FILLING USDA-FSIS FOOD SAFETY `KNOWLEDGE GAPS`: THE SAFETY OF AMBIENT TEMPERATURE AIR-DRIED BEEF (DROEWORS)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1030614
• Grant No.: 2023-67017-40046
• Cumulative Award Amt.: $328,947.00
• Proposal No.: 2022-09013
• Project Start Date: Aug 1, 2023
• Project End Date: Jul 31, 2026
• Grant Year: 2023
• Program Code: [A1332]- Food Safety and Defense

Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078

Performing Department
(N/A)

Non Technical Summary
South African-style air-dried beef (droëwors and biltong) is increasing in popularity in the USA. Unlike beef jerky that is heated to 160oF and higher, this style of dried beef is held at ambient temperatures of ~75oFand~55% relative humidity for 6-8 days. Droëwors is a sausage-like dried beef product generated from beef pieces left over from biltong processing.Since these products do not receive a heat lethality treatment, USDA-FSIS requires that they pass similar food safety pathogen lethality requirements as heat-treated shelf-stable beef jerky. USDA-FSIS regards air-dried beef products as having 'knowledge gaps' and their process safety is not adequately covered in the scientific literature for them to make policy decisions. Processors must use one of two USDA-FSIS process alternatives to make these products demonstrating lethality against a 'pathogen of concern' among raw beef: 1) test all lots of ingredients for Salmonella prior to use (must test negative) and have a process that delivers a 2-log reduction of Salmonella, or 2) have a process that provides >5-log reduction of Salmonella without testing. Because of the expense of Salmonella testing, the latter is preferred. In recent years, we have generated peer-reviewed research on the biltong process that now serves as USDA-FSIS validation for processors. We propose to examine conditions to achieve >5-log reductions with droëwors dried beef with 4-strain mixtures of Salmonella serovars, E. coli O157:H7, L. monocytogenes, and S. aureus during droëwors processing; evaluate a non-pathogenic biltong surrogate organism for in-plant validation of droëwors processing, and perform microbiome analysis of droëwors. As we performed withbiltong, successful completion of this study should provide ample scientific documentationfilling so-called USDA-FSIS 'knowledge gaps' ondroëwors process lethality,allowing them to make science-basedpolicy decisions on the droëwors process of manufacture.

Animal Health Component

25%

Research Effort Categories

Basic

65%

Applied

25%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3320	1100	65%
712	4099	1100	20%
712	4099	1040	15%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3320 - Meat, beef cattle; 4099 - Microorganisms, general/other;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

air-dried beef

biltong

coriander

droewors

knowledge gaps

pathogen

safety

spice

surrogate

Goals / Objectives
The major goals of our project are to determine the safety of air-dried beef (droëwors), a new South African dried beef product that is becoming increasingly popular in the USA. This falls within the program priority area ofvalidatinginnovative processes for food processing to effectively reduce the presence of enteric pathogens, including low-moisture foods. Air-dried beef such as biltong and droëwors areprocessed differently than traditional US dried beef (beef jerky) which is heated tolethal temperaturesof 160*F or more. Biltong (dried beef) and its sausage version (droëwors) are dried at ambient temperatures and moderate relative humidity (75oF, 55% RH).The USDA-FSIS hasdeclared that the lack of scientificdata in the literature quantifying the lethality of biltong/droëwors processes on enteric pathogens preventsthem from making decisions on biltong/droëwors processes from a scientific standpoint and therefore they have required processors to 'provide microbial validation' that the manufacture of these products is safe.The USDA-FSIS provided several alternative processes, one of which included demonstrating greater-than-or-equal-to a 5-log reduction of a pathogen of concern for biltong/droëwors (i.e., Salmonella), and claimed thatthis was a 'knowledge-gap' area and fertile ground for additional research. Based on this need, my lab provided additional research on biltong, publishing multiple peer-reviewed studies demonstrating >5-log reduction of Salmonella, STEC E. coli, Listeria monocytogenes, and Staphylococcus aureus on biltong;the use of alternative salts (CaCl2, KCl)that could be used instead of NaClto still achieve a >5-log reduction of pathogens and yet reduce sodium levels;and identify surrogate non-pathogenic bacteria that could serve as inoculumorganisms for in-plant validation. These studies have been recommended by USDA-FSIS to commercial processors for use as validation of their processes (if using the same conditions). However, droëwors sausage offersdifferent processing dynamics in whichpotential contaminating bacteria are internalized and coated in fat/protein, and are not on the surface as in biltong. The droëwors air-dried process remains to be scientifically validated for pathogen reduction and that is the intention of this project.

Project Methods
The methods used follow our statedobjectives.During this research, we will evaluate the 'log-reduction' of pathogenic and non-pathogenic surrogate organisms during processing in the manufacturer of air-dried sausage (droëwors).A) Culture-dependent and culture-independent microbiome analysis ofdroëwors processing. Culture-dependent microbiome analysis involves the actual isolation ofbacteria from samples throughout the droëwors process. It is universally accepted that you are not able to isolate the majority of organisms given the different media as well as the anaerobic vs aerobic requirements of different bacteria. But it allows you to generally recover representatives of the predominant organisms that are present. Culture-independent microbiome analysis involvesthe extraction oftotal DNA from whatever bacteria are presentand using PCR/16-S rRNA gene sequencing to determine the identities of the bacteria. The advantage is that detection/identification does not involve the ability to recover and grow the organism.Both of these methods of microbiome analysis will be performed withoutinoculation usingpathogens or surrogate bacteria, but simply evaluating the change of indigenous microbiota that occurs from raw beef through processing ofdroëwors.B) Surrogate analysis. Nonpathogenic surrogate validation of droëwors process to achieve 5-log reduction (in-lab validation of droëwors sausage process inoculated with surrogates). We previously examined the response of various nonpathogenic bacteria in biltong processing in order to identify one (or more) that mimics the process reduction obtained withpathogenic bacteria, so that they couldbe used for in-plant process validation using theactual equipment in a biltong processing facility. For biltong processing, we found that a predominant nonpathogenic organism on isolated from thefinal product (Carnobacterium spp.) resembled the reduction obtained with 4 different groups of foodborne pathogens and was implicated to be a good candidate asa surrogate organism for in-plant validation of the biltong process. However, this organism may not be a good surrogate candidate for droëwors sausage which hasdifferent processing dynamics droëwors since bacteria in droëwors sausage areinternalized. The search for a surrogate organism for in-plant validation ofdroëwors air-dried sausage would come from organisms isolated duringthe culture-dependent process described earlier, and then be assessed as a potential surrogate organism by using it as an inoculum of beef used in the droëwors sausage process.C) Pathogen reduction during droëworsprocessing. Microbial pathogen validation that the droëwors air-dried sausage process achieves >5-log reduction of foodborne pathogens typically associated with raw/dried beef (Salmonella, L. monocytogenes, S. aureus, E. coli O157:H7) involves inoculation of beef pieces with pathogens, following thedroëwors process of coarse grinding, marination, encasing in collagen casings, drying for 6-10 days,and enumerating the remaining pathogens at various stages of this process. Identification of multiple antibiotic resistances on all of our pathogenic inoculum bacteria allows us to recover them from background indigenous bacteria on media containing those antibiotics to determine their response to thedroëwors process. This experimentation will involve multiple individual trials with each of the various pathogens.Although USDA-FSIS only requires process validation with a single pathogen of concern (i.e.,Salmonella), corporate food safety managers ofvarious large supermarket chains often require additional safety assurances from processors they purchase from byrequiringdata from trials withadditional pathogens (i.e.,E. coliO157:H7,L. monocytogenes,S. aureus). Validation of the droëwors process with these additional pathogens will provide stronger confidence thatthe process provides a safe product for consumers.D) Staph enterotoxin analysis. Validation is also neededthat staphylococcal enterotoxin (SE) is not produced internally during the time course ofair-drying droëwors sausage at ambient temperatures. One of the concerns with microbial process validation ofbiltong was that vacuum-tumbling of meat during marination resulted in what USDA-FSIS considers as contributing to status as 'non-intact beef' whereby bacteria on the surface could be drawn into the beef during vacuum tumbling and the concern is whetherStaphylococcus aureusmay produceenterotoxins in the moist center of the beef before it is sufficiently dried. In the droëwors air-dried process, bacteria on beef pieces that arecoarsely ground and encased in collagen casing are internalized in thesausage and itcould be days beforethe point in the process where the product is sufficiently dried, and water activity reduced,to be considered safe from bacterial growth. This is especially so considering that there is no heat lethality step and the product is dried at a moderate~75oF. We will inoculate beef pieces with enterotoxigenic strains ofS. aureus(SEA, SEB)and use ELISA assay kits to detect if SEA/SEB enterotoxin levels increaseduring the processing of air-dried droëwors sausage. Should there be an increase in SEA/SEB production during processing, we will have to include a step to inhibit S. aureusearly in the process, perhaps at the stage before beef pieces are coarsely ground. However, in our biltong trials, the processing procedure was sufficient to inhibitS. aureusand we found lower enterotoxin levels than the residual culture carryover levels when we started.E) In-Plant Droëwors Validation using Surrogate Bacteria. Trials will be performed with nonpathogenic surrogate bacteria inoculated on beef to be used in the FAPC Meat Pilot Plant to verify in-lab data with commercial scale, in-plant validation of the droëwors sausage process to ensure adequate log reduction upon scale-up. USDA-FSIS has been actively supporting 'in-plant validation trials' by food processors because it is recognized that each processor has their own (different) equipment that may perform the same function but have different efficiencies and capabilities (temperature heating capacity, temperature come-up time, humidity generation, refrigerated chilling capacity, etc) that affect the performance of food processing. Although we have developed validation protocols in the lab with scientific equipment, not all processors have equipment capable of maintaining temperature/humidity with such precision. In-plant validations with non-pathogenic surrogate bacteria allow aprocessor to see how efficiently their commercial setup will perform towards their targeted processing conditions and microbial reduction goals.

Progress 08/01/23 to 07/31/24

Outputs
Target Audience:Project Title: Filling USDA-FSIS Food Safety `Knowledge Gaps`: The Safety of Ambient Temperature Air-Dried Beef (Droewors) Target Audience: This projectinvolves research into the safety of 'air-dried beef' (biltong, droewors). These are South African dried beef products that are gaining interest in the USA. In order to manufacture these products in the US, the USDA-FSIS regulatory agency requires that they follow manufacturing standards of US dried beef products, such as beef jerky.An obstacle to manufacturing and selling biltong and droewors in the US was that companies must achieve USDA-FSIS process validation established for traditional dried beef (i.e., beef jerky) and USDA-FSIS has identified these South African air-dried beefproductsas a 'knowledge gap' area that needs to be validated for safety. For instance, beef jerky is subject to a high heat lethality cook step (>185oF) with 90% humidity (steam) whereby biltong/droewors is simply dried at 75oF for 6-10 days. Therefore, USDA-FSIS required that air-dried beef processorsprovide either microbial validation studies or peer-reviewed scientific literature. The most relevant target audience would be potential beef processors who would manufacture and sell droewors/biltong in the US.This project targets a variety of audiences that include meat processors, consumers, students, food industry workers, professionals, scientific academicians, and the general public. Scientific presentations and journalarticles target scientific and academic professionals, food industry management, and the general public. The availability of alternate methods of ensuring the safety of air-dried beef should be well received by the meat processing industry, potential consumers, USDA-FSIS, and the scientific community. Changes/Problems:Changes: Based on our examination of 'acid-adapted' vs 'non-acid-adapted' cultures, we will use non-acid-adapted cultures as we believe the acid-adapted growth leads to stress in the cultures destined to be used as challenge organisms on droewors air-dried beef. What opportunities for training and professional development has the project provided?Graduate and undergraduate students who work on this project are located in the Robert M. Kerr Food & Agricultural Products Center. Aside from their involvement in this research, they have an opportunity to assist food processing companies through multiple projects that matriculate through ourlabs, or through the Center, to carry out our mission to help Oklahoma and national based companies with issues/problems they have regarding further processed foods. The PI's Food Microbiology labs arestructured so that graduate students not only work on their specific research projects but also provide company assistance by contributing efforts on industry projects to help solve food microbiology-related problems that localcompanies may be experiencing. This provides a great opportunity for undergraduate and graduate students to enhance theirproblem-solving abilities with actual problems incurred by the food industry. Because of this, >90% of ourMS/PhD-degreegraduates are well sought after and find placement in the food industry; several of ourPh.D. students have found academic faculty positions as well. I would say that our work provides good training and excellent opportunities for those students who spend the time to learn additional skills while earning their degree. In several recent years, wehad 3 undergraduates from ourlabs work on research that helped them land positions toward aveterinary medicine degree. Our center also puts on many food safety workshops each year (HACCP, Food Defense, Preventive Controls for Human Foods, SQF, FSMA Compliance) for industry, and both graduate and undergraduate students are encouraged to take these workshops to enhance their knowledge and capabilities. Students taking thesecourses/workshops get accredited by the appropriate organizations and they are well sought after by industry who would otherwise have to pay to send employees for these trainings. We also provide certificates for students (or industry workers) who have taken a sufficient course load of workshops that they are awardedthe "FAPC Certificate of Training as a Food Safety Professional". The certificate helps identify those employees who have achieved sufficient training that they should be considered strategic assets within their organization. Likewise, students who attain the certificate during their undergraduate/graduate degrees would be considered valuable job candidates by food companies that don't have to 'retrain' them once they are hired. The industry has responded well to our certificate program and we have now 'graduated' approximately 28people (total) who have achieved these certificates of training. How have the results been disseminated to communities of interest?The results of our research work are disseminated via peer-reviewed research publications (several publications have been submitted by the prior PI), departmental/university magazines, and the R.M. Kerr Food & Ag Product Center (FAPC) website. Also, information is disseminated through seminars or presentations: we had a poster presentation at the FAPC Food Science Research Symposium (April 2024) and additional updated poster presentation will be made at both the Project Directors' Mtg and the Annual Meeting of the International Association of Food Protection in Long Beach, 2024. We have an in-house communication specialist who helps us put out short bulletins/articles (FAPZ.biz website, Food Safety blog articles, and FAPC podcasts) and other extension-related publications. What do you plan to do during the next reporting period to accomplish the goals?Droewors and Biltong Air-Dried Beef Process Approval by USDA-FSIS.The USDA-FSIS provides 2 alternative processesfor acceptance of the manufacture ofair-dried beef since air-dried beef (biltong, droewors) does not conform to beef jerky manufacture, acceptance of the manufacturing process depends on obtaining validated studies (private lab, published peer-reviewed research). The acceptance can be either of the 2 alternative processes below: 1) Alternative 1: Test all lots of edible ingredients (meat, spices, salt) for the presence/absence of the pathogen of concern (i.e.,Salmonella) before use (must test negative), and demonstrate >2-log reduction of that pathogen during processing, or 2) Alternative 2: Validation needs to showthat the process yields >5-log reduction of the pathogen of concern during processing (and no need for Salmonellatesting). Note: In Alternative 1, testing each lot of ingredients forSalmonella(or other pathogen) is costly, and takes time,and if tested positive, the ingredientcan not beused, and if testing is missing, it could lead to a process deviation. Although the 2-log reduction ofSalmonellais an achievable process, theSalmonellatesting makes this cumbersome and less desirable for processors. In the Alternative 2process, a>5-log reduction of Salmonella (or other raw meat-associated pathogen) may not be easy to obtain, but onceconditions are identified to achieve that level, it would be a preferableprocess to use bymanufacturers. Currently, processors are using conditions identified for 5-log reduction of Salmonella for biltong air-dried beef for use as the process for droewors. The objective of our work is to validate the safety of droewors either by 5-log pathogen reductionor Salmonellatesting + 2-log process. Droewors process vs non-pathogenic surrogates for in-plant validation. In prior work with biltong, we demonstrated that non-pathogenicCarnobacterium sp.gave an equivalent 5-log reduction that we obtained withSalmonella, E. coliO157:H7,L. monocytogenes, andS. aureus.The use of non-pathogenic bacteria has been accepted by USDA-FSIS for process inoculation during in-plant process validation whereby the actual process is validated whereby conditions may differ greatly from those experienced in a laboratory setting. Droewors process vsSalmonella ser., E. coliO157:H7,L. monocytogenes,and/orS. aureus.We will also examine the droewors process against various 'pathogens of concern' for this type of product that may be contributed from theraw materials.If current processing using standard industry processes for droewors fails to achieve a 5-log reduction, we will examine organic aciddips of the meat (inoculated meat in our trials) and other allowable antimicrobials (i.e., peroxyacetic acid).

Impacts
What was accomplished under these goals? Acid-Adapted Cultures. Before initiating validation trials with 4-5 companies to be submitted to USDA-FSIS for validation of their process, our experimental protocols were submitted to USDA-FSIS to ensure they would accept the process by which the work was performed. One of USDA-FSIS' requirements was that the challenge cultures be 'acid-adapted' by growing them in media (TSB) with 1% glucose. With eitherSalmonella, E. coliO157:H7,L. monocytogenes, or S. aureus,this would reduce the pH of the media after culturingfrom ~6.8-6.9 in TSB with 0%glucoseto pH ~4.7-4.9 when cultured in media containing 1% glucose. This likely came from work done in the mid-1990s when meat processors were applying acidic rinses to beef carcasses and beef trimmings to reducethe prevalence ofE. coliO157:H7 andSalmonella in the finished product and because of the concept of pathogen acid-resistance, they wanted to ensure that the processes that manufacturers came up withwere sufficiently robust that they would reduce even acid-resistant pathogens. As we were awarded this grant, we wanted to validate this concept so we examined 'acid-adapted' vs 'non-acid-adapted' cultures in the biltong process. We used pathogens grown under both conditions and found that the acid-adapted cultures gave larger log reductions in biltong processing. We have hypothesized that this is the result of acid adaption causing acid stress to the cultures whereby they are more sensitive to process conditions (salt, desiccation) and recommend that growth under non-acid-adapted conditions (+1% glucose media, buffered to maintain pH 6.6-7.0) would provide cultures free from acid stress when cultured in media down topH 4.7 and directly comparable to acid-adapted cultures grown in media +1% glucose but without buffering. Microbial Reduction During Droewors Processing. We applied a typical droewors process (averageconditions from 3 different manufacturers) using a 4-strain cocktail ofCarnobacterium divergensandCarnobacterium gallinarum(non-pathogenic bacteria for in-plant validation) that we previously obtained >5-log reduction withthe biltong process. Using similar conditions for droewors (as averaged from the 3 commercial processes) as were used for biltong, through 4 trials we only obtained 2.6-2.9-log reductions.So far we believe we can achieve the Alternative 1 process (see the section on "what to do duringnext reporting period"), but preferable we would like to strive towards achieving >5-log reduction, if possible. Note:We believe it will be difficult to achieve the 5-log reduction as obtained with biltong without using additional antimicrobials since biltong, the bacterial inoculum, and marinade (vinegar, salt, spices) are adsorbed to the surface of solid meat during tumble marination and the surface inoculum is readily affected by processing conditions (salt increasing to 4% with >60% moisture loss, desiccation directly affecting surface bacteria. In droewors, the process includesgrinding of meat, bacteria, and marinade (vinegar, salt, spices) so the big difference is the distribution of bacteria and ingredients throughout the meat in a sausage-like product whereby the bacteria are encased in meat/protein and the antimicrobial ingredients are diluted throughout because of the grinding.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jade Wilkinson, K. Gavai, P. Muriana. The Effect of Acid Adaptation of Pathogenic Bacteria Used as Challenge Organisms for Microbial Validation of the Biltong Process. FAPC Research Symposium, Oklahoma State University, Stillwater, OK (March 28, 2023).
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jade Wilkinson, K. Gavai, P.M. Muriana. Effect of Acid Adaptation on Pathogenic Bacteria used as Challenge Organisms for Microbial Validation of the Biltong/Droewors Process. Undergraduate Res. Symposium, ConocoPhillips Alumni Center (April 18, 2023).
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Pratikchhya Adhikari, J. Wilkinson, K. Gavya, P. Muriana. Effect of Acid-Adaptation of Challenge Cultures on Pathogen Viability During Process Validation of Air-Dried Beef (Dro�wors, Biltong). FAPC Food Science Research Symposium, Stillwater, OK (April 11, 2024).