Progress 09/01/10 to 08/31/13
Outputs
Target Audience: Target audiences include meat/food processors, seafood/fish processors, students/researchers, outreach personnel. For SRD, target audiences may include investors and commercial funding sources. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Work done at the Robert M. Kerr Food & Ag Products Center (i.e., FAPC)constantly involves food companies asmost research is done in a manner addressing real world problems, whereby graduate students become aware of theseproblems and their research is directedin anattempt to address, correct, and solve those problems; opportunities exist for students to present their research findings locally at OSU research symposia and at national meetings. Problems are both practical (studentsgetexperience with how they affect food processors) as well as applied (students can apply molecular/biochemical methods to make food production safe).Students are also encouraged to attend and participate in workshops (HACCP, Basic and Advanced; GFSI; and others); some students have been given the opportunity to present workshop seminars.Therefore, training is directly involved in applied research activities performed at FAPC. How have the results been disseminated to communities of interest? Since SRD is a private company, they do not publish the results of their work as it might affect proprietary information. Information if disseminated by way of presentations at local/national scientific meetings, dissertations that are published for public analysis, and by way of research papers (some are in the process of development andsubmission). Practical data obtained from research is also presented during workshops to help elucidate the nature of research efforts to real world problems. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal: Improve Food Safety from Farm to Table Objectives: 1. Determine how ammonium hydroxide, when incorporated into a brine applied to fresh meat, impacts pathogenic microorganisms Escherichia coli O157:H7 and Salmonella serotype Typhimurium (previously completed; Dr. DeWitt left her position atOSUfor Oregon St. Univ. in Dec. 2011). 2. To develop a quantitative real-time immuno-PCR approach for rapid and sensitive detection of mycoflora in poultry feed/ingredients and airborne dust on the farm (previously completed in 2012; resulted in 1 publication and another is pending). 3. To determine the effect of surface treating pork with lactobacillus to reduce both Escherichia coli O157:H7 and Salmonella serotype Typhimurium (previously completed in 2012by Dr. Deb Vanoverbeke) 4. To advance the chemical detection technology, based on a solid-state sensor array, to autonomously detect, identify, and quantitate histamine, cadaverine, and putrescine in real-time (Dr. Brent Marquis). SRD has exposed the semi-conducting metal oxide (SMO) array to aging yellow fin tuna, optimized SMO sensing materials based on yellow fin tuna test results, expanded gas delivery system capabilities to include 3 main interferences from off-gassing yellow fin tuna as determined by gas chromatography and mass spectrometry (GC/MS) results, and retested optimized sensor arrays with interferences, designed sensor array measurement and support electronics, and designed sensor arrya's gas-sampling system. Because initial SRO sensor array test results with yellow fin tuna provided minimal responses, SRD decided to optimize the GC/MS methods to ensure that any levels of histamine, cadaverine, and putrescine would be measured to provide accurate evaluation and correlation of SMO sensor array response characteristics during yellow fin testing. During the testing with yellow fin tuna, SRD determined that the current GC/MS validation methods were not sufficient to accurately measure its off-gassing components. As a result, SRDinvestigated the use of carbamate derivatization procedures using propyl chloroformate as a derivatizing agent . For derivatization, all samples must be in liquid state requiring them to be bubbled and trapped into an impinger containing water as the solvent to facilitate further analysis via GC/MS. 5. To develop an inexpensive methodfor convenient delivery system for application of antimicrobials on the surface of rawmeats (Dr. Peter Muriana). We examined the use of atomizing- and liquid-impinging spray systems to spray antimicrobials (bacteriophage, antimicrobial chemicals)andoxidizing solutions (electrolyzed water, ozone) at raw beef and fat discswith the intention to reduce levels either on beef/fat trim prior to grinding, or reducing microbial levels on beef subprimals prior to blade tenderization. Although USDA (and FDA)allows ozone for use on raw and processed meats, andwherebyozone isconsidered a 'green solution' because it's short half lifeturns it into water and oxygen in a short time,the high organic load of meat itself quickly results in exhausting the antimicrobial effect of ozone, even when spraying surface inoculated beef samples. A similar scenario was observed with electrolyzed water (i.e., hypochlorous acid), another oxidant which is exhausted quickly with organic material. Oxidizing solutions might be more effective as a sanitizing rinse after an initial application ofdetergents to remove the organic debris from meat processing facilities. Bacteriophage were partially effective as antimicrobial treatments on E. coli- and Salmonella-inoculated beef discs, generating approximately 1.0 and 0.6-log reductions, respectively when pathogen-specific spray treatments were applied to surface inoculated meat samples. 6. To examine the molecular basis ofadherence of Listeria monocytogenes to surfaces. Several extraction methods were examined to best extract proteins from the surface of Listeria monocytogenes, especially because the extractions would be submitted for analysis by mass spectrometry. Various extractions methods were determined to be better or worse, based on the extent of molecular protein species identified after extraction. Ultimately, a urea-based method was found acceptable for use in identifying protein species by Orbitrap Mass Spectrometry. We utilized the results of the proteins identifiedas confirmation of protein expression,comparing proteins extracted and identified fromboth strongly- and weakly-adherent strains, as well as comparison of proteins obtained from planktonic cells (free floating in solution) vs those that are adhered to surfaces (glass beads) in an attempt to identify the molecular basis for attachment of Listeria monocytogenes. Significant differences in protein levels extracted from the surface of L. monocytogenes when subjected to different conditions (attached vs unattached, strongly-adherent vs weakly-adherent) were viewed as potential protein targets contributing to attachment. These analyses lead to identify 'internalin' as involved with strongly-adherent strains. When the internalin gene from weakly adherent strains was examinedby PCR amplification and sequence analysis, we found sequence deletions within the gene that could account for weakened attachment to surfaces. Internalin is a surface virulenceprotein that induces intestinal epithelial cells (non-professional phagocytic cells) to take in L. monocytogenes via receptor mediated entocytosis to begin an intracellular existance.We also used quantitative real-time PCR to examine expression of various protein-coding genes based on the differences in surfacepresence demonstrated byOrbitrap analysis from the various phenotypes and conditions examined. Hopefully, the identity of various proteins involved in surface adherence may provide additional information with which to eliminate L. monocytogenes as surface-adhering contaminants in food processing facilities.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Montalvo, C., and P.M. Muriana. 2013. Antimicrobial interventions for reducing E. coli 0157:H7 and Salmonella in Raw Meat. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Tiong, H.K., and P.M. Muriana. 2013. Orbitrap mass spectrometry and qRT-PCR analysis of gene expression to identify the molecular basis of bacterial adherence in Listeria monocytogenes. FAPC Research Symposium/OSU Research Week, Feb. 19, Oklahoma State Univ., Stillwater, OK.
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2013
Citation:
Christian Montalvo, M.S. (July, 2013). Evaluation of ozone, electrolyzed water, and bacteriophage as antimicrobial interventions for raw beef.
- Type:
Journal Articles
Status:
Other
Year Published:
2014
Citation:
High through-put direct extraction of surface proteins from phenotypic adherence variants of Listeria monocytogenes. Tiong, H.K., and P.M. Muriana. Manuscript submitted for internal review.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2012
Citation:
Amber Sharp, M.S. (2012). Effects of an Application of Lactobacillus on the Quality and Sensory Characteristics of Beef and Pork.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: The objectives of the various projects were to: Objective 1. Determine how ammonium hydroxide, when incorporated into a brine applied to fresh meat, impacts pathogenic microorganisms Escherichia coli O157:H7 and Salmonella serotype Typhimurium. Objective 2. To develop a quantitative real-time immuno-PCR approach for rapid and sensitive detection of mycotoxin (aflatoxin B1) in animal feed/ingredients. Objective 3. To determine the effect of surface treating pork with lactobacillus to reduce both Escherichia coli O157:H7 and Salmonella serotype Typhimurium. Evaluated the impact of antimicrobials on ground beef and ground pork relative to their merit on reducing microbial growth and still maintaining consumer acceptable color and organoleptic properties. Objective 4. To advance the chemical detection technology, based on a solid-state sensor array, to autonomously detect, identify, and quantitate histamine, cadaverine, and putrescine in real-time. Objective 5. To examine adherence of Listeria monocytogenes to processing surfaces. PARTICIPANTS: Principal investigator(s)/project director(s) (PIs/PDs) directed research conducted for each of the objectives, supervised data evaluation and interpretation and submitted progress reports; Graduate students conducted research and reported and analyzed data; Undergraduate students helped with data collection. Objective 1. Lina Ramirez (grad students). Objective 2. Objective 3. Deb VanOverbeke/Gretchen Hilton (co-PIs), Amber Sharp, Tricia Harlan (grad students; Andrea English, Kelly Vierck, Lakesha Hawkins, Shade Hughes, Jenna O'Danie (undergraduate student help). Objective 4. Brent Marquis/SRD (PI), Vincente Sy, Thomas Dunn (Test Engineers), Ravi Chilumula, Sathya Vasudevan (Signal Processors), Alexey Tomchenko, Yuan Gao (Material Chemists), Sagar Deshpande, Richard Leclerc (Analytical Chemists), and Aaron McCollough (Electrical Engineer). Objective 5. Peter Muriana (PI), Preetty Pranatharthiharan, Hung King Tiong (grad students), Samuel Munnangi, Roshan Revankar (undergraduate student help). TARGET AUDIENCES: Food/grain processors, meat processors, seafood/fish processors, students/researchers, outreach personnel. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 1. The ammonium hydroxide process was examined in actual processing and shelf life conditions with a commercial company manufacturing non-intact and marinaded beef. Except for some initial reduction in microbial counts, the ammonium hydroxide process did not significantly reduce microbial populations during shelf life of the products. Objective 2. Research project ended. Objective 3. Antimicrobials were effective in reducing microbial growth, however, the different antimicrobials could result in discoloration of product from the normal appearance (as accepted by consumers) and thus should be further evaluated to determine the concentration of antimicrobials when they are added to the product. Objective 4. SRD has worked to expose the semi-conducting metal oxide (SMO) sensor array to aging yellow fin tuna, optimize SMO sensing materials based on yellow fin tuna test results, expand gas delivery system capabilities to include 3 main interferents from off-gassing yellow fin tuna as determined via gas chromatograph and mass spectrometry (GC/MS) results and retest optimized sensor array with interferents, design sensor array measurement and support electronics, and design sensor array's gas-sampling system. SRD also tested the SMO sensor array to the off-gassing of fresh and aging yellow fin tuna in coordination with GC/MS analysis to determine the main compounds being emitted from yellow fin tuna, both fresh for reference baseline and aged to determine if histamine, cadaverine, and/or putrescine are emitted and indicative of aging fish. SRD is investigating the use of carbamate derivatization procedure using propyl chloroformate as a derivatizing agent whereby samples must be in liquid state requiring them to be bubbled and trapped into an impinger containing water as the solvent to facilitate further analysis via GC/MS. SRD began designing the sensor array measurement, control, and support electronics that target a handheld configuration. The sensor array gas-sampling system is being designed based on lessons learned with the gas delivery and mixing system. Objective 5. When cultures of E. coli O157:H7 or L. monocytogenes were applied to dead biofilm layers of either bacteria, they adhered quicker and at higher levels then the same bacteria applied to clean inert surfaces. Immature biofilm produced with L. monocytogenes immersed in 1000 ppm of quaternary amine sanitizer (BiQuat) took 2 min of immersion time for lethality to all cells in the biofilm whereas the same process with E. coli O157:H7 required 2 hrs of immersion. When boots from workers in meat slaughter processing were examined, recovery of bacteria imbedded in biofilm was recovered at high levels (10+E5 cfu/sq.cm) and were not significantly diminished when boots were dipped in 1000 ppm BiQuat for up to 1 hr. The use of proteolytic enzyme treatments were capable of removing the bacteria attached in biofilms to that sanitizer treatment could be more effective than pretreatment with ineffective detergents. The data suggests critical surfaces in meat processing facilities may need to be examined for the development of biofilms that are resistant to simple sanitation regimens.
Publications
- Objective 1. Lopez, L.R., P.M. Muriana, C.A.M. DeWitt. 2012. Antibacterial activities of phenolic compounds isolated from grape pomace (Vitis aestivalis ) on E. coli O157:H7, S. aureus, and L. monocytogenes. Ann. Meet. Inst. Food Technol., Las Vegas, NV (June 25-28), Abst. # 03523.
- Objective 3. Sharp, A. Effects of an application of Lactobacillus on the quality and sensory characteristics of beef and pork. Oklahoma State University, December, 2011. Food Science curriculum courses. 2012. Results from the study were added to two courses in the Food Science curriculum.
- Objective 4. SRD. 2012. SRD does not publish their chemical detection technology efforts or results to ensure intellectual property remains protected.
- Objective 5. Pranatharthiharan, P., and P. Muriana. 2012. Attachment of Listeria monocytogenes to surfaces is enhanced by the presence of nonviable cells remaining after treatment with BiQuat sanitizer. FAPC Res. Symp., Feb. 22, Stillwater, OK. Pranatharthiharan, Preetty. 2012. Attachment of Listeria monocytogenes and E. coli O157:H7 to surfaces is enhanced by the presence of nonviable cells remaining after treatment with Bi-Quat sanitizer.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The objectives of the various projects were to: Objective 1. Determine how ammonium hydroxide, when incorporated into a brine applied to fresh meat, impacts pathogenic microorganisms Escherichia coli O157:H7 and Salmonella serotype Typhimurium. Objective 2. To develop a quantitative real-time immuno-PCR approach for rapid and sensitive detection of mycotoxin (aflatoxin B1) in animal feed/ingredients. Objective 3. To determine the effect of surface treating pork with lactobacillus to reduce both Escherichia coli O157:H7 and Salmonella serotype Typhimurium. Objective 4. To advance the chemical detection technology, based on a solid-state sensor array, to autonomously detect, identify, and quantitate histamine, cadaverine, and putrescine in real-time. Objective 5. To examine adherence of Listeria monocytogenes to processing surfaces. PARTICIPANTS: Principal investigator(s)/project director(s) (PIs/PDs) directed research conducted for each of the objectives, supervised data evaluation and interpretation and submitted progress reports; Graduate students conducted research and reported and analyzed data; Undergraduate students helped with data collection. Objective 1. Christina DeWitt (PI). Claudia Cerruto-Noya, Lina Ramirez, Lin Koh, and Austin Lowder (grad students). Jordan Denton, Elis Monti, Alan Miller, Shirley Mack, Abbey Ankenman, and Jake Custer (undergrad students). Objective 2. Peter Muriana (PI), Dinesh Babu (grad student). Objective 3. Deb VanOverbeke/Gretchen Hilton (co-PIs), Amber Sharp (main grad student); Mary Katherine Henderson, Lacey Vedral, Blake Bloomberg, and Jackie Eager (grad students providing assistance). Lakesha Hawkins (undergrad). Objective 4. Brent Marquis/SRD (PI) Objective 5. Peter Muriana (PI), Preetty Pranatharthiharan/ H.K. Tiong (grad students). TARGET AUDIENCES: Food/grain processors, meat processors, students/researchers, outreach personnel. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 1. Results suggested that injection of beef with a brine formulated with 1% ammonium hydroxide instead of phosphate does not promote microbial growth and does not negatively impact meat quality. Results indicated there was an antibacterial effect against E. coli O157:H7 regardless of initial meat pH. Results also indicated that brines formulated with 2 and 3% ammonium hydroxide did not significantly differ from those formulated with 1% ammonium hydroxide in terms of their effectiveness against E. coli O157:H7. The impact of the results is they provided processors with a brine formulation alternative that significantly reduced sodium content (~50%) and produced a product as safe or more safe than the conventional commercial brine formulation. The impact is also that consumers are provided with healthier product. Objective 2. The RT-iqPCR assay that was developed was able to detect and quantify aflatoxin B1 from 10 ppb down to 0.1 ppb with high correlation (r2=0.97) and efficiency (99.5%). The approach also detected the high-dose hook effect phenomenon (excess antigen) which was overcome by the use of dilution protocols to eliminate false negatives that may occur at levels above quantification limits of the assay. Objective 3. Application of Lactobacillus by dipping was proven to have no significant treatment effect on the quality and sensory characteristics of beef strip steaks and pork loin chops. Lactobacillus bacteria displayed no adverse effects on off flavors, TBARS values, or color of beef and pork over 6 d of retail display. Objective 5. SEM analysis of strongly-adherent strains of L. monocytogenes and E. coli O157:H7 demonstrated confluent biofilm formation over entire surfaces. Biofilm formation was resistant to sanitizer treatment except after prolonged exposures. Surfaces containing dead layers of biofilm killed by prolonged exposure to sanitizer (BiQuat) allowed re-adherence of L. monocytogenes more quickly and to higher levels than clean surfaces. An enzymatic treatment using protease was able to remove the dead biofilm layer from surfaces. Strongly- and weakly-adherent strains were examined for surface protein expression using Orbitrap LC/MS method of analysis comparing the most prominent proteins extracted from the surface of bacterial cells. Molecular sequencing demonstrated sequence variation within the Internalin A gene (inlA) that could be involved with adherence and invasion.
Publications
- Objective 1. Cerruto-Noya, C. 2011. Evaluation of the microbial effects of 1% ammonium hydroxide in striploins. Oklahoma State University.
- Cerruto-Noya CA, Goad CL, Mireles DeWitt CA. 2011. Research Note: Antimicrobial effect of ammonium hydroxide when used as an alkaline agent in the formulation of injection brine solutions. J Food Protect 74(3):475-479.
- Cerruto-Noya C, Mireles DeWitt CA. 2011. Comparison of 1%, 2% and 3% Ammonium Hydroxide Solutions to Control Escherichia Coli O157:H7 on Beef Lean Surfaces. IFT Annual Meeting Technical Program Abstracts, Institute of Food Technologists, New Orleans, LA. Poster Session: 202-01.
- Cerruto-Noya C, Muriana P, Morgan JB, Mireles DeWitt CA. 2011. Evaluation of the Antimicrobial Effect of 1% Ammonium Hydroxide Brine Applied Through Needle Injection into Striploins. FAPC Research Symposium.
- Cerruto-Noya C, Muriana P, Morgan JB, Mireles DeWitt CA. 2011. Comparison of 1%, 2% and 3% Ammonium Hydroxide Solutions to Control Escherichia Coli O157:H7 on Beef Lean Surfaces. Whiteman Research Competition.
- Objective 2. Babu, D., and P.M. Muriana. 2011. Immunomagnetic bead-based recovery and real time PCR (RT iq-PCR) for sensitive quantification of aflatoxin B1. J. Microbiol. Meth. 86:188-194.
- Babu, D. and Muriana, P.M. 2011. Rapid and sensitive detection of aflatoxin in animal feeds and food grains using immunomagnetic bead based recovery and Real-Time immuno quantitative-PCR (RT-iqPCR) assay. Ann. Meet. Inst. Food Technol., New Orleans, LA (July 11-14), Abst. # 100-01.
- Objective 5. Pranatharthiharan, P., and Muriana, P.M. 2010. SEM Analysis of sanitizer effectiveness and enzymatic detachment interventions for strongly-adherent strains of L. monocytogenes from environmental surfaces. OSU Research Symposium, Oklahoma State University.
- Tiong, H.K., and Muriana, P.M. 2010. Quantitative analysis of listerial surface membrane proteins by spectrum counting. FAPC Res. Symposium, Oklahoma State University.
- Babu, D., and P.M. Muriana. 2011. Quantification of aflatoxin B1 in food and feed by real-time immunoquantitative PCR (RT iq-PCR). J. Microbiol. Meth. (manuscript in preparation).
- Objective 3. Sharp, A., 2011. Effects of an application of Lactobacillus on the quality and sensory characteristics of beef and pork. Oklahoma State University.
- Pranatharthiharan, P., and Muriana, P.M. 2010. SEM analysis of sanitizer effectiveness and enzymatic detachment interventions for strongly-adherent strains of E. coli O157:H7 and L. monocytogenes from environmental surfaces. FAPC Res. Symposium, Oklahoma State University.
- Pranatharthiharan, P., and Muriana, P.M. 2011. Scanning electron microscopic analysis of sanitizer effectiveness and enzymatic detachment interventions for strongly-adherent strains of E. coli O157:H7 and L. monocytogenes from environmental surfaces. Ann. Meet. Inst. Food Technol., New Orleans, LA (July 11-14), Abst. # 198-11.
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