Source: LOUISIANA STATE UNIVERSITY submitted to
CONTROL OF FOOD-BORNE PATHOGENS IN PRE AND POST HARVEST ENVIRONMENTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0212528
Grant No.
(N/A)
Project No.
LAB93876
Proposal No.
(N/A)
Multistate No.
S-1033
Program Code
(N/A)
Project Start Date
Oct 1, 2007
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Janes, M. E.
Recipient Organization
LOUISIANA STATE UNIVERSITY
(N/A)
BATON ROUGE,LA 70893
Performing Department
FOOD SCIENCE
Non Technical Summary
Several outbreaks have been linked to the consumption of seafood with foodborne pathogens. This project will develop methods for controling foodborne pathogens on the surface of seafood.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7120810110050%
7123721110050%
Goals / Objectives
Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments including meat, poultry, seafood, fruits and vegetables and nutmeats.
Project Methods
Inoculation of seafood and fish: Listeria monocytogenes or Salmonella spp. will be grown in BHI broth at 37degreeC for 24 h. The culture (5 ml) will then be added to a sterile dip cup and diluted in 45ml of PBS buffer. The seafood and smoked fish samples will be inoculated by dip method for 1 min. and allowed to sit for 1 h to ensure adhesiveness of cells to the sample surface. Following inoculation, each sample will be treated with either edible coatings containing antimicrobial agents, cetylpyridinium chloride (CPC) or acidified sodium chlorite (ASC). Samples will be stored at 4degreeC under simulated industry conditions and L. monocytogenes counts determined on modified Oxford (L. monocytogenes) or XLD (Salmonella spp.) media at day 0, 2, 4, 6, and 8. Preparation of sanitizers: Seven solutions of cetylpyridinium chloride (CPC) will be prepared in sterilized deionized water at concentration levels of 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%. All solutions will be made fresh prior to conducting experiment and will be used at room temperature within 1 h. Sodium chlorite solution (Keeper Professional Bio-Cide International, Inc.) will be mixed with citric acid and sterile distilled water to form the acidified sodium chlorite solution. The mixing process will be done according to manufacturer's instructions with sterile distilled water. The sodium chlorite concentrate (Keeper Professional) will be mixed with citric acid and allowed to activate for 10 minutes. The solution concentrate (pH 2.62) will be then diluted with sterile distilled water to 250, 500, 750 and 1000 parts per million (ppm). The various treatments will be placed in sterile specimen cups and held for one hour at room temperature before being used. Cetylpyridinium chloride treatment will be analyzed at concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0%. Edible coating treatment (LA): We will evaluate agar gels (0.75% or 1.25%), chitosan coatings, 15% whey protein films, 5% soy protein films or 0.5% calcium-alginate films with a combination of the most effective antimicrobial agents as determined above (Natrajan and Sheldon, 2000a-b). In addition, zein propylene glycol liquid film and zein prolamine powder preparations will be obtained from Freeman Industries, L.L.C., Tuckahoe, New York. The antimicrobial agents incorporated into the edible films will include nisin, lauric acid or lysozyme in combination with chelating agents needed to reduce the bacterial species inoculated onto the surface of food products. Nisin at a concentration of 500 IU/g, 1000 IU/g 1500 IU/g or 2000 IU/g of film solutions with and without added EDTA at 15 or 30 mM. Lysozyme will be tested at concentrations of 33, 66 and 99 mg/g of film solutions with and with out EDTA at 15 or 30 mM (Padgett et al., 2000). Lauric acid at concentrations of 4 and 8% (w/w) will be added to the test film solutions with and without added EDTA at 15 or 30 mM (Padgett et al., 2000).

Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: The results of this project were presented at the IAFP meeting in 2010 in Anaheim CA. as follows "G. Edwards, M.E. Janes, M. Gutierrez, S. Kerr and J. Young. 2010. Determining if the consumer method for boiling shrimp until floating effectively reduces Listeria and Salmonella species." Update results were presented at the 2011 Multi-State Project S-1033 at Fayetteville Ark. PARTICIPANTS: M. Janes, (PI), G. Edwards, LSU AgCenter. TARGET AUDIENCES: The scientific research that has been conducted on seafood and finfish for reduction of foodborne pathogens has been developed for the food processors, which is difficult for the average consumer to understand. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The purpose of this study was to determine whether the current consumer method of boiling shrimp until floating and pink in color is adequate in eliminating Listeria and Salmonella species. Shrimp samples were submerged into bacterial suspensions for 30 min then allowed to air dry for 1 h, and color parameters were measured using a colorimeter. Shrimp samples were separated into groups; day 0, 1, or 2, and stored at 4 degreeC. The samples were treated by placing into boiling water (100degreeC) on days 0 (inoculation day), 1 and 2. The shrimp were immediately removed from the boiling water once they floated to the surface and color parameters were measured. Bacterial counts were determined and Log CFU/g was calculated. The effect of sodium tripolyphosphate on the color change of cooked shrimp was also determined. Initial bacterial counts ranged from 3.0 to 5.4 Log CFU/g of shrimp. On day 0, 1, and 2, all bacterial counts were reduced to non-detectable levels for shrimp samples that floated. The bacterial counts remained at non-detectable levels during refrigerated (4 degreeC) storage of cooked shrimp. The redness (a*), yellowness (b*) and lightness (L*) were significantly higher (p<0.0001) in the cooked shrimp compared to uncooked for all days tested. The standard deviation for the redness (a*) in the cooked shrimp was large indicating a wide range of pink coloration for all days tested. The results suggest that boiling shrimp until they float will significantly reduce Listeria and Salmonella species, but color change will not and color variation can occur.

Publications

  • Edwards, Genevieve, Janes, Marlene, Lucina, Lampila, Supan, John. 2013. Consumer Methods to Control Salmonella and Listeria species in Shrimp. Journal of Food Protection 1:p59-64.
  • Edwards, Genevieve, Janes, Marlene, Lucina, Lampila, Supan, John. 2013. Consumer Methods to Control Salmonella and Listeria species in Shrimp. Master thesis, May 2012.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The results of this project were presented at the IAFP meeting in 2011 in Milwaukee, WI and used as the basis for graduate study for a Masters student. PARTICIPANTS: M.E. Janes (PI), and N. Hazard, LSU AgCenter. TARGET AUDIENCES: These results will be presented to consumers as easy, concise instructions for safe preparation of Louisiana blue crabs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Blue crabs (Callinectes sapidus) play an important role in Louisiana's economy. The increase in consumer popularity coupled with the ambiguity of cooking instructions available is portentous because of the tendency of some pathogenic bacteria to share the natural habitats of the Louisiana blue crab. While all seafood has the potential of being associated with foodborne illness, blue crabs are environmentally exposed to Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Listeria monocytogenes and Salmonella species. This study was designed to determine the least amount of time and temperature needed to reduce or eliminate each of the aforementioned bacteria from a single Louisiana blue crab with either boiling or steaming heat treatments. Once the single crab heat treatment studies were completed, the bacteria that showed the greatest thermal resistance-Listeria monocytogenes, and the bacteria most associated with foodborne illness in Louisiana blue crabs-Vibrio parahaemolyticus were inoculated into a serving size of crabs and subjected to heat treatments. The results were based on the bacterial log reduction of each heat treatment time point. The recommendations for safe cooking times were determined by the abundance of below detection limit or non-detectable level results for each bacterium tested, and the temperature was determined by the lowest temperature needed to achieve these conditions. Results of the heat treatment experiments were: boil one crab four minutes and cool one additional minute for an internal temperature of at least 79.5 degrees C and a total cooking time of five minutes; steam one crab for five minutes and cool two additional minutes for an internal temperature of at least 75 degrees C and a total cooking time of seven minutes; boil four crabs for 10 minutes and cool five additional minutes for an internal temperature of at least 85 degrees C and a total cooking time of 15 minutes; steam four crabs for 15 minutes and cool five additional minutes to reach an internal temperature of at least 85 degrees C with a total cooking time of 20 minutes. These results will be presented to consumers as easy, concise instructions for safe preparation of Louisiana blue crabs.

Publications

  • N.W. Hazard, J.D. Johnson, and M.E. Janes. 2011. Cooking times and temperatures for safe consumption of Louisiana Blue Crab. IAFP, Milwaukee, WI. (Abstract #P3-78).


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The results of this project were presented at the IAFP meeting in 2010 in Anaheim CA. Update results were presented at the 2010 Multi-State Project S-1033 at Estes Park, Colorado. PARTICIPANTS: This research project fully supported one M.S. student Nicole Hazard who graduated in December 2010. Dr. Janes was the lead PI on the project and assisted with the design of the research project, supervision of the student, and analysis of the data. TARGET AUDIENCES: The scientific research that has been conducted on seafood and finfish for reduction of foodborne pathogens has been developed for the food processors, which is difficult for the average consumer to understand. This research project will provided clear, science-based information to the consumers on the safest time and/or temperature needed for killing foodborne pathogens in boiled or steam crabs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Although always cooked before consumption, Louisiana blue crabs (Callinectes sapidus) are still a vehicle of foodborne outbreaks, particularly in private residences. Outbreaks of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the pathogens most associated with Callinectes sapidus. The purpose of this research was to determine the minimum temperatures necessary, either with boiling or steaming, for the safe consumption of Louisiana blue crab by consumers. Callinectes sapidus was inoculated with V. cholerae (ATCC 14035), V. parahaemolyticus (ATCC 33847), or V. vulnificus (ATCC 27562) and subjected to boiling or steaming with the internal temperatures recorded by ACR SmartButton temperature loggers. Once the crab cooled, it was picked and diluted with an equivalent of PBS. The homogenized samples were plated in duplicate on TCBS (V. cholerae and V. parahaemolyticus) or VVA (V. vulnificus) plates, incubated overnight at 37 degree C or 35 degree C, respectively, for 24 hr, and Log CFU/g was determined. Statistics showed significant differences between increasing temperature and bacterial reduction for each Vibrio tested. Temperatures that achieved non-detectable levels during boiling for three minutes or steaming for five minutes were: V. cholerae at 56.5degreeC or 57degreeC; V. parahaemolyticus at 50.5degreeC or 64degreeC; and V. vulnificus at 60degreeC or 53.5degreeC. Based on the data, it is recommended that a single blue crab be either boiled for three minutes at 60degreeC or steamed for five minutes at 65degreeC with an additional eight minutes to cool while picking to ensure that any V. cholerae, V. parahaemolyticus, and V. vulnificus present will be killed, with steaming being preferred over boiling.

Publications

  • N.W. Hazard and M.E. Janes, 2010. Boiling and Steaming Temperatures for Safe Consumption of Callinectes sapidus. Presented at the IAFP meeting in Anaheim CA, July 31 to August 4 (Abstract number P1 66).


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The results of this project were presented at the IAFP meeting in 2009 in Grapevine TX as follows "R. N. Senevirathne, M. A. Gutierrez, S. Datta, R. Scott, Sr., S. Chintagari, R. Jadeja, M. E. Janes. Effectiveness of different antimicrobial treatments on microbial populations on alligator carcasses." Update results were presented at the 2009 Multi-State Project S-1033 meeting, University of California, Davis. PARTICIPANTS: This research project fully supported one Ph.D. student Reshani Senevirathne who will graduate in May 2010. Dr. Janes was the lead PI on the grant and assisted with the design of the research project, supervision of the student, and analysis of the data. TARGET AUDIENCES: The combination of steam and ASC was the treatment that proved to be the most efficient in reducing coliforms, Enterobacteriaceae and Salmonella spp. on alligator carcasses. The results of this study can help the alligator industry to increase their yield and extend the shelf life of their by-products. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Alligator meat is mainly consumed in the southern United States however the industry wants to expand their market. To take advantage of this potential for increased market penetration and industry viability, the industry is also aware that the final product quality of alligator meat needs improvement. The purpose of this study was to evaluate the effects of different antimicrobial agents on alligator meat and to identify effective treatments. Four month old alligators were processed, and the carcasses were treated individually with different antimicrobial agents dissolved in an icy water bath for 5 minutes in order to find the most effective treatment. The antimicrobial agents were Lactic Acid (200PPM), Sodium Benzoate (200PPM), Calcium Lactate (200PPM), Chlorinated water (150PPM of Sodium Hypochlorite), and Acidified Sodium Chlorite (ASC) (50PPM). The two most efficient antimicrobial agents were Lactic acid and ASC which were combined with steam (60 seconds at 2 to 3 inches from surface). For the combined treatments, the samples were steamed before soaking in the antimicrobial solutions. The back, tail and ribs of alligator carcasses were swabbed (2 square inches) and then analyzed for total coliforms, total Enterobacteriaceae, Escherichia coli and Salmonella spp. Treatments with Lactic Acid, Calcium Lactate, Chlorinated water and the combinations of steam and Lactic Acid or ASC, significantly reduced total coliforms, total Enterobacteriaceae and Salmonella spp. counts by 1 log from control levels. Sodium Benzoate did not show significant reduction on any of the bacteria analyzed.

Publications

  • V. E. Burnham , M. E. Janes, L. A. Jakus, J. Supan, A. DePaola and J. Bell. 2009. Growth and survival differences of Vibrio vulnificus and Vibrio parahaemolyticus strains during cold storage. Journal of Food Science 74:M314-M318.
  • Stephenie L. Drake, Richelle Beverely, Amrish Chawla, Marlene Janes, John Supan, Jon Bell, Jay F. Levine, and Lee-Ann Jaykus. 2009. A simplified method to monitor internal oyster meat temperature on a commercial scale. Food Prot. Trends. May.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The effects of temperature on bacterial counts in shrimp were determined. Results were presented at the 2008 Multi-State Project S-1033 in Baton Rouge LA. PARTICIPANTS: This research project fully supported one M.S. student Sajida Plauche who will graduate in May 2009. Dr. Janes was the lead PI on the grant and assisted with the design of the research project, supervision of the student, and analysis of the data. TARGET AUDIENCES: This study is mainly intended to design a simple, easy and unbiased consumer guide for cooking shrimp to enhance the safety while handling and cooking them at home. This can also serve as a guide for manufacturers of ready-to-eat shrimp products while designing and planning the GMP's and HACCP plans during production. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Shrimp is among the most common seafood and favorite among consumers. Like any other food there are safety concerns about Shrimp. Listeria spp., Salmonella spp., Clostridium spp. and Vibrio spp. are among the pathogens of prime importance. Most of these pathogens can be eliminated by cooking. However, the extent of cooking and temperatures can greatly influence the safety of seafood. The current study is focused on the determination of minimum cooking temperatures for reducing Listeria spp., Salmonella spp. and Vibrio spp to non-detectable levels on the surface of shrimp. Shrimp were surface inoculated with the three different species mentioned above to about 5 Log CFU/g of shrimp and then incubated for two days. Shrimp samples were treated at five different temperatures on 0, 1 and 2 day by boiling in a water bath. The effects of temperature on bacterial counts were determined by plating and calculating the log CFU/g reduction for each temperature. The experiment was repeated with different temperatures for each bacterium until the bacterial load in the shrimp was at non-detectable levels. The internal temperature of 85degreeC was the least minimum temperature that was needed to kill all the bacteria tested. Vibrio spp. was less resistant to heat with bacterial counts reaching non-detectable levels at 55degreeC. For Salmonella spp. the minimum temperature required to reduce bacterial counts to non-detectable levels was 75degreeC, while Listeria spp. showed highest resistance up to 85degreeC.

Publications

  • S. Seo, J.M. King, W. Prinyawiwatkul, and M.E. Janes. 2008. Antibacterial activity of ozone-deolymerized crawfish chitosan. Journal of Food Scinece 73:M400-M404.
  • R. Beverly, M. E. Janes, W. Prinyawiwatkul, H. K. No. 2008. Edible chitosan films on ready-to-eat roast beef for the control of Listeria monocytogenes. Food Microbiology 25: 534-537.
  • K. Melody, R. Senevirathne, M. Janes, L. A. Jaykus, J. Supan. 2008. Effectiveness of Icing as a Post-Harvest Treatment for Control of Vibrio vulnificus and V. parahaemolyticus in the eastern oyster (Crassostrea virginicA). Journal of Food Protection, 71:1475-1480.
  • S. Datta, M. E. Janes, Q-G. Xue, J. Losso, and J. F. LaPeyre. 2008. Control of Listeria monocytogenes and Salmonella anatum on the surface of smoked salmon coated with calcium alginate coating containgin oyster lysozyme and nisin. Journal of Food Science, 73:M67-M71.