Progress 11/22/13 to 09/30/18
Outputs
Target Audience:This project covers a variety of audiences that are reached by my various 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 magazine articles ('Pathogen Patrol'/www.fapc.biz; FAPC Connect), and youtube videos cover a wide range of audiences that include 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' >2,700 times). My laboratory's facebook websitehttp://www.facebook.com/FAPCFoodMicroLab ;>1,250 facebook 'likes') and my page in Research Gate https://www.researchgate.net/profile/Peter_Muriana; >15,000 'reads' of posted research papers) covers 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 (Unitherm Food Systems, Michael Foods, 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. In addition, an undergraduate student (Mr.Cade Lemons) has received an Undergraduate Research Award (Dept. Animal Science UR Research Award) to perform research projects 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 (12 credits) requiring a minimum of 2 workshops in each of the 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 jobcandidates by food companies that don't have to 'retrain' them once they are hired. The industry has responded well to our certificate program. How have the results been disseminated to communities of interest?The results of our research work are disseminated via peer-reviewed research publications as well as through seminars such as 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, 2018) 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 magazine, Pathogen Patrol articles; FAPC Connect) and other extension-related publications. Some of our industry project reports are used by companies to provide as documentation to USDA 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?
Nothing Reported
Impacts
What was accomplished under these goals?
Major goals of the project. My lab continues to be a leader in microbial validation of industry food processes by application of thermal interventions (pre-package and post-package RTE meat surface pasteurization), chemical antimicrobialinterventions (antimicrobials applied to reduce E. coli/Salmonella in ground beef; antimicrobial spray prior to mechanical blade tenderization to mitigate translocation to the interior of tenderized beef), andbiological interventions (examination of bacteriophage to reduce E. coli / Salmonella in ground beef; use of bacteriocins to inhibit Listeria monocytogenes on RTE meats). What was accomplished under these goals? Bacteriocins as antimicrobial food preservatives. One long-term project that has been a staple in my lab has been the use of lactic acid bacteria (LAB)that produce antimicrobial peptides (bacteriocins, Bac+) that could function as effective food preservatives. We have identified numerous Bac+ LAB that is inhibitory to Listeria monocytogenes. I have generated spontaneous Bac-resistant strains of L. monocytogenes that can distinguish bacteriocins having different modes of action. When bacteriocins of different modes of action are combined, they make effective antimicrobial food preservative cocktails for use in foods against susceptible pathogenic (or spoilage) microorganisms. These bacteriocins are heat-stable and can even be added to foods that will be cooked and still retain antimicrobial activity. We are now examining their effect on other pathogens (Staphylococcus, Bacillus, Clostridium) to broaden the application range of these antimicrobials. Validation of food processes: Biltong process validation without atypical 'heat-kill' step. We have worked on a biltong process, which is aSouth African style beef jerky but without the heat-kill step. USDA-FSIS still requires a 5-log Salmonella reduction for process validation and we have performed testing with inoculated beef, vacuum-tumbling with spice-seasoned marinade, antimicrobial dips, and incubation in a humidity oven to demonstrate >5.5-log reduction after 4-6 days of drying. Use of Clostridium sporogenes as a surrogate for Cl. botulinum/perfringens for in-plant evaluation of celery nitrite. We have characterized the use of celery nitrite to inhibit Cl. sporogenes (3-strain spore mixture) in sous vide chicken breasts and we are currently examining the impact of celery nitrite to inhibit Cl. sporogenes germination in low- and high-fat beef franks. We have used permissive incubation temperatures after heat treatment to demonstrate germination and growth of spores in nitrite-negative control samples while inhibition germination/growthis demonstrated in nitrite-positive samples. We will be examining whether bacteriocins can function in the role of suppressing germination of Clostridium spores. Surface proteome of Listeria monocytogenes involved with adherence. We examined the use of Orbitrap Mass Spectrometry to screen proteins extracted from the surface ofstrongly- and weakly-adherent strains ofL. monocytogenes as a means of identifying differences in surface proteins that might correlate with the different adherence phenotypes. Differences in proteins were subject to gene expression analysis and several surface (and subsurface) genes were found to correlate to strong adherence. The use of sophisticated protein diagnostic tools is being more exploited to help identify molecular anomalies in foodborne pathogensthat can identify molecules that canimpact adherence that may lead to persistence in food processing plants that lead to contaminated foods, illness, and possibly deaths.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
National Advisory Committee on Microbiological Criteria for Foods (Muriana). 2018. Response to Questions Posed by the Department of Defense Regarding Microbiological Criteria as Indicators of Process Control or Insanitary Conditions. J. Food Prot., 81(1): 115-141. https://doi.org/10.4315/0362-028X.JFP-17-294.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Dennis Pletcher, A. Boeken, M. Aryal and P.M. Muriana. 2018. Use of Listeria innocua and Clostridium sporogenes as Surrogate Organisms for In-plant Validation of a Sous Vide Process for Chicken Breasts Using Celery Nitrite. P1-36. Intl. Assoc. Food Protection Annual Meeting (Salt Lake City, UT; July 8-11).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Manish Aryal and P.M. Muriana. 2018. Microplate Lethality Assay to Determine the Efficacy of Commercial Sanitizers for Inactivation of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. in Extended Biofilms. P1-63. Intl. Assoc. Food Protection Annual Meeting (Salt Lake City, UT; July 8-11).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Audrey Boeken and P.M. Muriana. 2018. Use of Green-label Bacteriocin-containing Microbial Fermentates for Control of Listeria monocytogenes in RTE Meat Applications. P3-220. Intl. Assoc. Food Protection Annual Meeting (Salt Lake City, UT; July 8-11).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Giovana Bodnar, P.M. Muriana, G. Nakazato, and L. Ma. 2018. Synergistic Antimicrobial Effect of Eugenol and Biologically Synthesized Silver Nanoparticles against Listeria monocytogenes. T2-10. Intl. Assoc. Food Protection Annual Meeting (Salt Lake City, UT; July 8-11).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Boeken, A., and P.M. Muriana. 2018. Application of a multiple mode-of-action bacteriocin cocktail as a natural antimicrobial for RTE meat applications. FAPC Research Symposium/OSU Research Week, Oklahoma State Univ., Stillwater, OK (Feb. 20, 2018).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Aryal, M., and P.M. Muriana. 2018. Evaluation of the efficacy of commercial sanitizers by microplate assay for inactivation of Listeria monocytogenes, E. coli O157:H7, and Salmonella sp. in biofilms. FAPC Research Symposium/OSU Research Week, Oklahoma State Univ., Stillwater, OK (Feb. 20, 2018).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Pletcher, D., A. Boeken, M. Aryal, and P.M. Muriana. 2018. In-plant validation of a sous vide process for chicken breasts with celery nitrite using Listeria innocua and Clostridium sporogenes (spores) as surrogate organisms. FAPC Research Symposium/OSU Research Week, Oklahoma State Univ., Stillwater, OK (Feb. 20, 2018).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Bodnar, G.C., G. Nakazato, P.M. Muriana and L.M Ma. 2018. Effect of eugenol and biologically synthesized silver nanoparticles against foodborne pathogen. FAPC Research Symposium/OSU Research Week, Oklahoma State Univ., Stillwater, OK (Feb. 20, 2018).
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Audrey Boeken (MS Food Science, May 2018). Characterization and use of bacteriocin-containing microbial fermentates for control of Listeria monocytogenes in RTE meat applications. Advisor: Peter Muriana. Department of Animal and Food Sciences, Oklahoma State University.
- Type:
Websites
Status:
Other
Year Published:
2018
Citation:
Facebook Food Micro Laboratory Page: https://www.facebook.com/FAPCFoodMicroLab.
- Type:
Websites
Status:
Other
Year Published:
2018
Citation:
Research Gate:
https://www.researchgate.net/profile/Peter_Muriana
- Type:
Websites
Status:
Other
Year Published:
2018
Citation:
Google Scholar Publication Citations:
http://tinyurl.com/googlescholarpmm
- Type:
Websites
Status:
Other
Year Published:
2018
Citation:
ORCID:
https://orcid.org/0000-0002-6448-1769
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:This project covers a variety of audiences that are reached by various 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, Farm-Focused Food Safety, and FSPCA workshops), symposia, lay magazine articles ('Pathogen Patrol'/www.fapc.biz), and youtube videos covers 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. My laboratory's facebook web sitehttp://www.facebook.com/FAPCFoodMicroLab ) and my page in Research Gate(https://www.researchgate.net/profile/Peter_Muriana) covers an audience of interested subscribers (i.e., >1,000 facebook'likes') from aroundthe world as well as technical and scientific-minded individuals. Extension/outreach activities that are often done with smallcompanies provides an audience of food-related businessmen and industry professionals (Unitherm Food Systems, Michael Foods). 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 PhD 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 time to learn additional skills while earning their degree. In addition, an undergraduate student (Ms. Alyssa Riggio)has received Undergraduate Research Award (Dept. Animal ScienceUR Research Award) to perform research projects in my lab. Our center also puts on many food safety workshops each year (HACCP, Food Defense, Preventive Controls for Human Foods, etc) for industry,and both graduate and undergraduatestudents areencourage to take these workshops to enhance their knowledge and capabilities. How have the results been disseminated to communities of interest?The results of our research work are disseminated via peer-reviewed research publicationsas well asthrough seminars such as presentations at the Nevada Food Safety Task Force (2015) or the FDA Western Regional Conference (2016), Annual Meeting of theInternationalAssociation for Food Protection (Tampa, FL; 2017)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 magazine, Pathogen Patrol articles) and other extension-related publications. Some of our industry project reports are used by companies to provide as documentation to USDA or FDA on validadation 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?I plan to continue our work with: 1. Bacteriocin antimicrobials as potential food preservatives, 2. Biofilm formation by L. monocytogenes on food processing equimentand evaluation of sanitizers effectiveness against biofilms. 3. Antimicrobial interventions on meat/poultry products in concernt with the use of plant-based nitrite (i.e., 'natural nitrite'). 4. Industry collaborations with various food companies.
Impacts
What was accomplished under these goals?
2017 Food Microbiological Research: 1. Bacteriocins as antimicrobials in foods. We have continued progress using mixtures of bacteriocins demonstrating different modes-of-action (MOA) for use as a topical agent in hotdogs during shelf life. The application of these antimicrobials inhibited Listeria monocytyogenes compared to control hotdogs and demonstrated favorable activity relative to several commercial antimicrobials and 'fermentates' currently in use. We will continue to examine the use of these cultures to co-commitantly produce bacteriocin while changing plant-based nitrates into nitrite during fermentation so that resulting fermentates contain both natural nitrite and natural bacteriocin inhibitors. 2. Analysis of antimicrobial activity in lysozyme unrelated to muramidase activity. In a project collaboration with a commercial company (Michael Foods), we examined heat-modified, chemical-modified, and proteoltically-hydrolzed lysozyme enzyme for antimicrobial activity unrelated to muramidase enzyme activity (i.e., hydrolysis of beta-1,4-linkage of peptidoglycan). Residual inhibitory activity unrelated to muramidase activity was difficult to determine from heat- and chemically-modified lysozyme, because of the potential for refolding of the partially denatured lysozyme molecule. However, we were able to identify significant antimicrobial activityagainst Listeria monocytogenesfrom clostripain-digested lysozyme that was unrelated to muramidase activity as determined by modified lysozyme activity assays. The prospects of internal bioactive (inhibitory) peptides residing within the lysozyme enzyme molecule provides potential for commercial exploitation for food safety, and possibly in combination with residual muramidase activity. 3. Flame grill for surface pasteurization of whole raw onions to eliminate Listeria and spoilage microorganisms prior to further processing. We also provided validation support for a commercial company (Unitherm Food Systems) that manufactures food processing equipment for the food industry. We used non-pathogenicListeria innocua (BSL-1) as a surrogate microoganism for surface inoculation of whole raw onions prior to passage through a gas flame oven (a process for which Unitherm Food Systems has a patent). Our data showed that surface flame peeling of onions significantly reduces the levels of total bacteria, yeast and molds, and our L. innocua inoculum indicating that this process would enhance food safety of processed onions antecedant to further processing (i.e.,reducing/eliminating the surface microbiota prior to further processing (slicing/dicing) would reduce the bacterial load contaminated the cut pieces and improve both food safety and quality by reducing both potential pathogens and spoilage organisms. This process is currently being used nationally and internationally.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Vijayakumar, P.P. and P.M. Muriana. 2017. Inhibition of Listeria monocytogenes on ready-to-eat meats using bacteriocin mixtures based
on mode-of-action. Foods 2017, 6, 22; doi:10.3390/foods6030022.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Aryal, M. and P.M. Muriana. Surface pasteurization of post-harvest raw whole onions to eliminate Listeria contamination prior to further processing. Poster # P1-123. Intl. Assoc. Food Protection, Annual Meeting, Tampa, FL, Jul 9-12, 2017.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Tiong, H.K. and P.M. Muriana. Identification of putative surface proteins involved in adherence of Listeria monocytogenes on abiotic surfaces. Poster # P2-153. Intl. Assoc. Food Protection, Annual Meeting, Tampa, FL, Jul 9-12, 2017.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2017
Citation:
Manish Aryal (MS Dissertation, Dept. Animal Science, Oklahoma State University, December, 2017). Microplate lethality assay to determine the efficacy of commercial sanitizers for inactivation of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella in biofilms.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Boeken, A., and P.M. Muriana. 2017. Identification and Characterization of a New Bacteriocin Mode of Action Against Listeria monocytogenes using Bacteriocin-Resistant Strains. FAPC Research Symposium/OSU Research Week, Feb. 16, Oklahoma State Univ., Stillwater, OK.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Tiong, H.K., and P.M. Muriana. 2017. Analysis of Surface Proteomics Involved in Adherence of Listeria monocytogenes to Abiotic Surfaces. FAPC Research Symposium/OSU Research Week, Feb. 16, Oklahoma State Univ., Stillwater, OK.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Aryal, M., and P.M. Muriana. 2017. Use of a Gas Flame Oven for Surface Pasteurization of Post-Harvest Raw Whole Onions to Eliminate Listeria Contamination. FAPC Research Symposium/OSU Research Week, Feb. 16, Oklahoma State Univ., Stillwater, OK.
- Type:
Websites
Status:
Published
Year Published:
2017
Citation:
Laboratory Facebook Page: https://www.facebook.com/FAPCFoodMicroLab.
Facebook page of laboratory activities of our food microbiology lab; has generated >1,000 'likes' nationally and internationally.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:This project covers a variety of audiences that are reached by my various 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, Farm-Focused Food Safety, andFSPCA workshops), symposia, lay magazine articles ('Pathogen Patrol'), and youtube videos covers 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. My laboratory's facebook web site http://www.facebook.com/FAPCFoodMicroLab ) and my page in Research Gate (https://www.researchgate.net/profile/Peter_Muriana) covers an audience of interested subscribers (i.e., 'likes') from around the world as well as technical and scientific-minded individuals. Extension/outreach activities that are often done with small companies provides an audience of food-relatedbusinessmen and industry professionals. 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 throughmultiple projects that matriculate throughmy 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 PhD students have found academic faculty positions as well.Iwould say that our work provides good training and excellent opportunities for those students who spendtime to learnadditional skillswhile earning their degree. In addition,an undergraduate student (Ms. Elizabeth Farney) whoreceive undergraduate research awards (Wentz Semester Scholar & CASNR-OAES UR Research Award) to perform research projects in my labwas accepted at the Kansas State Veterinary Schoolalthough Ioffered her a graduateassistantship in my lab. Our center also puts on many food safey workshops each year (HACCP, Food Defense,Preventive Controls for Human Foods,etc) for industry, butstudents are alsoencourage to take these workshops to enhance their knowledge and capabilities. How have the results been disseminated to communities of interest?The results of our research work are disseminated mainly via peer-reviewed research publications, but also through seminars such as presentations at the Nevada Food Safety Task Force (2015) or the FDA Western Regional Conference (2016), 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 (FAPC Flash articles), longer descriptions (FAPZ.biz magazine, Pathogen Patrol articles) and other extension-related publications. Some of our industry project reports are used by companies to provide as documentation to USDA or FDA on validadation 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?I plan to continue our work with: 1. Bacteriocin antimicrobials as potential food preservatives, 2. Biofilm formation by L. monocytogenes on food processing equiment and produce and vegetables. 3. Antimicrobial interventions on meat/poultry products, 4. Industry collaborations with various food companies. Ihave received sponsored research funding for 5 yearsfrom one company that has licensed our bacteriocin-producing cultures, obtained a graduate fellowship from anothercompany that has providedfunds for an MSgraduate fellowship for2 years for a graduate student to perform research providing microbial validation of food processing equipment they are involved with, and a 3rd company has providedresearch project funding for a post-doc to study antimicrobials derived from egg products..
Impacts
What was accomplished under these goals?
2016 Food Microbiological Research: Bacteriocins as antimicrobials in foods. Mixtures of bacteriocins of different modes-of-action (MOA) were examined for application in ice cream given the recent problems with Listeria monocytogenes in ice cream and resulting illnesses. Cell free culture supernatants of mixed MOA preparations worked best when added to liquid ice cream held at refrigeration temperatures for 1 to 24 hrs before freezing. We were able to achieve a 2-log reduction of L. monocytogenes in liquid ice cream containing bacteriocins when held2-4 hrs before freezing in comparison to control trials without bacteriocin. A greater affect was observed in low or reduced fat ice cream relative to regular (full-fat) ice cream indicating a tendency of the bacteriocin to partition into the fat phase. Analysis of surface proteins in Listeria monocytogenes acting asputative adhesins.We used Orbitrap LC-MS/MS as the basis for analysis of surface proteins extracted from L. monocytogenes.In prior work (2015) we examined 5 methods for extracting surface proteins that were all analyzed by Orbitrap Mass Spectrometry and found the UB-Ghost method asthe best for minimizing cytoplasmic proteins while yielding high counts ofsurface proteins. A prior protocol developed in our lab also allowedfacile screening of strongly- vs weakly-adherent strains.This extractionmethod was then used to compare surface extracted proteins fromplanktonicvs adhered (on glass beads)L. monocytogenes cells showing differences in recovered proteins that correlated to their phenotypic differences. We also observed differences in surface proteins recovered from the strongly-adherent strain grown in planktonic (free floating) culture vs. grown adhered to glass beads, suggesting differences in protein expression when grown in the two states. A final paper was used to examine the genetic expression of ~15 genes suggested by these prior studies and showed enhanced expression of several genes in the strongly-adherent strain, one of which was related to virulence. We hope to examine these types of protein differences in L. monocytogenes attached to produce compared to inert surfaces to see if biological-derived surfaces trigger expression of different genes. Studies of L. monocytogenes on both abiotic and biological surfaces may reveal information allowing improved removal from environmental food processing environments and sanitation of produce destined for consumption.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Tiong, H.K. and P.M. Muriana. 2016. Comparison of surface proteomes of adherence variants of Listeria monocytogenes
using LC-MS/MS for identification of potential surface adhesins. Pathogens, 5(2), 40; doi:10.3390/pathogens5020040.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Tiong, H.K. and P.M. Muriana. 2016. RT-qPCR analysis of 15 genes encoding putative surface proteins involved in
adherence of Listeria monocytogenes. Pathogens 2016, 5(4), 60; doi:10.3390/pathogens5040060.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Veasey, S., and P.M. Muriana. 2016. Evaluation of electrolytically-generated hypochlorous acid (electrolyzed water) for
sanitation of meat and meat-contact surfaces. Foods, 5(2), 42; doi:10.3390/foods5020042.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2016
Citation:
Hung King Tiong (PhD Dissertation, Dept. Animal Science, Oklahoma State University, June, 2016). Identification of putative surface adhesins by comparison of surface-adherence variants of Listeria monocytogenes using LC-MS/MS and RT-QPCR.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
FDA Western Regional Dairy Conference (Denver, CO). Present seminar on Listeria monocytogenes and Biofilms at the FDA Western Reg. Conf. where they would be having a forum discussing the Blue Bell ice cream Listeria outbreak-recall, May 3-4, 2016.
- Type:
Websites
Status:
Other
Year Published:
2016
Citation:
Laboratory Facebook Page:
https://www.facebook.com/FAPCFoodMicroLab
- Type:
Other
Status:
Other
Year Published:
2016
Citation:
Farney, E., Gautam, D., Aryal, M., Tiong, H.K., and P.M. Muriana. 2016. Application of bacteriocin antimicrobials from lactic acid bacteria for shelf life extension of refrigerated ground beef. FAPC Research Symposium/OSU Research Week, Feb. 16, Oklahoma State Univ., Stillwater, OK.
- Type:
Other
Status:
Other
Year Published:
2016
Citation:
Aryal, M., and P.M. Muriana. 2016. Analysis of Biofilm on Boots from Workers on a Meat Slaughter Floor and Validation of the Effectiveness of an Automated Boot Washer. FAPC Research Symposium/OSU Research Week, Feb. 16, Oklahoma State Univ., Stillwater, OK.
- Type:
Other
Status:
Other
Year Published:
2016
Citation:
Tiong, H.K., and P.M. Muriana. 2016. DNA array of select genes of two surface-adherent phenotypes of Listeria monocytogenes using PCR for identification of surface adhesins. FAPC Research Symposium/OSU Research Week, Feb. 16, Oklahoma State Univ., Stillwater, OK.
- Type:
Other
Status:
Published
Year Published:
2016
Citation:
Muriana, P.M. 2016. Food Microbiology: Health Officials and CDC Starting to Use Social Media to help Identify Food Safety Outbreaks. FAPC.biz Magazine, 'Pathogen Patrol', Vol. 11(1):17, Spring/Summer.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:This project covers a variety of audiences that are reached by my various efforts. Efforts in the teaching of formal classes (Food Microbiology FDSC3154/MICR 3154; Food Microbiology and Safety FDSC 5120) provides an audience of students and future professionals. Efforts made in presenting seminars, workshops (HACCP andFarm-Focused Food Safety workshops), symposia, lay magazine articles ('Pathogen Patrol'), and youtube videos covers 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. My laboratory's facebook web site (http://www.facebook.com/FAPCFoodMicroLab )and my page in ResearchGate (https://www.researchgate.net/profile/Peter_Muriana ) covers an audience of interested subscribers (i.e., 'likes') from around the world as well as technical and scientific-minded individuals. 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 Centerhave an opportunity to assist food processing companies through the multiple projects that we are involved with in order to carry outour missionto help Oklahoma food companies (as well as national-based companies) with issues/problems they have regarding further processed foods. My Food Microbiology lab is structured thatlaboratory 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 having. This provides a great opportunity for students, staff, post-docs to enhance their problem-solving abilities with actual problems incurred bythe food industry. Because of this, 90% of my MS-degree graduates are well sought and find placement in thefood industry; in addition, 2 recent PhD graduates (Dinesh Babu, 2010, and PaulVijayakumar, 2015)have accepted faculty positions at universities.I would say that our work provides good training and excellent opportunities for those students who spend some time to learn some skills and earn a degree. In addition, I currently havehad an undergraduate student (Ms. Elizabeth Farney)receive undergraduate research awards (Wentz Semester Scholar & CASNR-OAES UR Research Award)to perform research projects in my lab and although she has applied to Vet School, I am hoping she continuesas a graduate student in my lab.Our center also puts on a number of food safey workshop each year (HACCP, Food Defense, Audits, SQF, BRC, etc) and students are encourage to take these workshops to enhance their knowledge and capabilities. How have the results been disseminated to communities of interest?The results of our research workare disseminated mainly via peer-reviewed research publications, but also through seminars (presentation at the Nevada Food Safety Task Force, July,2015) 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 (FAPC Flash articles), longer descriptions (FAPZ.biz magazine, Pathogen Patrol articles) and other extension-related publications. What do you plan to do during the next reporting period to accomplish the goals?I plan to continue our work with: Bacteriocin antimicrobials as potential food preservatives, Research with molecular proteomics of the molecular basis of adherence in Listeria monocytogenes, Antimicrobial interventions on meat/poultry products, Industry collaborationswith various food companies.I am currently working withone company thathas contributed fundingforan MS-degree fellowship for up to 2 years for a graduate student to perform research providing microbial validation offood processing equipment they are involved with; another affiliationmight lead to a research project in 2016.
Impacts
What was accomplished under these goals?
2015 Food Microbiology Research: 1. Application of antimicrobial interventions on beef/pork trim to inhibit growth of E. coli O157:H7 or Salmonellain ground beef/ground pork. AFTEC 3000/Titon (buffered sulfuric acid)was tested ontrim for making ground beef/ground pork and on pork bellies for inhibition of E. coli O157:H7 in comparison with lactic acid, peroxyacetic acid (PAA), and PAA+Titon. Efforts were made to insure that liquid 'pick-up' did not exceed 0.5% by trim prior to grindingasspecified by USDA-FSIS requirements for ground meat products. In separate trials, AFTEC 3000, applied at 0.4% to beef trim that was mixed andheld at 5oC as treated trim(unground) or processed through a meat grinder ('ground'), treatment with AFTEC solution showed an immediate 0.5-1.0 log reduction in aerobic plate count (APC) relative to water-treated controls when tested at 0, 4, or 7 days when held at 5oC. Similar treatment of Salmonella-inoculated ground pork did not show significant difference from controls after 48 hrs post-treatment when held at 5oC.Surface-E. coli O157:H7-inoculated pork bellies were treated with 10-secondimmersion/dip treatments inwater or inTiton solution (pH 1.0 or pH 1.5). Water demonstrated minimal inhibition of E. coli O157:H7 (0.2-log reduction),while Titon (pH 1.0) showedequivalent levels of reduction (~0.5-log reduction) at 24-hr post-treatment as PAA, PAA+Titon, but not as high as 3% lactic acid (~0.75-log reduction). Perhaps longer time treatments and/or higher concentrations would provide equivalent or better reductions of E. coli O157:H7 (or Salmonella) on trim leading to reduced contamination of ground products. 2. Bacteriocins as antimicrobial preservatives in foods. Mixtures of bacteriocins having different Modes-of-Action (MOA) were tested against surface-challenge with Listeria monocytogenesin hotdog applications with finished productvia separate trials by adding bacteriocin cocktails of mixed-MOA suspensionsto the raw meat matrix prior to stuffing/cooking, or to spray hotdogs in their casings after cooking, or to add to hotdogs prior to vacuum packaging. All trials showed reduction of L. monocytogenes relative to control inoculations: after 14 weeks of storage, surface spraying hotdogs in casings showed the least reduction (3-log reduction); addition of mixed-MOA bacteriocin preparation to the meat matrix before cooking showed 6.5-log reduction after 14 weeks and a declining baseline from inoculation, but surface addition of small amounts of mixed-MOA Bac preps showed the greatest reduction (>6.5-log, below the limit of detection) that declined 0.5-log immediately (1.0-log reduction within 48 hrs)upon addition of bacteriocin into packages and was maintained near undetectable levels for the entire 14-weeks. As defined by USDA-FSIS, such an antimicrobial intervention may be considered an Alternative 1 process by providing for post-process lethality (at point of application) and control of growth of L. monocytogenes during shelf life.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Vijayakumar, P.P.; Muriana, P.M. A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action. Biomolecules 2015, 5, 1178-1194.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Vijayakumar, P.P., D. Bellmer, Muriana, P.M, and R. Huhnke. 2015. Microbial populations in sweet sorghum juice during fermentation. BAOJ Biotechnology, Vol. 1:1-9.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Tiong, H.K., S. Hartson, and P.M. Muriana. 2015. Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by Orbitrap mass spectrometry. J. Microbiol. Methods 110: 54-60. DOI:10.1016/j.mimet.2015.01.004.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Henning, C., D. Gautam, and P.M. Muriana. 2015. Identification of multiple bacteriocins in Enterococcus spp. using an Enterococcus-specific bacteriocin PCR array. Microorganisms 3: 1-16. DOI: 10.3390/microorganisms3010001.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Henning, C., P. Vijayakumar, R. Adhikari, B. Jagannathan, D. Gautam, and P.M. Muriana. 2015. Isolation and taxonomic identity of bacteriocin-producing lactic acid bacteria from retail foods and animal sources. Microorganisms 3:80-93. DOI: 10.3390/microorganisms3010080.
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Progress 11/22/13 to 09/30/14
Outputs
Target Audience: This project covers a variety of audiences that are reached by my various efforts. Efforts in the teaching formal classes provides an audience of students and future professionals. Efforts made in presenting seminars, workshops, symposia, lay magazine articles, and youtube videoscovers 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. My laboratory's facebook web site (http://www.facebook.com/FAPCFoodMicroLab covers an audience of interested subscribers (i.e., 'likes') from around the world. My page in ResearchGate (https://www.researchgate.net/profile/Peter_Muriana)attracts technical and scientific-minded individuals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Since my position is located in the R.M. Kerr Food & Agricultural Products Center, our mission is tohelp Oklahoma companies(as well asnational-based companies) with issues/problems they have regarding further processed foods. My Food Microbiology lab is structured that my laboratory personnel work on fundedresearch projects, but also support company assistance by working on projects to help solve food microbiology related problems companies may be having.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, my graduates are well sought out by food industry; my 2 recentMS graduate students were hired by Bar-S Foods (Elk City, OK)and Value Added Products (Alva, OK).My recent PhD graduate accepted a faculty position at University of Kentucky-Lexington (Dept. Animal Science). So, I would say that our work provides good training and excellent opportunities for those students who spend some time tolearn some skills andearn a degree. Our center also puts on a number of food safey workshop each year (HACCP, Food Defense, Audits, SQF, BRC, etc) and students are encourage to take these workshops to enhance their knowledge and capabilities. How have the results been disseminated to communities of interest? Results of our work has been disseminated by various modalities. Technical research has been submitted for peer-reviewed journal article publication. Additional technical data has been presented at various national (IFT)and regional meetings (OSU Research Week/FAPC Research Symposium). We have given updates at various workshops (HACCP, Farm-Focused Food Safety for Small Farms) where data was relevant as well as in-house newsletters distributed to state-wide industry clientel. What do you plan to do during the next reporting period to accomplish the goals? I plan to continue our work with bacteriocin antimicrobials as potential food preservatives and continue our research with molecular proteomics of the molecular basis of adherence in Listeria monocytogenes, as well as working with various projects in collaboration with food industry (I have several in progress: working to identify thermal death kinetics of contaminants of whole liquid pasteurized egg and antimicrobial interventions with raw beef/pork).
Impacts
What was accomplished under these goals?
2014 General Areas ofFood Microbiology Research: 1. Characterization of spoilage microorganisms. Identification of gas-producing organismsproblematic toa local commercial meat processor.Our objectiveswere to isolate and identify gas-producing organisms associated with package bloating of vacuum-packaged fresh beef products, examine incoming raw materials and niches in the meat processing workplace, and identify possible antimicrobial solutions interventions to reduce their occurrence in products on retail supermarket shelves. Samples taken from incoming package purge, environmental swabs or the processed meat samples themselves, were enriched in MRS broth and then inoculated into MRS-Durham tubes and incubated for the visual presence of gas production. Gas+ were definitively identified by 16S rRNA sequencing. Several gas-producing strains were used to inoculate lean beef discs for testing the efficacy of various commercially available antimicrobials (Zesti AM-5, BioVia-CDV, NovaGARD NR-100, Durafresh 2012, Durafresh 5924), organic acids, and combinations of laboratory-generated bacteriocin (Bac+) preparations from bacteriocinogenic LAB. The dominant Gas+ organisms were found to be Leuconostoc mesenteroides. These organisms were shown to be resistant to nalidixic acid and vancomycin which allowed us to generate selective media to enumerate them during antimicrobial assays on raw beef. Gas-producing organisms are intrinsically supplied to further raw beef processors from their incoming raw beef suppliers and may present problems to their retail products during refrigerated storage. Our study is attempting to identify antimicrobial interventions that will reduce or prevent the survival/growth of the Gas+ LAB that could be present on finished raw beef products. 2. Molecular characterization of pathogens. Molecular proteomics to identify the molecular basis of adherence in Listeria monocytogenes. Listeria contamination of food products has been connected to surface adherence and persistence in food processing environments and for the association of listeriosis with ready-to-eat products. Listeria strains isolated from environmental surfaces in meat processing facilities have shown strong, moderate, or weak adherence capabilities to abiotic surfaces. The genetic basis of the adherence is still obscure. Only three molecular determinants have been identified that are directly linked to biofilm formation and surface adherence. One biofilm-associated protein and two internalins are involved in adherence of Listeria to abiotic surfaces and data suggests that other genetic factors are involved in adherence. In the current study, we compared relative mRNA levels for a group of differentially expressed gene products, identified previously by LC-MS/MS, in surface extracts from strong- or weakly-adherent strains. We compared expression of targeted extracted/identified proteins between strongly- and weakly-adherent strains, and compared expression among cells grown at normal condition (planktonic, 30°C) vs adhered conditions (sessile, 30°C or planktonic, 42°C) using RT-qPCR. We found a number of proteins in L. monocytogenes functionally-associated with the cell envelop and cellular processes, intermediary metabolism, information pathway, vacuole escape, unknown proteins, or having no similarity to known proteins. The result revealed that twelve and fourteen genes were up-regulated during growth at high temperature (42°C) or as sessile cells, respectively. Of these, six gene products are known as virulence factors including invasins and autolysins and three members have been implicated as up-regulated genes during intracellular growth in mice. These results suggested a group of gene products, up-regulated either at 42°C, sessile growth (i.e., adhered), or both, in surface protein extracts of L. monocytogenes that are worth characterization for an adherence role to abiotic surfaces. Further characterization of these genes may be helpful in designing targeted sanitation regimens to fully control Listeria in food processing facilities. 3. Bacteriocins as antimicrobials in foods. a. Isolation of bacteriocin-producing lactic acid bacteria from retail foods and animal sources. b. Optimization of conditions to maximize the production of bacteriocins from select strains of lactic acid bacteria. c. Application of mixtures of bacteriocins of differentmodes-of-action to enhance anti-Listeria activity in ready-to-eat meats. Lactic acid bacteria (LAB) are generally-regarded-as-safe (GRAS) for use in foods by FDA as food ingredients. In addition to making lactic acid, some strains produce bacteriocins (antimicrobial peptides) that inhibit susceptible cells by forming pores in cell membranes. Interest in bacteriocins of LAB has increased as some inhibit foodborne pathogens which can lead to their potential use as natural food preservatives. Our objectives were to isolate, identify, and characterize bacteriocin-producing (Bac+) LAB for selection of bacteriocins to be used in food applications. Bac+ LAB were isolated from retail foods and other sources by direct plating or after enrichment. Phylogenetic identification was based on PCR amplification and sequencing of 16S rRNA genes and a 'bacteriocin PCR primer-array' of >40 known bacteriocin genes. Mode-of-action (MOA) of bacteriocins was determined against bacteriocin-resistant strains derived from sensitive strains. Microplate assays were performed with pH-neutralized and non-neutralized spent media to determine inhibition due to acid vs. bacteriocins, and the effect of mixing bacteriocins of different MOAs. Fermentor studies identified growth conditions allowing maximum yield of bacteriocins from either MRS, sweet whey, or corn steep liquor. The Bac+ strains identified in our study include members of the genera Pediococcus, Enterococcus, Lactobacillus, Lactococcus, Carnobacterium, Leuconostoc, and Staphylococcus. Bacteriocin-resistant (BacR) isolates of
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Babu, D.; Muriana, P.M. Sensitive quantification of aflatoxin b-1 in animal feeds, corn feed grain, and yellow corn meal using immunomagnetic bead-based recovery and real-time immunoquantitative-pcr. Toxins 2014, 6, 3223-3237.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Tiong, H.K.; Hartson, S.; Muriana, P.M. Comparison of five methods for direct extraction of surface proteins from listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry. Journal of Microbiological Methods 2015, 110, 54-60.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Henning, C.; Gautam, D.; Muriana, P.M. Identification of multiple bacteriocins in Enterococcus spp. using an Enterococcus-specific bacteriocin PCR array. Microorganisms 2015, 3, xxx-xxx.
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