total bacterial counts and onto PALCAM (Difco) agar for the selective enumeration of L. monocytogenes. Colonies formed on plates were counted after incubation at 30 °C for 48 h. After storage of sausage samples for 4 days at 10 °C and 15 h at 30 °C, the same procedure was followed to determine survival or growth of L. monocytogenes. 2.4. Preparation of experimental combinations containing rework and heating of samples Two replicate experiments were conducted for the following experimental combinations: (i) control (Bologna sausage emulsion—1 kg plus 10 ml of 24 h TSBYE mixed 10 strain culture of L. monocytogenes); (ii) control (Stranja sausage emulsion—1 kg plus 10 ml of the same TSBYE culture); (iii) stored (rework) Bologna cooked sausage inoculated with L. monocytogenes (20%) plus Bologna sausage emulsion (80%); (iv) stored (rework) Bologna cooked sausage, inoculated with L. monocytogenes (40%) plus uncooked Bologna sausage emulsion (60%); (v) stored (rework) Stranja cooked sausage, inoculated with L. monocytogenes (20%) plus uncooked Stranja sausage emulsion (80%); and (vi) stored (rework) Stranja cooked sausage, inoculated with L. monocytogenes (40%) plus uncooked Stranja sausage emulsion (60%). The frozen sausage emulsions (¡12 °C) were removed and refrigerated (4–5 °C) to defrost for 24 h. Cooked and inoculated Bologna and Stranja rework sausages were blended with thawed emulsions in a Waring Blender ML-12 Model (Waring Products Division, Corn, USA) for 15 min. The blended inoculated sausages were divided up and placed into sterile metal containers for preparation of the experimental combinations. Samples of experimented combinations were placed (38–40 g) into plastic thermostable tubes (length 10 cm and diameter 2.5 cm) reaching 1 cm below the upper edge of the tube. Heating of samples was accomplished in a water bath (Precision 120 V, 1550 watts, Jouan Inc., Winchester, VA, USA). Samples were all put into the water bath of 50 °C at the same time and the level of water in the bath was 0.5 cm below the top of the tubes. The temperature of the samples during heating was monitored using thermocouples (Type K beaded probes, Pico Technology Ltd, Cambridge, UK) and real-time data recording software (Pico Technology). Thermocouples were placed as follows: two in the two edges of the water bath and six in six product tubes through the length of the

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water bath. They were placed 2.5 cm deep in the emulsion of each tube. Heating times were as follows: from 50 °C to 68 °C, 38 min; to 70 °C, 43 min, and to 71.7 °C, 47 min. One-third of the samples were heated to 68, next third to 70 and the last third to 71.7 °C; after heating, samples from each treatment were taken oV the water bath and put in cold water (2–4 °C) for chilling. 2.5. Microbiological analysis of heated samples Samples from each experimental sausage combination were serially diluted and plated as follows. From the center of each test tube, 1 g samples were taken aseptically and put into 9 ml of sterile TSBYE. The samples were homogenized with Vortex-2 for 1 min. Samples were homogenized and serially diluted in 9 ml of BPW and appropriate dilutions were plated onto TSAYE and PALCAM, as explained before. Plates were incubated at 30 °C for 48 h, and colonies were counted and converted to log CFU/g. Homogenized samples in TSBYE were kept for 24 h at 4 °C for enrichment and use for analyses in case some samples showed no growth by direct plating. Samples of heated sausage combinations were removed aseptically from tubes and placed in Sterile 18-oz NASCO Sterile Whirl-Pack® Filter bags and stored 5 days at 10 °C. In sterile Whirl-Pack® Wlter bags with samples was added the same quantity of sterile BPW as the weight of the sample. Samples were homogenized (Masticator, IUL Instruments, Barcelona, Spain) for 2 min and 1 ml of homogenized sample was serially diluted in 9 ml of sterile 0.1% BPW and appropriate dilutions were plated onto TSAYE for determination of total bacterial counts and onto PALCAM (Difco) agar for the selective enumeration of L. monocytogenes. Colonies formed on plates were counted after incubation at 30 °C for 48 h. 2.6. Statistical analysis Two replicate experiments were conducted for each phase of the study, with Wve samples per treatment in each replicate. Microbiological data were converted to log CFU/g before being analyzed. Values for the mean log and standard deviation of each set of bacterial counts were calculated on the assumption of a log-normal distribution of microorganisms. Preliminary analysis of Wxed eVects using the general linear models (GLM) procedure of SAS® v 8.2 (31) indicated that log CFU/g populations were dependent on temperature of cooking (F-statistic D a; P < 0.0001), treatment (F-statistic D b; P < 0.0001) and day of analysis (F-statistic D c; P < 0.0001). The data regarding viable populations were evaluated using a 3 £ 6 £ 5 (temperature £ treatment £ day of analysis) factorial design. Least-squares means were separated using a protected pair wise t-test of SAS® (v 8.2). All

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diVerences were reported at a signiWcance level of alpha () D 0.05.

3. Results Data on physical parameters and chemical composition of the sausage products studied are presented in Table 1. Stranja sausage contained more lipids (20.2%) compared to Bologna sausage (11.03%) and less protein (12.47% vs. 16%). Water activity (aw) and pH values were similar for the two sausages (P > 0.05). Protein and fat contents of the Stranja sausage were similar to protein and fat contents of frankfurters produced by most of the sausage producers in USA (Wallace et al., 2003). Growth of L. monocytogenes inoculated on batches of Bologna and Stranja sausages used to prepare rework material are presented in Table 2. Levels of inoculation of the experimental batches of sausages were low (2.6 log CFU/g). Four days at 10 °C and 15 h at 30 °C were more than enough to allow increases in numbers of cells to levels higher than >8 log CFU/g of sausage. No diVerences in growth of the pathogen were observed between the two types of sausage. The chosen time and temperature (4 days at 10 °C plus 15 h at 30 °C), to simulate the worst possible storage conditions for sausages used as rework in meat producing plants, were adequate. Data showing survival and growth of L. monocytogenes in products containing rework are presented in Table 3. Control and experimental formulations before thermal treatment to 68, 70 or 71.7 °C were highly contaminated (>7 log CFU/g). Thermal treatment to 68 °C reduced contamination of L. monocytogenes in all sausage formulations by 3–4 log CFU/g; all samples heated to 68 °C contained L. monocytogenes. Heating to 70 °C reduced contamination by 4–5 log CFU/g in the sausage formulations. DiVerences of mean log CFU/g after thermal treatTable 1 Some physical and chemical parameters of laboratory-prepared batches of Bologna and Stranja sausages (n D 7–10) Parameters Water activity (aw) pH Protein (% in wet basis) Lipids (% in dry matter)

Bologna sausage

Stranja sausage

0.952 § 0.0062 6.21 § 0.02 16.0 § 0.98 11.03 § 3.68

0.960 § 0.003 6.26 § 0.02 12.47 § 0.79 20.2 § 2.86

Table 2 Growth of Listeria monocytogenes on experimental Bologna and Stranja sausages (PALCAM—mean log CFU/g; n D 10) used as rework Type of sausage Bologna sausage Stranja sausage

Inoculation level (mean log CFU/g sausage) 2.61 § 0.18 2.60 § 0.17

Mean log CFU/g sausage after storage for 4 days at 10 °C and 15 h at 30 °C 8.24 § 0.89 8.35 § 0.91

ment to 68 and 70 °C were not statistically signiWcant (P > 0.05). However, bacterial populations in sausages heated to 71.7 °C were reduced to 1 or less log CFU/g; cooking to 71.7 °C resulted in many samples having no detectable L. monocytogenes. As expected, mean log CFU/g of samples heated to 71.7 °C were lower (P < 0.05) compared to those of samples heated to 68 and 70 °C. Higher mean log CFU/g of L. monocytogenes after thermal treatment (68, 70 and 71.7 °C) were observed in control Stranja emulsion and the combination of 40% Stranja sausage rework plus 60% fresh emulsion; however, these results were not signiWcantly diVerent (P > 0.05) than those of other sausage formulations. Storage for 5 days at 10 °C of thermally (68, 70 and 71.7 °C) treated control and experimental formulations resulted in growth of L. monocytogenes by 1–2 log CFU/g. Increases observed were higher for products heated to 68 °C (up to 5.79 in control Stranja emulsion). Increases of L. monocytogenes in experimental formulations (20% or 40% Bologna sausage plus 80% or 60% emulsion; 20% or 40% Stranja sausage plus 80% or 60% emulsion) heated to 68, 70 and 71.7 °C were not signiWcantly (P > 0.05) diVerent. In control samples heated to 68 °C and stored for 5 days at 10 °C, L. monocytogenes increased (P < 0.05) signiWcantly. After 5 days of storage, growth of L. monocytogenes in control Stranja samples heated to 71.7 °C was statistically higher (P < 0.05) compared to the control Bologna samples.

4. Discussion Listeria monocytogenes has become an appreciable threat to the health of sensitive consumers and an important economic burden to the food industry. This pathogen causes more devastating sequelae than most other foodborne pathogens, and, at present, there is a “zero tolerance” regulatory requirements with regard to L. monocytogenes in cooked RTE foods in many countries (Ryser & Marth, 1999; Shank, Elliot, Wachsmuth, & LosikoV, 1996). Inclusion of rework in sausages is normal practice in some plants producing RTE foods, especially in developing countries. Cross-contamination with L. monocytogenes is probable in the process of selecting, collecting and keeping sausages to the time of their use as rework in new meat product formulation. Richmond (1990) noted that most of the contamination occurs during food handling following processing, as the product is exposed to contamination from the plant environment. Hygiene conditions, temperature and storage time inXuence the survival and growth of L. monocytogenes. In this experiment we simulated the worst possible storage conditions (4 days at 10 °C plus 15 h at 30 °C) for rework. Experimental sausages were contaminated with 24-h broth cultures of L. monocytogenes (10 strains) at low

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Table 3 Survival and growth of Listeria monocytogenes in sausages formulated with rework of inoculated sausages and fresh emulsion (PALCAM—mean log CFU/g; n D 10) Sausage formulation

Before heating (mean log CFU/g)

Heating (°C)

Control (Bologna emulsion)

7.47 § 0.11

60 70

3.38 § 0.70 3.14 § 0.9

5.04 § 0.75 4.32 § 0.32

71.7

1.18 § 1.19

2.18 § 1.26

68 70

4.14 § 0.31 3.83 § 0.35

5.79 § 0.82 4.93 § 0.36

71.7

2.59 § 0.66

4.31 § 0.70

68 70

3.32 § 1.77 2.57 § 1.99

4.31 § 1.94 3.82 § 1.53

71.7

1.14 § 1.45

2.02 § 1.81

68 70

3.53 § 1.80 3.02 § 2.04

4.43 § 1.60 3.41 § 1.88

71.7

0.65 § 1.12

1.89 § 1.33

68 70

3.79 § 1.49 2.93 § 2.10

4.77 § 2.14 3.71 § 2.15

71.7

0.45 § 0.77

0.91 § 1.49

68 70

3.67 § 2.02 3.53 § 1.71

5.23 § 1.44 3.87 § 2.30

71.7

1.72 § 1.16

2.77 § 1.69

Control (Stranja emulsion)

20% Bologna rework plus 80% emulsion

40% Bologna rework plus 60% emulsion

20% Stranja rework plus 80% emulsion

40% Stranja rework plus 60% emulsion

7.47 § 0.11

7.55 § 0.78

7.86 § 0.80

7.64 § 0.73

7.96 § 0.75

levels and stored to allow signiWcant growth of L. monocytogenes. Use of these contaminated sausages as rework in fresh sausage emulsions and processing to 68, 70 and 71.7 °C showed that some of the microorganisms can survive, especially when the internal temperature is 68 or 70 °C. However, at 71.7 °C L. monocytogenes, became undetectable in many samples, while some samples contained 1–2 log CFU/g. No statistically signiWcant diVerences of surviving cell numbers were found among formulations of Bologna sausage (low fat formulation) and Stranja sausage (high fat formulation). The results showed that L. monocytogenes survived better in the control Stranja samples and the experimental formulations containing Stranja sausage, as compared to control Bologna emulsion and the experimental Bologna formulation. Fain et al. (1991) reported that high fat (30.5%) ground beef was more protective of L. monocytogenes at 57.2 and 62.8 °C than low fat (2%) beef. Zaika et al. (1990) noted that L. monocytogenes was reduced by 3 log (2.82 § 1.01) at 71 °C (160 °F), using standard beef-pork emulsion (30% fat), inoculated with L. monocytogenes at 108 CFU/g. Destruction of the pathogen started above 45 °C (113 °F) and the average time to reach 71 °C (160 °F) was 70 § 11 min. The authors concluded that in the case of low Listeria contamination, frankfurter heat processing is enough to destroy the pathogen, but the margin of safety for the process is relatively small, particularly if higher levels of L. monocytogenes are encountered. In our study, the temperature was increased from

After heating (mean log CFU/g)

After storage for 5 days at 10 °C (mean log CFU/g)

45 °C to 68 °C in 48 min, to 70 °C in 54 min and to 71.7 °C in 57 min. Mean log CFU/g of L. monocytogenes in control and experimental formulations was more than 7 before thermal treatment. Reduction of the pathogen depended on the Wnal internal temperature. Review of the heat resistance of L. monocytogenes by Doyle, Mazzotta, Wang, Wiseman, and Scott (2001) showed that it is inXuenced by many factors, including age of culture, growth conditions, characteristics of food such as salt, aw, acidity, and others. Listeriae are more heat resistant than most other nonspore-forming foodborne pathogens. In our experiments heating of samples was slow (2 °C/min) and the main ingredient of the experimental sausages (Bologna and Stranja formulations) was pork trimmings. Kim, Murano, and Olson (1994) proved that slow heating (1.3 °C/min) of inoculated ground pork samples allowed survival of more L. monocytogenes than rapid heating (8 °C/min).

5. Conclusions Our results showed that stored sausages which may be contaminated with L. monocytogenes after manufacturing, can be hazardous if used as rework in fresh sausage. The number of cells that survived after thermal treatment depended mainly on internal temperature of heated sausage samples. A temperature of 71.7 °C resulted in major inactivation of the pathogen with many samples having

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no surviving cells of L. monocytogenes. Harris (2002) discussed the “zero tolerance” policy and action level for L. monocytogenes. The same object of attention “L. monocytogenes: low levels equal low risk” is noted by Chen, Ross, Scott, and Combas (2003). Our study indicated that in case of sausage contamination with L. monocytogenes through rework, low numbers of cells may survive in sausage samples even after heating to 71.7 °C. Acknowledgements This study was supported by the Colorado State University Agricultural Experiment Station and the United States Department of Agriculture. References Bedie, G. K., Samelis, J., Sofos, J. N., Belk, K. E., Scanga, J. A., & Smith, G. C. (2001). Antimicrobials in the formulation to control Listeria monocytogenes post-processing contamination on frankfurters stored at 4 °C in vacuum packages. Journal of Food Protection, 64, 1949–1955. Chen, Y., Ross, W. H., Scott, V. N., & Combas, D. E. (2003). Listeria monocytogenes: low level equal low risk. Journal of Food Protection, 66, 570–577. Doyle, M. E., Mazzotta, A. S., Wang, T., Wiseman, D. W., & Scott, V. N. (2001). Heat resistance of Listeria monocytogenes. Journal of Food Protection, 64, 410–429. Fain, A. R., Line, E., Jr., Moran, A. B., Martin, L. M., Lechowich, R. V., Carosella, J. M., et al. (1991). Lethality of heat to Listeria monocytogenes Scott A: D-value and z-value determinations in ground beef and turkey. Journal of Food Protection, 54, 756–761. Harris, L. J. (2002). Listeria monocytogenes. In D. O. Cliver & H. P. Riemann (Eds.), Foodborne diseases (2nd ed., pp. 137–150). Academic Press.

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