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359 Journal of Food Protection, Vol. 74, No. 3, 2011, Pages 359–364 doi:10.4315/0362-028X.JFP-10-294 Copyright G, International Association for Food Protection Effects of Sodium Citrate plus Sodium Diacetate and Buffered Vinegar on Escherichia coli O157:H7 and Psychrotrophic Bacteria in Brine-Injected Beef AMUDHAN PONRAJAN,1,2 MARK A. HARRISON,1 JACOB R. SEGERS,2 BRADLEY K. LOWE,2 RUSSELL O. MCKEITH,2 T. DEAN PRINGLE,2 KARINA G. MARTINO,1 JAKE H. MULLIGAN,1 AND ALEXANDER M. STELZLENI2* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/3/359/1685103/0362-028x_jfp-10-294.pdf by guest on 01 October 2021 1Department of Food Science and Technology and 2Department of Animal and Dairy Sciences, University of Georgia, Athens, Georgia 30602, USA MS 10-294: Received 15 July 2010/Accepted 4 December 2010 ABSTRACT The objective of this research was to examine the effects of sodium citrate plus sodium diacetate or buffered vinegar on Escherichia coli O157:H7 and psychrotrophic bacteria when incorporated in brine solutions for injected beef. Two experiments were conducted in which 30 top rounds and 30 top sirloins were injected (110%) to contain (i) 0.5% sodium chloride and 0.4% sodium tripolyphosphate as the control (CNT); (ii) CNT with a 1% solution of 80% sodium citrate plus 20% sodium diacetate (SCzD); or (iii) CNT with 2% buffered vinegar (VIN) in the final product. For the E. coli challenge, muscles were surface inoculated to target 6 log CFU/cm2. After injection and 10 days of storage in a vacuum package (4uC), one half of each muscle was sampled raw and the other half was cooked to an internal temperature of 60uC with a 12-min hold. For raw samples, a significant reduction of 0.6 and 1.0 log CFU/g of E. coli O157:H7 was observed in both SCzD- and VIN-injected top rounds and sirloins, respectively. All cooked samples were E. coli O157:H7 negative. For psychrotrophic analysis, subprimals were injected and vacuum packaged for 10 days at 0 ¡ 1uC. After 10 days of storage, steaks were fabricated and placed in aerobic display (4 ¡ 1uC) for 1, 7, 14, and 21 days. Psychrotrophic organism growth was restricted in SCzD and VIN samples when compared with CNT on all days except day 1. Sodium citrate plus sodium diacetate or buffered vinegar may improve the safety and shelf life of multineedle brine-injected beef. Escherichia coli O157:H7 was first identified as a process. Sporing (13) has shown that when subprimals were foodborne pathogen in 1982, when it was associated with contaminated with pathogens such as E. coli (K-12), the two outbreaks of hemorrhagic colitis caused by the pathogen could be internalized during multineedle injection. consumption of undercooked frozen ground beef patties Current research has focused on a two-step injection process (3, 7, 9). A 1993 multistate outbreak of E. coli O157:H7 to reduce or eliminate E. coli contamination, in which the associated with hamburgers at a national fast-food chain subprimal undergoes a surface decontamination step increased the awareness about this pathogen (2). In followed by multineedle injection (4, 6). Little research response, the U.S. Department of Agriculture, Food Safety has been conducted to streamline this process by examining Inspection Service (USDA-FSIS) declared E. coli O157:H7 the efficacy of including antimicrobials directly in the brine an adulterant in ground beef in October 1994 under the solution (11, 19, 20). Furthermore, thermal processing has Federal Meat Inspection Act (4, 16). In January 1999, the been proven to be an effective method of reducing or USDA-FSIS further clarified that E. coli O157:H7 was also eliminating internalized pathogens in whole muscle, non- considered an adulterant in nonintact and whole muscle, intact beef in experimental settings (10, 13). However, the nonintact beef (8, 15). However, three outbreaks of E. coli thermal destruction of internalized pathogens needs to be O157:H7 in August 2000, June 2003, and August 2004 evaluated under simulated commercial conditions for roast were associated with whole muscle, nonintact beef products. beef products. Therefore, the objectives of this research In 2005, the USDA-FSIS mandated that processors were to examine the efficacy of sodium citrate plus sodium producing whole muscle, nonintact beef products must diacetate or buffered vinegar in the brine solution (i) against reassess their hazard analysis and critical control point E. coli O157:H7 in beef top rounds and sirloins and (ii) on plans, specifically addressing the biological hazard E. coli psychrotrophic bacterial growth for beef steaks after O157:H7 (17). extended aerobic storage. E. coli O157:H7 adulteration of beef subprimals is MATERIALS AND METHODS primarily from surface contamination during the harvesting Experiment 1: E. coli O157:H7 cultures. Four strains of E. * Author for correspondence. Tel: 706-583-0398; Fax: 706-542-0399; coli O157:H7 (ATCC 43888, human fecal isolate; E0122, cattle E-mail: [email protected]. isolate; K3995, spinach isolate; and F4546, alfalfa sprout outbreak 360 PONRAJAN ET AL. J. Food Prot., Vol. 74, No. 3 isolate) were used for this study. E. coli O157:H7 strains were Sealed Air Corporation) vacuum packager and stored in incubators activated by three consecutive transfers from fresh stock cultures (LR5 TempGuard, MedRep, Inc., Newnan, GA) at 4 ¡ 2uC for (280uC) into 9 ml of tryptic soy broth (Difco, BD, Sparks, MD) 10 days to simulate transportation and storage. containing 50 mg/ml of ampicillin (ampicillin sodium salt, Sigma- Aldrich, St. Louis, MO) at 24 ¡ 2 h intervals incubated at 37 ¡ Experiment 1: subprimal sampling. After 10 days of 2uC. On the day of the experiment, 1 ml of the overnight culture storage, subprimals were removed from their vacuum package and from each strain was transferred into 9 ml of fresh tryptic soy broth placed in sterile stainless steel pans. The pH and weight of the plus ampicillin. Three-milliliter samples from each strain were samples were recorded. The subprimals were split into two halves, pooled together to form the initial inoculum cocktail (12 ml) with each top round half having one inoculation site (top round, 8 containing approximately 107 CFU/ml. by 8 cm; top sirloin, 6 by 6 cm). One half of each muscle was sampled raw; the other half was cooked to an internal temperature Experiment 1: meat procurement and inoculation. of 60uC (with a 12-min hold) following an eight-step commercial Institutional Meat Purchase Specifications 169A beef top rounds roast beef cycle using a commercial smokehouse (Alkar model (30) and 184B beef top sirloins (30) from cull cows were obtained 450, Alkar-RapidPak, Inc., Lodi, WI) and USDA-FSIS guidelines (FPL Food, LLC, Augusta, GA) and transported (0 ¡ 2uC) to the (14) for a 6.5 to 7.0 log reduction of Salmonella spp. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/3/359/1685103/0362-028x_jfp-10-294.pdf by guest on 01 October 2021 University of Georgia Meat Science and Technology Center Sampling was conducted by turning the subprimal upside (Athens). On the day of the experiment, 10 top rounds or top down and aseptically removing a thin layer (0.2 to 0.3 cm) from sirloins were placed in sterile stainless steel pans on sterile stainless the bottom surface. A sterile stainless steel block template (5 by steel wire racks inside a biological safety cabinet (Class II, Type 5 cm) was used to create an impression on the shaved surface. The A2, NuAire, Plymouth, MN). subprimal was then cut along the impression of the template using To calculate the inoculation level, overnight cultures were a sterile knife for each cut. The excised sample was placed on a enumerated to determine the number of E. coli O157:H7 colonies sterile cutting board and divided into three sections based on height for each strain, and then calculations were made to determine how (top, middle, and bottom). Each section was aseptically separated many dilutions were needed and how many microliters of the from the sample block. Sample sections were then placed into master inoculum were required to target 6.0 log CFU/cm2 for each stomacher bags. The cooked roast beef was sampled as early as surface area. The calculated amount of E. coli O157:H7 was spot possible after thermal processing to prevent residual cooking. inoculated on the lean surface of the muscle. Top rounds were inoculated on three surfaces (two 8 by 8 cm and one 5 by 5 cm). Experiment 1: E. coli O157:H7 analysis. To analyze Top sirloins were inoculated on three surfaces (two 6 by 6 cm and surface inoculation counts, the samples (5 by 5 cm) were placed one 5 by 5 cm). Sterile aluminum templates were used as an into sterile stomacher bags with 225 ml of 0.1% peptone (Difco, inoculation guide, and the corners of the designated area were BD). The samples from each third of the raw and cooked beef were marked using dye (GL #80 Purple Brite, Great Lakes Meat weighed and placed in stomacher bags with 200 ml of E. coli Branding Ink, Koch Supplies Inc., Kansas City, MO). The medium (EC medium), modified (Difco, BD) containing novobi- inoculated beef was allowed to rest for 30 min, allowing the E. ocin antimicrobic supplement (Difco, BD). All samples were coli O157:H7 to attach. To calculate the initial E. coli O157:H7 stomached (Stomacher 400 circulator, Seward Laboratory Systems population on the inoculated beef surface, a portion (5 by 5 cm) of Inc., Bohemia, NY) for 2 min at 230 rpm. Serial dilutions were each muscle was aseptically excised using sterile scalpel handles made using 9 ml of 0.1% peptone.

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