Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
DETECTION, CHARACTERIZATION, AND INHIBITION OF FOODBORNE PATHOGENIC AND SPOILAGE MICROORGANISMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0178073
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2007
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
FOOD AND AGRICULTURE PRODUCTS CENTER
Non Technical Summary
A. Certain pathogens are persistant in meat processing environments and contaminate ready-to-eat (RTE) meat products. B. Rapid detection and microbial interventions are important to insure food safety. A. This project examines microbial interventions to reduce pathogen contamination on processed meats. B. The project will examine rapid methods for detection, and biochemical and genetic characteristics of Listeria.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
50150101150100%
Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
5010 - Food;

Field Of Science
1150 - Toxicology;
Goals / Objectives
I. To apply nucleic acid-based methods for detection of foodborne pathogens (i.e., L. monocytogenes) II. To genetically subtype foodborne pathogens by PFGE, Ribotyping, or Sequence analysis. III. To develop a biofilm assay for, and analysis of biofilm formation by, L. monocytogenes. IV. To develop methods to eliminate pathogenic & spoilage organisms from RTE meats.
Project Methods
I. Two fluorecence-based PCR approaches will be examined for detection of L. monocytogenes, 1) Amplifluor Universial PrimersTm, and 2) generic fluorescence-based primers with amplimer capture and end-point fluorescence. II. Genetic subtyping will be performed on pathogen isolates (L. monocytogenes) obtained from the screening of RTE meat processing facilities to show relatedness among recurring isolates and on isolates in our culture collection that are identified as biofilm-formers. III. A Carboxy Fluorescein Diacetate (5,6-CFDA) microplate biofilm assay will be developed to facilitate the screening of hundreds of strains (pure culture) of L. monocytogenes we have collected for further studies on the mechanism of biofilm formation by L. monocytogenes. IV. Thermal surface pasteurization (pre-package pasteurization, post-package pasteurization) and antimicrobials (bacteriocins, chemicals) will be used to reduce pathogens on the surface of ready-to-eat meat products.

Progress 10/01/03 to 09/30/07

Outputs
OUTPUTS: Activities:

Industry Outreach Projects. Major projects that involved 1-10 weeks of laboratory testing and analysis involving practical research for 8 local and 20 national meat/food processors. The laboratory work was focused on improving quality and safety related to meat/food processes. We ran various validation and shelf life studies with pathogen-inoculated products, examining lethality to the pathogens using antimicrobial chemicals and ingredients, as well as thermal treatments using submersed water and radiant heat pasteurization of deli meats, evaluation of jerky processing conditions, evaluation of microbial spoilage organisms on commercial products, as well as validation of natural antimicrobials as replacements for chemical preservatives. Overall, these activities provided validation to commercial processors that the intended process was sufficient in reducing or eliminating foodborne pathogens. Our studies with meat surface thermal pasteurization have been adopted by industry and cited by USDA-FSIS as examples of post-process lethality steps for reducing risk and enhancing the safety of processed meats.

Events:

HACCP Workshops (Advanced HACCP, Basic HACCP, Non-meat HACCP). HACCP workshops (all 3 categories) were held approximately 3 times each per year during this time period; each lasted 2-3 days. Participants were from meat/food processing industries from Oklahoma and neighboring states and included management, company HACCP team members, and plant and line workers. External participants included State and Federal USDA personnel (http://fapc.biz/PAGES/haccp.htm).

Food Safety RoundTables. The FSR was held quarterly during this time period. The routine was a technical presentation in the morning (I averaged two technical presentations of the 4 quarterly meetings) followed by discussion of the presentation. USDA inspectors would then leave the conference room whereby industry personnel could voice opinions, comments, questions, that would later be posed FAPC personnel hosting the RoundTable to USDA-FSIS technical representative by conference call in anonymous fashion. We felt this would be the best way to draw critical commentary out of industry meat/poultry processing personnel (http://fapc.biz/PAGES/roundtable.htm).

FACP Research Symposium. An annual research symposium was initiated in 2001 that included an invited keynote speaker, graduate student research oral presentations (4-12), and graduate student poster presentations (20-30), as well as a Food Center tour and luncheon (http://fapc.biz/PAGES/researchsymposium.htm).

Food Industry Trends (FIT) Conference. An annual national conference was initiated that involved approximately a dozen invited speakers on various topics important to the meat/food industry including representatives from USDA, FDA, industry, and academia. Cooperation of organizing members included representatives from OSU, OK Dept. Health, OK Dept. Commerce, and OK Dept. Agriculture (http://fapc.biz/PAGES/fitconference.htm). PARTICIPANTS: Principal investigator and project director for most of the projects listed. -Peter Muriana

Laboratory Technicians employed during this project: -Catherine Davidson -Bill Quimby -Will Robertson -Brad Jordan

Graduate students employed on this project: -Saritha Gedella -Nanditha Gande -Sunita Macwana -Suparna Mitra -Kalpana Kushwaha -Rachel Wright -Shawnna Veasey -Will Robertson -Bill Quimby

Collaborators (individuals) on this project: -Roy Escoubas (OSU) -Tim Bowser (OSU) -David Howard (Unitherm Foodsystems) -Mike Benson (Jennie-O Turkey Store) -Bob Riley (Bar-S Foods)

Collaborators (companies) on this project: -West Liberty Foods -Vector Intl. -J. Frierich Co. -Snowball Foods -Perdue Farms -Charlie's Pride -V. Giordano Corp. -Dietz & Watson -Boar's Head -Lopez Foods Inc -Vaughan Foods Inc -Allison's Gourmet Kitchens -Ralph's Packing TARGET AUDIENCES: Target Audiences: -Food Processors, management -Technical personnel in food processing -Consumers -Food safety personnel

Efforts: -workshops -seminars, discussions -roundtables -symposia, meetings -presentations at annual meetings -research publications -project reports -presentations made at company offices -newsletters, fact sheets, flash bulletins, flyers -quarterly magazine distributed to OK processors

Impacts
Adherence assay for Listeria monocytogenes. This study helped to identify a method to detect and quantify strongly-adherent strains of L. monocytogenes from various strains we collected from RTE and raw meats, and meat processing plants. Strongly-adherent strains of L. monocytogenes had 100,000-fold greater levels of adherence than weakly-adherent strains. Adherence was demonstrated to glass, stainless steel, plastic, and rubber and at 10, 20, 30, and 40oC. Although all strains of L. monocytogenes are considered pathogenic to humans, but those that adhere more strongly are better able to develop biofilms, contaminate surfaces, and cross-contaminate foods. This study emphasizes the need for greater environmental sanitation and postprocess lethality on RTE meat products.

Surface pasteurization of RTE meats. We continued our investigations with surface pasteurization of RTE meats by examining conditions for radiant heating of exposed RTE deli meats that would be packaged immediately after heating (i.e., prepackage pasteurization). This process, along with our previously studied submersed water postpackage pasteurization, helped to achieve the reduction of potential surface contamination of RTE meats with Listeria monocytogene. These processes were accepted by USDA-FSIS as evidenced by their citing of our work and the process in their guidance documents for the industry as evidence of post-process lethality steps that could be used by industry to reduce risk of L. monocytogenes contamination on RTE products. The process was accepted within the RTE meat processing industry as evidenced by the many companies that implemented the process that helped them to achieve safer RTE processed meats that could be used to change their Alternative process to a reduced-risk status that is acknowledged by USDA-FSIS with reduced testing.

Antimicrobial effect of liquid smoke extracts on RTE meats. My lab worked with a liquid smoke extract manufacturer as well as two major RTE meat manufacturers (frankfurters and deli turkey) to examine the effect of low acid/low phenolic liquid smoke extracts on survival of L. monocytogenes on RTE meats. The liquid smoke extracts were able to reduce initial levels of Listeria and prevent their growth on RTE meats for up to 10 weeks achieving either an Alternative 1 or 2 USDA-FSIS process. We also examined the inhibition when combined with surface (thermal) pasteurization of RTE meats. The data suggests that inhibitory liquid smoke extracts can provide inhibition of Listeria when used alone, or in combination with other Postprocess lethality steps.

Multi-locus sequence typing of Listeria monocytogenes. We used multi-locus sequence typing (MLST) as a method of typing L. monocytogenes. Using PCR primers targeting 5 virulence factors in L. monocytogenes, amplimers were sequenced, added together as a contiguous, artificial gene, and compared to sequences obtained from other strains by multiple sequence alignment. Comparison of DNA sequence is more easily accommodated by software and data is readily transferable between labs.

Publications

  • Journal Articles (2003-2007):
  • Muriana, P.M., Bowser, T., Davidson, C., Tilahun, M., and D.E. Gibbs. 2003. Flash pasteurization of contaminated streams using a direct-contact gas-fired water heater. Food Microbiol. 20:77-82
  • Gande, N., and P.M. Muriana. 2003. Pre-package surface pasteurization of ready-to-eat deli meats for reduction of Listeria monocytogenes. J. Food Prot., 66:1623-1630.
  • Robertson, W., and P.M. Muriana. 2004. Reduction of Salmonella by two commercial egg white pasteurization methods. J. Food Prot. 67:1177-1183.
  • Veasey, S., Yerramsetty, P., Wright, R., and P.M. Muriana. 2006. Efficacy of electrolyzed water on Listeria monocytogenes and other foodborne pathogens. Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #054A-34.
  • Veasey, and P.M. Muriana. 2007. The use of hypochlorous acid (electrolyzed water) for reduction of pathogens and spoilage microorganisms in RTE meat processing environments and meat products. Ann. Meet. Inst. Food Technol., Chicago, IL (July 27-Aug 1), Abst. #058-12.
  • Kushwaha, K., and P.M. Muriana. 2007. Virulence analysis of surface-adherent strains of Listeria monocytogenes in tissue culture assays. Ann. Meet. Inst. Food Technol., Chicago, IL (July 27-Aug 1), Abst. #058-13.
  • Wright-Gamble, R., and P.M. Muriana. 2007. A microplate fluorescence assay for the measurement of the ability of strains of Listeria monocytogenes from meat and meat processing plants to adhere to abiotic surfaces. Appl. Environ. Microbiol. 73: 5235-5244.
  • Muriana, P.M., Gande, N., Robertson, W., Jordan, B., and S. Mitra. 2004. Effect of prepackage and postpackage pasteurization on postprocess elimination of Listeria monocytogenes on deli turkey products. J. Food Prot. 67(11):2472-2479.
  • Gedela, S., J.R. Escoubas, and P.M. Muriana. 2007. Effect of inhibitory liquid smoke fractions on Listeria monocytogenes during long-term storage of frankfurters.. J. Food Prot. 70:386-391.
  • Gedela, S., R. Wright, S. Macwana, J.R. Escoubas, and P.M. Muriana. 2007. Effect of inhibitory extracts derived from liquid smoke combined with postprocess pasteurization for control of Listeria monocytogenes on ready-to-eat meats. J. Food Prot. 70: In Press.
  • Abstracts (2003-2007):
  • Gande, N., and P.M. Muriana. 2003. Radiant heat (prepackage), alone and in combination, with submersed water (postpackage) pasteurization for reduction of Listeria monocytogenes on the surface of roast duck, turkey bologna, deli ham, and roast beef. Ann. Meet. Inst. Food Technol., Chicago, IL (July 12-16), Abst. #60C-18.
  • Gande, N., Muriana, P.M., Robertson, W., Mitra*, Suparna, and B. Jordan. 2003. Pre- and postpackage pasteurization of deli turkey products for pathogen reduction. Ann. Meet. Inst. Food Technol., Chicago, IL (July 12-16), Abst. #60C-16.
  • Robertson, W., and P.M. Muriana. 2004. Reduction of Listeria monocytogenes on hotdogs using liquid smoke extracts. Ann. Meet. Inst. Food Technol., Las Vegas, NV (July 12-16), Abst. #67E-25.
  • Robertson, W., and P.M. Muriana. 2004. Prepackage pasteurization of hotdogs for reduction of L. monocytogenes using a hot water deluge system. Ann. Meet. Inst. Food Technol., Las Vegas, NV (July 12-16), Abst. #67E-26.
  • Robertson, W., Jordan, B., Gande, N., Mitra, S., Juneja, V., and P.M. Muriana. 2004. Lethality of Listeria monocytogenes, Salmonella sp., and E. coli O157:H7 during heating of sausage emulsion. Ann. Meet. Inst. Food Technol., Las Vegas, NV (July 12-16), Abst. #67E-27.
  • Wright, R., and P.M. Muriana. 2004. Development of a microplate fluorescence-based assay to screen isolates of Listeria monocytogenes for ability to form biofilms. Ann. Meet. Inst. Food Technol., Las Vegas, NV (July 12-16), Abst. #114D-28.
  • Mitra, S., and P.M. Muriana. 2004. Detection of Listeria monocytogenes from meats using fluorescent-based universal realtime PCR primers. Ann. Meet. Inst. Food Technol., Las Vegas, NV, (July 12-16), Abst. #99A-36.
  • Muriana, P.M., and R. Wright. 2006. Identification of strong- and weakly-adherent strains of Listeria monocytogenes and enzymatic detachment from surfaces. Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #039H-07.
  • Macwana, S., and P.M. Muriana. 2006. A novel approach for rapid identification and sequencing of different bacteriocins produced by lactic acid bacteria based on a practical immunity class. Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #020B-06.
  • Kushwaha, K., and P.M. Muriana. 2006. Phylogenetic typing of Listeria monocytogenes by multilocus sequence typing (MLST). Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #039H-06.
  • Gedela, S. 2005. OSU, M.S. Dec. 2005. Application of liquid smoke alone and in combination with pre- and post-package pasteurization against Listeria monocytogenes on ready-to-eat meats.
  • Dissertations (2003-2007):
  • Macwana, S. 2007. OSU, Ph.D., Aug. 2007. Bacteriocins: Unique approaches for the characterization of peptide antimicrobials in lactic acid bacteria.
  • Wright, R. 2006. OSU, M.S. Jan. 2006. Development of a fluorescence-based microplate assay to screen strains of Listeria monocytogenes for attachment to environmental surfaces.
  • Quimby, B. 2004. OSU, M.S. Dec., 2004. DNA fingerprinting of Listeria spp. isolated from feedlot and slaughter facilities using pulsed-field gel electrophoresis.
  • Mitra, S. 2004. OSU, M.S. Summer, 2004. Real-time PCR using universal primers (UniPrimer) for detection of Listeria monocytogenes in food.
  • Gande, N. 2003. OSU, M.S. July, 2003. Pre- and post-package pasteurization of ready-to-eat meats to control Listeria monocytogenes.
  • Will Robertson. 2003. Reduction of Salmonella spp in liquid egg white. OSU, M.S. July, 2003.
  • FAPC Flash Newsletters, FAPC.Biz Magazine Articles, and Extension Fact Sheets (2005-2006):
  • Muriana, P. 2006. FAPC/IFT-OK to Host Research Symposium Feb.22. (http://fapc.biz/fapcflash/researchsymposium06.pdf)
  • Muriana, P. 2006. E. coli in Spinach. (http://fapc.biz/fapcflash/ecolispinach.pdf)
  • Muriana, P. 2006. E. coli in spinach deals big blow to spinach and fresh-cut salad industries (Fall, 2006, p.4). http://fapc.biz/magazine/fall2006.pdf
  • Muriana, P. 2006. FAPC receives electrolyzed water equipment donation from Unitherm (Fall, 2006, p.5). http://fapc.biz/magazine/fall2006.pdf
  • Muriana, P. 2006. Making a splash with electrolyzed water (Summer, 2006, p.5). http://fapc.biz/magazine/summer2006.pdf
  • Muriana, P. and K. Kushwaha. 2005. Extension Fact Sheet FAPC#136. Food Pathogens of Concern: Listeria monocytogenes. http://fapc.biz/factsheets/fapc136.pdf


Progress 10/01/05 to 09/30/06

Outputs
Development of a Microplate Assay to Determine Adherence Properties of Individual Strains of Listeria monocytogenes Isolated from Fresh Ground Beef, Ready-to-Eat Meats, and Meat Processing Environments. A microplate assay was developed to examine strains of Listeria monocytogenes isolated from fresh meats, RTE meats, and meat processing plants. The assay was based on allowing a period of attachment in microplate wells, washing away planktonic cells with a plate washer, uptake of added substrate (5,6-carboxyfluorescein) by the attached cells that is hydrolyzed by internal cellular esterases to a fluorescent derivative, then washed again in a microplate washer, and finally reading fluorescence from attached cells in a fluorescent plate reader. We identified the optimal incubation time of attached cells with substrate to be 15 min at 25oC. Strongly- and weakly-adherent cells were confirmed by visual observation using fluorescence microscopy and scanning electron microscopy. Protease treatment was also used to recover and quantify attached cells from microplate wells or from glass, plastic, stainless steel, and rubber substrates for further comparison of weak and strong-adherent strains. Our data show that strongly adherent strains were attached at 5-logs higher attachment levels to surfaces (108 cfu/well) than weakly adherent strains (103 cfu/well), that they attached equally well to the four substrates tested, and demonstrated strong adherence at 10o, 20o, 30o, or 40oC. Analysis of surface adherence of L. monocytogenes from substrates commonly found in RTE meat processing environments may provide a better understanding of mechanisms involved with pathogen attachment in sensitive food processing areas.

Impacts
Listeria monocytogenes has been a persistent problem to meat processors manufacturing ready-to-eat meats such as frankfurters and deli sandwich meats. L. monocytogenes is known to form biofilms on surfaces which may become harborage points to further contaminate food, equipment, or spread by human cross contamination. Because of these characteristics, many processors find it difficult to completely eradicate L. monocytogenes from meat processing environments once it becomes established in niche harborage location on various surfaces in a plant. We examined strains of L. monocytogenes that were associated with meat and meat processing facilities and found that there were significant differences among various strains in regard to their ability to adhere to different types of surfaces that could be found in meat processing plants (plastic, stainless steel, rubber, and glass). Those strains that were found to be strongly-adherent, were able to adhere equally well to all surfaces and at all temperature ranges likely to be found in processing plants. We also identified methods of removing the adhered bacteria using protease treatment. Although this was done to allow us to quantify levels of cells recovered from surfaces in our experimental trials, the protease process could potentially be used as a commercial sanitation regimen for facility sanitation.

Publications

  • Gedela, S., Escoubas, J.R., and Muriana, P.M. 2007. Effect of liquid smoke extracts on the shelf life of frankfurters inoculated with Listeria monocytogenes. J. Food Prot. 70:100-105.
  • Muriana, P.M., Wright, R. 2006. Identification of strong- and weakly-adherent strains of Listeria monocytogenes and enzymatic detachment from surfaces. Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #039H-07.
  • Macwana, S., and Muriana, P.M. 2006. A novel approach for rapid identification and sequencing of different bacteriocins produced by lactic acid bacteria based on a practical immunity class. Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #020B-06.
  • Kushwaha, K., and Muriana, P.M. 2006. Phylogenetic typing of Listeria monocytogenes by multilocus sequence typing (MLST). Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #039H-06.
  • Veasey, S., Yerramsetty, P., Wright, R., and Muriana, P.M. 2006. Efficacy of electrolyzed water on Listeria monocytogenes and other foodborne pathogens. Ann. Meet. Inst. Food Technol., Orlando, FL (June 24-28), Abst. #054A-34.
  • Kalpana, K., and Muriana, P.M. 2006. Subtyping of Listeria monocytogenes by Multilocus Sequence Typing and Pulsed- field gel electrophoresis (PFGE). Dept. Animal Science Res. Rpt. Available at: http://www.ansi.okstate.edu/research/2006rr .
  • Macwana, S., and Muriana, P.M. 2006. A Novel Approach for Rapid Identification and Sequencing of Different Bacteriocins Produced by LAB Based on a Practical Immunity Class. Dept. Animal Science Res. Rpt. Available at: http://www.ansi.okstate.edu/research/2006rr .


Progress 10/01/04 to 09/30/05

Outputs
Application of Liquid Smoke to Reduce and/or Control Listeria monocytogenes in Ready-to-Eat Meats. In challenge studies of retail franks, low levels of L. monocytogenes were able to increase to significantly higher levels on 5 of 8 brands tested containing lactate/diacetate when held at 1.6oC (35oF). Liquid smoke was able to reduce and control growth of L. monocytogenes for up to 10 weeks on franks treated for as little as 1 sec (dip application) or when dropped through an atomized mist of liquid smoke. Hotdogs were also manufactured without lactate/diacetate by a commercial manufacturer and sprayed with liquid smoke using a commercial spray device as they exited the peeler. When inoculated at 3 different levels (101, 102, 103 CFU) with a 4-strain cocktail of L. monocytogenes and stored at abuse temperatures of 6oC (43oF), the smoke-treated samples demonstrated effective control of L. monocytogenes. Thermal surface pasteurization was examined in combination with liquid smoke extracts to reduce and prevent growth of L. monocytogenes during shelf life of sensitive RTE meat products. When tested on frankfurters inoculated at 108 CFU, a liquid smoke extract demonstrated a 0.25-log reduction of L. monocytogenes, whereas 1 min in-bag pasteurization (73.9oC/165oF) of inoculated franks gave a 2.9 log reduction. However the combination of liquid smoke and pasteurization gave a 5.25-log reduction that resulted in no detectable Listeria by week 3 when tested weekly for 6 weeks of storage at 10oC (50oF). In comparison tests, a 1 sec dip in a clear liquid smoke extract with reduced smoke flavor gave a 5-log reduction on frankfurters when heated a 73.9oC (165oF) for 1 min with no recoverable Listeria detected for 10 weeks when stored at 10oC (50oF). A similar study combining liquid smoke treatment with postpackage pasteurization of deli turkey chubs demonstrated a 2-3 log reduction of L. monocytogenes with no grow out during 10 weeks of storage at 6.1oC (43oF).

Impacts
Listeria monocytogenes has been troublesome for many ready-to-eat (RTE) meats, including hotdogs, which have had among the highest incidence of Listeria contamination among fully-cooked RTE meat products. The most recent USDA-FSIS policy provides incentives for processors who accommodate greater levels of safety by employing either a post-process lethality step or antimicrobial chemicals (Alternative 2), or both (Alternative 1), to control Listeria in RTE meats. The data shows that surface application of liquid smoke extracts by dipping or spraying may inhibit the growth of L. monocytogenes on hotdogs during shelf life and should facilitate a claim as an Alternative 2 (and possibly Alternative 1) process for HACCP purposes. When liquid smoke extracts we examined were combined with a thermal process, the control of L. monocytogenes that was obtained should be sufficient to achieve a USDA-FSIS Alternative 1 process category.

Publications

  • Gedela, S. Application of liquid smoke alone and in combination with pre- and post-package pasteurization of against Listeria monocytogenes on ready-to-eat meats. M.S. Thesis, December 2005.
  • Gedela, S., W. Robertson, and P.M. Muriana. 2004. Effect of liquid smoke extracts on frankfurters inoculated with Listeria monocytogenes. Dept. Animal Science Res. Rpt. Available at: http://www.ansi.okstate.edu/course/2004rr/20/20.htm.


Progress 10/01/03 to 09/30/04

Outputs
Surface pasteurization of deli turkey products. Surface pasteurization for inactivation of Listeria monocytogenes was evaluated for radiant heat prepackage pasteurization, submersed water postpackage pasteurization, and combinations of the two on various ready-to-eat deli turkey products. Products were inoculated with a 4-strain mixture of L. monocytogenes either by in-package liquid inoculum or surface sponge-contact inoculation with approximately 109 CFU. Additional testing of radiant heat pasteurization was performed with low-level inoculation of product undersides with circa 100 CFU of L. monocytogenes followed by enrichment recovery after pasteurization as an attempt to examine the most difficult surface to pasteurize since the product sits on a stainless steel mesh belt as it travels through the radiant heat oven. Prepackage pasteurization provided 2.0-2.8 log reductions when processed for 60 sec and 2.8-3.8 log reductions when processed for 75 sec. An improved radiant oven provided up to 3.53- (60 sec) and 4.76 log (75 sec) reductions of L. monocytogenes. No positive samples were detected after enrichment when 40 samples of deli turkey (4-kg) undersides were inoculated at low levels and processed for 75 sec. Submersed water postpackage pasteurization provided 1.95-3.0 log reductions when processed for 2, 3, 4, or 5 min and combinations of the two processes gave 3.0-4.0 log level inactivation of L. monocytogenes using either 60+60 sec, or 60+90 sec, for the prepackage and postpackage pasteurization processes, respectively. These processes, either individually or in combination, can provide postprocess lethality for the manufacture of safe RTE deli meats.

Rapid detection of L. monocytogenes by labelled real-time PCR probes. We optimized conditions for use of a commercial real-time PCR probe (UniPrimerTm) for PCR detection of L. monocytogenes from raw and processed meat products. Primer target sequences for PCR were based on the listeriolysin hemolysin gene (hlyA). Real-time PCR was performed after initial inoculation, and after 1o and 2o enrichments of various raw/ground and processed meats inoculated at 100-106cfu/25gm. We were able to detect L. monocytogenes from pure culture tubes having Listeria levels >104 cfu/ml. In both raw and RTE meats, UniPrimerTM real-time PCR was able to detect meat samples inoculated with as few as 1 cfu/25gm after 2o enrichment. Analysis of direct PCR, and PCR after 1o and 2o enrichment of live and dead (autoclaved) cell cultures of L. monocytogenes at 109 cfu/ml showed that both live and dead cells gave equivalent detection by real-time PCR when detection was examined without enrichment, whereas the detection of dead cells was reduced after 1o enrichment and undetectable after 2o enrichment. This indicates the benefit of enrichment procedures to eliminating the detection of dead cells from consideration.

Impacts
Surface Pasteurization of RTE meats. The data we have obtained on the efficacy of surface pasteurization immediately prior to packaging (prepackage pasteurization) or after packaging (postpackage pasteurization) is currently used by various commercial meat processors and accepted by USDA-FSIS as HACCP validation. Our work with prepackage and postpackage pasteurization has been cited by USDA-FSIS as examples of post-process lethality treatments that may allow change of food processing category from an Alternative 3 to an Alternative 2 process. These processes are currently being used by large and medium-sized processors throughout the U.S.

Rapid detection of L. monocytogenes by real-time PCR (UniPrimerTm).Improved detection methods for L. monocytogenes will enable processors to implement detection more efficiently and perhaps sample environmental surfaces more frequently if results can be obtained more rapidly. The novelty of the Amplifluor UniprimerTm real-time PCR probe is that the same probe can be used for different genetic targets by simply adding a short sequence (i.e., Z-tail) to the 5'-end of one of the primers. Other real-time probes require each one to be synthesized and optimized specifically for the intended target. The UniPrimerTm probe in combination with our primers, provided specific detection of L. monocytogenes from meats following primary and secondary enrichment. Further research to shorten these enrichment periods could further enhance rapid detection using this method.

Publications
  • Muriana, P.M., Gande, N., Robertson, W., Jordan, B., and S. Mitra. 2004. Effect of prepackage and postpackage pasteurization on postprocess elimination of Listeria monocytogenes on deli turkey products. J. Food Prot. 67(11):2472-2479.
  • Gande, N. Pre- and post-package pasteurization of ready-to-eat meats to control Listeria monocytogenes. M.S. Thesis, December 2003.
  • Mitra, S. Applications of a real-time 'Universal Primer' for PCR detection of Listeria monocytogenes from meat products. M.S. Thesis, July 2004.


Progress 10/01/02 to 09/30/03

Outputs
Surface pasteurization of ready-to-eat (RTE) meats: A pilot plant model of a radiant heat oven was obtained from a local manufacturer and examined for prepackage surface pasteurization of Listeria monocytogenes on large RTE deli meats (turkey bologna, roast beef, corned beef, and ham). We examined both dip- and contact-inoculation methods for inoculating the surface of these products with a four-strain cocktail of L. monocytogenes. With the radiant heat oven, we obtained 1.25-to-3.5 log reductions of L. monocytogenes with treatment times of 60-120 sec at air temperatures of 246-399oC (475-750oF). Prepackage pasteurization (60 sec) was also combined with postpackage pasteurization by hotwater submersion (45, 60, or 90 sec) resulting in a 2.0-3.9 log cycle reduction of L. monocytogenes.

Pasteurization of liquid egg white: The effect of pH, processing temperature, and the preheating step was examined in two commercial egg white pasteurization methods (Amour and Standard Brands methods) that utilize hydrogen peroxide as a processing aid using a five-strain cocktail of Salmonella spp. A bench-top continuous flow pasteurization system was devised that would allow the introduction of hydrogen peroxide after the initial preheat step that is specified for both processes. Both processes were evaluated with egg white at three pH levels (pH 8.2, 8.6, and 9.0), at four temperatures (51.7o, 53.1o, 54.4o, and 55.8oC), and at four residence times (different for either process) so that D-values could be calculated at each temperature. Both methods were compared at the minimum time/temperatures allowed by USDA-FSIS for these methods and demonstrated at least a 1-log greater reduction (P < 0.05) for the Standard Brands method than the Armour method in 10 out of 12 of the pH and temperature combinations tested. Analysis of the preheat portion of the two methods showed that there was no contribution (P > 0.05) towards Salmonella reduction when compared with the identical process without the preheating step.

Impacts
Surface Pasteurization of RTE meats. The data we have obtained on the efficacy of surface pasteurization immediately prior to packaging (prepackage pasteurization) or after packaging (postpackage pasteurization) has already been used by various medium/large commercial processors and accepted by USDA-FSIS as HACCP validation of the particular surface pasteurization method. This is important to the RTE meat and poultry processing industry in regard to recent USDA Directives (10,240.3) and final rules (July 6, 2003) that provide for reduced product risk categories and reduced USDA testing for processors who implement postprocess lethality measures. Our work with prepackage and postpackage pasteurization has been cited by USDA-FSIS as examples of such treatments.

Egg white pasteurization. Data for egg white pasteurization methods at the time of our work was based on pre-1968 industry data for which most liquid egg pasteurization was done "off-line" in which the egg white pH reaches pH 9.0. Current modern egg pasteurization facilities are mostly "in-line" whereby eggs are broken and egg white is pasteurized while still fresh (pH ~8.0-8.2). Our work demonstrates the lack of contribution to the pasteurization process for the preheat step of two widely used commercial processes as well as differences in reduction of Salmonella that may be obtained. Recent publication of a new Egg Pasteurization Manual for the U.S. and international egg pasteurization industry has made recommended changes in egg white pasteurization guidelines based on data we obtained.

Publications
  • Gande, N., and P.M. Muriana. 2003. Pre-package surface pasteurization of ready-to-eat deli meats for reduction of Listeria monocytogenes. J. Food Prot., 66:1623-1630.

  • Robertson, W., and P.M. Muriana. 2004. Reduction of Salmonella by two commercial egg white pasteurization methods. J. Food Prot. 67:000-000(In Press).

  • Froning, G.W., Peters, D., Muriana, P.M., Brashears, M., and K. Eskridge. 2002. International egg pasteurization manual. United Egg Association, Alpharetta, GA.


Progress 10/01/01 to 09/30/02

Outputs
A commercial direct contact gas-fired water heater was modified for in-lab evaluation of microbial reduction and temperature consistency at various water flow rates. The water heater was modified so that a contaminated input stream could be treated using 50-gal (189.3-liter) bulk feed tanks. A final reservoir tank was bypassed using 6 sampling ports placed in pairs along 3 locations in the vertical heating column with which to test the efficacy of a single pass at different positions in the heating zone. Temperature probes were utilized to follow water temperature at 8, 10, and 12 gallons/min (30.3, 37.9, and 45.4 liters/min). When using inoculated water streams, we obtained 4-7 log-cycle reductions of Lactococcus lactis, Lactobacillus curvatus, Enterococcus faecalis, Listeria ivanovii, Listeria welshimeri, and Escherichia coli suggesting that this may have potential use in situations where food safety issues involved in water recycling are of interest in food processing environments.

Impacts
Food processing activites are generally water-intensive in regards to water utilization. Water useage limits may be established for various municipalities where water availability is limited or where there are significant seasonal variabilities in availability. Therefore, methods that reduce water consumption, or, allow for recycling are becoming more appealing to many food processors. We have worked with a local (Oklahoma) company to demonstrate the pasteurization efficacy of their water heater for potential use in food processing markets where water recycling/reutilization is being contemplated.

Publications

  • Muriana, P.M., Quimby, W., Davidson, C., and J. Grooms. 2002. Post-package pasteurization of RTE deli meats by submersion heating for reduction of Listeria monocytogenes. J. Food Prot. 65:963-969.
  • Muriana, P.M., Bowser, T., Davidson, C., Tilahun, M., and D.E. Gibbs. 2003. "Flash pasteurization" of contaminated streams using a direct-contact gas-fired water heater. Food Microbiol. 20:77-82.


Progress 10/01/00 to 09/30/01

Outputs
Fluorescent-tagged primers for PCR-based non-gel detection of Listeria monocytogenes. The Amplifluor UniPrimerTm system was optimized for the detection of L. monocytogenes. The UniPrimer system consists of a molecular beacon-type primer possessing an extra Z-tail; the same sequence is added to the 5'-end of one of the primers. During PCR amplification, the UniPrimer gets incorporated into the amplified product, the hairpin is opened, and the fluorophore becomes distal to the quencher and generates high-level fluorescence. We examined 4 primer sets by both agarose gel and fluorescence analysis using a fluorescence plate reader. We first examined and optimized L. monocytogenes in pure culture and then worked with inoculated food samples. Listeria selective broth media (i.e., UVM, Fraser broth) both contain acriflavine and high NaCl levels which causes excessive background fluorescence and interferes with the PCR reaction, respectively. These interferences were eliminated by a) centrifugation and resuspension of enrichment samples, and b) precipitation of Listeria DNA from cell lysates with isopropanol (which extracts and eliminates acriflavine from DNA) prior to PCR. On pure cultures, the Amplifluor UniPrimerTm system gave high-level fluorescence detection of Listeria monocytogenes with very little signal with 4 other species of Listeria and was confirmed by agarose gel analysis. When used with inoculated meat products, L. monocytogenes did not show as dramatically high fluorescence signals when compared to other Listeria species, although agarose gel analysis demonstrated amplified products only with L. monocytogenes. Future efforts to improve this approach will pursue immunomagnetic bead capture and cell removal from interfering food components.

Impacts
The processed meat industry has increased product and environmental testing under HACCP regulations. This has prompted many companies to use a test-and-hold approach to sample testing whereby product is not released to commerce until test results come back negative. This places a burden on the company to maintain refrigerated storage facilities and reduces the shelf life of the tested lot. The development of user-friendly rapid methods for detecting targeted pathogens should prove beneficial to the industry.

Publications

  • Hannah Nayakanti. Application of fluorescent-tagged p rimers for PCR-based non-gel detection of Listeria monocytogenes. M.S. thesis, Dec. 2001.
  • Muriana*, P.M., Quimby, W., Davidson, C., and J. Grooms. 2001. Post-Package Pasteurization of RTE Deli Meats by Submersion Heating For Reduction of Listeria monocytogenes. J. Food Prot. (in press).
  • Mao, Y., Muriana*, P.M., and M.A. Cousin. 2001. Molecular characterization and transpositional inactivation of lacticin FS92, a broad spectrum bacteriocin produced by Lactococcus lactis. Food Microbiol. 18:165-175.
  • Grooms, J., Quimby, W., and P. M. Muriana. 2001. PFGE genomic subtyping of Listeria spp. from cattle holding, slaughter, and processing facilities. Ann. Meet. Inst. Food Technol., New Orleans, LA, Abst.59F-33.


Progress 10/01/99 to 09/30/00

Outputs
Genomic subtyping of Listeria spp. from cattle, slaughter, and processing facilities. More than 500 isolates of Listeria spp. were obtained from cattle holding facilities at OSU's Willard Sparks Research Center, from cattle/carcasses before and after slaughter at OSU's Food & Ag Products Center slaughter facility, and from 3 commercial meat processors of ready-to-eat (RTE) meat products. Isolates were obtained by direct fecal sampling, from surface swabs of hides and finished beef carcasses, and from environmental swabs of non-food contact surfaces in processing facilities. The Listeria isolates were then analyzed by pulsed-field gel electrophoresis (PFGE) with the Bio-Rad CHEF DR using the restriction enzymes, ApaI and SmaI. Gel image analysis of the profiles with the Bio-Rad Gel Doc and DNA fingerprinting system provided degree relatedness and dendrogram trees of the isolates. In the cattle-holding facilities sampled, the same genomic subtypes were repeatedly found, in both environmental and animal fecal samples, as much as 9 months apart, indicating retention of specific strains for long periods in an outdoor animal-holding environment. Additional isolations of the same pattern were found on hides of animals brought to slaughter and also recovered from finished carcasses and within the slaughter environment. Similar situations were detected among the isolates from RTE meat processing facilities: the same genomic subtype found in the raw meat ingredients were also found elsewhere in plant and some of these subtypes were also found in different facilities that receive raw ingredients from the same suppliers.

Impacts
Pulsed-field gel electrophoresis (PFGE) has been instrumental in epidemiological investigations to find sources of outbreak strains and to determine relatedness. Future analysis of the 'recurring' strains isolated from meat processing environments may identify important characteristics that have contributed to the persistance of those strains and provide insight into improving current sanitation practices such that potentially deadly Listeria spp. can be eliminated from processing environments.

Publications

  • Mao, Y., Muriana, P.M., and M.A. Cousin. 2000. Molecular characterization and transpositional inactivation of lacticin FS92, a broad spectrum bacteriocin produced by Lactococcus lactis. Food Microbiology (in press).
  • Quimby*, W. and Muriana, P.M. 2000. Incidence of Listeria monocytogenes and E. coli O157:H7 in cattle holding facilities, feces, and on finished carcasses. Ann. Meet. Inst. Food Technol., Dallas, TX (June 10-14), Abst. #78F-19.
  • Davidson, C., Quimby, W., Grooms*, J., Muriana, P.M., and Lou, Y. 2000. Post-package pasteurization of RTE deli meats by submersion heating for reduction of Listeria monocytogenes. Ann. Meet. Inst. Food Technol., Dallas, TX (June 10-14), Abst. #65C-28.
  • Davidson, C., Muriana*, P.M., Tilahun, M., Bowser, T., and Gibbs, D.E. 2000. Potential application of the Webco QuikWaterTm heater for `flash pasteurization' of microbially-contaminated food processing streams. Ann. Meet. Inst. Food Technol., Dallas, TX (June 10-14), Abst. #65C-29.
  • Ma, L., Muriana, P.M., and Cousin, M.A. 2000. Sequencing and chimeric expression of the gene(s) encoding curvaticin FS47, an antilisterial bacteriocin produced by Lactobacillus curvatus FS47. Ann. Meet. Inst. Food Technol., Dallas, TX (June 10-14), Abst. #78D-12.


Progress 10/01/98 to 09/30/99

Outputs
Post-package pasteurization. A steam-injected submersed water heating bath was used to identify time and temperature process parameters for post-package pasteurization of ready-to-eat (RTE) deli meats (i.e., 8-12 lb fully-cooked turkeys, hams, roasts) for reduction of surface-contaminated Listeria monocytogenes. A 4-strain cocktail of L. monocytogenes was mixed with product purge and added to products at approximately 10+E7 CFU/ml prior to vacuum packaging. The Listeria strains used were made resistant to streptomycin and rifamycin and cell counts were obtained by plating on non-selective media (Tryptic Soy agar) containing these antibiotics; this allows heat-injured cells to grow that may otherwise be inhibited on Listeria-selective media. Products were then pasteurized for various time and temperature regimens ranging from 195-210 degrees F and for 2-10 minutes. Temperature of product was recorded using thermocouple temperature probes through the vacuum packaging film, along the product surface and under product skin or surface where appropriate. Microbial reduction levels on L. monocytogenes ranged from approximately 2-log reductions for 195 degrees F with 2 minute holding time to 5-log cycle reduction at 210 degree F with 8 minute holding time. Typical cook-in-bags designed for traditional commercial process have now been changed to accommodate higher-temperature cook cycles that may be desired in post-package pasteurization of RTE deli meats. Purge volume was quantitated so that plate counts could be adjusted for additional purge that developed during the heating process.

Impacts
Impact. Recent outbreaks from consumption of contaminated deli luncheon meat and frankfurters suggests that these products had post-packaging contamination with L. monocytogenes. The current processing data provides parameters with which processors may perform post-package pasteurization to reduce potential incidental contaminatin with L. monocytogenes on RTE deli meats

Publications

  • Wu, F.M., and P.M. Muriana. 1999. Cloning, sequencing, and characterization of genomic-subtracted sequences from Listeria moncytogenes. Appl. Environ. Microbiol. 65(12) :5427-5430.27.
  • Davidson*, S.S. Reilly, E. Harp, S.E. Gilliland, and P.M. Muriana. 1999. Incidence of Esherichia coli, Listeria monocytogenes, Campylobacter spp., and Salmonella spp. in ground beef and on beef carcasses in Oklahoma. Ann. Meet. Inst. Food Technol., Chicago, IL (July 24-28), Abst.#79C-17.
  • Y. MAO, P. M. Muriana, and M. A. Cousin. 1999. Generation and characterization of bacteriocin deficient mutant of Lactococcus lactis FS92 by using Tn917 transposon mutagenesis. Ann. Meet. Inst. Food Technol., Chicago, IL (July 24-28), Abst. #22A-22.
  • Ying Mao (Ph.D. dissertation, Purdue University, Aug. 1999). Molecular and genetic characterization of broad-spectrum bacteriocins produced by Lactococcus lactis FS56 and FS92.
  • Li Ma (Ph.D. dissertation, Purdue University, Oct. 1999). Cloning and genetic characterization of curvaticin FS47, a bacteriocin produced by Lactobacillus curvatus FS47.


Progress 10/01/97 to 09/30/98

Outputs
New project began 10/01/98. No progress to report.

Impacts
(N/A)

Publications

  • No publications reported this period