1260 Journal of Food Protection, Vol. 59, No. 12, 1996, Pages 1260-1266 Copyright ©, International Association of Milk, Food and Environrnantal Sanitarians Validation of Pepperoni Processes for Control of Escherichia coli 0157:H7+ JAY C. HINKENS,lt NANCY G. FAITH,2 TIMOTHY D. LORANG,! PHILLIP BAILEY,l DENNIS BUEGE,3 CHARLES W. KASPAR,2 and JOHN B. LUCHANSKy2,4* IDoskocil Companies 1nc., Jefferson, Wisconsin 53549; and 2Department of Food Microbiology and Toxicology, Food Research 1nstitute, 3Muscle Biology Laboratory, and4Department of Food Science, University of Wisconsin, Madison, Wisconsin 53706, USA (MS# 96-18: Received 7 February 1996/Accepted'l April 1996) ABSTRACT break another outbreak of E. coli 0157:H7 was linked to meat, this time, a dry fermented pork and beef salami (8). The outbreak of Escherichia coli 0157:H7 linked with The salami outbreak involved 20 individuals in Washington dry-cured salami in late 1994 prompted regulatory action that (age range, 23 months to 77 years; median age, 6 years; 3 required manufacturers of fermented products to demonstrate a hospitalized; one 6-year-old with hemolytic uremic syn- 5-log unit reduction in counts of this pathogen during processing. Therefore, pepperoni batter (75% pork:25% beef with a fat content drome [HUS]) and 3 individuals in California (age range, 4 of ca. 32%) was inoculated with a pediococcal starter culture and a to 75 years; 3 hospitalized; one 4-year-old with HUS) (8). E. five-strain mixture of E. coli 0157:H7 (~2 X 107 CFU/g) and coli 0157:H7 was found on presliced product from delicates- stuffed into 55-mm diameter fibrous casings 47 cm in length. The sen counters (8). In early 1995 there was another outbreak of viability of the pathogen was monitored before stuffing, after HUS linked to semidry (uncooked) fermented sausage (9). fermentation, after thermal processing, and/or after drying. Chubs This outbreak involved 23 patients (age range, 4 months to were fermented at 96°F (36°C) and 85% relative humidity (RH) to 12 years; median age, 4 years; 23 with HUS) in South pH :0; 5.0 and then dried at 55°F (l3°C) and 65% RH to a Australia and was attributed to E. coli 0111 :NM, another moisture/protein ratio of :0;1.6:1 (modified method 6 process). Counts of the pathogen decreased about 1.2 log units after serotype of E. coli that produces a Shiga-like toxin(s). As a fermentation and drying. In subsequent experiments, heating chubs result of the California and Washington salami outbreak the after fermentation to internal temperatures of 145°F (63°C) instan- U.S. Department of Agriculture (USDA) Food Safety and taneous or 128°F (53°C) for 60 min resulted in a ~5-log unit Inspection Service (FSIS) developed guidelines (24) for decrease in numbers of strain 0157:H7 without visibly affecting sausage manufacturers to validate processes to ensure a the texture or appearance of the product. These data revealed that a 5-log unit reduction in counts of E. coli 0157:H7. traditional nonthermal, process for pepperoni was only sufficientto Among the many different categories of fermented eliminate relatively low levels (ca. 2 log CFU/g) of E. coli sausage, pepperoni is one of the most popular varieties, with 0157:H7, whereas heating to internal temperatures of 145°F (63°C) instantaneous or 128°F (53°C) for 60 min delivered a 5 to 6 annual consumption in the United States exceeding 370 log unit reduction in counts of the pathogen in pepperoni. million pounds (Gerry Durnell, personal communiciltion). Pepperoni types and sizes vary greatly, but three basic Key words: Escherichia coli 0157:H7, pepperoni, pathogen, categories predominate. First, there is the traditional, small- fermentation diameter (28 to 36 mm) product found in the deli case or on pegboards in supermarkets. The second type, representing the largest volume consumed, is pepperoni stuffed into 49- The Western United States outbreak of Escherichia coli 0157:H7 in 1993 due to consumption of contaminated to 54-mm diameter casings and then sliced (1 to 2 mm thick) hamburger patties at a fast-food restaurant chain (7) brought and sold to pizza restaurants. A small portion of this type of considerable focus on this pathogen. Despite the heightened product is also sold as sticks and subsequently used as a awareness, approximately 1 year after the hamburger out- topping for frozen pizza. The third category of pepperoni is the large-diameter (60 to 80 mm) product used on sand- wiches. Differences among manufactures and categories of * Author for correspondence. Tel: 608-263-7777: Fax: 608-263-1114; pepperoni notwithstanding, the use of various combinations Email: [email protected]. of salts, pH, heating, cooling and/or drying ensure that t Present Address: Silliker Laboratories of Wisconsin, 3688 Kinsman Blvd., pepperoni is wholesome and safe relative to the parasite Madison, WI 53704. Trichinella spiralis and to bacterial pathogens such as t Portions of this research were presented at the Annual Meeting of the International Association of Milk, Food, and Environmental Sanitarians, Staphylococcus aureus. In contrast, to date, there has been 30 June to 3 July 1996 in Seattle, Washington, USA( 14). no information available to certify that pepperoni is safe E. COLI 0157:H7 IN PEPPERONI 1261 relative to E. coli 0157:H7. To this end, the objective of the dextrose (0.16 Ib/25 Ib of batter; A. E. Staley, Decatur, IL), 3% present study was to validate the effect of different process- spice mix (0.75 Ib/251b of batter; Doskocil house blend), and 2.0% ing conditions on the fate of E. coli 0157:H7 in pepperoni. cure mixture (0.5 Ib/25 Ib of batter; Doskocil house blend). The cure and spice mixtures were formulated to contain garlic, anise, MATERIALS AND METHODS red pepper, clove, and oleoresin of paprika, BHA (28 ppm; Eastman Chemical, Kingsport, TN), BHT (butylated hydroxytolu- Bacterial strains ene) (28 ppm; Eastman), and citric acid (28 ppm; Fuyang Pharma- Five strains of E. coli 0157:H7 were used in this study to ceutical, China), and to provide a final batter concentration of 156 inoculate pepperoni: (i) strain SLH21788, human isolate from the ppm NaN02 and ca. 3.33% NaCl. The batter was inoculated with Wisconsin daycare outbreak of 1994 (22); (ii) strain C7927, human the five-strain cocktail of E. coli 0157:H7 (final concentration of isolate from the Massachusetts apple cider outbreak of 1991 (6); 2:2 X 107 CFU/g of batter) and mixed using a Buffalo mixer (iii) strain F-90, sausage isolate from the California dry-cured (model 2VSS; John E. Smith's and Sons Co., Buffalo, NY) for 2 salami outbreak of 1994 (8); (iv) strain EC204P, pork isolate from min. Six milliliters of thawed pediococcal starter culture in 42 ml of 8 the Food Research Institute Culture Collection; and (v) strain sterile dH20 were added per 25 Ib of batter to deliver ca. 10 CFU/g C9490, human isolate from the Western States hamburger outbreak of batter. The commercial spice and cure were added, and mixing of 1993 (7). The E. coli strains were maintained at -70°C in 2X was continued for an additional 2 min. A noninhibitory, food grade, Trypticase soy broth (TSB) (Difco Laboratories Inc., Detroit, MI) green dye (FD&C yellow 5 and FD&C blue 1, McCormick & Co., plus 10% glycerol. Prior to inoculation into pepperoni batter, the Inc., Hunt Valley, MD) was added to the batter as an aid to monitor cultures were transferred twice in TSB supplemented with. 1% for uniform mixing. A control batter was prepared prior to the glucose at 37°C overnight before use as outlined by the USDA experimental batter that was identical to the latter except for the FSIS (24). A commercial Pediococcus acidilactici starter culture presence of the serotype 0157:H7 cocktail. Chemical analyses (Lactacel 115; Quest International, Sarasota, FL) was maintained were performed (see below) on representative chubs prepared from and propagated according to the manufacturers instructions. the control batter. The inoculated batter was removed from the mixer and ground through a 0.125 in. (ca. 0.32 cm) die plate using a Preparation ofE. coli 0157:H7 inoculum Hobart grinder (model 84142; Hobart Manufacturing Co., Troy, Each of the five strains of E. coli 0157:H7 were separately OH). The batter was then stuffed (ca. 694 g per chub) using a hand grown overnight in 250 ml of TSB plus I% glucose at 37°C with stuffer (Koch Supplies, Inc., Kansas City, MO) into 55-mm shaking (100 rpm). The cells were harvested by centrifugation at diameter fibrous casings (TeePak, Inc., Westchester, IL) to a final 1,880 X g for 20 min at 4°C, and washed and resuspended in a length of 47 cm and the resulting chubs were sealed using a casing small volume of 0.1 % peptone (Difco). The five cell suspensions clipper (Poly-clip model SCH 7210; Niedecker, West Germany) were combined and the final volume was adjusted to 50 ml with loaded with series 7000- VSCC staples (U.S. Clip Corporation, 0.1 % peptone such that each strain contributed about 20% to the Mundelein, IL). Casings were soaked in tap H20 at room tempera- final inoculum of 1 X 109 CFU/ml. The five-strain cocktail was ture for 10 min prior to use. held on ice for ~30 min before addition to sausage batter as The chubs were transferred to a smokehouse (model 1000; described below. The viable count of the cocktail was confirmed by Vortron, Inc., Beloit, WI) and fermentation was conducted at 96°F spread plates on MacConkey sorbitol agar (MSA) (Difco) just prior (36°C) with a relative humidity (RH) of 85 to 90% until reaching a to inoculation of the batter.