Shelf life evaluation for ready-to-eat sliced uncured turkey breast and cured ham under probable storage conditions based on Listeria monocytogenes and psychrotroph growth

International Journal of Food Microbiology 126 (2008) 49–56

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Shelf life evaluation for ready-to-eat sliced uncured turkey breast and cured ham under probable storage conditions based on Listeria monocytogenes and psychrotroph growth Amit Pal, Theodore P. Labuza, Francisco Diez-Gonzalez ⁎ Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, Minnesota 55108, United States

A R T I C L E

I N F O

Article history: Received 9 January 2008 Received in revised form 24 April 2008 Accepted 29 April 2008 Keywords: Listeria monocytogenes Shelf life Baranyi model Sliced turkey breast Sliced ham Potassium lactate Sodium diacetate

A B S T R A C T The growth variability of three Listeria monocytogenes ribotypes in ready-to-eat (RTE) sliced uncured turkey breast and cured ham was studied under storage conditions that RTE foods are likely to encounter. Three product treatments studied were: (1) a control; (2) a formulation subjected to high pressure processing to reduce initial microbial load (HPP); (3) a formulation containing 2.0% potassium lactate and 0.2% sodium diacetate (PL/SD). After separate inoculation with individual L. monocytogenes ribotypes and packaging each treatment under air and vacuum, the packages were stored at 4, 8, or 12 °C and the counts of L. monocytogenes and psychrotrophic bacteria (PPC) were determined for several weeks. The Baranyi model was used to estimate lag times and growth rates. Significant effect of strain difference was noted in both sliced products (P b 0.05). In the absence of antimicrobials (HPP and control), the growth rate (GR) of L. monocytogenes strains increment from 4 to 8 °C and from 8 to 12 °C was approximately 10 and 2 fold, respectively. The addition of PL/SD was effective in restricting the growth of L. monocytogenes and PPC at 4 °C, but at 8 and 12 °C significant growth was observed (more than 100-fold increase) (P b 0.05). In PL/SD samples, vacuum packaging slowed down the onset and the rate of growth of L. monocytogenes at 12 °C in sliced ham and at 8 and 12 °C in sliced turkey breast. Generally, the time to increase by 2-logs was greater in control samples than as observed in HPP-treated samples. When antimicrobials were present, the current results showed that L. monocytogenes was able to grow more than 100-fold within the typical quality-based shelf life of 60 to 90 days at 8 and 12 °C. The findings of this study should be useful in setting the duration of a safetybased shelf life for RTE sliced meat and poultry foods. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Consumption of foods contaminated with Listeria monocytogenes, poses significant health risks to humans (Siegman-Igra et al., 2002). Listeriosis is a relatively rare infection in humans but the severity and mortality rate can reach up to 50% (Low and Donachie, 1997). The risks of getting infected with listeriosis are greatest for individuals with compromised immune systems, pregnant women, children, and elderly persons (Farber and Peterkin, 1991). According to the listeriosis risk assessment report by the U. S. Food and Drug Administration and the U.S. Department of Agriculture's Food Safety and Inspection Service, the consumption of non-reheated ready-to-eat (RTE) deli meat and poultry products foods pose the greatest relative public health risk potential for listeriosis (USFDA/CFSAN-USDA/FSIS-CDC, 2003). Current incidences of 3.1 foodborne listeriosis cases per million population in the U.S. (CDC, 2007) is significantly lower than those from the 1980s (N5 cases per million population), when L. monocytogenes was rated as

⁎ Corresponding author. Tel.: +1 612 624 9756; fax: +1 612 625 5272. E-mail address: [email protected] (F. Diez-Gonzalez). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.04.028

the most important in food safety risk (Slutsker and Schuchat, 1999). However, the Healthy People 2010 goal of reducing foodborne listeriosis by 50% (2.5 cases per million populations) has yet to be achieved (CDC, 2007). The number of foodborne listeriosis cases reported in the Europe is also comparable to the cases in the U.S. (Slutsker and Schuchat, 1999). L. monocytogenes can be easily killed by thermal treatments such as cooking, therefore the contamination of RTE meat and poultry products with this pathogen is known to occur after the lethality treatment step (Cox et al., 1989; Tompkin, 2002). Vorst, Todd, and Ryser (2006) demonstrated the likelihood of cross contamination from sequential transfer of Listeria cells between delicatessen slicer blade and slices of turkey breast, bologna, and salami. Since Listeria is ubiquitous in nature, as RTE foods are exposed to the environment, Listeria can easily adhere to food products at any stage from manufacturing to retail or at domestic handling (Gombas et al., 2003). In addition, L. monocytogenes is capable of surviving antimicrobial treatments applied to the processing facility surfaces and manufacturing process (Cox et al., 1989; Norwood and Gilmour, 2000). Recent surveys across the world have reported an incidence of L. monocytogenes in RTE foods at retail levels from 0.89% to 23.4%

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A. Pal et al. / International Journal of Food Microbiology 126 (2008) 49–56

(Gombas et al., 2003; Van Coillie et al., 2004). Even with the smallest contamination levels, there is a potential for a large number of people to become infected with L. monocytogenes from RTE foods (Mead et al., 2006), mainly due to widespread market access and high consumption volume. Several studies have documented the handling abuses of RTE meat and poultry foods in terms of storage temperature and packaging conditions of leftovers during product shipment and at home storage, respectively (Audits International, 1999; Sergelidis et al., 1997; USDAFSIS, 2003; Godwin et al., 2007). The Food Safety and Inspection Service recommends that RTE foods are stored at 4.4 °C or below to avoid temperature-abuse conditions. Storage at higher temperature will result in spoiled or unsafe food relatively early due to faster multiplication of microorganisms (USDA-FSIS, 2006). However, according to survey data on refrigeration temperatures, only 73% of retail and 40% of home refrigerators maintain temperature at or below 5 °C, respectively (Audits International, 1999). In the same report, approximately 3% of commercial grocery refrigerators also were found to be operating at temperatures in the range of 15–17 °C. Growth evaluation under the probable storage conditions is important since it can help develop shelf life model to predict pathogen growth for consume-by date labels and help to control listeriosis incidences (NACMCF, 2005). The practice of storing RTE foods at refrigeration temperature is common for controlling the growth of L. monocytogenes and maintaining product quality; however, low temperature can only reduce the growth rate of this psychrotrophic microorganism. Another common practice to control Listeria in RTE meats includes addition of antimicrobials in the product formulation, especially salts of lactate and diacetate. These compounds have proven to be effective in maintaining low numbers of L. monocytogenes at temperatures below 5 °C. (Blom et al., 1997; Stekelenburg, 2003; Samelis et al., 2002). Previous microbial challenge studies in foods are conducted with a single strain or cocktail strains although this latter contamination would be a rare occurrence in the real world. It is well known from broth studies that strains of L. monocytogenes vary widely in their growth behavior (Barbosa et al., 1994; Begot et al., 1997; De Jesus and Whiting, 2003; Lianou et al., 2006). Using multiple strains allows for the inclusion of strains representing diverse backgrounds, but also assumes no intra-species competition and deviations from behavior when grown independently. However, this hypothesis has not been proven. The purpose of this study was to determine growth kinetic parameters of three strains of L. monocytogenes with distinct ribotypes in cooked uncured sliced turkey breast and cooked cured sliced ham. The results of this study could be useful for establishing safety-based ‘sell by’ or ‘consume by’ shelf life models for RTE sliced turkey breast and ham. 2. Materials and methods 2.1. Strains and preparation of inoculum Three distinct ribotypes of L. monocytogenes used in this study were DUP-1044A (4b serovar isolated from frankfurters linked to a multistate outbreak in 1998), DUP-1038 (4b serovar isolated from Mexicanstyle cheese in 1985), and DUP-1030A (1/2a serovar isolated from a sporadic case in 1998). All three strains were kindly provided by Dr. Martin Weidmann from Cornell University, Ithaca, NY. Selection of these three strains was based on their growth behavior in tryptic soy broth (TSB) and slurries of frankfurters and uncured sliced turkey breast at 4, 8, and 12 °C as previously conducted in our laboratory. Ribotypes DUP-1030A and 1044A were the two fastest growing strains and ribotype DUP-1038 was one of the slowest growing isolates among a total of 19 strains (Pal et al., 2008). The strains were stored in vials containing TSB (Neogen Corp., Lansing MI) and 10% glycerol (Sigma Chemical Co., St. Louis, MO) at −55 °C. For inoculum preparation, frozen suspensions were streaked on tryptic soy agar (TSA; Neogen Corp., Lansing MI) and TSA plates

were incubated at 37 °C for 24 h. Single colonies from TSA plates were transferred to tubes containing 10 ml TSB and allowed to grow at 37 °C for 24 h. Following incubation, counts of L. monocytogenes were enumerated by serially diluting in 0.1% peptone water (Neogen Corp., Lansing MI) and spread plating on TSA, which were incubated similar to as stated earlier. Working cultures were maintained at 4 °C for no more than four weeks on TSA slants. 2.2. Product description L. monocytogenes strains were inoculated onto cooked uncured sliced turkey breast and cooked cured sliced ham. The sliced meat and poultry products were supplied by a local producer and shipped in vacuum-sealed bulk packages to the laboratory in ice coolers in less than 24 h. Three types of sliced turkey breast and ham were used: 1) a control standard formulation containing no additional treatment/ additives, 2) standard formulation subjected to a high pressure processing treatment of 400 MPa for 15 min (HPP), and 3) standard formulation with the addition of a combination of 2.0% potassium lactate (PL) and 0.2% sodium diacetate (SD). The purpose of HPP was to reduce the initially present microflora in sliced meats and evaluate the impact of microbial competition effect on L. monocytogenes growth between sliced meats with greater background microflora (control) and reduced microflora (HPP). As reported by the manufacturer, sliced turkey breast contained 60 to 70% moisture, 18 to 22% protein, 1 to 3% fat, 1.8 to 2.2% salt, and included sugar, sodium bicarbonate, and carrageenan. Sliced ham had similar moisture content, 18 to 24% protein, 2 to 5% fat, and was brine cured with formulation containing 2.3 to 2.8% salt, 0.8% sugar, 0.5% phosphates, 500 ppm sodium erythorbate, and an ingoing amount of 190 ppm sodium nitrite. The turkey breast and ham samples were stored at −20 °C until the day of inoculation. Inoculation with L. monocytogenes was done within a week after receiving the turkey and ham products. 2.3. Initial microbial testing of turkey breast and ham products On the date of receipt, three random 25-g samples from each type of sliced turkey breast and ham from different batches were selected for enumerating the background psychrotrophic plate counts (PPC) and confirming the absence of any typical Listeria colonies. For measuring initial PPC, the samples were mixed with 225 ml of 0.1% peptone water and homogenized for 3 min. The homogenates, when required, were serially diluted in 0.1% peptone water, and 0.1-ml aliquots were plated on plate count agar (PCA, Sigma Chemical Co., St. Louis, MO). Counts were enumerated after 10 days incubation of PCA plates at 8 °C and expressed as log10 (CFU/g). For testing background Listeria, two replicates of 25 g samples of the meat and poultry products were homogenized with 225 ml Listeria enrichment broth (Sigma Chemical Co., St. Louis, MO) inside stomacher bags (Fisher Scientific®, Labplas Inc., Quebec, Canada) for 2 min using a stomacher (Tekmar Company, Cincinnati, OH). The homogenized samples were incubated at 37 °C for 24 h. Following enrichment, 0.1 ml of triplicate samples from each bag was spread plated on PALCAM agar (Neogen Corp., Lansing MI). 2.4. Inoculation experiments The frozen meat and poultry products were thawed at 8 °C for 24 h. After thawing, slices of turkey breast (mean weight: 27.9 g; mean radius: 5.2 cm; thickness: 0.3 cm) or ham (mean weight: 29.5 g; mean radius: 5.2 cm; thickness: 0.3 cm) from each product type were placed on the sterile surface of stomacher bags. The stomacher bags were aseptically cut open from three sides and kept inside a biosafety cabinet. A stationary phase inoculum of L. monocytogenes cultures incubated at 37 °C for 24 h in TSB was serially diluted in 0.1% peptone water and 1 ml containing 3–4 log10 CFU was separately inoculated on

A. Pal et al. / International Journal of Food Microbiology 126 (2008) 49–56

top surface of each product type. The inoculum was spread over the entire circular surface of turkey or ham slices using sterile glass rods. The slices were left for 5 min at room temperature to insure adhesion of cells on the product surface. Two slices from each product treatment that were inoculated with an individual L. monocytogenes strain were aseptically transferred using sterile forceps into nylon-polyethylene packages (3 mil standard barrier, 15.2 cm × 20.3 cm, Koch Industries, Kansas City, MO) with an uninoculated side of one slice placed over an inoculated side of the second slice. The packages were heat sealed with or without vacuum (95 kPa) using a vacuum packaging unit (Kramer Grebe, Compack, model D3560, Biedenkopf–Wallau, Germany) and then incubated at 4, 8, or 12 °C. 2.5. Sampling and microbiological analysis At least twenty packages containing two inoculated slices were stored at 4, 8, or 12 °C at each test level (strain, product type, temperature, and package) of the experiment and duplicate packages were analyzed at each sampling time. The packages containing turkey breast or ham were sampled periodically nine times for the counts of L. monocytogenes and four times for PPC after 90, 60 and 45 days incubated at 4, 8 and 12 °C, respectively. Samples were analyzed within 2 h after inoculation for day 0 counts. Before sampling, packages were surface wiped on the outside with a paper towel soaked in 70% (vol/ vol) ethanol. The microbial recovery method was modified from Luchansky, Porto, Wallace, and Call (2002). A slit was made aseptically to open the package for the microbial counts and 30 ml of 0.1% peptone water was aseptically added. The open end of the package was folded to close it and shaken vertically for at least 40 s, serially diluted in 0.1% peptone water, and 0.1 ml in single plate or 1 ml in four separate plates was surface plated. L. monocytogenes was enumerated on PALCAM agar and PPC were measured on PCA. The lowest detection limits for measuring L. monocytogenes and psychrotrophic counts of the analysis was 30 CFU/package (1.47 log10 CFU/package). The temperature/time parameters for incubation of PALCAM and PCA plates were 37 °C/ 48 h and 8 °C/10 days, respectively. On PALCAM agar L. monocytogenes was recognized as grey-colored colony with black precipitate. The microbial growth at each level was measured by replicating the experiments twice. 2.6. Curve fitting and statistical analysis Microbial counts from the two trials (n = 2) were averaged and converted to log10 CFU/package and plotted as a function of time for all the treatment levels. A composite curve of PPC growth data was plotted considering no influence from L. monocytogenes strain types. The curve fitting on L. monocytogenes growth was done using the Baranyi– Roberts model (Baranyi and Roberts, 1994) using DMFit 2.0 as kindly provided by Dr. Józef Baranyi. The model estimated the lag time (days) and growth rate (GR = 2.303 × slope, days− 1) with standard errors. The initial population (No, log10 CFU/package) and maximum population density (MPD, log10 CFU/package) were reported as observed from the experiments. The statistical difference between the growth parameters at P = 0.05 was estimated by comparing the 95% confidence intervals calculated from the average ± (1.96 × standard error). The time during lag and exponential phases were combined to calculate the time a strain would take for 2-log increment (TTR100). The difference in L. monocytogenes growth was tested separately on sliced turkey breast or ham with (PL/SD) and without (HPP and control) antimicrobials at each temperature. Analysis of variance mixed model procedure (Mixed-Proc) of SAS® (SAS, 2004) was used for the significant effects of fixed effects and their interactions at P ≤ 0.05. The model contained the effect of strain, sliced meat, package, time as fixed effects. Replication was used as a random effect to account for variance due to different batches between the replicates. Standard deviations from the average microbial counts were calculated and least squares

51

means were separated using pairwise comparison at the 95% confidence level. 3. Results Before inoculation, the absence of Listeria cells was confirmed on all sliced turkey breast and ham product batches. Initial PPC (average ± standard error) in the PL/SD containing meats were 1.69 ± 0.04 log10 CFU/g, in HPP were 1.25 ± 0.02 log10 CFU/g, and in control samples were 2.90 ± 0.05 log10 CFU/g for sliced turkey breast before inoculation with L. monocytogenes. Similarly in sliced ham, the counts of initial psychrotrophic bacteria were 1.32 ± 0.10 log10 CFU/g in PL/SD, 1.07 ± 0.10 log10 CFU/g in HPP, and 2.51 ± 0.02 log10 CFU/g in control samples. The initial L. monocytogenes counts on the inoculated sliced products at the three temperatures ranged from 2.28 to 4.87 log10 CFU/ package, but in approximately 75% of the measurements this number was between 3.20 and 3.40 log10 CFU/package (Tables 1–6). The Baranyi's model predicted that the average counts on day 0 ranged from 2.20 to 4.69 log10 CFU/package (not reported). From mixed-proc ANOVA analysis, the overall growth of L. monocytogenes in sliced products without PL/SD was significantly affected by strain type but packaging treatment (air or vacuum), and HPP (P b 0.05) effects were not consistent. In the presence of PL/SD, no Listeria growth was observed at 4 °C in sliced turkey breast (Table 1) and in sliced ham (Table 4). At 8 °C only DUP-1044A was able to show growth (N2-log) in PL/SD containing sliced ham (Table 5) and at same temperature all three strains were able to grow in uncured sliced turkey breast (Table 2). At the highest studied temperature (12 °C), growths of all three strains were observed in the presence of antimicrobials but the rates of growth were significantly lower than as noted in HPP-treated and in control samples (Tables 3 and 6) (P b 0.05). Therefore, when antimicrobials were included, the effect of strain type on growth behavior of L. monocytogenes was significant only at 8 and 12 °C in sliced turkey breast and in sliced ham.

Table 1 Growth kinetics parameters of Listeria monocytogenes ribotypes on different cooked uncured sliced turkey breasts packaged in air (A) or vacuum (V) at 4 °C Strain

Sliced turkey typea

Growth rate ± SD (day− 1)b

Lag time ± SD (days)

Nod

MPDe

TTR100f (days)

DUP-1044A

PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A)

0.00 ± 0.00 −0.01 ± 0.00 0.27 ± 0.01 0.27 ± 0.01 0.19 ± 0.01 0.23 ± 0.01 0.01 ± 0.00 0.02 ± 0.00 0.18 ± 0.01 0.18 ± 0.01 0.15 ± 0.01 0.16 ± 0.01 −0.01 ± 0.00 −0.01 ± 0.00 0.20 ± 0.01 0.23 ± 0.01 0.21 ± 0.01 0.22 ± 0.01

–c

3.11 4.08 3.91 3.91 3.98 3.49 3.70 3.51 3.98 4.27 4.17 3.73 2.92 3.89 3.73 3.61 3.86 3.73

3.76 3.65 9.35 9.16 9.33 8.68 3.95 4.10 9.07 7.79 7.54 8.81 3.91 3.80 9.07 8.80 8.94 8.80

–g

DUP-1038

DUP-1030A

8.0 ± 2.2 5.6 ± 2.5

16.9 16.9 24.6 20.1

25.9 25.9 37.9 34.8

22.5 20.2 22.1 20.6

Results are the average of at least two independent experiments. The growth rate and lag time were estimated using Baranyi–Roberts model (Baranyi and Roberts, 1994). a PL/SD: combination of 2% potassium lactate and 0.2% sodium diacetate; HPP: high pressure processed with 400 MPa for 15 min; Control: no treatment. b Negative value indicates inhibition or listericidal effects. c No lag observed. d No: observed average initial count in log10 (CFU/package). e MPD: observed maximum population density in log10 (CFU/package). f Time to reach 100 times any initial Listeria population. g No growth observed.

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A. Pal et al. / International Journal of Food Microbiology 126 (2008) 49–56

Table 2 Growth kinetics parameters of Listeria monocytogenes ribotypes on different cooked uncured sliced turkey breasts packaged in air (A) or vacuum (V) at 8 °C

Table 4 Growth kinetics parameters of Listeria monocytogenes ribotypes on different cooked cured sliced ham packaged in air (A) or vacuum (V) at 4 °C

Strain

Sliced turkey typea

Growth rate ± SD (day− 1)b

Lag time ± SD (days)

Nod

MPDe

TTR100f (days)

Strain

Sliced ham typea

Growth rate ± SD (day− 1)b

Lag time ± SD (days)

Nod

MPDe

TTR100f (days)

DUP-1044A

PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A)

0.26 ± 0.06 0.24 ± 0.05 1.86 ± 0.11 2.14 ± 0.10 1.76 ± 0.15 2.00 ± 0.19 0.02 ± 0.00 0.06 ± 0.01 1.16 ± 0.14 1.31 ± 0.16 0.97 ± 0.12 1.02 ± 0.11 0.23 ± 0.05 0.22 ± 0.06 1.78 ± 0.16 1.83 ± 0.12 1.63 ± 0.18 1.68 ± 0.17

35.3 ± 4.3 31.4 ± 4.3 –c

3.89 3.89 3.82 3.82 3.63 3.63 3.89 3.70 2.41 2.41 3.20 2.41 3.77 3.65 3.22 3.22 3.11 3.11

6.65 6.85 9.77 10.29 9.92 10.44 5.04 5.19 9.29 9.29 10.73 9.14 3.49 3.37 9.94 10.46 10.09 10.61

53.2 50.5 2.5 2.2 2.6 2.3 –g 76.4 8.0 6.0 8.3 7.0 55.0 56.3 2.6 2.5 2.8 2.7

DUP-1044A

PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A)

− 0.01 ± 0.00 − 0.01 ± 0.00 0.16 ± 0.02 0.16 ± 0.01 0.16 ± 0.01 0.17 ± 0.02 − 0.01 ± 0.00 −0.02 ± 0.00 0.13 ± 0.02 0.14 ± 0.02 0.13 ± 0.01 0.15 ± 0.02 − 0.01 ± 0.00 −0.02 ± 0.00 0.17 ± 0.02 0.18 ± 0.02 0.15 ± 0.01 0.18 ± 0.01

–c

3.29 3.07 4.37 4.34 4.37 3.49 3.70 3.76 4.40 4.51 4.17 3.73 3.70 3.03 3.53 4.40 3.53 3.46

3.18 3.10 9.06 8.98 9.06 8.68 3.95 3.21 8.36 8.65 7.54 8.81 3.29 2.90 7.48 9.15 8.50 8.22

–g

DUP-1038

DUP-1030A

4.0 ± 1.2 2.5 ± 1.1 3.6 ± 1.4 2.4 ± 1.1 35.4 ± 4.2 35.5 ± 5.2

DUP-1038

DUP-1030A

22.9 ± 5.0 15.7 ± 4.5 21.9 ± 4.0 17.4 ± 3.9

36.7 ± 4.4 27.5 ± 5.4 34.1 ± 2.5 29.5 ± 2.2

29.0 ± 6.1 33.6 ± 4.7 29.1 ± 4.7 23.6 ± 7.4

51.8 44.1 50.5 44.3

71.8 59.5 69.0 60.6

56.1 59.3 59.5 49.3

Results are the average of at least two independent experiments. The growth rate and lag time were estimated using Baranyi–Roberts model (Baranyi and Roberts, 1994). a PL/SD: combination of 2% potassium lactate and 0.2% sodium diacetate; HPP: high pressure processed with 400 MPa for 15 min; Control: no treatment. b Negative value indicates inhibition or listericidal effects. c No lag observed. d No: observed average initial count in log10 (CFU/package). e MPD: observed maximum population density in log10 (CFU/package). f Time to reach 100 times any initial Listeria population. g No growth observed.

Results are the average of at least two independent experiments. The growth rate and lag time were estimated using Baranyi–Roberts model (Baranyi and Roberts, 1994). a PL/SD: combination of 2% potassium lactate and 0.2% sodium diacetate; HPP: high pressure processed with 400 MPa for 15 min; Control: no treatment. b Negative value indicates inhibition or listericidal effects. c No lag observed. d No: observed average initial count in log10 (CFU/package). e MPD: observed maximum population density in log10 (CFU/package). f Time to reach 100 times any initial Listeria population. g No growth observed.

The average GR of all three strains in both sliced products consistently increased with an increase in temperature. In most cases, when no antimicrobials were present (HPP-treated and control), the growth rate increment from 4 to 8 °C and from 8 to 12 °C was approximately 10 and 2 fold, respectively (Tables 1–6). For example, at

4 °C the average growth rate of DUP-1044A in sliced HPP ham under vacuum packaging was 0.27 day− 1, but this parameter increased to 1.87 and 2.60 day− 1 respectively at 8 and 12 °C under similar conditions (Tables 1–3). Comparing GR of L. monocytogenes in uncured sliced

Table 3 Growth kinetics parameters of Listeria monocytogenes ribotypes on different cooked uncured sliced turkey breasts packaged in air (A) or vacuum (V) at 12 °C Strain

Sliced turkey typea

Growth rate ± SD (day− 1)b

Lag time ± SD (days)

Nod

MPDe

TTR100f (days)

DUP-1044A

PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A)

0.46 ± 0.11 1.09 ± 0.13 2.60 ± 0.13 2.72 ± 0.09 2.58 ± 0.43 4.10 ± 0.46 0.57 ± 0.12 0.44 ± 0.06 2.61 ± 0.19 2.31 ± 0.15 1.07 ± 0.10 1.29 ± 0.16 0.56 ± 0.11 0.91 ± 0.43 2.19 ± 0.25 2.22 ± 0.23 2.16 ± 0.10 2.57 ± 0.17

10.1 ± 3.7 –c

2.74 2.74 4.23 4.23 4.18 4.18 3.08 4.08 2.98 3.01 2.39 3.39 4.87 4.08 3.29 3.29 3.65 3.65

6.37 9.92 9.32 9.77 8.92 9.20 6.66 8.48 10.19 10.30 8.59 9.76 9.92 8.48 9.14 9.42 9.61 9.76

20.1 4.2 1.8 1.7 2.3 2.7 24.4 10.4 4.0 3.4 8.3 5.9 11.8 8.7 2.1 2.1 2.1 1.8

DUP-1038

DUP-1030A

0.5 ± 0.5 1.6 ± 1.7 16.2 ± 2.3 2.2 ± 0.3 1.4 ± 0.3 4.0 ± 1.5 2.3 ± 1.3 3.6 ± 3.5 3.7 ± 4.1

Results are the average of at least two independent experiments. The growth rate and lag time were estimated using Baranyi–Roberts model (Baranyi and Roberts, 1994). a PL/SD: combination of 2% potassium lactate and 0.2% sodium diacetate; HPP: high pressure processed with 400 MPa for 15 min; Control: no treatment. b Negative value indicates inhibition or listericidal effects. c No lag observed. d No: observed average initial count in log10 (CFU/package). e MPD: observed maximum population density in log10 (CFU/package). f Time to reach 100 times any initial Listeria population.

Table 5 Growth kinetics parameters of Listeria monocytogenes ribotypes on different cooked cured sliced ham packaged in air (A) or vacuum (V) at 8 °C Strain

Sliced ham typea

Growth rate ± SD (day− 1)b

Lag time ± SD (days)

Nod

MPDe

TTR100f (days)

DUP-1044A

PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A)

0.22 ± 0.02 0.22 ± 0.06 1.00 ± 0.20 1.08 ± 0.25 0.92 ± 0.25 0.96 ± 0.14 − 0.54 ± 0.50 −0.01 ± 0.00 0.84 ± 0.12 0.87 ± 0.10 0.86 ± 0.17 0.90 ± 0.29 −0.01 ± 0.00 −0.01 ± 0.00 0.92 ± 0.33 1.00 ± 0.12 0.80 ± 0.10 0.93 ± 0.15

36.5 ± 3.1 35.5 ± 5.2 5.1 ± 0.1 4.6 ± 0.1 6.2 ± 0.1 7.4 ± 0.1 –c

4.08 4.08 4.07 4.26 3.83 3.95 3.90 3.90 3.65 3.70 3.59 3.59 3.53 3.41 3.63 3.95 3.76 3.76

6.50 6.70 8.93 9.04 9.15 9.04 3.23 3.61 9.04 8.96 9.12 9.12 3.26 3.15 9.16 9.04 9.04 9.04

57.3 56.3 9.7 8.8 11.2 12.2 –g

DUP-1038

DUP-1030A

9.1 ± 1.2 9.9 ± 1.1 8.0 ± 1.9 7.5 ± 1.1

9.2 ± 2.0 6.4 ± 1.2 7.2 ± 1.2 4.4 ± 1.3

14.6 15.2 13.3 12.6

14.2 11.0 13.0 9.3

Results are the average of at least two independent experiments. The growth rate and lag time were estimated using Baranyi–Roberts model (Baranyi and Roberts, 1994). a PL/SD: combination of 2% potassium lactate and 0.2% sodium diacetate; HPP: high pressure processed with 400 MPa for 15 min; Control: no treatment. b Negative value indicates inhibition or listericidal effects. c No lag observed. d No: observed average initial count in log10 (CFU/package). e MPD: observed maximum population density in log10 (CFU/package). f Time to reach 100 times any initial Listeria population. g No growth observed.

A. Pal et al. / International Journal of Food Microbiology 126 (2008) 49–56 Table 6 Growth kinetics parameters of Listeria monocytogenes ribotypes on different cooked cured sliced ham packaged in air (A) or vacuum (V) at 12 °C Strain

Sliced ham typea

Growth rate ± SD (day− 1)b

Lag time ± SD (days)

Nod

MPDe

TTR100f (days)

DUP-1044A

PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A) PL/SD (V) PL/SD (A) HPP (V) HPP (A) Control (V) Control (A)

0.32 ± 0.05 0.72 ± 0.06 1.99 ± 0.10 2.18 ± 0.17 1.50 ± 0.10 1.66 ± 0.19 0.13 ± 0.02 0.48 ± 0.17 1.18 ± 0.11 1.42 ± 0.12 0.89 ± 0.11 1.45 ± 0.17 0.65 ± 0.11 0.65 ± 0.07 2.10 ± 0.25 2.31 ± 0.12 1.45 ± 0.09 1.60 ± 0.08

5.7 ± 3.1 –c

3.63 3.63 2.94 3.82 3.82 2.94 2.84 3.89 2.64 3.14 2.66 2.66 3.87 4.08 2.93 3.32 2.93 2.28

6.80 9.77 9.86 8.34 8.83 8.98 5.25 7.10 9.19 9.24 7.70 7.81 9.92 9.92 8.56 10.24 9.05 10.22

20.2 6.4 2.3 2.1 3.1 2.8 34.4 26.2 7.7 3.2 7.1 5.4 14.3 7.1 2.2 2.0 3.2 2.9

DUP-1038

DUP-1030A

16.7 ± 3.8 3.8 ± 0.9 1.9 ± 1.4 2.2 ± 0.7 7.2 ± 2.4

Results are the average of at least two independent experiments. The growth rate and lag time were estimated using Baranyi–Roberts model (Baranyi and Roberts, 1994). a PL/SD: combination of 2% potassium lactate and 0.2% sodium diacetate; HPP: high pressure processed with 400 MPa for 15 min; Control: no treatment. b Negative value indicates inhibition or listericidal effects. c No lag observed. d No: observed average initial count in log10 (CFU/package). e MPD: observed maximum population density in log10 (CFU/package). f Time to reach 100 times any initial Listeria population.

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all studied variables. For example, at 4 °C in sliced turkey breast significant differences for GRs of DUP-1044A in vacuum packaged samples were observed (P b 0.05) but not in samples that were packaged in air (Table 1). Similarly, there were no significant difference between GRs of DUP-1038 between HPP and control in either packaging type, but control samples had significantly longer lag times than HPPtreated samples, which resulted in greater TTR100 values in HPP than in control samples (P b 0.05) (Table 1). During the challenge studies, generally the psychrotrophic microbial population in HPP samples remained significantly lower than in the control at similar time points (P b 0.05) (Figs. 1 and 2). The antimicrobial combination PL/SD not only affected the growth of L. monocytogenes but also delayed growth of psychrotrophs and lowered their MPD by at least 2 logs during the experiments at each temperature (Figs. 1 and 2). However, MPD of psychrotroph in slices with PL/SD increased in air packaging and at higher storage temperature. In turkey breast slices without PL/SD, MPD of PPC reached 8.0 log10 CFU/package by at least 50, 10, and 5 days at 4, 8, and 12 °C, respectively (Fig. 1). At 4 °C in the PL/SD treated turkey slices, MPD did not increase more than 6.0 log10 CFU/package, while an increase of PPC to at least 8.0 log10 CFU/package at 8 °C was reached in 60 days. At 12 °C, a similar MPD increment was reached in vacuum packages at 30 days and in air packages at 20 days (Fig. 1). In cured slices of ham with PL/SD, irrespective of packaging treatment, PPC did not increase to more than 6.0 and 8.0 log10 CFU/package when stored at 4 °C for 90 days and for 60 days at 8 °C, respectively (Fig. 2). The psychrotroph counts reached more than 8.0 log10 CFU/package only when ham slices with PL/SD were kept at 12 °C. 4. Discussion

turkey breast and cured sliced ham, the rates were greater on turkey breast slices at all studied temperatures. The sliced product type had an effect on both the lag and growth phases. In samples, without PL/SD, the minimum time to increase by 2 log10 (TTR100) in sliced turkey breast at 4, 8, and 12 °C was 16.9, 2.2, 1.7 days, respectively (Tables 1–3) and in sliced ham was 44.1, 8.8, 2.0 days, respectively (Tables 4–6). No growth occurred at 4 °C in turkey slices with added antimicrobials; however TTR100 values in the range of 50.5 to 76.4 days at 8 °C and 4.2 to 24.4 days at 12 °C were calculated (Tables 2 and 3). Similarly for cured sliced ham with added PL/SD, the range of TTR100 at 8 and 12 °C varied from no-growth to 57.3 days and 6.4 to 34.4 days, respectively (Tables 5 and 6). In both sliced products without PL/SD in their formulation, the shortest TTR100 of 1.7 days was determined for ribotype DUP-1044A in air packaged turkey slices at 12 °C previously treated with HPP (Table 3). Growth inhibition from antimicrobials was also temperature, packaging, and strain dependent. Compared to other two strains, a slower growth (longer lag phases or lower growth rates) of DUP-1038 was noted at all growth conditions (P b 0.05). In general, the strains DUP-1044A (serotype 4b) and DUP-1030A (serotype 1/2a) had shorter lag times and greater GR than the strain DUP-1038 (serotype 4b). For example, the range of average lag time at 4 °C in control sliced ham was 27.5 to 36.7 days for DUP-1038 as compared to the range of 15.7 to 22.9 days for DUP-1044A (Table 4). Under similar product and temperature, GR varied from 0.15 to 0.18 day− 1 in fast growing strains versus 0.13 to 0.15 day− 1 in a slower growing strain (Table 4). At 12 °C, DUP-1044A reached an MPD of only 6.37 log10 CFU/package under vacuum packaging with PL/SD, but this parameter was 9.92 log10 CFU/ package in the air package sliced turkey breast (P b 0.05) (Table 3). Under similar conditions MPD values of strain DUP-1038 were 6.66 and 8.48 log10 CFU/package, respectively (Table 3) (P b 0.05). In the absence of PL/SD, MPD of L. monocytogenes was always able to reach greater than 7.40 log10 CFU/package at all temperatures for the two products. In the HPP-treated samples the growth of all three L. monocytogenes strains had longer lag times, greater GRs, or MPDs as compared to controls treatment, but significant differences were not consist under

The current study showed that the application of a lactate– diacetate combination can effectively inhibit the growth of L. monocytogenes on RTE foods as long as the product is not subjected to temperature-abuse conditions. At temperatures when the PL/SD was able to inhibit growth of L. monocytogenes, the type of packaging conditions had little impact on its growth. The current results stress the importance of avoiding temperature-abuse conditions during storage and handling. Usually refrigerated RTE meat and poultry are marked with a quality-based shelf life of 60 to 90 days depending on the manufacturer. As estimated by Gombas et al. (2003), the contamination levels of L. monocytogenes in luncheon meats could be from 0.04 to 104 CFU/g. Our results showed that in the event of contamination of deli meat and poultry foods even with a lowest detectable level, strains of L. monocytogenes were capable of exhibiting growth especially under temperature-abuse conditions. This can be illustrated using the growth parameters of DUP-1044A at 8 and 12 °C in sliced turkey breast, which showed that the time to reach a 100-fold increase for Listeria would occur in less than 60 days, even with an initial load at 0.04 CFU/g (i.e. the detection limit of 1 cell/25 g). In sliced ham, when L. monocytogenes did grow, this limit was also reached in less than 60 days. This observation further supports the need for a post packaging treatment to ensure safety. Lianou et al. investigated the growth of Listeria on vacuum packaged ham (2007a) and turkey breast (2007b) for several time periods when stored at 4 °C followed by aerobic storage at 7 °C for 12 days to simulate a consumer handling and poor refrigeration conditions. Their results showed that L. monocytogenes reached more than 107 CFU/cm2 from initial levels of 40 CFU/cm2 within 12 days when sliced products were stored at 7 °C. This is similar to our results with control samples at 8 °C under air packaging. The average growth rate of L. monocytogenes was 0.9 day− 1 in sliced cured ham in Lianou et al. study (2007a) and closely matched with our results (0.80 to 0.96 day− 1) in control samples at 8 °C with the three strains. However, with PL/SD treated sliced ham, our results showed no growth at 8 °C; whereas,

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A. Pal et al. / International Journal of Food Microbiology 126 (2008) 49–56

Fig. 1. Growth of psychrotrophic microflora (log10 CFU/package, n = 2) on vacuum- or air-packaged sliced turkey breast processed with — combination of 2% potassium lactate (PL) and 0.2% sodium diacetate (SD) in the final formulation (▲), high pressure with 400 MPa for 15 min prior to inoculation, HPP (□), and control (○) at (A) 4 °C, (B) 8 °C, and (C) 12 °C. The figure contains psychrotrophic counts obtained from three independent Listeria monocytogenes inoculation studies on sliced turkey breast. Each data point represents the average value of two random samples at each replicate.

growth rates of approximately 0.48 day− 1 were observed by Lianou et al. (2007a). Such a difference in L. monocytogenes growth could be due to variation in initial levels of PL/SD, which were not reported in Lianou et al.'s study. At 12 °C, Listeria cells were able to grow to a level of 100 CFU/g (or 2500/25 g sample) in the range of 2.1 to 7.7 days. This is of greater concern especially to countries where a tolerance of L. monocytogenes of 100 CFU/g at the time of consumption is allowed in delicatessen foods (Todd, 2007). It has been reported that the growth of L. monocytogenes is reduced with the rise in competing background microflora present in the broth or meat medium (Tsigarida et al., 2000; Pleasant et al., 2001). In this study, HPP was used to reduce the background flora before inoculation. Although the difference in PPC between HPP and control were significant in most cases, the corresponding effect on the growth of L. monocytogenes was not consistently significant. It can be hypothesized that PPC differences between HPP and control samples in this study were not sufficient to influence the growth behavior of L. monocytogenes strains. Listeria contamination of deli meats after high pressure treatment of packages has not been reported, nevertheless for certain packaging, temperature or strain types, it still reflects the possibility of an increased pathogen growth in samples with reduced background microflora, something that can occur at home or food service establishments when the package is opened and left for future use.

Mataragas and Drosinos (2007) suggested that the end of qualitybased shelf life can precede the time for L. monocytogenes to grow to the microbiological criterion for RTE meat products and thus consumers would likely discard a product before L. monocytogenes can reach a risk level. In their study, Mataragas and Drosinos (2007) used 102 CFU/g as a safety objective level as established by the International Commission on Microbiological Specifications for Foods (1994). This value is as also used in some countries (Todd, 2007). As reflected from the current results, the growth of background microorganisms was less in HPP-treated samples, thus it could delay the on set time for microbial spoilage to be visual. Usually the signs of microbial spoilage in RTE meat or poultry products occur when the count of background microorganisms is 106 CFU/g (7.5 log10 CFU/package in our study) or greater (Korkeala et al., 1989), and our findings indicated that PPC reached this level by 60 days for products both with and without PL/SD above 4 °C. Thus during this time, Listeria cells would be able to grow to a significant risk level before the time for visual spoilage by background flora or end of the quality-based shelf life date, especially in uncured lower initial microbial counts (HPP-treated) products in which case the safety-based shelf life may override quality-based shelf life. In order to develop predictive food microbiology models or quantitative microbial risk assessments for L. monocytogenes in RTE foods,

A. Pal et al. / International Journal of Food Microbiology 126 (2008) 49–56

55

Fig. 2. Growth of psychrotrophic microflora (log10 CFU/package, n = 2) on vacuum- or air-packaged sliced ham processed with — combination of 2% potassium lactate (PL) and 0.2% sodium diacetate (SD) in the final formulation (▲), high pressure with 400 MPa for 15 min prior to inoculation, HPP (□), and control (○) at (A) 4 °C, (B) 8 °C, and (C) 12 °C. The figure contains psychrotrophic counts obtained from three independent Listeria monocytogenes inoculation studies on sliced ham. Each data point represents the average value of two random samples at each replicate.

the fastest growing strain at refrigeration temperature is needed as a conservative index for the shelf life models (Scott et al., 2005). Therefore, another focus of this study was to compare the growth of 3 different strains of L. monocytogenes under similar treatments. Obviously one cannot predict the strain type that may contaminate a particular batch of food products, yet it is known that half of listeriosis cases are implicated to the 4b and 1/2a serotypes but similar serotype dominance is non-matching in environmental samples and processed foods (Vitas and Garcia-Jalon, 2004). Inoculation with the 4b serotype is common in challenge studies; however current results showed that the strains, DUP-1044A and DUP-1038, despite having a common serogroup, showed a different growth behavior. The third strain DUP1030A that belonged to the 1/2a serotype had similar fast growth characteristics as DUP-1044A. These three strains also had similar relative differences in their growth kinetics in broth medium (Pal et al., 2008). The difference in history of these strains could explain the growth differences amongst them, e.g. DUP-1044A was isolated from frankfurters from a listeriosis outbreak while DUP-1038 was an isolate from Mexican-style cheese outbreak. The information about the isolation source of DUP-1030A was incomplete. Prospective research should be directed in understanding the growth differences in strains at the molecular level, so that a marker can be targeted to select the fastest growing strain.

Determination of the time period during which a product will remain safe and stable can be accomplished using inoculation testing and shelf life evaluations (Betts, 2006). L. monocytogenes has been recognized as safety indicator in RTE foods (NACMCF, 2005), therefore safety-based shelf life can be determined by prediction of the time the fastest growing pathogen requires to reach a level practical for regulatory and industrial purposes (Mataragas et al., 2006). We estimated the kinetic parameters and specific extent of growth (lag time, growth rate, and TTR100) within the environmental conditions that RTE foods are exposed. A further validation step would be assessing growth under dynamic temperature storage conditions; however, predicting a pattern of temperature fluctuation has always been a challenge. To control Listeria growth from post-lethality contamination, processed meat manufacturers are using sodium or potassium salts of lactate and sodium diacetate (Stekelenburg, 2003). Interests in natural and low sodium food products due to nutritional concerns are also on rise (Matthews and Strong, 2005). With the intent of providing a safety-based shelf life (i.e. “use by XX-XX”) for both cured and uncured RTE meat and poultry products, the results of this study present a comprehensive evaluation of the growth behavior of both L. monocytogenes and spoilage microflora under treatments and conditions that packaged RTE meat and poultry products can encounter.

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Using these data from the fastest growing strains, processors can decide the shelf life period on their products depending on the formulation. For a safety-based shelf life label, a ‘sell-by’ date can be marked based on our data from vacuum packaged products, better yet one should also include the “Use by” date based on the time after consumer opening of the package and storing in air at temperatures above 4 °C. The growth parameters from storage in air packaging reflect its utility in ‘consume-by’ date prediction. The outcomes of this study signify the importance of adequate temperature handling of RTE meat and poultry products. The risks in products formulated with 2.0% PL and 0.2% SD are lower, but L. monocytogenes has potential to grow particularly under conditions of temperature-abuse. 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