Validation of Pathogen Surrogate Escherichia coli strains for Systemic Microbial Reductions in Fresh Beef Harvest and Processing

This in-plant study was conducted to determine the overall level of bacterial reduction by multiple interventions over a two-day period. ATCC non-pathogenic surrogate strains of E.coli were utilized in this study to mimic the behavior of Salmonella and E.coli. Foreshanks of beef carcasses were inoculated a total of two times per each repetition. A total of three repetitions were conducted, each over a two-day period. On day one, samples were collected post initial inoculation and post day one interventions. On day two, samples were collected from previously inoculated and treated carcasses, post reinoculation, and post day two treatments. Sample collection was performed utilizing 25 mL BPW pre-hydrated sponge swabs, sampling the outside of the foreshank each time covering a total surface area of 100 cm2. Microbial loads were determined using spread plating technique and counting. The average level of attachment on day one achieved for the three repetitions was 7.43 logs. Post intervention, the level of microbial load remaining was 1.67 log, resulting in an average 5.76 log reduction. The average level of attachment of day two achieved for the three repetitions was 7.54 logs. Post intervention, the level of microbial load remaining was 5.3 log, resulting in an average 2.24 log reduction. To summarize the reduction between the three repetitions and day one and day two, an 8-log average reduction was demonstrated when combining all interventions performed by the plant. This standard of operation is replicable industry wide for antimicrobial treatment validation. Introduction: With beef being the third most consumed meat globally (18), and the United States being the top producer of such (19), maintaining safe production lines is critical for maintaining a dependable food supply. On average, Americans consume 55 pounds of beef per person, per year (11), with the United States also being a net beef importer, as lower quality and cheaper meats are often purchased and processed domestically (20). Global production has more than doubled since 1961 (16), with beef production taking up an estimated 3.7% of yearly greenhouse gas emissions (12). With this production at peak levels and showing no signs of slowing, large scale producers employ multiple interventions to reduce microbial populations and ensure a non-contaminated product. Most pre-harvest interventions used to reduce risk of pathogen contamination occur at the feedlot level, as it is the most manageable and manipulatable (2). Probotics, such as Probicon or Bovamine, are employed as well, as they have been shown to reduce levels of pathogens detected in lymph nodes in cattle (8). Antibiotics are additionally used as well, as they change the gut microbiome of the rumen in cattle and allow for more compounds to be produced in the gut of cattle that are beneficial (7). When it comes to treatments administered at time of harvest, dehairing is a process that removes hair on the hide of the carcass, resulting in a visually cleaner carcass and reduces level of trimming of fecal contamination required (9). This physical intervention occurs within the production facility, and producers also employ vacuuming of treatment regions of carcasses to remove excess liquid after washing, which has been shown previously to reduce bacterial counts by 1.0 log CFU/100 cm2 (3). Ozonated water is often employed as well, in which water is created with passing of an electric current through oxygen gas, creating the water that has antimicrobial properties (10). In addition, beef processors use multiple antimicrobial interventions to reduce microbial loads including a lactic acid spray, a hot water spray treatment, and blends of acids. The lactic acid spray is commonly used industry wide, and this plant utilized it during the initial stages immediately after dehiding on pre-evisceration carcasses. In a study conducted by Castillo et al., researchers investigated the microbial reduction of Escherichia coli O157:H7 and Salmonella Typhimurium effectuated by a lactic acid intervention. Researchers applied a 500 ml spray of lactic acid at 4% concentration for a total of 30 seconds both prechill and post chill, as they also wanted to study if reductions are different on a hot carcass vs a cold carcass. Researchers found that prechill, treatments reduced the counts from 3.3-3.4 log cycles with a water wash alone, to 5.2 log cycles when employing a lactic acid treatment in addition to a water wash, almost a 2-log increase. Post chill, the lactic acid treatment showed a 2.0 to 2.4 log reduction in E. coli O157:H7 and a 1.6 to 1.9 reduction in Salmonella Typhimurium. (5) A multitude of studies have demonstrated a strong ability for lactic acid to kill bacteria and prevent potential contamination and this intervention is commonly used industry wide within beef production. Researchers at Texas Tech University in 2005 conducted a study to determine the ability of four strains of lactic acid bacteria (LAB) to inhibit Escherichia coli O157:H7 and Salmonella in ground beef. Additionally, researchers were looking to determine if these bacteria had any sensory effects on the product and would decrease the overall favorability of consumption for consumers. Samples of lactic acid bacteria were stored and pathogen populations were counted on days 0,4,8, and 12. After 4 days of storage, researchers found that there was significant pathogen reduction, with an average of 1.5 log cycle reduction of Escherichia coli O157:H7, and 3-log cycle reduction of Salmonella. Researchers concluded that addition of LAB to ground beef at refrigeration temperatures can be a critical and effective intervention for controlling foodborne pathogens. Within the production process, most of the contamination on carcasses comes from the hides of the cattle. This occurs when animals defecate and spread bacteria from the excrement onto their hide or from the pens, the trailer or other animals. Once hides are removed, the legs, or shanks, are one of the most highly contaminated regions of the carcass, thus is an area of concern when application of interventions is administered to reduce microbial load (6) Hot water treatments are also commonly used, and as addressed previously in the study conducted by Castillo et al., are an effective way at helping lower the microbial load of carcasses. In this plant, both a hot water cabinet on the whole carcass and a steam cone were used to on shanks of the carcass, in which the steam cone was specially designed and fitted to slide directly over the fore and hind shanks of the carcass. Finally, Citrolow is a blend of acids that are classified as GRAS (generally recognized as safe), creating a low pH environment that is non-conducive to the survival and growth of bacteria. Broadly speaking, these mixtures of acids are generally the same ingredients, serving as an antimicrobial treatment that can be utilized at various stages within the production process. A study published in January of 2018 showed that spray washing with Citrolow reduced Enterobacteriaceae counts by 2.14-2.57 log cycles, 3.06-4.08 log cycles, and 4.65-4.99 on the belly, head, and butt regions of cattle carcass, respectively (14). These treatments, in conjuncture with lactic acid sprays, hot washes, and removal of visible debris by employees, are all effective and safe methods for reducing potential for pathogenic bacteria and produce a safer packaged product. These interventions have been validated as effective antimicrobial interventions against both Escherichia coli O157:H7 and Salmonella, and have been tested a multitude of times within the plant utilized in this specific study. Portillo et.al bio mapped this environment in 2024, demonstrating clear microbial reductions. Samples were collected at three phases in the production chain: the lairage area, harvest floor, and fabrication floor. Researchers at Texas Tech showed a statistical difference in reduction of pathogens using the TEMPO system, demonstrating a clear and effective hurdle approach employed at the plant being studied(15). . While these interventions are effective, researchers must find ways to test these interventions in a non-laboratory setting. This is where surrogate bacteria can be utilized. Surrogate strains of E. coli that are non-pathogenic and mimic the behavior of pathogenic E. coli and Salmonella were developed by researchers to test the effectiveness of in-plant treatments in beef production facilities (12). While treatments can be tested for their efficacy within a laboratory setting, testing within the actual processing facility gives a much more accurate representation of overall cleanliness and effectiveness. Every possible condition within a processing facility cannot be accurately replicated within a laboratory, so it is critical to verify treatment effectiveness within the processing facility that will be utilizing the intervention. Pathogens, however, can’t be introduced into a facility, so there is a need to utilize pathogens surrogates to achieve the goal of in plant behavior. The strains created and utilized for this study are ATCC E. coli strains BAA 1427,1428,1429,1430,and 1431 (1). The objective of this study was to develop an industry standard for measuring a 5-log reduction in raw beef products throughout the processing chain. While there exist previous studies that demonstrate the effectiveness of the strains used as effective surrogates for Salmonella and E. coli (19), research is still needed to demonstrate a 5-log reduction within a beef processing plant from harvest to final beef trimmings. This project looked to validate the usage of mitigation treatments applied to beef carcasses to reduce microbial loads.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Food Science

Grantor

Texas Tech University

Year dc:date.issued

2025

Author dc:creator

• Starnes, Michael

Chair dc:contributor.committeechair

• Brashears, Mindy

Committee members dc:contributor.committeemember

• Miller, Mark
• Echeverry, Alejandro

• Surrogate
• Validation
• Beef
• Interventions

Handle dc:identifier.uri

https://hdl.handle.net/2346/102360

OAI identifier oai:identifier

oai:ttu-ir.tdl.org:2346/102360