Irradiation of Ready-to-Eat Meats: Eliminating Listeria monocytogenes

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Chapter 5

Irradiation of Ready-to-Eat Meats: Eliminating Listeria monocytogenes While Maintaining Product Quality 1

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C h r i s t o p h e r H. Sommers , N a t a s h a Keser , X u e t o n g Fan , F . M o r g a n Wallace , J o h n S. Novak , A. Philip Handel , a n d B r e n d a n A. N i e m i r a 1

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Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, P A 19038 Department of Bioscience and Biotechnology, Drexel University, 3141 Chestnut Street, Philadelphia, P A 19104 2

Listeria monocytogenes, a food-borne pathogen, is a common contaminant on ready-to-eat (RTE) meat products such as frankfurters, bologna, ham and deli turkey meat. A number of food-borne illness outbreaks have been attributed to this microorganism. Since 1998, over 90 million pounds of R T E meats have been recalled due to contamination with L. monocytogenes. Ionizing radiation can eliminate L. monocytogenes from R T E meat products. The radiation resistance of L. monocytogenes is dependent on the R T E meat formulation and the genetic characteristics of the contaminating strain. Ionizing radiation can also impact product quality factors including color, lipid oxidation, and generation of volatile sulfur compounds and hydrocarbons. As with elimination of microorganisms, effects of ionizing radiation on product quality are also product specific.

© 2004 American Chemical Society Komolprasert and Morehouse; Irradiation of Food and Packaging ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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Incidence and Radiation Resistance Listeria monocytogenes causes an estimated 2,500 cases of food-borne illness, and 500 deaths, annually in the United States (1). The mortality rate due to listeriosis, among susceptible populations, is approximately 20% (/). Many of these illnesses have been associated with consumption of contaminated readyto-eat meat products such as frankfurters and deli meats (2-6). L. monocytogenes is capable of growth at refrigerated temperatures and in high salt environments, and such growth produces no apparent signs of spoilage in food products (7). Ready-to-eat (RTE) meat products are cooked as a processing step, with contamination occurring between the cooking and packaging steps. Because L. monocytogenes is capable o f growth at low temperatures, postprocess contamination with a relatively small number of microorganisms (10 CFU/g at the end of a 4 week refrigerated storage period (8-10). Due to the high mortality rate associated with listeriosis the U S D A ' s Food Safety Inspection Service (FSIS) has instituted a zero tolerance policy for L. monocytogenes in R T E meat products (11). Post-process contamination o f R T E meat products with L. monocytogenes is well documented. In 1998 approximately 2.5% of ready-to-eat meat products tested by the U S D A ' s FSIS were positive for L. monocytogenes (12). In a recent survey of frankfurters obtained from several commercial plants, approximately 1.6% tested positive for L. monocytogenes (13). In a review o f microbiological testing programs for the years 1990 to 1999, approximately 1.31% of small diameter sausages (frankfurters) and 5.16% of ham and sliced luncheon meats tested positive for the presence ofL. monocytogenes (14). 2

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Table I. Large Recalls (Over 100,000 lbs) of R T E Meat Products Due to Contamination with Listeria monocytogenes (5) Year Pounds Recalled Case No. Product 2002 28,000,000 090-2002 R T E Turkey, Various 2002 4,200,000 098-2002 R T E Turkey, Various 2000 16,895,000 076-2000 R T E Poultry, Various 900,000 2000 065-2000 Weiners 2,100,000 1999 046-99 Beef Frankfurters 126,739 1999 035-99 Bacon Chips 35,000,000 1999 005-99 R T E Meats, Various 35,000,000 1998 044-98 Hot Dogs/Packaged Meats 1998 1,734,002 035-98 Frankfurters

Komolprasert and Morehouse; Irradiation of Food and Packaging ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


79 A risk assessment completed by the U . S. Food and Drug Administration (FDA), the U S D A ' s FSIS, and the Centers for Disease Control and Prevention found that 7.6% of frankfurters tested positive for L. monocytogenes (2). In that same study, non-reheated frankfurters represented the top risk for listeriosis among the 20 product categories evaluated on a per serving basis. Since 1998 over 90 million pounds of R T E meat products have been recalled due to contamination with L. monocytogenes (5). A listing of the larger recalls of R T E meats is shown in Table I. Products including bacon bits, beef jerky, roast beef, frankfurters (hot dogs), ham, and turkey have tested positive for L. monocytogenes and have been recalled as a result (5). O f the 14 serotypes currently identified for L. monocytogenes serotypes l/2a, l/2b, and 4b account for 95% of illness in humans, with serotype 4b being responsible for most illnesses in North America (75). In a comprehensive survey on the recovery o f L. monocytogenes from commercial hot dog packs, Wallace et al. (13) found that approximately 90% of the isolates were l/2a while the remainder were primarily serotype 4b. It should be noted that the high percentage of positive packs (16%), primarily serotype l/2a, from Plant 133 (Table II) raised the overall positive rate in that survey (13). While some hot dog packs were found to contain L. monocytogenes strains of more than one serotype, the vast majority of packs contained a homogeneous population (13). L. monocytogenes detection rates, and serotype information are presented in Table II.

Table II. L. monocytogenes Recovery Rates and Strain Characteristics Frankfurter Type Facility Serotype % Packs Predominant Code Ribotype(%)* Positive 94 A (100) l/2a Turkey 0.07 133 Turkey A (100) l/2a 16.0 172 Beef 0.11 A (100) l/2a 344 F(30) 4b Beef, Pork & Chicken 0.16 4b B(30) NT O(20) 4b N(10) 367 Pork l/2a 1.5 A (82) 385 NT Pork and Beef G (100) 0.08 439 Pork and Beef A (100) l/2a 2.2 * Predominant ribotype was assigned a letter code based on similarity between isolates for the basis for the study (13). Facility codes were generated randomly for anonymity (13).



80 R T E Meat Formulation. There is relatively little data available pertaining to elimination of L. monocytogenes from R T E meat products using ionizing radiation prior to 1999. Frankfurters and bologna are fine emulsion sausages that can vary greatly in formulation (13, 16). Meats and meat mixtures used in frankfurters and bologna can include (but are not limited to) beef, pork, chicken, and turkey. Additives can include sodium nitrite, sodium chloride, phosphates, erythorbate, ascorbate, etc. Extenders and binders, used to increase product firmness and to reduce purge (fluid loss), can include products such as lactose free whey, soy protein concentrate, various flours, carrageenan, yeast lysate, etc. Sweeteners can include anything from glucose to high fructose corn syrup. Antimicrobial compounds including organic acids, sodium or potassium lactate, and sodium diacetate can be added to the product emulsion or applied to the product surface to inhibit the growth of L. monocytogenes. In short, there is no "standard" frankfurter or bologna formulation, but rather a complex family of widely differing formulations within a class of products. Other types of R T E meats exhibit the same variability in product formulation. L. monocytogenes D value. Recent studies have elucidated the phenomenon of variability in L. monocytogenes radiation resistances when inoculated onto different R T E meat products. The radiation resistance of food-borne pathogens is typically expressed as either a D j value, the ionizing radiation dose required to eliminate one logi o f the pathogen, or as a 5 logio reduction dose. Sommers and Thayer (17) found that D i values for a mixture of four L. monocytogenes strains surface inoculated onto commercially available frankfurters ranged from 0.48 kGy to 0.71 kGy (Table III). Radiation doses o f 2.45 to 3.55 k G y are therefore needed to eliminate 5 logio of the pathogen from hot dogs. Niemira et al. (18) found D i values ranging from 0.62 kGy to 0.77 kGy when L. monocytogenes strain H7762 was surface-inoculated onto beef frankfurters or soy-based imitation meat products (Table III). Foong et al. (19) found that doses ranging from 2.5 kGy to 3.0 kGy were required to eliminate 5 logio of the microorganism from four different types o f R T E meat products. Thayer et al. (20) found a D of 0.69 to 0.70 kGy for L. monocytogenes inoculated into cooked ground turkey meat or cooked turkey nuggets. The role of specific additives on the radiation resistance of L. monocytogenes inoculated into R T E meats was investigated. Sommers et al. (21) found that soy protein concentrate, an extender that contains phytates and isoflavones with antioxidant activity, increased the radiation dose required to eliminate 5 logio of the pathogen from 3.1 to 3.75 kGy. In other work, application of citric acid, a p H reductant and antimicrobial, to frankfurter ! 0

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81 Table III. Dip Values of L. monocytogenes Inoculated onto R T E Meats Product (Study) D (kGy) Reference Beef Frankfurter #1 0.52 17 Beef Frankfurter #2 0.52 17 Mixed Meat Frankfurter #1 17 0.71 17 Mixed Meat Frankfurter #2 0.71 Poultry Frankfurter #1 17 0.49 Poultry Frankfurter #2 17 0.70 Poultry Frankfurter #3 17 0.64 0.60-0.62 21 Beef Bologna #1 22 Beef Bologna #2 0.66-0.71 Turkey Bologna This Study 0.58 Cooked Turkey 20 0.68-0.70 18 Soy Hot Dog 0.77


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surfaces increased the radiation sensitivity of L. monocytogenes (8). The inclusion of sodium diacetate, or sodium diacetate and potassium lactate mixtures, in the formulation increases the radiation sensitivity, and prevents post-irradiation growth, of L. monocytogenes inoculated onto cooked beef bologna (9, 10). The phenomenon of variability in the radiation resistance of L. monocytogenes on R T E meats could be reproduced using commercially used additives.

Mechanism of Lethality. Ionizing radiation induces D N A strand-breaks, transition mutations, transversion mutations, frameshift mutations, and deletions in bacterial cells (22 - 25). Mudgett et al. (26) observed that gamma radiation induced mutagenesis in Escherichia coli began when post-irradiation survival reached 1.5%, or a dose of 0.6 kGy. Wijker et al. (27) recommended 0.25 kGy, a gamma radiation dose that decreased survival to 2%, for proper selection and characterization of mutants in E. coli strain EC919. In L monocytogenes, a radiation dose of 2.0 to 2.5 k G y is required for a 1 to 2% single gene inactivation rate, as determined by mutation of the microorganism's hlyA (hemolysin) gene (Figure 1). Ionizing radiation also disrupts cell membrane associated with D N A complexes that are required for plasmid partitioning and active sites for the D N A repair process (28-31), and also induces loss of plasmids that carry genes required for food-borne pathogen virulence (32). Disruption of the L. monocytogenes cell membrane by ionizing radiation can also lead to increased sensitivity to organic acids and antimicrobial compounds such as diacetate (9).



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Figure 1. Radiation resistance and mutagensis of L. monocytogenes H7762 that was surface-inoculated onto beeffrankfurters. Each experiment was conducted independently three times. Mutants were selected by plating on blood agar plates. Individual logio reduction points and 95% confidence limits are shown for the Dj value while error bars are shown for hlyA mutation rate at each radiation dose (This Study). 0

Product Chemistry and Quality L i p i d Oxidation. Oxidation of lipids in meat products, an autocatalytic and temperature dependent process, is enhanced by the presence of oxygen, and can be induced by free radical generators including ultraviolet light and ionizing radiation (33). Ionizing radiation induced lipid oxidation also results in the formation of aldehydes, ketones and diacyglycerols (33-36). A number of studies have examined lipid oxidation in R T E meats. Radiation doses of 3 to 4 kGy result in a statistically significant doubling of lipid oxidation in beef bologna (15% fat) (8-10, 21, 37). Nam et al. (38) found increased lipid oxidation in precooked turkey, pork, and beef patties irradiated under aerobic conditions, which was ameliorated by vacuum packaging. Lipid oxidation in R T E meats can be influenced by product formulation. D u et al. (39) found that inclusion of antioxidants into the emulsion of sausages made with turkey leg meat lessened radiation induced lipid oxidation while Sommers and Fan (37) found that inclusion of excess glucose (>4%) increased the likelihood of lipid oxidation in beef bologna. Volatile Sulfur Compounds (VSC's). Previous studies of irradiated raw meats have indicated that off-odors can be generated as a result of the irradiation process (34, 35, 40, 41). The off-odor has been called 'irradiation' odor, and has been characterized as 'wet dog', 'sulfide', 'metallic', 'wet grain', 'goaty' or 'burnt' (41). The changes in off-odors are primarily due to formation of volatile



83 compounds including hydrocarbons, alcohols, aldehydes and ketones, which are generated from lipids (38-45). Several V S C ' s , derived from radiolysis of sulfur containing amino acids, include methyl sulfide, hydrogen sulfide, sulfur dioxide, dimethyl disulfide, methanethiol, (methylthio) acetic acid and carbon disulfide, are produced in irradiated raw and R T E turkey meat (39,42,43) (Figure 2). Turkey muscle is one of the meats most sensitive to irradiation in terms of offflavor development (46). While these compounds can be produced in all meats, off odors have not been noted to be problematic following irradiation of beef and mixed meat fine emulsion sausages (8-10, 21, 37).

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Figure 2. Generation of volatile sulfur compounds in vacuum-packaged RTE turkey meat by irradiation. RTE turkey meat as determined by pulsed flame photometric detection (PFPD) (43). Results are the average of nine (n-9) samples and represent the sum of hydrogen sulfide, sulfur dioxide, methanethiol, and dimethyl disulfide peak area square roots. Standard error bars are shown at each radiation dose (This Study).

Color. Ionizing radiation can induce color changes in R T E meat products (47). R T E poultry meat has been problematic for ionizing radiation induced color changes. In poultry, especially turkey, this manifests itself as a radiation induced increase in redness (a-value). Nam et al. (38, 45) suggested that carbon monoxide heme pigments could be responsible for the increased redness. A s with V S C ' s , and other volatile compounds, the use of antioxidants and vacuum packaging were capable of reducing the induced color change (39, 42). Ionizing radiation has also been shown to induce loss of redness in beef bologna and frankfurters (8-10, 21, 37). A s with lipid oxidation and V S C generation color change in irradiated meats can be influenced by product formulation, with loss


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of redness being enhanced by excess carbohydrate and lessened by inclusion of commonly used coloring agents such as paprika oleoresin (8, 37). Elimination of L. monocytogenes versus V S C Generation. A number of approaches can be used to ameliorate the problem o f V S C generation in R T E turkey meat. One such approach could include the use of antioxidants in the R T E meat formulation prior to cooking and irradiation (39). Another approach, as mentioned earlier, would be to include antioxidants in the diets of animals prior to slaughter (42). Still another approach is to lower the ionizing radiation dose required to eliminate L. monocytogenes, so as to not affect product quality. Thayer et al. (48) demonstrated that application of heat following irradiation increased the l o g reduction of Salmonella on chicken meat. Sommers et al. (*) demonstrated that reducing the pH by use of a citric acid dip could increase die ability of ionizing radiation to eliminate L. monocytogenes from vacuum-packed frankfurters. Juneja and Eblen (49) demonstrated increased sensitivity of L. monocytogenes to heat in the presence of acidulant. In this study, the use of citric acid applied to the turkey deli meat surfaces, ionizing radiation, and heat (75°C) was evaluated for the ability to eliminate L. monocytogenes H7762 and reduce the generation of V S C ' s . The ability of the combination treatments to eliminate L. monoctyogenes from the surface of R T E turkey is shown in Figure 3. 10

Figure 3. Elimination ofL. monocytogenes H7762 from the surface of vacuumpacked RTE turkey meat by a combination of citric acid (CA) applied to the product surface, irradiation (to 1.0 kGy) and thermal treatment (75 °C). Each experiment was conducted independently three times (This Study).


85 The order of treatments was application of sterile deionized water or 5% citric acid to product surfaces (10 g pieces), vacuum packaging, irradiation to a dose of 1.0 kGy, and submersion in a water bath (75°C) for a period of 1 minute. The L. monocytogenes (10 CFU) was applied to the product surface following application of acidulant and prior to vacuum-packaging. Following treatments (acid, irradiation, heat, or combinations of the three) the samples were processed for microbiology (8-10). Each experiment was conducted independently three times. Use of acidulant alone resulted in a 0.5 logio reduction o f L. monocytogenes. Use of irradiation in combination with heat, without acidulant, resulted in a 2 logio reduction of L. monocytogenes. However, use of 5% citric acid (pH 4.5) in combination with irradiation and heat resulted in a 5 logio reduction of the microorganism (Figure 3).


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Figure 4. The generation of volatile sulfur compounds in irradiated (1.0 kGy), heated (75 °Cfor 1 min.), and acid treated (5% citric acid) ready-to-eat turkey meat. The treatments were (A) Unirradiated, unheated and non-acid treated samples (B) Unirradiated, nonheated, acid treated samples (C) Irradiated, heated, and non acid treated samples (D) Irradiated, acid treated, and heated samples. V S C ' s were measured using pulsedflame photometric detection (PFPD) (15). Values represent the sum of hydrogen sulfide, sulfur dioxide, methanethiol, and dimethyl disulfide peak area square roots (n-9). Standard error bars are shown for each treatment (This Study).



86 Following determination of the conditions required to eliminate 5 logio of Lmonocytogenes from the R T E turkey meat surfaces, the generation o f V S C ' s was measured using uninoculated product. The treatment of 1 kGy, 0% acidulant, and 75°C for 1 minute reduced the V S C ' s to the 0 kGy level, possibly due to decreased thermal instability o f the compounds. The reduction in V S C generation did not correlate with a significant reduction of L. monocytogenes (Figure 4). In contrast, the generation o f V S C ' s was reduced significantly ( A N O V A , n=9, a=0.05) from that obtained at the 1.0 to 3.0 kGy doses. No differences in product color or lipid oxidation were noted in the 5% citric acid, 1.0 kGy, and 75°C treated samples was noted.

Conclusions What little data is available suggests that it should be possible to produce L. monocytogenes free, organoleptically acceptable, R T E meat products. Sensory studies of vacuum packaged frankfurters irradiated to doses of 8.0 and 30 kGy, at subfreezing temperatures, originally conducted for the purpose of providing rations for military personnel, have been conducted (50-55). In those studies a radiation dose of 8.0 kGy produced undesirable sensory traits in 3 of 18 categories while frankfurters irradiated at a dose of 30 kGy were scored as being less palatable in 8 of 18 categories (50-53). Sensory studies conducted with vacuum packaged turkey frankfurters irradiated to doses of 5.0 and 10.0 kGy, at temperatures of 2°C and -30°C, indicated it was possible to obtain product which was not significantly different than non-irradiated frankfurters (50-53). While unknown at the time, the use of turkey frankfurters was important because of issues concerning volatile sulfur compounds generated by ionizing radiation. What then, is the threshold for generation of V S C ' s in actual products, as applies to consumer satisfaction, versus detection by analytical chemistry equipment by trained scientists? In more recent work A l Bachir and Mehio (54) irradiated R T E beef luncheon meat to a dose of 4 kGy and found that the product was organoleptically acceptable, with the shelf-life being extended from 10 to 14 weeks. The judge and jury for irradiated R T E meats will ultimately be the consumer. Unfortunately, despite a plethora of instrumental analysis of irradiated R T E meats, relatively little work has been published pertaining to consumer preferences for these products when irradiated to doses of

Irradiation of Ready-to-Eat Meats: Eliminating Listeria monocytogenes

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