95% of consumer exposure to nitrite is from consumption of vegetables, they modified their stance on nitrite that if companies used 'natural nitrite' (i.e., use of microbial fermentation of vegetable extracts to generate nitrite from vegetable nitrate), that they could label their food product as having 'no added preservatives' on the label (but can not call the product 'cured'). This is attracting numerous companies because of the change in stance on labelling. We will be validating the use of surrogate Clostridium spp. (Clostridium sporogenes) that could be used for in-plant validation of commercial processes as well as examining some of our culturecollections for bacteria thatcanmodifynitrate into nitrite. A final aspect of my project is to characterize notable attributes offoodborne pathogens and/or spoilage organisms that could be helpful to food safety of consumers or beneficial to food processors. Some of these attributes may be the microbial basis of adherence of Listeria monocytogenes or other bacteria involved in biofilms; the use of sanitizers/antimicrobials to kill bacteria entrapped in biofilms (biofilms provides protection to the enclosed bacteria); characterization of gas- or slime-producting bacteria that result in spoiled meat products. Understanding the basis of biofilm formation or biological activities resulting in gas or slime production will better allows us to implement interventions to make foods safe and prevent spoilage, both of which have significant economic impacts in the food industry.' />
Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
DETECTION, CHARACTERIZATION, AND INHIBITION OF FOODBORNE PATHOGENS, TOXINS, AND SPOILAGE MICROORGANISMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1016158
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2018
Project End Date
Sep 30, 2023
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Food And Agriculture Products Center
Non Technical Summary
The proposed project is specifically involved with various facets of food safety: bacteriocins (antimicrobial peptides) produced by lactic acid bacteria for use as food preservatives and antimicrobial interventions against foodborne pathogens. Since we have identified different bacteriocins that inhibit targeted pathogens (i.e., Listeria monocytogenes) by different modes of action, our approach is to use a mixture of bacteriocins comprising these different modes of action in order to obtain the best efficacy against the targeted pathogen. Part of this project will also be looking to screen for more/new bacteriocin-producing strains as we might find some that are better than those currently in our collection. We will also be looking to identify potentialprobiotic bacteria that could bebeneficial to food production animals if provided in animal feed by evaluating various biochemical reactions that they mightpossess.Another aspect of this project is to evaluate the use of 'natural nitrite' against sporeforming Clostridia. Chemical nitrite is a food preservative that must be listed on the ingredient label; label-conscious consumers often to not like the presence ofnitrite in their food because of the potential development of carcinogenicnitrosaminesand consciously look for alternative products (a noted product line that uses nitrite is cured meats such as hotdogs).In recent years it has becomewidely publicized that vegetables are known sources of nitrate and when consumed, the microorganisms in our intestinal tract convert the nitrate to nitrite.Recently, FDA and USDA-FSIS, considering that >95% of consumer exposure to nitrite is from consumption of vegetables, they modified their stance on nitrite that if companies used 'natural nitrite' (i.e., use of microbial fermentation of vegetable extracts to generate nitrite from vegetable nitrate), that they could label their food product as having 'no added preservatives' on the label (but can not call the product 'cured'). This is attracting numerous companies because of the change in stance on labelling. We will be validating the use of surrogate Clostridium spp. (Clostridium sporogenes) that could be used for in-plant validation of commercial processes as well as examining some of our culturecollections for bacteria thatcanmodifynitrate into nitrite. A final aspect of my project is to characterize notable attributes offoodborne pathogens and/or spoilage organisms that could be helpful to food safety of consumers or beneficial to food processors. Some of these attributes may be the microbial basis of adherence of Listeria monocytogenes or other bacteria involved in biofilms; the use of sanitizers/antimicrobials to kill bacteria entrapped in biofilms (biofilms provides protection to the enclosed bacteria); characterization of gas- or slime-producting bacteria that result in spoiled meat products. Understanding the basis of biofilm formation or biological activities resulting in gas or slime production will better allows us to implement interventions to make foods safe and prevent spoilage, both of which have significant economic impacts in the food industry.
Animal Health Component
65%
Research Effort Categories
Basic
25%
Applied
65%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123320110020%
7123330110020%
7123260110010%
7123520110010%
7124010110010%
7124099110015%
7127299110015%
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; 3260 - Poultry meat; 3520 - Meat, swine; 3330 - Other beef cattle products; 4010 - Bacteria; 7299 - Research equipment and methods, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Major goals and specific objectives of this project:To examine the use of biological interventions (bacteriocins of lactic acid bacteria; bacteriophage) to inhibit pathogens and spoilage organisms in foods.Multiple Mode-of-Action (MOA). Comprise a 'bacteriocin cocktail' containing as many MOAs as possible (4-5?).Bac+ culture isolations and identifications using bacteriocin PCR array, Mass Spec.Bacteriocins: surface applications. Use of bacteriocin-containing fermentates as surface antimicrobials to inhibit L. monocytogenes on RTE meats as multi-MOA mixtures.Bacteriocins: added into foods. Use of MOA mixtures or with other antimicrobials.Bacteriocins: use of bacteriocin-producing LAB (Bac+ LAB) cultures. to inhibit pathogens/spoilage organisms in food (using the cultures themselves in foods).Bac+ LAB cultures to modify vegetable nitrate into nitrite for applications against Clostridium spp. in vacuum packaged foods.Generate 'commercialized bacteriocins' in a form that can be used by companies to test in their products (liquid concentrate, freeze-dried powders).2. 'Natural Nitrite'. To use 'natural nitrite' (i.e., vegetable-source nitrate → microbial fermentation conversion to nitrite) for control of Clostridia spp. in vacuum-packaged products.Identify the effectiveness of Bac+ LAB to inhibit Clostridium sporogenes (and/or Cl. perfringens). To use 'green label' culture fermentate preparations derived from GRAS LAB to inhibit and reduce survival of Cl. sporogenes, Cl. perfringens.Identify the effectiveness of Bac+ LAB to inhibit spore germination of Clostridium sporogenes (and/or Cl. perfringens). To use 'green label' culture fermentate preparations derived from GRAS LAB to inhibit spore germination from Cl. sporogenes, Cl. perfringens.Compare Bac+ LAB fermentates with 'natural nitrite ' commercial fermentates. To compare our mixed-mode-of-action culture fermentates with the effectiveness of other commercial culture fermentates for inhibition of L. monocytogenes on hotdogs.Examine Bac+ LAB for the ability to modify vegetable-nitrate into nitrite. To examine the ability of our 'green label' anti-listerial/anti-clostridial LAB to convert vegetable-sourced nitrate to nitrite while simultaneously producing the inhibitory bacteriocins in their fermentates.Examine Bac+ LAB fermentates to inhibit Clostridium sporogenes spores/cells in processed RTE and vacuum-packaged meats. To examine the effect of our mixed-MOA LAB culture fermentates on preventing germination of Clostridium spores, or inhibition of Clostridium vegetative cells, in vacuum-packaged cooked meats.3. Probiotic bacteria. Analysis of probiotic bacteria that may be beneficial to animals.Culture collection 1 (Muriana). Several hundred cultures have been isolated from retail/raw foods and from animal sources and evaluated for bacteriocin production. We will also examine a number of characteristics that might contribute to ruminant (or non-ruminant) digestion such as starch-, lipid-, and protein-hydrolysis, bile resistance, adherence to tissue culture cells as a potential indication to adherence to intestinal epithelia, and other characteristics.Culture collection 2 (Gilliland). Another culture collection is available from which were derived the largest IP revenue for Oklahoma State University (cultures licensed to Chrs. Hansen for 'BovamineTm ' microbial product for cattle.New animal isolates (FAPC slaughter). The R.M. Kerr Food & Ag Products Center routinely harvests ~160 cattle, 360 swine, 40 goats, 5 sheep annually in their slaughter facility. These present an opportunity to collect bacterial isolates from freshly harvested animals.4. Foodborne pathogens and spoilage organisms. Characterization of foodborne pathogens and spoilage organisms.Various pathogenic and spoilage bacteria have special attributes that contribute to their problematic nature with foods. Strains of L. monocytogenes that are strongly-adherent persist in food processing establishments; spoilage organisms that produce 'slime' (dextrans) or gas become problematic to meat processors. Molecular or biochemical examination of special attributes (adherence, biofilm formation, gas/slime production) of pathogens or spoilage organisms may help find solutions to alleviate problems with organisms.
Project Methods
Methods. The general scientific methods applied to our laboratory research are basic food microbiology protocols. Specific methods will be required in performing a number of experimental approaches such as the use of tissue culture to evaluate the adherence of bacteria that might implicate a culture that could be a good 'probiotic' bacteria. Other microbial testing will be targeted towards the identification of beneficial biochemical activity of select bacteria (proteolytic, lipolytic, saccharolytic, bacteriocinogenic) that could also augment probiotics for cattle/animal feed or use as food preservatives. Isolation of new bacterial strains will be identified by PCR amplification of 16S rRNA and submitted to our Core Facilities for sequencing, and then searching the NCBI database for matching identities. Our activities withantimicrobial interventions has drawn interest and support from the food industry, some of which have provided us with unique equipment (I.e., Unitherm Food Systems) that has facilitated our industry outreach and research impact. Academic classroom methods of evaluation are the various 'tests' given throughout the course and even duringworkshops we implement a pre- and post-workshop testing to measure the impact of our workshops as indicators of success.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:This project covers a variety of audiences that are reached through various efforts. Efforts in the teaching of formal classes provide an audience of students and future technology professionals. Efforts made in presenting seminars, workshops (HACCP workshops), symposia, lay emagazine articles (www.fapc.biz: FAPC Connect; FAPC Food Safety blog, FAPC youtube channel), and youtube videos cover a wide range of audiences that includes consumers, students, industry workers, professionals, managers, academicians, state legislators, and the general public. Scientific presentations and journal articlestarget scientific/academic professionals, administrators, legislators, food industry management,and the general public.Interested subscribers from around the world as well as technical and scientific-minded individuals. Extension/outreach activities that are often done with small companies provide an audience of food-related businessmen and industry professionals and sometimes leads to new research areas Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Aside from academically-oriented food microbiology research, students and staff in the food microbiology program at theRobert M. Kerr Food & Agricultural Products Center have an opportunity to assist food processing companies through multipleprojects that matriculate through my lab in order to carry out our mission to help Oklahoma food companies (and nationallybased companies) with issues/problems they have regarding further processed foods. My Food Microbiology lab is structuredthat graduate students not only work on their specific research projects but also support company assistance by contributing effort on industry projects to help solve food microbiology related problems that local companies may be experiencing. This provides a great opportunity for undergraduate and graduate students to enhance their problem-solving abilities with actual problems incurred by the food industry. Because of this, 90% of my MS-degree graduates are well sought and find placement in the food industry; several of myPh.D. students have found academic faculty positions as well. I would say that our work provides good training and excellentopportunities for those students who spend the time to learn additional skills while earning their degree. This year I have had3 undergraduates work in my lab. Our center also puts on many food safety workshops each year(HACCP, Food Defense, Preventive Controls for Human Foods, etc) for the industry, and both graduate and undergraduate students are encouraged to take these workshops to enhance their knowledge and capabilities. FAPC Certificate for Training as a "Food Safety Professional". I lead a team to initiate the 'FAPC Certificate for training as a Food Safety Professional' for industry and students who accumulate sufficient credits of workshop training (12credits) requiring a minimum of 2 workshops in each of three workshop categories: Basic, Advanced, and Regulatory. Thecertificate helps identify those employees who have achieved sufficient training that they should be considered strategicassets within their organization. Likewise, students who attain the certificate during their undergraduate/graduate degreeswould be considered as valuable job candidates by food companies that don't have to 'retrain' them once they are hired. Theindustry has responded well to our certificate program and we have now 'graduated' approximately 20people that haveachieved these certificates of training (http://fapc.biz/workshops/food-safety-professional). How have the results been disseminated to communities of interest?The results of our research work are disseminated via peer-reviewed research publications, departmental/universitymagazines, the R.M. Kerr Food & Ag Product Center (FAPC) web site. Also, information is disseminated through seminars orpresentations at the Nevada Food Safety Task Force (2015) or the FDA Western Regional Conference (2016), AnnualMeeting of the International Association for Food Protection (Tampa, FL, 2017; Salt Lake City, UT, 2018; Louisville, KY, 2019; virtual-online, 2020) or workshops where industry-applicable research is presented and discussed. We have an in-house communication specialistwho does well to help us put out short bulletins/articles (FAPZ.biz website, FAPC Connect, Food Safety blog articles,and FAPC podcasts) and other extension-related publications. Some of our industry project reports are used by companies toprovide documentation forUSDA process validation. What do you plan to do during the next reporting period to accomplish the goals?1. Biltong (dried beef) processing: a) Validation of biltong processing (5-log reduction) withListeria monocytogenes, E. coliO157:H7, andStaphylococcus aureus. b) Microbiome and metagenomic analysis of organisms during the processing of biltong, from raw beef to dried beef product. 2. Industry collaborations with various food companies.

Impacts
What was accomplished under these goals? Accomplishments under the above-stated goals: Objective #2.'Natural Nitrite'. To use 'natural nitrite' (i.e., vegetable-sourcenitrate→ microbial fermentation conversion tonitrite) for control ofClostridiaspp. in vacuum-packaged products. a)Isolation of nitrate-reducing bacteria (NRB's) and quantification of nitrate/nitrite by HPLC analysis. We developed an 'on-agar' colony-screening assay to detect the conversion of nitrate to nitrite on agar plates using M17 agar base plates (1.5%). After incubation and colonies developed embedded in the sandwich overlay, nitrite was detected using a thin (plain) overlay agar layer containing sulfanilic acid followed by a second overlay agar layer containing alpha-naphthyl amine; the appearance of red color zones above colonies indicated the presence of nitrite. C8 reversed-phase ion-pairing HPLC analysis showed thatStaphylococcus caprae and Panteoa agglomerens,isolated with the M17-Nitrite agar assay,were able to reduce nitrate (ferment) to nitrite in broth to 915 ppm and 866 ppm nitrite, respectively.Examination of new isolates will allow the more efficient applicationofnatural vegetable nitrite in the processed meat industry. b) Spores ofClostridium sporogenes wereused as a '(pathogen-surrogate) challenge organism' for inoculation of low-fat and high-fat frankfurters. A three-strain spore crop from Clostridium sporogenes (ATCC 3584, ATCC 19404, and ATCC BAA-2695) was applied during ingredient formulation of low and high fat hotdogs that were divided into 3 batches (control without nitrite, hotdogs with sodium nitrite, hotdogs with celery nitrite). In both processes, sodium nitrite was compared to comparable levels of celery nitrite.In our shelf life assays, growth was inhibited at both 5oC and 15oC, even if nitrite was absent; however, spore germination and outgrowth readily occurred at 35oC. Comparison of nitrite effects is best evaluated at 35oC as a permissive condition to examine the effects of nitrite treatments. Celery nitrite was as good as sodium nitrite in both low and high-fat hotdogs and spore outgrowth was only observed at 35oC abuse temperature conditions. The nitrite validation assay described herein allows easy determination if nitrite levels can prevent spore germination underpermissive conditions to help keep processed meat safe. Objective #4. Foodborne pathogens and spoilage organisms. a) Evaluation of a selective agar medium for enumeration ofSalmonellaserovars used as challenge organisms after conditions of stress.SalmonellaThompson 120,SalmonellaHeidelberg F5038BG1,SalmonellaHadar MF60404,SalmonellaEnteritidis H3527, andSalmonellaTyphimurium H3380 were characterized for antibiotic resistance and acid adaptation in Tryptic Soy Broth containing 0%, 0.25%, or 1.0% glucose. Sodium pyruvate was evaluated for recovery after stress but no enhancing effect was observed, possibly because the strains were acid-adapted. Selenite Cystine Broth, traditionally used as a selective enrichment broth, was used as the basis for Selenite Cystine Agar (SCA) in combination with three antibiotics to which ourSalmonellais resistant. Serovars ofSalmonellawere enumerated on TSA, SCA, Xylose Lysine Desoxycholate (XLD), and Hektoen Enteric (HE) selective agars (all containing the same antibiotics) after conditions of nutrient starvation, desiccation, acid stress, and thermal stress. The data show that quantitative enumeration of ourSalmonellaserovars on SCA was not significantly different (p> 0.05) than those achieved on TSA for all tested stress categories. Levels ofSalmonellaenumerated on XLD and/or HE were significantly different (p< 0.05) than on TSA and SCA and often more than 1-2-log lower, consistent with the inhibition of injured cells. b) Biltong (dried beef) processing to achieve USDA-FSIS validation.Our objectives were to examine processes for the manufacture of biltong that achieves a 5-log reduction ofSalmonellawithout a heat lethality step and with, or without, the use of additional antimicrobials. Beef pieces (1.9 cm × 5.1 cm × 7.6 cm) were inoculated with a 5-serovar mixture ofSalmonella(SalmonellaThompson 120,SalmonellaHeidelberg F5038BG1,SalmonellaHadar MF60404,SalmonellaEnteritidis H3527, andSalmonellaTyphimurium H3380), dipped in antimicrobial solutions (lactic acid, acidified calcium sulfate, sodium acid sulfate) or water (no additional antimicrobial), and marinaded while vacuum tumbling and/or while held overnight at 5 °C. After marination, beef pieces were hung in an oven set at 22.2°C, 23.9°C, or 25°C depending on the process and maintained at 55% relative humidity. Beef samples were enumerated forSalmonellaafter inoculation, after dip treatment, after marination, and after 2, 4, 6, and 8 days of drying. Water activity was generally <0.85 by the end of 6-8 days of drying and weight loss was as high as 60%. Trials also examined salt concentration (1.7%, 2.2%, 2.7%) and marinade vinegar composition (2%, 3%, 4%) in the raw formulation. Nearly all approaches achieved a 5-log10reduction ofSalmonellaand were attributed to the manner of microbial enumeration eliminating the effects of microbial concentration on dried beef due to moisture loss. This is the first published report of a biltong process achieving >5.0 log10reduction ofSalmonella. c)Process lethality (5-Log Reduction) ofSalmonellaand salt concentration during sodium replacement in biltong marinade.This study evaluated the use of alternative salts, potassium chloride (KCl), and calcium chloride (CaCl2) in the biltong marinade to achieve a ≥ 5-log reduction ofSalmonella, a pathogen of concern in beef products. Beef pieces (1.9 cm × 5.1 cm × 7.6 cm) were inoculated with a five-serovar mixture ofSalmonella(SalmonellaThompson 120,SalmonellaEnteritidis H3527,SalmonellaTyphimurium H3380,SalmonellaHeidelberg F5038BG1, andSalmonellaHadar MF60404), vacuum-tumbled in a traditional biltong marinade of salt, spices, and vinegar containing either NaCl, KCl or CaCl2(2.2% concentration) followed by an 8-10 day drying period at 23.9 °C (75 °F) and 55% relative humidity. Microbial enumeration ofSalmonellawas conducted following inoculation, after marination, and after 2, 4, 6, 8, and 10 days of drying in a humidity/temperature chamber. Biltong produced with CaCl2, NaCl, or KCl achieved a > 5-log reduction ofSalmonellaafter 6, 7, and 8 days, respectively. As expected, the biltong made with the corresponding salt had the most abundant ion in the sample. A sampling of several commercial brands of biltong for sodium content showed that some were significantly above the allowable level of claims made on package ingredient statements. The substitution of NaCl with KCl or CaCl2during biltong processing can also provide a 5-log reduction ofSalmonellato produce a safe product. d) Evaluation of new generation QAC sanitizer for effectiveness against Pseudomonas and Staphylococcus biofilms.A sanitizer waseffective against E. coli O157:H7, Salmonella spp., and L. monocytogenes was applied against enhanced biofilms of strains of Staphylococcus spp. and Pseudomonas spp. (as required by EPA) in 96-wells microplates. Additionally, worker boots were swabbed with a trypsin solution and then treated with the sanitizer spray solution. Bacteria isolated (before treatment) were identified by 16S rRNA PCR and DNA sequencing. All treatments were carried out in triplicate replication and analyzed by RM-ANOVA using the Holm-Sidak test for pairwise multiple comparisons to determine significant differences (p < 0.05).There was a ~4-5 log reduction of bacterial strains (microplate assay) within the first 1 min of treatment and also greater > 3-log reduction in bacterial population from encrusted biofilms from workers' boots.Rotation of sanitizer chemistries may prevent selective retention of chemistry-tolerant microorganisms if they may occur.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Karolenko, C.; Muriana, P. Quantification of Process Lethality (5-Log Reduction) of Salmonella and Salt Concentration during Sodium Replacement in Biltong Marinade. Foods 2020, 9, 1570.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Karolenko, C.E.; Bhusal, A.; Nelson, J.L.; Muriana, P.M. Processing of Biltong (Dried Beef) to Achieve USDA-FSIS 5-log Reduction of Salmonella without a Heat Lethality Step. Microorganisms 2020, 8, 791.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Karolenko, C.E.; Bhusal, A.; Gautam, D.; Muriana, P.M. Selenite Cystine Agar for Enumeration of Inoculated Salmonella Serovars Recovered from Stressful Conditions during Antimicrobial Validation Studies. Microorganisms 2020, 8, 338.
  • Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Bhusal, A. Vegetable nitrate: Isolation of nitrate reducing bacteria for fermentation of nitrate to nitrite and use of vegetable-derived nitrite to inhibit germination of Clostridium spores in RTE meats. MS dissertation, Oklahoma State University, July 2020 (Peter Muriana, advisor).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Karolenko, C.; Bhusal, A.; Gavai, K.; Muriana, P. Novel processing of dried beef products (Biltong) without antimicrobial intervention to achieve USDA-FSIS validation of Salmonella (5-log reduction). Annual Meeting (virtual), International Association of Food Protection, October 26-28, 2020, Poster P2-51.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Karolenko, C.; Bhusal, A.; Muriana, P. Selenite cystine agar as a selective enumeration media for Salmonella serovars used in antimicrobial intervention studies incurring conditions of metabolic stress. Annual Meeting (virtual), International Association of Food Protection, October 26-28, 2020, Poster P2-93.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Shah, K.; Muriana, P. Validation of sanitizer effectiveness against Staphylococcus and Pseudomonas biofilms, natural biofilms from workers boots, and selective correlation of biofilm bacteria to sanitizer chemistry. Annual Meeting (virtual), International Association of Food Protection, October 26-28, 2020, Poster P3-169.
  • Type: Websites Status: Published Year Published: 2020 Citation: https://www.facebook.com/FAPCFoodMicroLab
  • Type: Theses/Dissertations Status: Accepted Year Published: 2020 Citation: Shah, K. Application of next generation quaternary ammonium chloride sanitizer against Staphylococcus and Pseudomonas laboratory biofilms and natural biofilms found on worker's boots in a meat processing plant. MS dissertation, Oklahoma State University, December 2020 (Peter Muriana, advisor).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Bhusal, A.; Muriana, P. Isolation, characterization, and HPLC quantitation: nitrate reducing bacteria and their fermentation of nitrate to natural vegetable nitrite. Annual Meeting (virtual), International Association of Food Protection, October 26-28, 2020, Poster P2-103.
  • Type: Websites Status: Published Year Published: 2020 Citation: https://www.researchgate.net/profile/Peter_Muriana


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:This project covers a variety of audiences that are reached by a variety of efforts. Efforts in the teaching of formal classes (Food Microbiology FDSC3154/MICR 3154; Food Microbiology and Safety FDSC 5120; Advanced Food Microbiology FDSC 4153) provides an audience of students and future professionals. Efforts made in presenting seminars, workshops (HACCP and FSPCA workshops), symposia, lay emagazine articles (www.fapc.biz: FAPC Connect; FAPC Food Safety blog, FAPC youtube channel), and youtube videos cover a wide range of audiences that includes consumers, students, industry workers, professionals, managers, academicians, state legislators, and the general public. Scientific presentations and journal articles target scientific/academic professionals, administrators, legislators, and the general public (Google Scholar shows that my peer-reviewed research papers have been 'cited' >2,850 times). My laboratory's facebook website http://www.facebook.com/FAPCFoodMicroLab has >2,798 facebook 'likes') and my page in Research Gate https://www.researchgate.net/profile/Peter_Muriana has >19,636 'reads' of posted research papers) covering an audience of interested subscribers from around the world as well as technical and scientific-minded individuals. Extension/outreach activities that are often done with small companies provide an audience of food-related businessmen and industry professionals and sometimes lead into new research areas (Clements' Foods, A&G Organics, Decon7, Unitherm Food Systems/Marlen, Wayne Farms, Florida Foods LLC, etc). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Aside from academically-oriented food microbiology research, students and staff in the food microbiology program at the Robert M. Kerr Food & Agricultural Products Center have an opportunity to assist food processing companies through multiple projects that matriculate through my lab in order to carry out our mission to help Oklahoma food companies (and national based companies) with issues/problems they have regarding further processed foods. My Food Microbiology lab is structured that laboratory personnel work on funded research projects, but also support company assistance by working on projects to help solve food microbiology related problems companies may be experiencing. This provides a great opportunity for students, staff, post-docs to enhance their problem-solving abilities with actual problems incurred by the food industry. Because of this, 90% of my MS-degree graduates are well sought and find placement in the food industry; several of my Ph.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. This year I have had 4 undergraduates work in my lab and currently have 2 that will continue into the spring semester (one is pursuing the possibility of graduate/MS program under my advisorship). Our center also puts on many food safety workshops each year (HACCP, Food Defense, Preventive Controls for Human Foods, etc) for the industry, and both graduate and undergraduate students are encouraged to take these workshops to enhance their knowledge and capabilities. FAPC Certificate for Training as a "Food Safety Professional". I have lead a team to initiate the 'FAPC Certificate for Training as a Food Safety Professional' for industry and students who accumulate sufficient credits of workshop training (12 credits) requiring a minimum of 2 workshops in each of three workshop categories: Basic, Advanced, and Regulatory. 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 as 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 12 people that have achieved these certificates of training (http://fapc.biz/workshops/food-safety-professional). How have the results been disseminated to communities of interest?The results of our research work are disseminated via peer-reviewed research publications, departmental/university magazines, theR.M. Kerr Food & Ag Product Center(FAPC) web site.Also, information is disseminated through seminarsor presentations at the Nevada Food Safety Task Force (2015) or the FDA Western Regional Conference (2016), Annual Meeting of the International Association for Food Protection (Tampa, FL, 2017; Salt Lake City, UT, 2018;Louisville, KY, 2019) or workshops where industry-applicable research is presented and discussed. We have an in-house communication specialist who does well to help us put out short bulletins/articles (FAPZ.biz website,FAPC Connect,Food Safety blogarticles, andFAPC podcasts) and other extension-related publications. Some of our industry project reports are used by companies to provide as documentation to USDA-FSIS or FDA on validation studies that we perform and/or to provide to other companies interested in their products. What do you plan to do during the next reporting period to accomplish the goals?1. Bacteriocin antimicrobials as potential food preservatives, 2. Analysis of Biofilms, biofilms in food processing environments, and the effective application of sanitizers that can address biofilms. 3. Antimicrobial interventions on meat/poultry products (i.e., 'natural nitrite', biltong processing to reduce Salmonella, STEC E. coli) 4. Industry collaborations with various food companies.

Impacts
What was accomplished under these goals? 'Natural Nitrite'. To use 'natural nitrite' (i.e., vegetable-source nitrate → microbial fermentation conversion to nitrite) for control of Clostridia spp. in vacuum-packaged products. Spore crop of Clostridium sporogenes as a surrogate organism for pathogenic Clostridium spp. Spore crops were obtained by the growth of 4 different strains of Cl. sporogenesin broth or on agar plates (cultured anaerobically) such that final spore crop levels were approximately 108 spores/ml. Testing of vegetable nitrite vs sodium nitrite in sous vide Chicken and all-beef frankfurters. Spores were usedon the surface ofchicken breast(sous vide)or in the meat matrix ofall-beef hotdogs prior to cooking, Products consistedof meat products inoculated without nitrite (control), with sodium nitrate (chemical preservative), or vegetable (celery) nitrite (i.e., 'natural nitrite').Products were sampled before and after heating/cooking, daily, and then weekly to determine whether outgrowth occurs. Vegetable nitrite performed similarly to sodium nitrite whereas products without nitrite resulted in high levels of outgrowth when incubated at abuse temperatures. The data shows that vegetable nitrite can prevent spore germination similar to sodium nitrite. Nitrite Agar using M17 broth. Commercial nitrate broth was converted into nitrite agar (1.5%) and tested for the detection of nitrate-reducing bacteria using a 2-layer overlay method similar to the in-tube nitrate broth reduction test. Initial isolatesof nitrate-reducing bacteria (NRBs) appeared to alwaysbe Gram(-) bacteria. We, therefore, developed an 'M17 Nitrate Agar' which allowed us to recover Gram(+) NRBs.When tested, we observed strains that could convert nitrate to nitrite while others converted nitrate to nitrite and then to other nitrogenous compounds. HPLC assay for Nitrate/Nitrite. An HPLC system (reversed-phase C8 column, diode array detector, automated sample injector, quad pump) was used with ion-pairing to quantify nitrate and nitrite from vegetable and meat samples. We are using this system on extracts of raw/cooked food samples that contain nitrite to follow the fate of nitrite during the shelf life of food products. Probiotic bacteria. Analysis of probiotic bacteria that may be beneficial to animals. Microbial agar assays were developed to detect lipolytic, proteolytic, cellulolytic, and sacharolytic activity by bacterial colonieson agar plates. Culture collection and animal sample testing. Over >500 cultures from the Gilliland/FAPC culture collection and Muriana culture collection and approximately24cattle and swineintestinal samples were screened to detect bacteria withthe enzymatic activities described above. We have isolated 4 bacteria that demonstrate all 4 activities listed above (other bacteria have one or several activities, but 4 have all 4 activities). These would make good candidates as potential dietary adjuncts to facilitate rumen activity (cattle) or digestive abilities of swine. We continue to look for support to perform animal trials with these cultures. Foodborne pathogens and spoilage organisms. Characterization of foodborne pathogens and spoilage organisms. Biltong validation. We have been working with processors to provide USDA-FSIS 5-log reduction of Salmonella during the processing of biltong (no heat lethality step). We use acid-adapted serovars of Salmonella to inoculate raw beef (~1-in x 2-in x 3-in)that is vacuum-marinaded in spices/salt/vinegar and incubated at 70-80*F at ~55% RH for 6-8 days. Water activity of internal beef portions is determined as a measure of safety. We have achieved a 5-log reduction of Salmonellaafter 6-8 days with Aw < 0.85. Selective media for Salmonella. Salmonella serovar strains were characterized for antibiotic resistance and all 5 serovars were resistant to the same 6 antibiotics and 3 antibiotics were usedintryptic soy agar as a selective medium. However, background bacteria grew up against the 3 antibiotics and we have examined other selective media that could complement the antibiotic resistance. Results - Achieving 5-log reduction allows those processors to manufacture and sell biltong products under USDA-FSIS inspection. We hope to publish our data soon so that other processors could benefit and make use of the published data. Biofilms and Sanitizers. A microplatebiofilm assay platform was developed. Strains of 3 differentfoodborne pathogens were screened using the 5,6-CFDA fluorophore to identifythe strongest-adhering strains of L. monocytogenes, Salmonella, and E. coli O157:H7. These strains were used to develop biofilms to examine the efficacy of 5 commercial sanitizers. Decon7 was the most effective against L. monocytogenes, Salmonella, and STEC E. coli (see research journal publications) and has now shown efficacy against Pseudomonas spp. and Staphylococcus aureus as well. We will further examine Decon7 in apractical processing environment.

Publications

  • Type: Other Status: Published Year Published: 2019 Citation: Lock, Tori. FAPC assists with validation of South African dried meat product, Feb. 2019. https://news.okstate.edu/articles/agricultural-sciences-natural-resources/2019/biltong_project.html
  • Type: Other Status: Published Year Published: 2019 Citation: Muriana, Peter. The Food Files Podcast: Food Trends - Probiotics. https://www.podbean.com/eu/pb-bfdgx-bd5699