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April 2016 | Volume 4, Issue 2

STEC CAP News CONTROLLING SHIGA TOXIN-PRODUCING E. coli TO IMPROVE BEEF SAFETY

Director’s Update

The Annual Meeting of the International Association for Food Protection (IAFP) will be held in St. Louis Missouri on July 31-August 3 (https://www. foodprotection.org/annualmeeting/). As we have done in past years, we will have a STEC CAP breakfast there on Monday August 1. The breakfast will be Room 100 at the America’s Center Convention Center and start at 7:00 AM. All collaborators should have received the Year 5 funding by this point. The grant

is formally scheduled to end on 12/31/2016. The request for a Year 6 no-cost time extension will be submitted in the next few months. As part of the submittal, we will be judged on our accomplishments to date. Please make sure that all your Points on the Board (journal papers, conference presentations, interns,…) are up to date at the reporting hub (http://www.stecbeefsafety.org/ points-board). As part of this process, we are having each of the Objective Team leaders make presentations at the Annual Conference summarizing their team’s accomplishments, key impacts, and what is left to finish. The team leaders have received the template each group will be using. Please work with them to identify our accomplishments and to justify the funding we have received.

Dan Gallagahar

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reetings from the EMT. As usually, there are several important upcoming events we all need to be aware of. The 2016 Annual STEC conference will be held June 14-16 at the Embassy Suites in Lincoln NE. Registration is open at http://www. stecbeefsafety.org/2016-steccap-collaborators-meeting. The site also contains a link to the draft agenda. Registration is free, but we do need confirmation of your attendance. Poster session abstracts are due on May 2, 2016, so if you haven’t submitted your abstract please do so as soon as possible. All interns are expected to present a poster. Feel free to contact Jill Hochstein (jhochstein2@unl.edu; 402-472-8564), our Project Manager, with questions or for more details.

On the administrative side, we would like to thank Dan Schaefer of Cargill for his work with the Stakeholders Advisory Board. Mr. Schaefer has stepped down and so we would like to welcome Dr. John Ruby, Vice President of Technical Services

...continued page 6 STEC CAP Team 126D VBS Lincoln, NE 68905 PHONE: 402-472-8564 FAX: 402-472-9690

stecbeefsafety.org

A new face at STEC CAP

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s part of the education and outreach component of Objective 5 of the STEC CAP, 74 internships have been offered to students across the country since 2013. These internships provide students with the opportunity to gain knowledge, skills, and abilities in all aspects of food safety by partnering with a faculty mentor and their research team. In turn, this helps the STEC CAP team to recruit, mentor, engage, and train the next generation of food safety researchers. Biological Science Laboratory Technician (BSLT) Laura Shane is one of the first of these interns to be selected. Since 2013 Shane has worked with Anna Porto-Fett and John B. Luchansky and their team at the USDA Agricultural Research Service, Eastern Regional Research Center (ERRC) in Wyndmoor, Pennsylvania. Shane was selected for one of these highly-competitive STEC CAP internships while in college, and was then hired as a temporary BSLT in 2013 after graduating from Delaware Valley University with a B.Sc. in Food Science and Technology. Once a permanent

...continued page 5 Inside this issue Director’s Update ...........................................1 New Face ..........................................................1 Pepperoni..........................................................2 Student Perspective........................................6 STEC Conference coming ..............................6


STEC CAP News Validation of Post-Fermentation Heating Times and Temperatures to Control Shiga Toxin-Producing Cells of Escherichia coli (STEC) in a Pepperoni-Type Product

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ry-fermented sausage have been produced and consumed for centuries, largely without untoward consequences until some 30 years ago with the emergence of acid-tolerant and Shiga toxin-producing serotypes of Escherichia coli (Beauchamp and Sofos, 2010; Griffin et al., 2003). Our group and other investigators have reported on the fate of STEC in a variety of both short- and long-term ripened cured-dry sausage (Calicioglu et al., 2002; Chikthimmah and Knabel, 2001; Faith et al., 1997; Faith et al., 1998a; Faith et al., 1998b; Glass et al., 1992; Glass et al., 2012; Heir et al., 2013; Holck et al., 2011; Hinkens et al., 1996; Riordan et al., 1998; Rode et al., 2012). Most of these studies established that fermentation alone is only sufficient to deliver about a 1- to 2-log reduction of STEC in products such as soudjouk, pepperoni, genoa salami, and Norwegian dry-sausage. However, producers are required by USDA-FSIS (USDA, 2011) to validate a 2- or 5-log reduction of the regulated serotypes of STEC during manufacture of fermented meats (Reed, 1995a, Reed, 1995b). As part of a Blue Ribbon Task Force commissioned by the National Cattlemen’s Beef Association we validated processing parameters, commonly used by the industry, for lethality towards STEC during fermentation, curing, drying, and storage of dry-fermented sausage (Nickelson et al., 1996). As one outcome of this initiative, our team and subsequently other researchers reported that post-fermentation heating is quite likely the only effective and reliable method to achieve a 5-log reduction of STEC in certain products, notably pepperoni, without causing adverse effects on product quality. The two most widely accepted and practiced postfermentation heating parameters are arguably heating to internal temperatures of 145°F (63°C) instantaneous or 28°F (53°C) for 60 min (Hinkens et al.,1996; Nickelson et al.,1996). Thus, the purpose of the present study was to validate the lethality of post-fermentation heating times and temperatures for lethality towards single strains representing the regulated STEC serotypes to provide manufacturers with additional processes/options for ensuring the safety of dry-fermented sausage while maintaining quality and managing costs. We developed an experimental matrix and recipe/process for a non-dried pepperoni-type product to be inoculated with STEC at the USDA ARS Center for Excellence in Process

STEC CAP News Validation of Post-Fermentation Heating Times and Temperatures to Control STEC in Pepperoni-Type Sausage Fermentation parameters Target final pH Post-fermentation heating temperatures

96˚F and 85% RH pH 4.6 and pH 5.2 100˚, 110˚, 120˚, 130˚F 0.5 h to 12.5 h at 95% RH

Validation in Wyndmoor, PA (See Figure 1). In brief, coarse ground meat (25:75% beef:pork and 30:70% fat:lean) was mixed with non-meat ingredients and starter culture (Pediococcus acidilactici) and then inoculated with the STEC-8 cocktail (ca. 7.0 log CFU/g). Batter was fine ground, stuffed into fibrous casings, and then fermented to ca. pH 4.6 or pH 5.2 before being heated to target internal temperatures of 100° to 130°F over 0.5 to 12.5 h (See Figure 1). In general, the endpoint pH after fermentation ranged from ca. pH 4.5 to pH 4.7 and ca. pH 4.8 to pH 5.1. As expected, fermentation alone delivered a 0.3- to 1.2-log CFU/g reduction in pathogen numbers, albeit in less time when fermented to pH 4.6 than pH 5.2. Fermentation to ca. pH 4.6 or ca. pH 5.2 followed by post-fermentation heating to 100° to 130°F and holding generated total reductions of ca. 2.0 to 6.7 log CFU/g. Representative data for sausage heated to 120°F are shown in Figure 1. To achieve the required 2- or 5-log lethality of STEC it may be necessary to develop additional ingredients or starter cultures to inhibit this pathogen and/or to validate post-fermentation interventions, including heat and high pressure processing (HPP), to deliver the required lethality. The validated post-fermentation time/temperature heating processes described herein provide manufacturers with additional options to produce wholesome products while maintaining the highest standards of safety and without introducing appreciable capital or end-product costs. Although “drying” per se was not addressed in this study, it is highly likely that further reductions in pathogen levels would be realized following a typical drying regimen for a pepperoni-type sausage. Achieving further reductions during drying may also allow for lower temperatures and shorter times for post-fermentation heating and/or lower pressures and shorter times for HPP and is the primary objective of a companion study that is underway. These data will be useful for manufacturers of dry-fermented sausage to validate/ achieve the required reduction of STEC while producing a high-quality and wholesome product. These data also confirm that processes previously validated as effective for serotype O157:H7 strains of E. coli will likely be as effective towards strains of the other six regulated serotypes of STEC.

[NOTE: This research was conducted by Laura Shane for the STEC CAP Student Internship Program. Portions of this research were presented at the STEC CAP Annual Meeting in 2013 and the Reciprocal Meat Conference in 2014. A manuscript describing this research has been submitted for peer review]

In general, higher post-fermentation heating temperatures and lower pH delivered greater reductions of STEC

Processing

Post-Fermentation Heating Also evaluated HPP as a postfermentation kill step (87,000 psi for 0.5 to 3.0 min)


STEC CAP News

STEC CAP News

A new face at STEC CAP ...continued from page 1 position became available in 2015, Laura applied and was selected for full-time employment as a BSLT at ERRC. Shane’s interest in the field came from her involvement in her high school’s vocational Culinary Program, which taught her basic cooking techniques and piqued her interest in learning more about food science/technology. “It was kind of like a light bulb moment for me – the realization of how much science there actually is behind the foods we eat,” said Shane. “And that’s when I knew I needed to study food science.”

The project involved evaluating both the “effectiveness of fermentation” as well as different post-fermentation heating parameters, such as time and temperature. This experience helped to teach Shane about the different facets of developing and working on a scientific research project.

“[The student researchers were able to] connect with all of the other interns and collaborators and see what other amazing research is being done by the grant,” said Shane.

“Working as a STEC CAP intern was a really great experience,” said Shane. “It was truly educational to be able to participate in all aspects of the project from the planning, to the lab work, to the data entry, and scientific poster.”

Currently, Shane is working on various projects with Porto-Fett, Luchansky, and other researchers on their team, including STEC CAP Student Interns, involving numerous pathogens and their impact on food safety in addition to her “primary focus” of working with STEC in beef.

According to Shane, the STEC CAP internship program initially appealed to her because it “sounded like a really good opportunity to get involved.”

These projects are very important for public safety, as many people are occasionally exposed to potentially harmful foods due to improper cooking, storage, and/or handling practices. The results are used to help find alternate methods to safely process, store, and/or prepare foods, as well as to raise public awareness of the dangers of things like undercooked or inadequately prepared/handled beef.

It was kind of like a light bulb moment for me – the realization of how much science there actually is behind the foods we eat,” said Shane. “And that’s when I knew I needed to study food science. Her former position as an undergraduate student hourly at the ERRC, where she worked with Drs. Luchansky and Porto-Fett, helped familiarize her with the STEC CAP internship program, which she then applied to. Her interest in the field and dedication to food safety helped to land her the position. “I like to think I was chosen because I’m hard-working, responsible, and really interested in food safety, and how I can make a difference… which I think are all really important qualities that a STEC CAP intern should possess,” said Shane. Shane’s passion has been noticed by those she works with. “I saw a lot of potential in [Shane] from the get go,” said Luchansky. “[She] is enthusiastic, eager to learn, curious by nature, and very much a team player.” The research that Shane worked on during her internship evaluated “the fate of STEC following fermentation and [heating] of a pepperoni-like dry/fermented sausage.”

She also liked interacting with other researchers and discussing different projects.

“One reason it’s important to work on these projects is because a lot of the products we work on are consumed raw [that is after sometimes being cooked to less than ‘done’] or are considered readyto-eat,” said Shane. “It’s important for us to determine whether food preparation processes, such as fermentation, are sufficient in killing harmful pathogens and [making the products] safe.” Porto-Fett enjoys working with Shane on these projects and watching her gain experience. “It is very nice to see [Shane] maturing in her career as a food scientist,” said Porto-Fett. “I am very glad to have her as a team member at ERRC.” Luchansky agrees. Many of the things that Shane learned through the internship are applicable to her current work. “Participating in this internship program has taught me so much and helped me grow as a scientist,” said Shane. “I gained skills both in and out of the lab that apply to the research I do every day.” Shane was able to present the project from her internship, titled “Validation of fermentation and heating of a dryfermented sausage for control of Shiga-toxin producing Escherichia coli,” at the 2013 STEC CAP annual conference in Lincoln, Nebraska, which was an experience she enjoyed. Her research was also presented at the 2014 Reciprocal Meat Conference in Madison, Wisconsin. “[Presenting at the STEC CAP conference] was an opportunity to really take ownership for your research, and was something I’m really proud of and thankful that I was selected to participate in,” said Shane.

“I have enjoyed watching her mature both personally and professionally these past three years,” said Luchansky. “It is a real pleasure to have her on our team.” by Angelica Savoca Co-Editor-in-Chief, The Arrowhead, Souderton Area High School

...Directors Update ...continued from page 1 at JBS USA LLC, to the SAB. Finally, some well-deserved congratulations to our own Dr. Rodney Moxley on being named Charles Bessey Professor at the University of LincolnNebraska. To quote from the UNL news release: The Charles Bessey Professorship recognizes faculty members who have established exceptional records of distinguished scholarship or creative activity. Dr. Moxley is nationally and internationally recognized for his pioneering efforts and original discoveries on Escherichia coli (E. coli). Dr. Moxley’s research on bacterial cell adherence, immune responses to adherence proteins in challenged cattle, and subsequent field efficacy studies led to licensing of the first vaccine in the world for E. coli O157:H7 in cattle. Other studies in gnotobiotic piglets as a model for E. coli O157:H7 infection were the first to provide evidence that Shiga toxin 2 in particular caused severe vascular damage which could be protected against by passive immunization against the toxin. His research on E. coli as a foodborne pathogen and how it is transmitted within the food chain has significantly increased the understanding of how to mitigate this important public health concern. His work has, and continues to, benefit both veterinary and human medicine. He is an outstanding teacher who is highly regarded by the veterinary students in the Professional Program in Veterinary Medicine. His effective leadership in professional organizations, on editorial boards and grant panels, and on major research initiatives have contributed toward his outstanding reputation. Congratulations Rod! Well deserved!


STEC CAP News

STEC CAP News

Student Perspective

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hen Katherine Phetxumphou and I were invited to join the STEC-CAP grant, we were both enthusiastic to work on a project that was not necessarily our area of expertise. Up until this time, our main focus has been on environmental issues concerning water and air pollution and had no previous experience with microbial contamination on cattle or even food manufacturing. At first glance, it seemed like food science students would have been more qualified than us to tackle the project’s goals, but we quickly realized that the food industry needs experts with our skillsets as well. We have to admit that we were slightly anxious given the magnitude of the project, but were encouraged by the fact that we could use our background to help food scientists evaluate effective controls for food borne pathogens. Food scientists are specialists at monitoring for pathogens, beef production, meat processing, and consumer consumption topics, but what about topics that involve extensive farm-to-fork modeling, Monte Carlo simulations, or other statistical techniques? This part of the project was where we were able to utilize our quantitative and programming skills. It has been an opportunity that other young professionals in the environmental engineering field do not usually get. When I meet other professionals and discuss the project, I often joke that I am an environmental engineer by training, but deep down, I am a “Jack-of-all-trades” and often wish I was a food scientist. We believe this project is the epitome of interdisciplinary research. We have broadened our understanding of how to improve public health away from the standard water and air quality issues in our field. Not surprisingly, undertaking a project that is not particularly in the scope of our training has had its challenges. For example, most of our environmental engineering colleagues and professors have little background on food issues so it has been frustrating at times to solve simple, yet not-soobvious R-coding problems when no one around us knew what in the world was going on. Fortunately, we have been grateful to collaborate with food safety specialists outside the grant and abroad. Through our experiences and obstacles, we have learned to effectively communicate across disciplines and improved our technical skills.

I was able to develop a working model of a generic cattle processing plant. A model that can be assigned various input and factors to see how the system changes in “what if” scenarios. I have presented my work at the STEC-CAP annual conference in Manhattan, Kansas and IAFP conference in Portland, Oregon. These trips are at the top of my best experiences list during my whole STEC project involvement as I was able to meet people from all corners of the field who are working towards the same goal. It was exciting to see how enthusiastic and supportive they were of my work. Katherine’s part of the STECCAP puzzle involves modeling human dose-response using STEC outbreak data available in the literature. We certainly motivate each other to keep moving along whenever we hit a road block in our research. Having her perspective on particular issues, and vice-versa, has definitely help me think like a multidisciplinary researcher. Plus, it is always nice to have someone that listens to my frustrating rants of why R-studio is being uncooperative today. Overall, we are appreciative and extremely satisfied with our experience thus far. It has been a time for us to critically think and learn about issues that we were not initially comfortable with. Whether we become professionals in the engineering field or food industry, this project has been a stepping stone for our future work as interdisciplinary researchers and professional collaborators. We have met countless professionals who helped us advance our technical ability while also making lasting memories and meaningful connections. by Samson Zhilyaev and Katherine Phetxumphou

Check us out on the Web! Visit us at: www.stecbeefsafety.org Subscribe to the listserv. Send an email to: listserv@unl.edu In the message field: subscribe stecbeefsafety This project was supported by Agriculture and Food Research Initiative Grant No. 2012-68003-30155 from the USDA National Institute of Food and Agriculture, Prevention, Detection and Control of Shiga Toxin Producing Escherichia coli (STEC) from Pre-Harvest Through Consumption of Beef Products Program –A4101.

STEC CAP April Edition 2016 Vol. 4 Issue 2  
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