Skip to main content
ARS Home » Southeast Area » Stoneville, Mississippi » Warmwater Aquaculture Research Unit » Research » Research Project #144153

Research Project: Umbrella Project for Food Safety

Location: Warmwater Aquaculture Research Unit

2015 Annual Report


1a. Objectives (from AD-416):
Research will address methods to determine the presence of pathogens in catfish/catfish products and to maximize elimination methods. Detection techniques will be developed to aid in processing and packaging operations, which should further enhance product safety. Specifically the new objectives are: 1)Optimize safety of aquaculture products through innovative processes for reducing microbiological, physical and chemical hazards in seafood/aquaculture products. 2)Determine the mechanisms influencing microbial survival of selected pathogens in seafood/aquaculture products. 3)Optimize the market value of seafood/aquaculture products through enhanced food safety and quality.


1b. Approach (from AD-416):
Catfish. Determine optimum rates of microbial reduction through innovative processing in catfish products including evaluation of consumer acceptance. Determine viable methods of hazard reduction (smoking, acidulants, antimicrobials, etc) in catfish products during processing and storage. Determine the methods by which these methods reduce hazards within the products evaluated. Enhance the physical safety of catfish fillets with innovative analysis technology. Seafood/Produce. Determine the efficacy of IQF freezing, irradiation, and high pressure processing and other technologies on the safety and quality of oysters, shrimp and produce. Objective 2: Catfish/ Seafood/Produce. Determine the mechanistic approach by which the certain pathogenic bacteria may be reduced in aquatic species. Utilize PCR analysis and other assays to determine the sensitivity and specificity of various isolates in response to innovative treatments. Objective 3: Catfish. Enhance product value through innovative smoking and further processing of catfish fillets. Value-added analysis will compared products to commodity value for product enhancement addition. Evaluate value-added products to address potential food safety issues. Seafood/Produce. Evaluate consumer acceptance of products enhanced through various processing methods. Preparation techniques, ingredient inclusion, packaging and storage methods will be evaluated at various time frames and inclusion rates to determine specie specific parameters limitations. Analyze economics of various market potentials. Catfish. Determine optimum rates of microbial reduction through innovative processing in catfish products including evaluation of consumer acceptance. Determine viable methods of hazard reduction (smoking, acidulants, antimicrobials, etc) in catfish products during processing and storage. Determine the methods by which these methods reduce hazards within the products evaluated. Enhance the physical safety of catfish fillets with innovative analysis technology. Seafood/Produce. Determine the efficacy of IQF freezing, irradiation, and high pressure processing and other technologies on the safety and quality of oysters, shrimp and produce. Objective 2: Catfish/ Seafood/Produce. Determine the mechanistic approach by which the certain pathogenic bacteria may be reduced in aquatic species. Utilize PCR analysis and other assays to determine the sensitivity and specificity of various isolates in response to innovative treatments. Objective 3: Catfish. Enhance product value through innovative smoking and further processing of catfish fillets. Value-added analysis will compared products to commodity value for product enhancement addition. Evaluate value-added products to address potential food safety issues. Seafood/Produce. Evaluate consumer acceptance of products enhanced through various processing methods. Preparation techniques, ingredient inclusion, packaging and storage methods will be evaluated at various time frames and inclusion rates to determine specie specific parameters limitations. Analyze economics of various market potentials.


3. Progress Report:
At Mississippi State University (MSU), researchers have identified a murine norovirus (strain MNV-1), which can be propagated in vitro. Classified as a genogroup V virus, MNV-1 has more biochemical, pathological, genetic, and morphological similarities to human noroviruses than other surrogates. Currently, Food and Drug Administration (FDA) approves doses of up to 5.5 kilo grays (kGy) to control food-borne bacterial pathogens in seafood. However, whether this dose range effectively inactivates food-borne viruses is not reported yet and is being investigated at MSU by using X-ray doses (1.0 to 5.0 kGy) to inactivate MNV-1. Vibrio vulnificus is the deadliest pathogen in the consumption of raw oysters grown in warm estuarine waters. Pocket size rapid detection kits for detecting Vibrio vulnificus and Vibrio parahaemolyticus has been developed and is being validated and a patent application is pending for this technology. With the assistance of researchers at the USDA-ARS Warmwater Aquaculture Research Unit in Stoneville, MS, the genomes of Edwardsiella (E.) tarda (FL95-01), Edwardsiella (E.) piscicida (S11-285), E. piscicida-like species (sp.) (Synonym (syn.) E. anguillarum; LADL 05-105) and E. hoshinae (ATCC 35051) have been sequenced, cloned, annotated and are ready for release. Researchers have identified that E. piscicida is much more virulent to channel catfish than either E. tarda or E. piscicida. Using gold nanoparticles for pathogen detection, MSU researchers have found that the nanoparticle (NP) localized surface plasmonic resonance is a reliable predictor of the optimal NP aggregation states for the Surface enhanced raman spectroscopy (SERS) acquisitions. It also has been found that the structure of the backfilled organothiols have tremendous impact on the biological activities of the protein molecules immobilized onto the gold nanoparticle (AuNP) surfaces. MSU has identified three primers which were effective in distinguishing serotypes 1/2a, 1/2c, 3a, and 3c but not 4b, 4d and 4e Listeria pathogens that may contaminate channel catfish and other sea foods. Matrix-assisted laser desorption lionization –time of flight (MALDI-TOF) was therefore used for intraspecies differentiation of Listeria strains and may be able to lead to the identification of novel biomarkers that distinguish high-risk Listeria (L.) monocytogenes serotypes from low-risk serotypes. Researchers at MSU have successfully constructed an Alk-D-like protein (adlp) mutant by deleting an adlp encoding for alkylbase deoxyribonucleic acid (DNA) glycosylase-like protein. The deletion of adlp L. monocytogenes enhanced the flagellar motility and decreased biofilm formation. The adlp mutant strain also impaired the virulence in mice. The proteome from catfish infected or not infected with E. ictaluri was determined and results showed protein patterns revealed several important differences between infected and non-infected animals. Various edible proteins have been used to stabilize nanoemulsions. Stable mono-dispersed nano-vesicular emulsion carrier systems are being generated in the desired nano-scale (<100 nanometer (nm)). The stable nanoemulsion system will provide a system for carrying bioactive ingredients for fish processing. More characterizations of muscadine grape grown by USDA-ARS scientist at the ARS Southern Horticultural Laboratory in Poplarville, MS, were analyzed for juice quality and health promotion components. Results consistently showed that black varieties had the highest phenolic substances. Food-grade soybeans grown by USDA-ARS scientist at the Crop Genetics Research Unit in Stoneville, MS, were analyzed for protein quality for making foods. Results showed some varieties have potential to be utilized for foods. In addition, research found trypsin inhibitors in soybean protein whey may be used to maintain catfish fish protein integrity for making surimi products. In addition to the above described collaborations with USDA-ARS researchers, MSU researchers have recently initiated collaborations with scientists of the USDA-Southern Regional Research Center in New Orleans, LA, to conduct research on fish waste utilization or identification of allergens in various peanut cultivars grown in Mississippi.


4. Accomplishments
1. Researchers at Mississippi State University have obtained results from X-Ray technology processing on tuna fillet that showed more than a 6 log colony forming unit (CFU) reduction of Salmonella population being achieved with 0.6 kilo grays (kGy) X-ray treatment. Furthermore, treatment with X-ray significantly reduced the initial inherent microbiota on raw tuna fillets and inherent levels were significantly (probability (p) < 0.05) lower than the control samples throughout the shelf-life storage at 5, 10 and 25°C for 25, 15 and 5 days, respectively. There was a significant effect of X-ray treatment on tuna color after treatment (day 0). However, no significant differences (p > 0.05) in color or texture of control and treated samples were observed after processing. In addition, researchers at the Mississippi State University identified a murine norovirus (strain Murine norovirus (MNV)-1), which can be propagated in vitro. Classified as a genogroup V virus, MNV-1 has more biochemical, pathological, genetic, and morphological similarities to human noroviruses than other surrogates. Research is being continued to eliminate noroviruses in sea foods.

2. Development of rapid assays for detection of Vibrio species. Scientists at Mississippi State University (MSU) have validated the test kit for detection of Vibrio vulnificus and Vibrio parahaemolyticus, which are deadly pathogens in the consumption of raw oysters grown in warm waters. A patent application has been submitted for this technology.

3. In the collaboration with a scientist at the USDA-ARS Warmwater Aquaculture Research Unit, Stoneville, MS, the genomes of Edwardsiella (E.) tarda (FL95-01), E. piscicida (S11-285), E. piscicida-like species (sp.) (synonym (syn.) E. anguillarum; LADL 05-105) and E. hoshinae (ATCC 35051) have been sequenced by, cloned, annotated and are ready for release by Mississippi State University researchers. They have identifed that E. piscicida is much more virulent to channel catfish than either E. tarda or E. piscicida.

4. In research using gold nanoparticles for pathogen detection, researchers at the Mississippi State University have found that the nanoparticle (NP) localized surface plasmonic resonance is a reliable predictor of the optimal NP aggregation states for the surface enhanced raman spectroscopy (SERS) acquisitions. They also found that the structure of the backfilled organothiols have tremendous impact on the biological activities of the protein molecules immobilized onto the gold nanoparticle (AuNP) surfaces. This will provide foundation for the development of sensitive pathogen detection systems using gold nanoparticles.

5. Researchers at Mississippi State University (MSU) have found three primers were effective in distinguishing serotypes 1/2a, 1/2c, 3a, and 3c but not effective for differentiating 4b, 4d and 4e Listeria pathogens that may contaminate channel catfish and other sea foods. Maxtrix-assisted laser desorption lionization-time of flight (MALDI-TOF) was therefore used for intraspecies differentiation of Listeria strains. Preliminary results showed that using spectroscopic proteomic methods may be able to lead to the identification of novel biomarkers that distinguish high-risk Listeria (L.) monocytogenes serotypes from low-risk serotypes.

6. Researchers at MSU have successfully constructed an Alk-like protein (adlp) mutant by deleting an adlp encoding for alkylbase deoxyribonucleic acid (DNA) glycosylase-like protein. The deletion of adlp Listeria (L.) monocytogenes enhanced the flagellar motility and decreased biofilm formation. The adlp mutant strain also impaired the virulence in mice.

7. The proteome from catfish infected or not infected with Edwardsiella ictaluri was determined and results showed protein patterns revealed several important differences between infected and non-infected animals. Therefore, proteomic techniques may lead to the discovery of new biomarkers in catfish relevant to foodborne disease.

8. Persistence, adherence, and survival of acid-, alkali-, heat-, oxidative-, and salt-stress adapted cells of high-risk Listeria (L.) monocytogenes serotypes under normal catfish processing conditions and their prevention/destruction were researched at the Mississippi State University. Researchers have identified a high diversity of heat tolerance within strains of L. monocytogenes serotypes. Differences in survival of heat stress adapted cells of L. monocytogenes serotype 1/2a in various disinfectants/sanitizers and essential oils were examined. The survival of heat stress adapted cells of L. monocytogenes was decreased by 2.2 - 2.7 logs in lethal acid, alkali, chlorine and quaternary ammonium compounds. However, the survival of heat stress adapted cells of L. monocytogenes cells was increased by 3.5 - 4.0 logs in two essential oils, carvacrol and bay oil. This research is important for developing improved hazard analysis and critical control points (HACCP) practice in the future.

9. Various edible proteins, including whey protein concentrate, bovine serum albumin and ß-lactoglobulin, have been used to stabilize nanoemulsions. Stable mono-dispersed nano-vesicular emulsion carrier systems are being generated in the desired nano-scale (<100 nanometer (nm)). Stability is being assessed for the different physical and chemical conditions. The stable nanoemulsion system will provide a system for carrying bioactive ingredients for fish processing to improve food safety and quality.

10. A research project has been initiated to manufacture biodegradable packaging films that can enhance food safety and extending quality by using chitosan from shrimp and other shell fish waste and with the combination of plant cellulosic materials. The research will provide a set of data for integrated chemical and processing techniques for preparing novel antimicrobial biodegradable films for seafood packaging from totally post-harvest agricultural and shrimp wastes available in Mississippi. Another expected outcome from this project will be the significant reduction in the amounts of hazardous wastes associated with using synthetic polymers.

11. Detection and characterization of chemical residues in aquatic foods and other foods produced in Mississippi. An inductively-coupled plasma mass spectroscop7 (ICP-MS) for detection and quantification of heavy metals and trace minerals in fish species, and an automated digestion system for preparation of samples have been purchased and are in the stage of installation, which is anticipated to be completed in July of 2015.


Review Publications
Narayanan, L.A., Edelmann, M. 2014. Ubiquitination as an efficient molecular strategy employed in salmonella infection. Frontiers in Immunology. 5:558.

Qian, S., Kamlesh, S., Ramakrishna, N. 2014. Influence of temperature on acid-stress adaptation in Listeria monocytogenes. Foodborne Pathogens and Disease. 11:43-49.

Shen, Q., Jangam, P.M., Soni, K., Nannapaneni, R., Schilling, W., Silva, J. 2014. Low, medium and high heat tolerant strains of Listeria monocytogenes and increased heat stress resistance after exposure to sublethal heat. Journal of Food Protection. 77:1298-1307.

Zhang, D., Perera, G.S., Lacour, A., Emerson, J.P., Henderson, K.L., Zou, S. 2015. Iodide-induced organothiol desorption and photochemical reaction, gold nanoparticle (AuNP) fusion, and SERS signal reduction in organothiol-containing AuNP aggregates. Journal of Physical Chemistry. 119:4261-4267.

Zhang, D., Gadogbe, M., Chen, M., Zhao, X., Saebo, S., Beard, D.J. 2015. Can para-aryl-dithiols cross-link two plasmonic noble nanoparticles as monolayer dithiolate spacers. Journal of Physical Chemistry. 119:6626-6633.

Zhang, D., Nettles, C.B. 2015. A generalized model on the effects of nanoparticles on fluorophore fluorescence in solution. Journal of Physical Chemistry. 119:7941-7948.

Zhang, D., Nettles, C.B., Hu, J. 2015. Using water raman intensity to determine the effective excitation and emission path lengths of fluorophotometers for correcting fluorescence inner filter effect. Analytical Chemistry. 87:4917-4924.

Zhang, D., Wang, A., Vangala, K., Vo, T., Fitzkee, N.C. 2014. A three-step model for protein-gold nanoparticle adsorption. Journal of Physical Chemistry. 118:8134-8142.

Zhang, D., Siriwardana, K., Gadogbe, M., Ansar, S.M., Zou, S., Walters, K.B. 2014. Ligand adsorption and exchange on pegylated gold nanoparticles. Journal of Physical Chemistry. 118:11111-11119.

Reichley, S.R., Ware, C., Greenway, T.E., Wise, D.J., Griffin, M.J. 2015. Real-time assays for detection and quantification of Edwardsiella tarda, Edwardsilla piscicida and Edwardsiella piscicida-like sp. in catfish tissues and pond water. Journal of Veterinary Diagnostic Investigation. 27(2):130-139.

Mahoud, B.S., Chang, S., Wu, Y., Nannapaneni, R., Sharma, C., Coker, R. 2015. Effect of X-ray treatments on salmonella enterica and spoilage bacteria on skin-on chicken breast fillets and shell eggs. Food Control. 57:110-114.

Feugang, J.M., Liao, S.F., Crenshaw, M.A., Clemente, H., Willard, S.T., Ryan, P.L. 2015. Lectin-functionalized magnetic iron oxide nanoparticles for reproductive improvement. Journal of Fertilization:In Vitro. 3(2).

Allen, P.J., Mithell, Z., Devries, R.J., Aboagye, D., Ciaramella, M., Ramee, S.W. 2014. Salinity effects on Atlantic sturgeon growth and osmoregulation. Journal of Applied Ichthyology. 30:1229-1236.