1. Rinse & Chill  Technology: A Novel Intervention for Reduction of Undesirable Bacteria on Beef Carcasses and On-going Protection from E. coli O157:H7 in Vacuum- packaged Ground Beef

2. ABSTRACT Rinse and ChillTM Technology, developed by MPSC, Inc., St. Paul, Minnesota involves rinsing a chilled solution of sugars and salts through the cardiovascular system. The solution removes most of the residual blood as it circulates throughout the carcass and drains. Rinse and ChillTM Technology is a process, which ensures a consistent reduction in pH and internal temperature and has been demonstrated to significantly reduce the presence of microorganisms, particularly coliforms and generic Escherichia coli. In addition, Rinse and ChillTM Technology provides on-going protection for vacuum-packaged ground beef. Data collected from two separate commercial beef slaughtering facilities demonstrated a 40.3% (n=180; p value = 0.039) and 41.2% (n = 100; p value = 0.009) reduction for aerobic microorganisms on rinsed carcasses when compared to controls. More importantly, the two commercial facilities demonstrated a 99.3% (n = 180; p value = 0.125) and 67.8% (n = 100; p value = 0.002) reduction in coliforms on the rinsed carcasses versus the controls. One of the commercial slaughter facilities also demonstrated an 83.7% (n = 100; p value 0.0008) reduction in generic E. coli on the rinsed carcasses versus the controls. Research was conducted that demonstrated that vacuum- packaged ground beef made from Rinse and ChillTM carcasses provided a 4 log reduction in E. coli 0157:H7 as compared to only a 2 log reduction in the control carcasses.

3. INTRODUCTION Due to increased consumer awareness and recent changes in the regulation of meat inspection, there has been a move by the meat industry to improve sanitary conditions and the microbiological status of meat in slaughtering and processing plants. In 1993, the “Zero Tolerance” policy, which requires knife- trimming for removal of all visible physical contamination from beef carcasses prior to washing and chilling, was enacted. Since 1996, the Pathogen Reduction/HACCP Act requires meat and poultry slaughter establishments to implement sanitation standard operating procedures (SSOPs) and a hazard analysis critical control (HACCP) system. The establishment of microbiological performance criteria with standards for generic Escherichia coli and Salmonella as a means of verification of HACCP also were implemented. These regulations have led to more research, development and application of meat decontamination technology with the objective of helping the industry to meet or exceed regulatory requirements, and to provide the consumer with a microbiologically cleaner and safer product. The objectives of this study were to evaluate the effectiveness of Rinse and ChillTM Technology as 1) a novel intervention method for reduction of undesirable bacteria on beef carcasses and 2) a method to control or destroy the growth of E. coli 0157:H7 in vacuum-packaged ground beef.

4. MATERIALS AND METHODS Slaughter Cattle were slaughtered humanely in each commercial facility. They were assigned randomly to two groups, control or rinsed. For this study, animals were slaughtered and rinsed on seven sampling events in Plant X; in Plant Y, animals were slaughtered and rinsed on three sampling events. Cattle assigned to be rinsed were bled by severing both jugular veins. Near completion of the bleeding, an incision was made in the left carotid artery, and a catheter was inserted into the artery for the rinsing process (MPSC, Inc., St. Paul, MN). The rinsing solution consists of a mixture of dilute sugars and salts. Control groups were bled using the traditional methods. Carcass Sampling Meat/Turkey Carcass Supply Kits from NASCO (Fort Atkinson, WI) were used to collect the carcass sponge samples. Carcass sampling by the sponge method followed the procedure described in the U.S. Meat and Poultry Inspection Regulation. Ten milliliters of buffer was used to hydrate a sterile sponge. Another 15 ml was added to the sponge in the bag, to bring the total volume to 25 ml, after swabbing the sample area with the sponge. Swabbing consisted of 10 horizontal strokes and 10 vertical strokes in the template area of the brisket, flank and rump. Carcass sponge samples were taken either 2 hours (Plant X) or 24 hours (Plant Y) after carcasses were washed and placed in the coolers. The carcass sponge samples were immediately refrigerated (<4ºC) prior to shipping overnight to the laboratory in a Styrofoam insulated shipping container with freezer packs.

5. MATERIALS AND METHODS Microbiological Analysis Sponge samples were analyzed for aerobic plate count (APC) using 3MTM PetrifilmTM Aerobic Count Plates (St. Paul, MN). Coliforms and generic E. coli were analyzed using 3MTM PetrifilmTM E. coli Count Plates. Each sample was stomached for 2 min. One millimeter of broth was removed from the sample bag after stomaching and placed onto the respective PetrifilmTM plate. The samples were plated in duplicate and the plates incubated at 37ºC for 48 h. Vacuum-packaged Ground Beef Sampling Ground beef was prepared from a 50/50 mixture of shoulder and chuck (w/w). Rinsed and Control ground beef was inoculated with approximately 104 CFU/g of E. coli 0157:H7. Twenty-five gram inoculated samples were vacuum- packaged and stored at refrigerated temperatures (4-10ºC) until microbiological analysis. Triplicate samples were analyzed for the presence of APC and E. coli O157:H7 at 1, 17, 31, 50, 70 and 92 days. Statistical Analysis All bacterial counts were converted to log10 CFU/ml for statistical analysis. The statistical test used in this study was the student t-test (paired, two-tailed) and the level of significance was considered p< 0.05. The calculations were performed with Microsoft® Excel Version 2002, statistical functions (Microsoft Corp., Redmond, Washington).

6. Analysis of APC and coliforms from 2 hour cooler sponge samples over 7 sampling dates from Plant X APCNAve. CFU/cm2 % Reduction P value (95% = <0.05) Control901224 R&C9073140.3%0.039 ColiformsNAve. CFU/cm2 % Reduction Frequency of coliforms P value (95% = < 0.05) Control909722/90 R&C900.799.311/900.125

7. Analysis of APC and coliforms from 2 hour cooler sponge samples over 7 sampling dates from Plant X

8. Analysis of APC, coliforms and generic E. coli from 24 hour cooler sponge samples over 3 sampling dates from Plant Y APC NAve. CFU/cm2 % Reduction P value (95% = <0.05) Control50580 R&C5034141.2%0.009 Coliforms NAve. CFU/cm2 % Reduction Frequency of coliforms P value (95% = < 0.05) Control5034541/50 R&C5011167.831/500.002 Generic E. coli NAve. CFU/cm2 % Reduction Frequency of generic E. coli P value (95% = < 0.05) Control5029431/50 R&C504883.716/500.0008

9. Analysis of APC, coliforms and generic E. coli from 24 hour cooler sponge samples over 3 sampling dates from Plant Y

10. Rinse & Chill  Technology On-going Protection Significant on-going protection in further processing of carcasses, such as in ground beef Inoculated with E. coli 0157:H7; Vacuum packaged; 40F; 2 trials; triplicate Ave. (n=6) Day 1 Log10 CFU/g Day 17 Log10 CFU/g Day 31 Log10 CFU/g Day 50 Log10 CFU/g Day 70 Log10 CFU/g Day 92 Log10 CFU/g Control4.653.813.062.682.772.27 R&C4.763.392.051.812.070.82 P value (95% -.05) 0.1410.005*0.007*0.002*0.0006*0.021*

11. Vacuum-packaged ground beef; 40 F E. coli 0157:H7 Log10 CFU/g E. Coli O157:H7 Day

12. Rinse & Chill  Technology Conclusions Carcass swabs w/Rinse &Chill TM Reduction in APC –40.3% –41.2% Reduction in coliforms –99.3% –67.8% Reduction in generic E. coli –83.7% Vacuum-packaged Ground Beef (shoulder/chuck - 50/50) Control – 2.38 log reduction in E. coli 0157:H7 in 91 days Rinse & Chill – 3.94 log reduction in E. coli 0157:H7 in 91 days

13. Research Conducted and Supported by: University of Minnesota –Department of Food Science and Nutrition, College of Agriculture, Food and Environmental Sciences Joellen M. Feirtag, Ph.D. –Department of Clinical and Population Sciences, College of Veterinary Medicine Michael M. Pullen, DVM Agricultural Utilization Research Institute –D.T. Bartholomew, B.J. Reuter, D. Timmerman MPSC, Inc.