Viability of Listeria monocytogenes was monitored during refrigerated (4°C) and/or frozen (i.e., deep chilling at −2.2°C) storage on casing-cooked hams that were commercially prepared with and without potassium lactate and sodium diacetate (1.6%), buffered vinegar (2.2%), buffered vinegar and potassium lactate (1.7%), or a blend of potassium lactate, potassium acetate, and sodium diacetate (1.7%). A portion of these hams were subsequently surface treated with lauric arginate ester (LAE; 44 ppm). In phase I, hams (ca. 3.5 kg each) were sliced (ca. 0.7 cm thick, ca. 100 g), inoculated (ca. 4.0 log CFU per slice), surface treated with LAE, and stored at either 4°C for 120 days or at −2.2°C for 90 days and then at 4°C for an additional 120 days. In phase I, without antimicrobials, the population of L. monocytogenes increased by ca. 5.9 log CFU per slice within 120 days at 4°C; however, pathogen levels increased only slightly (ca. 0.45 log CFU per slice) for hams formulated with potassium lactate and sodium diacetate and decreased by ca. 1.2 log CFU per slice when formulated with the other antimicrobials. For slices held at −2.2°C and then stored at 4°C, but not treated with LAE, L. monocytogenes increased by ca. 4.5 log CFU per slice for controls, whereas when formulated with antimicrobials, pathogen levels decreased by ca. 1.4 to 1.8 log CFU per slice. For product treated with LAE, L. monocytogenes increased by ca. 4.0 log CFU per slice for controls, whereas when formulated with antimicrobials, pathogen levels decreased by ca. 0.9 to 1.9 log CFU per slice. In phase II, whole hams (ca. 1.0 kg each) containing antimicrobials were inoculated (6.8 log CFU per ham) and then stored at −2.2°C for 6 months. Pathogen levels decreased by ca. 2.0 to 3.5 log CFU per ham (without LAE treatment) and by ca. 4.2 to 5.2 log CFU per ham (with application of LAE via Sprayed Lethality in Container) when product was held at −2.2°C. In general, deep chilling hams was listericidal, and inclusion of antimicrobials in the formulation suppressed outgrowth of L. monocytogenes during extended cold storage.
As the etiological agent of several recent recalls and related illnesses attributed to contaminated and/or improperly prepared or stored food, Listeria monocytogenes remains an appreciable threat to public health (26, 27). Ready-to-eat (RTE) foods with an extended (refrigerated) shelf life, such as vacuum-packaged, delicatessen-type red meat and poultry products, are of particular concern because L. monocytogenes is both psychrotrophic and facultatively anaerobic. The growth temperature of this bacterium ranges from −1.5 to 45°C, but this can vary somewhat depending on the strain and on the intrinsic and extrinsic parameters of a food or the composition of a synthetic medium (7, 10, 11, 18, 20, 33). Regardless, L. monocytogenes can survive, and even grow, on most RTE meats despite conditions of higher salt, inclusion of preservatives, and/or stringent control of temperature and atmosphere within which such products are stored.
In comparison to other foodborne pathogens, L. monocytogenes presents a particular challenge to food processors because it is widely distributed in nature, persistent on occasion in some processing plants and in the refrigerators of consumers, and/or resistant to or tolerant of food-relevant conditions of pH, NaCl, atmosphere, and temperature. In general terms, the prevalence of this pathogen in cooked RTE red meat and poultry products has declined appreciably over the past 20 years, down from ca. 4.61% in the early 1990s to ca. 0.32% in 2010 (35). More recently, the recovery rate of L. monocytogenes from some 25,000 higher-volume, higher-risk RTE food samples regulated by the U.S. Department of Health and Human Services, U.S. Food and Drug Administration and the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service purchased between 2010 and 2012 at retail groceries within four FoodNet sites ranged from 0.049 to 1.0% (5, 15); this is appreciably lower than a recovery rate of ca. 1.6 to 1.8% reported over a decade earlier in two studies of similar magnitude, scope, and depth that sampled many of these same food categories and types and that were also purchased at grocery stores (9, 36).
Several advances related to the development and delivery of biological, chemical, and physical interventions, as well as implementation of strict regulatory guidelines, have quite arguably contributed directly to the steady decline in the recovery rate of L. monocytogenes from RTE foods over the past 20 years (7, 26, 34). Some of these advances were made to extend the shelf life of meat and poultry products while in distribution, at retail, or in the home to accommodate the extended anticipated shelf life. Other advances were necessitated by consumer preference and attendant trends toward so-called clean-label products and foods that are lower in salt and preservatives that receive minimal processing. Among the most effective and most widely used intervention is the inclusion of organic acids, notably lactates, as an ingredient in the formulation of RTE red meat and poultry products. Likewise, among the most widely practiced approaches for postprocessing treatment of RTE meats is the use of the Sprayed Lethality in Container (SLIC) (12, 24, 25) and electrostatic spray assisted (ESS) (16, 28) methods for delivery of antimicrobials into product containers or directly onto the product surface, respectively. The efficacy of such interventions and delivery methods can be potentiated when used in combination with proper handling of products and appropriate control of temperature and/or atmosphere during storage and further processing and handling.
In the present study, we evaluated the antilisterial potential of various food-grade chemicals as ingredients in commercial, water-added, boneless hams in combination with extended (3 to 6 months) storage at refrigeration (4°C) and freezing (−2.2°C) temperatures. The objective was to quantify both the safety and quality of boneless hams that may be stored for an appreciable time at near-frozen conditions (aka “deep chilling hams” at −2.2°C) while still under the control of the processor.
MATERIALS AND METHODS
Approximately equal numbers of a five-strain cocktail of L. monocytogenes (strains MFS-2, MFS-102, MFS-104, MFS-105, and MFS-110) were used to surface inoculate boneless, water-added hams as described previously (22) and as detailed below.
One batch of freshly manufactured, casing-cooked vacuum-packaged hams was formulated with and without potassium lactate and sodium diacetate (PM; 1.6%; Galimax ProMeat Plus, Galactic Inc., Milwaukee, WI), buffered vinegar (BV; 2.2%; e(Lm)inate V, Hawkins Inc., Roseville, MN), buffered vinegar and potassium lactate (VL; 1.7%; e(Lm)inate VL, Hawkins), or potassium lactate, potassium acetate, and sodium diacetate (LAD; 1.7%; e(Lm)inate LAD, Hawkins). Hams were formulated to a protein fat free target of 17.0 and exhibited an average finished product moisture level of 72.35%. The finished product salt concentration averaged ca. 2.35% and a final pH before inoculation of about pH 6.0. Hams were formulated to contain ca. 200 ppm of ingoing nitrite by formulation, with ca. 10 to 30 ppm of residual nitrite typically remaining after cooking. These products were obtained directly from a producer-collaborator (Clemens Food Group, Hatfield, PA), transported on ice to the USDA, Agricultural Research Service (ARS) Eastern Regional Research Center (Wyndmoor, PA), and stored at 4°C for up to 2 days prior to being inoculated with the five-strain cocktail of L. monocytogenes.
Hams (ca. 3.5 kg each) were aseptically removed from their original package, sliced (ca. 0.7 cm thick; ca. 100 g; ca. 13 cm in diameter), repackaged into nylon-polyethylene bags (Koch Supplies, Kansas City, MO), and then surface inoculated with 1 ml (500 μl per each side or face) of the five-strain cocktail of L. monocytogenes, using a pipet to achieve a target level of ca. 4.0 log CFU per slice essentially as described (23). Each package was then massaged by hand for ca. 20 s to ensure for adequate coverage of the inoculum. Next, 1.7 ml of ethyl-N-dodecanoyl-L-arginate hydrochloride (LAE; 44 ppm; CytoGuard LA, A&B Ingredients Inc., Fairfield, NJ) was delivered (or not) into each package via the Sprayed Lethality In Container (SLIC) (12) delivery method. Packages were then vacuum sealed to 950 mbar using a Multivac A300/16 vacuum-packaging unit (Sepp Haggemuller KG, Wolf-ertschwenden, Germany) and were stored for up to 120 days at 4°C or at −2.2°C for 3 months and then at 4°C for 120 days.
Whole, casing-cooked, water-added hams (ca. 1.0 kg each) were aseptically removed from their original package and placed into nylon-polyethylene bags (Koch Supplies) before a pipet was used to introduce 2 ml of the L. monocytogenes cocktail into each bag to achieve a target level of about 6.8 log CFU per ham, as mentioned above. Packages containing inoculated chubs were massaged by hand for ca. 20 s for inoculum distribution and then were surface treated (or not) with 4.5 ml of LAE (44 ppm; A&B Ingredients Inc.). Packages were vacuum sealed as described above and were stored for up to 6 months at −2.2°C.
The pathogen was recovered from ham slices and hams using the USDA-ARS package rinse method (14) and was enumerated by adding 25 ml (slices) or 40 ml (hams) of either 0.1% peptone water or Dey-Engley neutralizing broth (Difco, BD, Franklin Lakes, NJ) to samples that were not treated or were treated with LAE, respectively. The rinsate was direct plated onto duplicate modified Oxford agar plates (Difco), which were subsequently incubated at 37°C for 48 h (detection limit = ≤1.56 log CFU per ham or ≤1.35 log CFU per slice). Two trials and three replicates per each sampling point for each trial for each product formulation were conducted.
Data were analyzed using version 9.1.3 of the SAS statistical program (SAS Institute, Cary, NC). Analysis of variance was used to determine the effects of type and concentration of antimicrobial on the viability of L. monocytogenes during extended refrigerated or frozen storage. Mean separations were performed using the Bonferroni least significant difference method to determine the significant differences (P ≤ 0.05) among treatments.
RESULTS AND DISCUSSION
From a regulatory perspective, L. monocytogenes is arguably the bacterial foodborne pathogen that has received the most attention over the past 25 years. Although the pathogen is eliminated from RTE products during processing, the bacterium can be reintroduced onto the surface of RTE red meat and poultry products if the finished product is exposed to the food processing environment before packaging and/or during handling at retail and food service establishments, as well as in the home (18, 25, 26). Postprocessing contamination, along with the current trend toward minimally processed, clean-label foods, provides justification for continued research to develop and validate interventions to manage the threat of listerosis in RTE meats. Interventions such as low-temperature storage and inclusion of food-grade antimicrobials into the formulation of RTE meats can substantially slow the growth of L. monocytogenes in and on red meat and poultry products. Germane to the present study, the average generation time of L. monocytogenes at an ideal (4°C) refrigeration temperature was estimated at 43 h, with the bacterium being only somewhat adversely affected by temperatures ≤0°C (3, 26). In fact, among foodborne pathogens, L. monocytogenes is considered to be one of the most resistant to freezing (1, 19). Further studies are needed to evaluate the effect of low-temperature storage of meats in combination with food-grade antimicrobials as ingredients or with surface-applied agents as an economically favorable and efficacious strategy to better manage L. monocytogenes in and on RTE meats.
As one objective of the present study, we monitored the fate of L. monocytogenes on ham slices during storage at 4°C, with and without prior deep chilling (storage at −2.2°C for 3 months). We observed that inclusion of PM, BV, VL, or LAD in the formulation of hams was significantly (P ≤ 0.05) more effective at suppressing outgrowth of L. monocytogenes compared to hams formulated without these food antimicrobials over the anticipated refrigerated shelf life of 120 days. However, inclusion of BV, VL, or LAD in the formulation was significantly (P ≤ 0.05) more effective at inhibiting outgrowth of the pathogen than PM over 120 days of storage at 4°C. In addition, regardless of whether or not slices of ham were formulated with antimicrobials, surface treatment with LAE delivered less (P ≥ 0.05) than an anticipated initial lethality toward the pathogen when compared with otherwise similar hams (sliced or chubs) that were not surface treated with LAE. More specifically, when ham slices were stored at 4°C without being previously held for 3 months at −2.2°C, in the absence of antimicrobials as an ingredient or as a surface-applied agent, pathogen numbers increased by ca. 5.9 log CFU per slice during the refrigerated shelf life of the ham slices (Fig. 1). When product was formulated with antimicrobials but was not surface treated with LAE, pathogen levels remained relatively unchanged on ham slices containing PM (ca. 0.45 log CFU per slice increase) but decreased by ca. 1.2 log CFU per slice for hams formulated with BV, LAD, or VL (Fig. 1). In the absence of antimicrobials as an ingredient, but when surface treated with LAE, after an initial reduction of ca. 0.4 log CFU per slice compared with otherwise similar product that was not initially treated with LAE, pathogen numbers increased by ca. 4.9 log CFU per slice during the refrigerated shelf life of the ham slices. When product was formulated with PM and was surface treated with LAE, pathogen levels were initially about 0.3 log CFU per slice lower than similar product that was not surface treated with LAE, but then pathogen levels increased slightly (ca. 0.5 log CFU per slice increase) over the remainder of the refrigerated storage period. For products formulated with the other antimicrobials and surface treated with LAE, pathogen levels were initially about 0.47 log CFU per slice lower than similar product that was not surface treated with LAE, but then pathogen levels decreased by an additional ca. 0.85 log CFU per slice over the remainder of the storage period.
When slices of ham were held at −2.2°C and then stored at 4°C for up to 120 days, regardless of whether or not hams were formulated with antimicrobials and/or surface treated with LAE, no statistical differences (P ≥ 0.05) were observed in pathogen numbers after 3 months of storage at −2.2°C (Fig. 2). Nonetheless, inclusion of PM, BV, VL, or LAD in the formulation, alone or in combination with surface treatment with LAE, was significantly (P ≤ 0.05) more effective at inhibiting outgrowth of the pathogen when compared to hams that were formulated without antimicrobials that were surface treated (or not) with LAE over 120 days of storage 4°C. However, all four antimicrobials tested were equally effective (P ≥ 0.05), alone or in combination with LAE, after holding or deep chilling at −2.2°C and then storage at 4°C for up to 120 days. More specifically, when slices of ham were held at −2.2°C and then were stored at 4°C for up to 120 days, in the absence of antimicrobials as ingredients or as a surface-applied agent, pathogen numbers decreased by ca. 0.6 log CFU per slice after 3 months of storage at −2.2°C, and then pathogen numbers increased by ca. 4.5 log CFU per slice compared to starting levels during storage at 4°C for 120 days (Fig. 2). Similar results were obtained when hams were formulated without added antimicrobials but were surface treated with LAE via SLIC (Fig. 2): pathogen numbers decreased by ca. 0.5 log CFU per slice after 3 months of storage at −2.2°C and then increased by ca. 4.0 log CFU per slice compared with starting levels during storage at 4°C for 120 days. For slices formulated with antimicrobials but not surface treated with LAE, L. monocytogenes numbers decreased by ca. 0.5 log CFU per slice after 3 months of storage at −2.2°C and then decreased by an additional ca. 0.4 to 0.9 log CFU per slice after subsequent storage at 4°C for 120 days (Fig. 2). Likewise, when hams were formulated with PM, BV, LAD, or VL and were treated with LAE via SLIC and subsequently were held for 3 months at −2.2°C, regardless of the formulation, pathogen numbers decreased by ca. 0.5 to 0.9 log CFU per slice as a consequence of applying LAE via SLIC (Fig. 2). After subsequent storage at 4°C for 120 days, an additional decrease of 0.2 to 1.1 log CFU per slice was observed.
Inclusion of salts of organic acids (i.e., lactate and diacetate) and buffered vinegar in the formulation of RTE meats reportedly suppressed outgrowth of L. monocytogenes during extended refrigerated storage (1, 4, 17, 24, 26). Hence, under the conditions tested herein, we expected that pathogen numbers would remain relatively unchanged over the refrigerated shelf life of hams formulated with PM, BV, LAD, or VL. In addition, studies established that LAE applied via SLIC onto the surface of RTE meats was effective at delivering an initial lethality (ca. 1.1 to 5.0 log CFU) of L. monocytogenes within 2 to 24 h at 4°C (12, 13, 17, 25, 31), but was not effective at inhibiting outgrowth during storage. However, there has been little information published on the combined effect of using organic acids as ingredients and surface-applied LAE for products, such as hams, that for practical and logistic reasons may be subjected to an extended frozen storage, that is, that may be held for 3 to 6 months at −2.2°C.
In phase II of the present study, we evaluated the effect of deep chilling whole hams in combination with antimicrobial ingredients and a surface-applied food-grade surfactant on viability of L. monocytogenes. Over 6 months of storage at −2.2°C, no statistical differences (P ≥ 0.05) were observed between hams that were formulated with PM, BV, VL, or LAD compared with those that were formulated without antimicrobials (Fig. 3). However, a significant (P ≤ 0.05) initial lethality was observed on hams that were surface treated with LAE via SLIC compared with otherwise similar hams that were not surface treated with LAE on day 0 of refrigerated storage and during 6 months of storage at −2.2°C (Fig. 3). For whole hams formulated with or without food-grade chemicals, but not surface treated with LAE, that were held at −2.2°C for over 6 months, pathogen numbers decreased by ca. 2.0 to 3.5 log CFU per ham compared with starting levels. However, when hams were surface treated with LAE via SLIC, the initial numbers of L. monocytogenes were reduced by ca. 3.5 to 4.5 log CFU per ham on day 0 of storage compared with otherwise similar product not treated with LAE via SLIC (Fig. 3). Thereafter, an additional decrease of ca. 0.7 log CFU per ham was observed after 6 months of storage at −2.2°C for all products treated with LAE (Fig. 3). These data confirm that deep chilling of hams at −2.2°C is appreciably listericidal, especially when used in combination with the intrinsic properties of the ham formulations tested herein.
As expected, in the present study, we observed that, regardless of the formulation, storage of water-added, casing-cooked hams at −2.2°C for up to 6 months significantly affected the viability of L. monocytogenes. Geornaras et al. (8) also evaluated prior cold storage (i.e., 1.7°C for up to 180 days) of noninoculated, nonsliced deli meats on subsequent potency of lactate-diacetate for controlling L. monocytogenes introduced after opening the packages and slicing the product. The authors concluded that product age did not affect the antilisterial activity of the lactate-diacetate. Other investigators (21, 22, 30) reported survival of L. monocytogenes on the surface of frankfurters during frozen storage for up to 3 months. Although freezing temperatures may affect L. monocytogenes viability on RTE meats during storage, pathogen numbers can appreciably increase upon subsequent thawing or storage at 4°C (11, 21). Thus, in addition to temperature, the most effective strategy to control L. monocytogenes in RTE meats is inclusion of food-grade chemicals as ingredients, notably, surfactants such as LAE, salts of organic acids such as lactate and diacetate, and clean-label ingredients such as buffered vinegar (2–4, 6, 12, 18, 29, 31, 32). As noted by Zhang et al. (37), the antilisterial efficacy of food-grade chemicals can be appreciably affected by the brand or batch of a given antimicrobial, product pH, and/or the initial and evolving types and levels of the indigenous flora. However, a balance must be reached between adopting effective interventions to enhance safety and address regulatory requirements and, at the same time, maintaining the quality and sensory attributes of the final product at a reasonable cost. Also, given that the demand for hams is generally seasonal as opposed to flat throughout the year, processors need to build an inventory of hams in anticipation of meeting the always looming cyclic demand for such products. Hams typically have an extended refrigerated shelf life of >120 days at 4°C. At first glance, this may seem quite lengthy, but in reality it might not be sufficient time for some processors to build adequate inventory for peak demand. As demonstrated in the present study, deep chilling of hams provides appreciable anti-listerial activity for at least 6 months at −2.2°C. When used in combination with food-grade and/or clean-label ingredients, with or without surface treatment with LAE, the deep chilling of hams can further enhance product safety and extend shelf life during (subsequent) extended storage at 4°C without appreciably affecting product quality (data not shown). Thus, as an added benefit to the antilisterial potency of storing hams at −2.2°C, the practice of “deep chilling” (aka “sleeping hams”) the product will help processors insure that sufficient hams are available throughout the year, and especially during peak periods, and will also enhance product safety and quality.
Special thanks are extended to Nelly Osoria, Laura Shane, and Laura Stahler of the USDA-ARS-ERRC (Wyndmoor, PA) for their assistance and technical expertise. We also express our gratitude to Bryan Vinyard (USDA-ARS, Beltsville, MD) for statistical analyses of the data herein. We are particularly thankful for the resources, support, and assistance of our long-standing collaborators at Clemens Food Group (Hatfield, PA). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.