Progress 09/01/08 to 08/31/11
Outputs
OUTPUTS: The results from three projects were delivered at the following conferences: Institute of Food Technologist Conference, Chicago 2010; OSU Research Days, FAPC; OSU Research Days, Whiteman Competition. The following outlines the different outputs from the stated objectives from this grant: For the detection of soy allergens in food, a DNA based protocol using Real-Time PCR was developed. This involved identifying the proper DNA extraction kit. Identifying and developing the primer targeting the lectin gene (Le1). Sensitive methods for detection of staphylococcal enterotoxins A (SEA) and B (SEB) and aflatoxin B1 using immunomagnetic bead capture in combination with Real-Time PCR. The method was able to detect enterotoxins at levels 1 thousand to 1 million times lower than commercial kits, providing robust detection even from solid and semi-sold foodstuffs (i.e., ground turkey). The method for aflatoxin B1 had a detection limit of 0.1 ppb which is 200x more sensitive than current ELISA based technology. Current work is underway to identify, develop and demonstrate accurate delivery of target biogenic amines (cadaverine, putrecine and histamine) to the vapor phase of a sensor. Established protocol of GC detection. Obtained cadaverine and putrescine in standard high-pressure gas cylinders for sensor development. Developing a gas delivery system for histamine. First prototype had been built and is in the process of being validated. PARTICIPANTS: DeWitt was the project director (PD) and was responsible for management, preparation and submission of all grant reports. In addition, she was also responsible for budget management and managed allergen and ammonium hydroxide project objectives. Muriana, Gilliland, and Marquis (PIs) were responsible for enterotoxin, spinach, and sensor objectives, respectively. They were responsible for management, preparation and submission of all objective reports to the PD. Cuesta Alonso (post-doc) was responsible for data collection for the spinach objective. Cerruto-Noya and Babu were Ph.Ds. assigned to the ammonium hydroxide and enterotoxin objectives, respectively. They were responsible for obtaining data, evaluating and reporting results to PIs. Wellings, Parsons, Hopkins, Hayden, Vasan, Prasad were M.S. students who provided lab support. Lowder and Ramirez were Ph.D. students who provided lab support. Dry was an undergraduate student who provided lab support. Partnerships: The University of Maine partnered with SRD and provided in-kind support in obtaining product (tuna) for testing. Creekstone Farms allowed access to facilities to obtain product for the ammonium hydroxide project. They also provided some in-kind support for the product. The Robert M. Kerr Food & Agricultural Products Center (FAPC) provided labor for maintenance and cleaning of meat processing facilities where ammonium hydroxide project was conducted. Labor included the meat manager's time, the assistant manager's time, and the time of two to three undergraduate students to maintain and clean facilities. Since this is a USDA inspected facility, they also carried the cost of the inspector. The facility also provided some of the supplies for meat processing. The FAPC also provided lab staff support: 3 laboratory technicians (J. Gruntmeir, L. Koh, S. McCoy) and one post-doc, K. Kushwhana all provided laboratory support in the form of supply acquisition and data analysis and evaluation. The National Institute for Microbial Forensics and Food & Agricultural Biosecurity (NIMFAB) also partnered with Dr. S. Gilliland for completion of the spinach objective. They provided a graduate student and time from three faculty. Fresh Express also partnered with Dr. S. Gilliland and the director of NIMFAB to provide financial support for the spinach project objectives. The objectives of this project provided training and professional development for two post-doc, 4 Ph.D.'s, 7 M.S., and 1 undergraduate. In addition, three undergraduate students who came to OSU as interns from Brazil (Monti, Blois, and Miller) and three OSU undergraduate students (Denton, Ankeman, and Mack) also received training from the efforts from this grant. TARGET AUDIENCES: Horticultural Food Product Producers Horticultural Food Product Processors Meat Processors Food Safety Regulators Food Safety Researchers PROJECT MODIFICATIONS: Drs. Benner, DeWitt (new job), and Gilliland (deceased) left the project at various timeframes during this period.
Impacts
A real-time PCR protocol was developed to detect soy in food. The detection limit was 50 ppm. Optimum extraction was achieved using QIAamp DNA stool mini kit. Various primers were evaluated and it was found Lectin forward: 5- TCC ACC AAATCC ACA CAT C -3 and reverse : 5- GAA GCA AAA GAC CAA GAA AGC AC -3 were developed. Investigations into the microbiological impact of using 1% ammonium hydroxide (AH)as an alkaline processing aide in beef injection brines has indicated that psychrotrophic, mesophilic, and gram-negative bacteria are more negatively impacted when compared to conventional brines. Spray application of brines containing AH at 1, 2, and 3% on beef with E. coli O157:7 was evaluated. These results demonstrated that brines containing AH had higher log reductions than phosphate containing brine. AH formulated brine was compared to brine without AH for its impact on E. coli O157:H7 when injected in beef. Analysis was completed on day 0 and 1. Result were similar to the spray study (~0.5 log cfu/g reduction). However, this level of reduction occurred for both AH containing and no AH brines. Previous research has demonstrated that similar differences in meat pH can reduce microbial counts. A protocol for Aflatoxin B1 was developed and optimized. PureProteomeTm protein G magnetic beads and anti-AFB1 monoclonal antibody AFC13 were used for toxin capture. Toxin detection was done using polyclonal secondary antibody A-8679 tethered with a 563-bp DNA oligomer. Protocols for preparation of reporter DNA, conjugation of reporter DNA with polyclonal anti-aflatoxin antibodies for detection and real-time PCR amplification of the reporter DNA were optimized. Signal amplification of the DNA conjugated onto the detector antibody was done using an internal DNA segment (101-bp) of the larger 563-bp fragment. Detection of scombroid poisoning based on solid-state sensor arrays was developed. Analytical validation of biogenic amine gases associated with scombroid poisoning, putrescine, cadaverine and histamine, in the vapor phase was accomplished by integrating an oven into a gas delivery system. Heated gas delivery lines (75C) were also added. More than 32 different SMO sensing materials were synthesized, deposited onto Micro Electromechanical System (MEMS) platforms and evaluated. Critical sensor response features have been identified to establish the classification algorithm's architecture. An immunomagnetic PCR signal amplification assay (iPCR-SA) was developed for recovery and detection of staphylococcal enterotoxin A and B (SEA, SEB) in foods. Anti-SEA or anti-SEB primary antibodies were coated onto COOH-modified magnetic beads using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide reagent. Secondary antibodies were covalently linked to amino-modified reporter DNA oligonucleotides (563 bp) via the linker molecule succinimidyl-4[N-maleimidomethyl]-cyclohexane-1-carboxylate. An internal 159-bp portion of the reporter DNA retained by the captured toxin molecule was then amplified by real-time PCR. SEA and SEB were detected in tryptic soy broth, milk, lemon cream pie, tuna salad, deli turkey, and ground turkey at levels as low as 7.5 fg/ml.
Publications
- No publications reported this period
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: For this grant period, results from three projects were delivered at the following conferences: Institute of Food Technologist Conference, Chicago 2010; OSU Research Days, FAPC; OSU Research Days, Whiteman Competition. The following outlines the different outputs from the stated objectives from this grant: For the detection of soy allergens in food, a DNA based protocol using Real-Time PCR was developed. This involved identifying the proper DNA extraction kit. Identifying and developing the primer targeting the lectin gene (Le1). Sensitive methods for detection of staphylococcal enterotoxins A (SEA) and B (SEB) and aflatoxin B1 using immunomagnetic bead capture in combination with Real-Time PCR. The method was able to detect enterotoxins at levels 1 thousand to 1 million times lower than commercial kits, providing robust detection even from solid and semi-sold foodstuffs (i.e., ground turkey). The method for aflatoxin B1 had a detection limit of 0.1 ppb which is 200x more sensitive than current ELISA based technology. Current work is underway to identify, develop and demonstrate accurate delivery of target biogenic amines (cadaverine, putrecine and histamine) to the vapor phase of a sensor. Established protocol of GC detection. Obtained cadaverine and putrescine in standard high-pressure gas cylinders for sensor development. Developing a gas delivery system for histamine. First prototype had been built and is in the process of being validated. PARTICIPANTS: DeWitt was the project director (PD) and was responsible for management, preparation and submission of all grant reports. In addition, she was also responsible for budget management and managed allergen and ammonium hydroxide project objectives. Muriana, Gilliland, and Marquis (PIs) were responsible for enterotoxin, spinach, and sensor objectives, respectively. They were responsible for management, preparation and submission of all objective reports to the PD. Cuesta Alonso (post-doc) was responsible for data collection for the spinach objective. Cerruto-Noya and Babu were Ph.Ds. assigned to the ammonium hydroxide and enterotoxin objectives, respectively. They were responsible for obtaining data, evaluating and reporting results to PIs. Wellings, Parsons, Hopkins, Hayden, Vasan, Prasad were M.S. students who provided lab support. Lowder and Ramirez were Ph.D. students who provided lab support. Dry was an undergraduate student who provided lab support. Partnerships: The University of Maine partnered with SRD and provided in-kind support in obtaining product (tuna) for testing. Creekstone Farms allowed access to facilities to obtain product for the ammonium hydroxide project. They also provided some in-kind support for the product. The Robert M. Kerr Food & Agricultural Products Center (FAPC) provided labor for maintenance and cleaning of meat processing facilities where ammonium hydroxide project was conducted. Labor included the meat manager's time, the assistant manager's time, and the time of two to three undergraduate students to maintain and clean facilities. Since this is a USDA inspected facility, they also carried the cost of the inspector. The facility also provided some of the supplies for meat processing. The FAPC also provided lab staff support: 3 laboratory technicians (J. Gruntmeir, L. Koh, S. McCoy) and one post-doc, K. Kushwhana all provided laboratory support in the form of supply acquisition and data analysis and evaluation. The National Institute for Microbial Forensics and Food & Agricultural Biosecurity (NIMFAB) also partnered with Dr. S. Gilliland for completion of the spinach objective. They provided a graduate student and time from three faculty. Fresh Express also partnered with Dr. S. Gilliland and the director of NIMFAB to provide financial support for the spinach project objectives. The objectives of this project provided training and professional development for two post-doc, 4 Ph.D.'s, 7 M.S., and 1 undergraduate. In addition, three undergraduate students who came to OSU as interns from Brazil (Monti, Blois, and Miller) and three OSU undergraduate students (Denton, Ankeman, and Mack) also received training from the efforts from this grant. TARGET AUDIENCES: Horticultural Food Product Producers Horticultural Food Product Processors Meat Processors Food Safety Regulators Food Safety Researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A real-time PCR protocol was developed to detect soy in food. The detection limit was 50 ppm. Optimum extraction was achieved using QIAamp DNA stool mini kit. Various primers were evaluated and it was found Lectin forward: 5- TCC ACC AAATCC ACA CAT C -3 and reverse : 5- GAA GCA AAA GAC CAA GAA AGC AC -3 were developed. The cycle protocol was 1. Step 1: 95C for 5:00 min; 2. (45 repeats): Step 1: 95C for 0:30 min, Step 2: 61C for 0:30 min, Step 3: 72C for 0:30 min (Real Time Detection); 3. (81 repeats): Step 1: 55C for 0:30 min (Melt Curve) 0.5C temperature change, 95C end point. The optimized PCR reaction for real-time detection was as follows: Biorad IQ SYBR supermix (7.5uL), 15uM Lectin Reverse Primer (0.1uL), 15uM Lectin Forward Primer (0.1 uL), DNA template (2.0uL), H2O (5.3uL). Investigations into the microbiological impact of using 1% ammonium hydroxide (AH)as an alkaline processing aide in beef injection brines has indicated that psychrotrophic, mesophilic, and gram negative bacteria are more negatively impacted when compared to conventional brines. Spray application of brines containing AH at 1, 2, and 3% on beef with E. coli O157:7 was evaluated. These results demonstrated that brines containing AH had higher log reductions than phosphate containing brine. However, increasing AH from 1 to 3% did not increase log reduction. AH formulated brine was compared to brine without AH for its impact on E. coli O157:H7 when injected in beef. Analysis was completed on day 0 and 1. Result were similar to the spray study (~0.5 log cfu/g reduction). However, this level of reduction occurred for both AH containing and no AH brines. Final meat pH (no AH was ~0.5 log lower at 5.27) was different. Previous research has demonstrated that similar differences in meat pH can reduce microbial counts. A protocol for Aflatoxin B1 was developed and optimized. PureProteomeTm protein G magnetic beads and anti-AFB1 monoclonal antibody AFC13 were used for toxin capture. Toxin detection was done using polyclonal secondary antibody A-8679 tethered with a 563-bp DNA oligomer. Protocols for preparation of reporter DNA, conjugation of reporter DNA with polyclonal anti-aflatoxin antibodies for detection and real-time PCR amplification of the reporter DNA were optimized. Signal amplification of the DNA conjugated onto the detector antibody was done using an internal DNA segment (101-bp) of the larger 563-bp fragment. Scombroid poisoning is closely associated with spoiled fish from the Scombroidae and Scomberesocidae families, but non-scombroid species can be involved. Detection of scombroid poisoning based on solid-state sensor arrays was developed. Analytical validation of biogenic amine gases associated with scombroid poisoning, putrescine, cadaverine and histamine, in the vapor phase was accomplished by integrating an oven into a gas delivery system. Heated gas delivery lines (75C) were also added. More than 32 different SMO sensing materials were synthesized, deposited onto Micro Electromechanical System (MEMS) platforms and evaluated. Critical sensor response features have been identified to establish the classification algorithm's architecture.
Publications
- Cerruto-Noya CA, Goad CL, Mireles DeWitt CA. 2010. Research Note: Antimicrobial effect of ammonium hydroxide when used as an alkaline agent in the formulation of injection brine solutions. Under revision for J Food Protect.
- Cerruto-Noya,CA, Goad, CL, Mireles-Dewitt, CA. 2010. Antimicrobial effect of ammonium hydroxide when used as an alkaline agent in the formulation of injection brine solutions. Available at http://www.ansi.okstate.edu/research.
- Mitra, R., Cuesta-Alonso, E., Wayadande, A., Talley, J., Gilliland, S., Fletcher, J. 2009. Effect of route of introduction and host cultivar on the colonization, internalization, and movement of the human pathogen escherichia coli O157:H7 in spinach. J Food Prot 72(7):1521-1530.
- Paneerseelan L, Muriana PM. 2009. An immunomagnetic PCR signal amplification assay for sensitive detection of Staphylococcus aureus enterotoxins in foods. J Food Prot 72(12):2538-2546.
- Paneerseelan L. Detection of Staphylococcus aureus enterotoxins and enterotoxin producing strains, Ph.D. Thesis, Oklahoma State University, 2008 , 162 pages; AAT 3341757.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Activities performed for: Objective 1 - conducted a phase 1 evaluation of the impact brine containing ammonium hydroxide has on microbial flora when in injected in beef loins. Microbial analysis included aerobic plate counts (mesophilic and pyschrotrophic), gram negative, lactobacillus and coliforms and was conducted on d 1, 5, 12, and 19. Preliminary indications are that there is an initial reduction of APC microbial populations immediately following injection up to d 5. However, at d12 differences are not perceivable. Analysis at d 19 demonstrates ammonium hyroxide has higher counts than the control (commercial phosphate based brine). The was also a significant impact on gram negative survival and growth. Objective 2 - Spinach plants were inoculated with E. coli O157:H7 using 4 different techniques for inoculation. Detection was reported for up to 2 weeks for all treatments. Stab inoculation 22%, Leaf-drop 38%, Rhizosphere drench 47%, Pressure application through abaxial leaf surface 100%. In addition, 3 cultivars with different leaf surface morphology were compared for colonization. Leaf surface morphology appears to impact bacterial colonization. Objective 3 - Measured volatile organic vapors from degrading yellowfin tuna at different temperatures by developed GCMS method. Optimized signal processing and pattern recognition algorithms based on sensor array response characteristics. Selection of sensors and the development of sensor array for the detection of histamine, cadaverine and putrescine from the materials selected using optimized algorithms. Tested and validated performance of the sensor array performance using the optimized algorithms. Synthesized, developed and tested improved sensor materials with the use of zeolites for increased chemical specificity and rapid, reversible and reproducible responses that yield high sensitivity and quantitative information. Objective 4 - Optimization of the bead coupling procedure was done using anti-aflatoxin monoclonal and polyclonal antibodies. Polyclonal antibodies were selected for both capture and detection antibodies. A 16-24 hour coupling time for polyclonal anti-aflatoxin antibodies and the paramagnetic beads was decided as per bead manufacturer's instructions. Protocols for preparation of reporter DNA, conjugation of reporter DNA with polyclonal anti-aflatoxin antibodies for detection and real-time PCR amplification of the reporter DNA were optimized. Optimization has now overcome problem with a supplier's SYBR green mix as well as the generation of primer-dimers during PCR (new primers have been designed to overcome the problem). Objective 5 - PCR primer was successfully redesigned to remove ghost peaks which turned out to be very late forming primer-dimers. Products include: Objective 3 - Developed GCMS methods and verification techniques for measuring histamine, putrescine and cadaverine in the vapor phase. Developed novel metal oxide materials for the detection of histamine, putrescine and cadaverine. Development of hit detection, classification and concentration estimation of histamine, putrescine and cadaverine algorithms. Objectives 4 and 5 - PCR primers developed. PARTICIPANTS: Individuals: (1) 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. (2) Graduate students conducted research and reported and analyzed data. (3) Undergraduate students helped with data collection. Partner Organizations: SRD PI and Scientists were responsible for conducting Objective 3. Collaborators and contacts: PI responsible for objective 2 collaborated with OSU collaborators in Plant Pathology and Horticulture. SRD collaborated with scientist from the University of Maine to obtain fish samples (objective 3). PI responsible for objective 1 collaborated with biotechnology scientist within her department, Animal Science, for primer development. Training or professional development: Project is providing training for 4 Ph.D. students, 3 Master's students, and 5 undergraduate students. TARGET AUDIENCES: These projects are targeted to improving food safety for producers and processors in the following areas: Objective 1: Meat processors Objective 2: Plant breeders, Spinach Growers and Processors Objective 3: Fisherman and Fish processors Objective 4: Grain Storage facilities, Food processors that utilize grain, Commercial Testing Laboratories Objective 5: Food processors that utilize soy products, Commercial Testing Laboratories PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 1 - Results suggest that inclusion of ammonium hydroxide in injection brines may improve safety in fresh meat products since important pathogens for meat (E. coli O157:H7 and Salmonella spp) are gram negative. Objective 2 - Results suggest leaf surface morphology does impact surface bacterial colonization. This knowledge can be utilized to produce a safer food supply by leading plant breeders to optimize those selection characteristics for leaf morphology that do not provide protected niches for bacterial growth.
Publications
- Babu, D., and P.M. Muriana. 2009. Rapid and sensitive detection of aflatoxin in animal feeds, cereal grains, and food products using immunomagnetic bead-based recovery and real-time PCR assay. FAPC Research Symposium, February, Oklahoma State University, Stillwater, OK.
- Panneerseelan, L., and P.M. Muriana. 2009. An immunomagnetic PCR signal amplification (iPCR-SA) assay for sensitive detection of Staphylococcus aureus enterotoxins in foods. J. Food Prot. 72: (in press).
- Kushwaha, K., and P.M. Muriana. 2009. Adherence characteristics of Listeria strains isolated from 3 ready-to-eat meat processing plants. J. Food. Prot. 72:2125-2131.
- Kushwaha, K., and P.M. Muriana. 2009. Comparison of invasiveness among surface-adherent variants of Listeria monocytogenes in Caco-2 cell culture assays. Intl. J. Food Microbiol. (in press).
- Cuesta Alonso, E., J. Fletcher, A.C. Wayadande, R. Mitra, and S.E. Gilliland. 2008. Internalization of Escherichia coli O157:H7 in growing spinach plants. Inst. Food Technol. Abstract, New Orleans, LA.
- Mitra, R., P. Cuesta-Alonso, S. Leyman, A. Wayadande, S. Gilliland and J. Fletcher. 2008. Microscopic surveillance of fluorescently tagged Escherichia coli O157:H7 in spinach plants. APS Centennial Meeting Abstract, Minneapolis, MN.
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