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Survival and Cooking Inactivation of Escherichia Coli O157:H7 in Nonintact Beef Products

Sofos, John
Colorado State University
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Escherichia coli O157:H7 is considered as an adulterant of raw ground and other nonintact beef products. Due to the severity of the illnesses caused by this pathogen, its inherent acid tolerance, and its low infectious dose, especially in young children, regulatory agencies and industry have an interest in evaluating processes that may contribute to the spread of the pathogen through beef products including ground beef, nonintact (tenderized and restructured) beef steaks or roasts, and products made from trimmings, as well as procedures for its control in such products. In addition to the pathogen being potentially internalized during processing of such products, its destruction rate during heating may be altered if meat is subjected to marination, tenderizing or restructuring procedures. During tenderization, transfer of E. coli O157:H7 may occur from: (i) the surface to the interior of contaminated meat; and, (ii) contaminated meat to previously non-contaminated meat (e.g., cross-contamination via blade- or needle-tenderization, and/or recycling of injection solutions or brines). E. coli O157:H7 may survive cooking in these products and cause illness among consumers, especially if the injected ingredients interfere with thermal inactivation or increase the heat resistance of the pathogen. With regards to restructured beef steaks or roasts, which consist of mixtures of cuts and/or trimmings of meat, contamination from the surface may be introduced into the interior during their formation. However, due to their nature, restructured beef products may be perceived as intact steaks or roasts, and as such be cooked at temperatures inadequate to destroy contamination that may have become internalized. The goal of this work is to determine risks of E. coli O157:H7 associated with processing and preparation of marinated, nonintact beef steaks or roasts and restructured beef products, and provide information for minimizing such risks using novel or alternative marination and processing technologies. Thus, specific major objectives of the work are to:
  1. Determine E. coli O157:H7 survival/growth during storage (refrigeration, freezing, or retail) as well as thermal destruction rates during cooking at various temperatures of nonintact, tenderized or restructured beef steaks and roasts marinated with existing marinades (e.g., calcium chloride, organic acids, sodium chloride), or substitutes (such as calcium lactate/ascorbate for calcium chloride), as well as novel marinades (e.g., herbs and antioxidants), and various marination strategies;
  2. Investigate whether product preparation procedures that minimize E. coli O157:H7 survival during storage and subsequent cooking are also effective against stress-adapted cells (e.g., acid tolerant);
  3. Use the data generated to develop fact sheets and presentations that will be disseminated through Colorado State University Cooperative Extension and presented during educational workshops at local, state and national processor/industry meetings. Modifications in formulations and processing procedures developed will minimize potential risks and enhance product safety.
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NON-TECHNICAL SUMMARY: Escherichia coli O157:H7 is an adulterant of raw ground or otherwise nonintact beef products, and because of its inherent acid tolerance and low infectious dose, especially in young children, there is a need to identify processes and factors that may contribute to spread of disease through beef products including ground beef, intact (non-tenderized) beef steaks, nonintact (tenderized) beef steaks and products made from meat trimmings. The goal of this work is to determine risks of E. coli O157:H7 associated with processing and preparation of marinated, nonintact, or restructured beef products, and to provide information for minimizing such risks using novel or alternative marination and processing technologies. Based on the data collected, educational materials will be developed to assist the industry and consumers in the control of the pathogen through application of modified marination, tenderization, restructuring and cooking formulations and procedures.

APPROACH: Extensive screening work will be done with ground beef to evaluate marination, tenderizing and restructuring ingredients and procedures for their effects on pathogen survival during storage and cooking. Subsequently, effective treatments will be evaluated on fresh beef steaks and roasts, intact or nonintact, inoculated (5 log CFU/cm2, or higher) with E. coli O157:H7 and marinated either by immersion in marinades or by needle-injection for varying lengths of time (0-240 h). Marination mixtures will be made by mixing ingredients including: antimicrobial agents, herbs, antioxidants, calcium salts, phosphates and sodium chloride. Marinated samples will be stored for 30-90 days at -20 degrees C, 20-30 days at 4 degrees C and 8-12 days at 12 degrees C in air-permeable films, vacuum packages or a staged combination of both packaging conditions to simulate vacuum-packaged cuts moved to retail display packaging (vacuum-retail). Furthermore, inoculated and marinated or nonmarinated beef steaks/roasts will be subjected to mechanical tenderization using blades. Populations of E. coli O157:H7 and the natural flora will be monitored during storage. At different time intervals during storage, samples will be subjected to cooking to three target temperatures (55, 60 or 65 degrees C). For those marinades with altered antimicrobial effectiveness due to mechanical tenderization, independent experiments will be performed to identify the ingredient(s) in the marinades that are responsible for inhibition and resistance or sensitivity of E. coli O157:H7 during storage and exposure to heat (cooking), respectively. Specifically, inoculated meat samples will be marinated with mixtures of ingredients or with each ingredient separately and will be subjected to cooking, as described above. Additional studies will monitor the effect of stress-adaptation of E. coli O157:H7 on survival/growth during storage and evaluate subsequent heat tolerance in tenderized or restructured beef steaks and roasts subjected to marinades or marination techniques shown to minimize survival of the pathogen. Adaptation of cells will be performed with exposure to sublethal stressing that may potentially be encountered in the environment, or on the products, as a result of acidification, depression of water activity or other changes in intrinsic factors of products generated by marination, tenderization, or restructuring. Warner-Bratzler shear force determinations of uninoculated, nonintact beef steaks and roasts marinated using novel marination formulations or techniques identified to be effective against E. coli O157:H7 will also be determined. Data will be used to develop and validate mathematical models for predicting the fate of the pathogen as a function of processing variables, such as water activity, organic acid concentration and storage temperature in marinated/tenderized beef subprimals. The data will also be used to develop educational materials for processors, foodservice and consumers, guiding them to effective product processing for pathogen control and acceptable product quality. Also, workshops and pilot demonstrations for small and mid-sized processors will be conducted.

PROGRESS: 2007/09 TO 2008/08
OUTPUTS: The tenderness, flavor and overall eating quality of under-valued whole muscle cuts of beef can be enhanced by injecting them with tenderizers and marination ingredients; such products are then classified, for regulatory purposes, as nonintact beef products. A microbiological food safety concern associated with these products is that they may contain internalized Escherichia coli O157:H7 cells, which may potentially survive thermal destruction if these products are undercooked (intentionally or accidently). An additional concern is that the tenderization and marination ingredients may interfere with thermal inactivation of pathogen cells by increasing their heat resistance or otherwise protecting them from thermal inactivation. A study was performed to evaluate the effect of tenderizing/marinating ingredients on thermal inactivation of E. coli O157:H7 in a lean (5 percent fat) ground beef model, simulating nonintact products. The ground beef was inoculated (7 log CFU/g) with a 5-strain mixture of E. coli O157:H7, and then mixed with the following ingredients: nothing (control), water, a mixture of flavoring agents (FA; black pepper, garlic and onion powder, old hickory smoked salt, Worcestershire sauce), or mixtures of 0.23 percent calcium chloride (CC)-FA, CC-FA-0.3 percent acetic acid (AA), 0.5 percent sodium chloride (SC)-0.25 percent sodium tripolyphosphate (ST), SC-ST-FA, SC-ST-1.8 percent potassium lactate (PL), SC-ST-PL-FA, SC-ST-PL-AA, and SC-ST-PL-AA-FA. Product portions (30 g) were extruded into tubes (2.5x10 cm) and were stored at 4 degrees C overnight. The samples were then heated in a circulating water bath to an internal temperature of 60 or 65 degrees C, simulating rare or medium-rare doneness of beef, respectively. Samples were analyzed for weight, fat and moisture losses, pH, and total bacterial and E. coli O157:H7 populations after inoculation, storage (4 degrees C), and heating. Cooking of control samples to 60 or 65 degrees C reduced total bacterial counts by 0.8 and 5.0 log CFU/g, respectively. Reductions of E. coli O157:H7 in product cooked to 60 degrees C were higher (P less than 0.05) in samples treated with AA than in those not treated with the organic acid. Thermal destruction of the pathogen at 65 degrees C in samples treated with SC and ST, but without AA (3.4-4.0 log CFU/g), was lower than in those not containing these ingredients (5.1-5.8 log CFU/g). However, when AA was included in formulations containing SC and ST, thermal inactivation of E. coli O157:H7 was enhanced (5.1-5.9 log CFU/g). The pH values of samples with and without AA were 4.85-5.38 and 5.33-5.97, respectively. Fat and moisture losses were not affected by treatment and temperature. Cooking losses of CC-FA-AA samples were higher (60 degrees C, 10.7 percent; 65 degrees C, 17.6 percent) than others but decreased when SC and ST were added. These findings suggest that sodium chloride and sodium tripolyphosphate may protect internalized E. coli O157:H7 cells from thermal inactivation when nonintact beef products are undercooked, but inclusion of organic acids in marination recipes may enhance inactivation of the pathogen in such products.
PARTICIPANTS: Nothing significant to report during this reporting period.
TARGET AUDIENCES: Audiences targeted were the meat industry and regulatory agencies. Data were presented at the 61st Reciprocal Meat Conference, American Meat Science Association (June 22-25, Gainesville, FL), the 68th Annual Meeting of the Institute of Food Technologists (June 28-July 1, New Orleans, LA), the 95th Annual Meeting of the International Association for Food Protection (August 3-6, Columbus, OH), and FoodMicro 2008, 21st International ICFMH Symposium (September 1-4, Aberdeen, Scotland), and nine abstracts were published.

IMPACT: 2007/09 TO 2008/08
An estimated 62,000 cases of symptomatic Escherichia coli O157:H7 infections occur every year in the United States due to the consumption of contaminated foods, resulting in an estimated 1,800 hospitalizations and 52 deaths. Approximately 3,000 of these cases may result in hemolytic uremic syndrome. Ingestion of as few as 10 cells can result in illness, and surveillance data indicate that children under the age of 5 and immunocompromised individuals are more susceptible to infection. Epidemiological evidence from outbreak and sporadic cases of infection with E. coli O157:H7 indicates that ground beef is a major foodborne source of exposure. In recent years, however, nonintact beef products other than ground beef have also been implicated in E. coli O157:H7-related outbreaks. Nonintact beef products include intact meat cuts (e.g., chucks, ribs, tenderloins, striploins, top sirloin butts, rounds) that have been mechanically tenderized, injected with solutions (e.g., brines, marinades) to enhance tenderness and/or flavor, and those restructured into formed entrees. This category also includes any comminuted product that has been processed by chopping, flaking, grinding or mincing. A microbiological food safety concern associated with these processes is that it may lead to contamination of interior parts of meat products through transfer of cells from the surface to the interior, cross-contamination from equipment surfaces (e.g., solid- or hollow-needle injectors or blades) and recirculating brines, or by entrapment through mixing of trimmings or cuts to form larger items. Another concern is that ingredients used for marination, tenderization, brining and restructuring may interfere with thermal inactivation of E. coli O157:H7 by increasing its heat resistance. It is very likely that consumers and foodservice establishments will not be aware of the dangers associated with nonintact products and will cook them to the same degree of doneness as intact products. The project evaluates the relative contribution of existing and novel marination, tenderization, brining and restructuring ingredients on the thermal inactivation of E. coli O157:H7 during cooking of nonintact beef products. The goal is to identify ingredients that protect or enhance pathogen destruction during cooking to the rare and medium-rare state (as preferred by many consumers in the United States). Research data will be used by the industry to develop or modify current commercial marination and brining formulations, and tenderization, marination, brining or restructuring procedures in order to enhance pathogen inactivation and to improve product safety even when cooking at lower temperatures. Data to this point indicate that sodium chloride, sodium tripolyphosphate, calcium salts, and potassium lactate may protect E. coli O157:H7 from inactivation when nonintact products are undercooked, while inclusion of organic acids with the other ingredients may enhance pathogen destruction. The generated data will also be useful to regulators in conducting risk assessments that will assist them in determining whether intact and nonintact products should continue to be regulated differently.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Escherichia coli
Sanitation and Quality Standards