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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

2016 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:
The effect of X-ray doses on reducing a human norovirus surrogate (MNV-1) in pure culture and in half-shell oyster, salmon sushi, and tuna salad at a neutral pH were studied using the range of X-ray power ranging from 0 to 5 kGy (kilo grays). The D-values (90% reduction of the virus) of the X-ray sterilization against virus in these foods were determined. The survival of the norovirus was determined. The X-ray radiation effect on firmness of oyster was also determined. For utilization of carp, invasive species in the Mississippi River system, Mississippi State University researchers (MSU, MS State, MS) visited the Moon River Carp Processing company in Indianola, MS. Carp surimi was prepared from carp fillet using different protocol for making gels with desirable texture. Carp meat was minced and cryoprotectants were added. MSU researchers have extracted functional proteins from catfish by-products after filleting. The by-products were ground into various particle sizes, and extracted with alkaline solution and salt solution. The products obtained were characterized using electrophoresis and gel making methods. MSU researchers have studied the technologies to create antimicrobial packaging materials using seafood shellfish waste and cellulose. Researchers have isolated cellulose from corn stalks to prepare nanofibers, which have been combined with chitosan to prepare antimicrobial packaging films. The packaging films were tested for their properties using various physical-chemical tests, and their ability to inhibit the growth of Listeria, Salmonella and E-Coli. Investigations are underway for testing the effect of water quality and temperature-oxygen levels on catfish fillet color and product quality. Linkages have been made with commercial catfish farms, and processing industries. Catfish will be raised to commercial size and tested for various quality factors of fish (including color, texture and taste of the product) in experimental tanks. Researchers at MSU have determined the contribution of a gene fragment, LMOf2365_2464 to adhesion and intracellular replication of non-virulent L. monocytogenes, and characterized the role of hydrolase LMOf2365_2464 (renamed PdeE) in adhesion and intracellular replication, and have determined the virulence regulon controlled by LMOf2365_2464 during intracellular replication. Researchers at MSU have begun to characterize the serine hydrolase profiles in the HD-11 cells before and after infections with Salmonella Typhimurium using a chemoproteomic approach with a specific proteomic probe, fluorophosphonate-biotin, which targets the serine hydrolase superfamily of enzymes and enables activity-based protein profiling (ABPP) of the serine hydrolase class of enzymes. At MSU, researchers have identified conducted experiments to test the transfer of Listeria monocytogenes from biofilms formed on the stainless steel blade to cut slices of bologna. The effect of biofilm types on the growth of the transferred bacteria were characterized. MSU researchers have determined the growth rates, survival and biofilm formation by Listeria monocytogenes strains at high and low concentrations of catfish mucus at 22°C and 10°C. In 0.375 miligrams (mg)/milliliter (ml) of mucus, Listeria monocytogenes Bug600 reached 9 log /ml at 22°C in 32 h whereas it grew to 7 log CFU (colony forming unit) /ml at 10°C in 72 h. In 0.026 mg/ml of mucus, L. monocytogenes Bug600 remained at ~3.5 CFU/ml at both 22°C and 10°C in 72 h. One hundred and twenty-two experimental lines and one commercial peanut variety were analyzed for their protein subunit compositions using one dimensional and two-dimensional electrophoreses for molecular differences, and tested for their allergen reactivity. Research has been underway to study differential expression of Aspergillus flavus miRNA (micro ribonucleic acid)-like small RNAs (ribonucleic acids) in aflatoxin resistant and susceptible maize inbred lines. Small RNAs with sizes smaller than 200 nucleotides were isolated using the Sigma mirpremier isolation kit. Analysis of the isolated RNAs by Agarose gel electrophoresis indicated that there were no DNA and large RNA contamination. A Nanodrop 2000c Spectrophotometer was also used to determine the concentration and quality of the isolated small RNAs. The small RNAs had an A260/A280 ratio of 1.98, suggesting their high quality. Research also has begun to determine the levels of microcystin-LR, a heat-stable cyanotoxin, in channel catfish muscle, liver, and water from 43 fish disease diagnostic cases and from 10 healthy ponds. More samples from up to 150 cases are being collected and will be analyzed. Research is also being initiated to implement a newly developed method for measuring the quantity of Botulism toxin in infected catfish.


4. Accomplishments
1. In the work to eliminate norovirus infection on seafoods, the results showed D-values of 1.1, 1.8, 2.0, and 2.4 kGy for half-shell oyster, salmon sushi, tuna salad, and whole shell oyster, respectively. X-ray power of 5 kGy reduced virus by about 2.5 log magnitude in tuna salad and reduced virus to less than 100 PFU (plague forming unit) per gram in oyster and salmon samples. No significant effect of X-ray on color and firmness of oyster was found. MSU scientists have met with operators of oysters purging and processing facilities and blueberry packers to discuss implementation of x-ray irradiation to reduce viral load and highlighted advantages of x-ray irradiation over gamma irradiation currently being used by some facilities.

2. In the work on value-added utilization of the invasive carp species for nutritional protein products, the results showed that carp meat without skin and dark meat made whiter color surimi gels. Carp surimi gels had a firm texture as compared to commercial surimi products made from Alaska Pollock showing useful functional properties of carp proteins. Mississippi State University scientists met with representatives of Moon River Foods, a newly established carp processing facility in Indianola, Mississippi to discuss use of this technology in their carp processing operation.

3. In the work to utilize seafood waste (from shell of shrimp) for making value-added antimicrobial packaging materials, four types of cellulose nanofibers, prepared by bleaching, acid hydrolysis and oxidation process had different physical structures. A specific film produced by oxidation had the best nanostructure with 3 to 15 nm (nano meters) in width. The chitosan-cellulose complex films reduced oxygen permeation and had good thermal properties and antimicrobial activity. Additional work is being conducted to further develop the chitosan-cellulose films with the goal of development and adoption of the films by commercial food processors and distributors.

4. In the work on targeting the Endocannabinoid System to Enhance Immunity, at least 8 different serine hydrolases were detected and identified. These enzymes can catabolize endocannabinoids by catalyzing their hydrolysis and, thus, can regulate their levels in cells. A small-molecule inhibitor was found to selectively inhibit hydrolase activities in intact cells. Inactivating the metabolism of endocannabinoids with small molecules could also augment the phagocytic activity of immunity-macrophages.

5. In the work on the understanding of biofilm formation and transferability of Listeria monocytogenes on the surfaces of food and processing equipment. The results showed concrete biofilms had more transfer than static biofilm type after multiple slicing of meat, but the bacteria once transferred had similar growth rate on the contaminated meat. Based on this research it is recommended that processors follow strict adherence to cleaning of all surface areas on processing equipment to prevent formation of concrete biofilms which will result in reduced transfer of bacteria from equipment to the meat.

6. Commonly used cleaners and sanitizers are not effective in killing Listeria monocytogenes in catfish processing environments. Therefore, Mississippi State University researchers are studying other factors that influence growth and biofilm formation in Listeria monocytogenes. It was determined that increased concentrations of catfish mucus on processing equipment prolongs Listeria monocytogenes survival and enhances biofilm formation. In addition, surface of equipment made from buna rubber surfaces had lower Listeria monocytogenes biofilm formation than stainless steel, polyethelyene and polyurethane surfaces. MSU researchers are determining methods to reduce Listeria monocytogenes on catfish processing equipment by reducing accumulation of catfish mucus and substitution of other materials with buna rubber when possible. This information will be transferred to catfish processors.

7. In the work on study of screening of peanut allergens and selection of lowest allergen variety for processing to further reduce allergens in the final food products, the results showed 11 lines had lower allergens than others. The thermal resistant activities are being tested by food processing. The food processing effect on allergen and food quality will be further studied. The biotechnologies learned from this study can be applied to catfish allergens identification in the future. Fish is one of the big eight group of food sources of allergens.


5. Significant Activities that Support Special Target Populations:
Catfish production is the most important aquaculture in the United States, and particularly concentrated in Mississippi, Alabama, Arkansas and Louisiana. The USDA Census of Aquaculture in 2000 classified 84% of catfish farms as small businesses, with annual sales of less than $500,000. Of the less than 348 catfish farms in the United States, 40% or so are considered small in size. The total production value of Mississippi agricultural and aquacultural products in 2013 was about 7.5 billion. Mississippi catfish production ranks number one in the nation, and with $189 million value in 2015. According to USDA agricultural statistics of 2014, national catfish production has declined by about 55% since 2003’s production peak due to strong foreign competition and high cost of fish feed. In addition, fish diseases are an increasing challenge for production. Under these strenuous conditions, the catfish fillet processing industries still employ many local people and have produced a great impact on the economy of many small rural communities. The quality of catfish, in terms of off-flavor remains a major challenge for marketing fillet. In addition, value-added utilization of by-products, which account for approximately 60% of total fish mass, from fillet processing plant is badly needed for enhancing the total value of catfish. During the last ten years, other agricultural and horticultural products are increasing overtime with chicken, corn and soybeans being the three largest food crops in Mississippi. The study on the interactions of other food crops with catfish, carp and sea foods also will mutually benefit food safety and quality. For the US catfish growers to compete for the market share, it is important to conduct research for the development of safe, high quality domestic catfish products. MSU scientists have expanded our research to utilize the invasive carp species, which have outgrown the native fish in the Mississippi river system, and is negatively affecting the ecology of the river. Eliminating carp by making into acceptable foods can provide an important source of nutritious animal proteins for human utilization. In the last year, Dr. Chang, the program director at the Mississippi State University, had visited several USDA-ARS research centers interested in food safety and visited with several catfish processing industries and a carp company in Mississippi to discuss how ARS-university collaborative research provides science and technology to support the agricultural, aquacultural and seafood industries. Dr. Chang also has visited several regional USDA-ARS centers to seek collaborations with ARS scientists to help achieve the goals of ARS in the Southern and Southeastern US regions. As the results of these visits, almost every subward funded to the researchers has a USDA-ARS collaborator. The research findings on the mechanisms for pathogen biology, transmissions, and their control by X-ray, coating with antioxidants and other food processing methods have been presented in various scientific meetings, including the 2015-16 annual meetings of the Institute of Food Technologists in Chicago, IL. This research project will advance science which plays an important role in maintaining viable aquaculture and seafood industries in Mississippi and the United States and in assuring safe and high-quality food products to the consumers. In addition, this research will contribute to the post-harvest utilization of the abundant Mississippi agricultural products to produce value-added end products to enhance the economy of the overall food and agricultural communities.


Review Publications
Nho, S., Abdelhames, H., Reddy, S., Karsi, A., Lawrence, M.L. 2015. Identification of high-risk Listeria monocytogenes serotypes in lineage I (serotype 1/2a, 1/2c, 3a and 3c) using multiplex PCR. Journal of Applied Microbiology. 119(3):845-852.

Mukherjee, D., Haque, Z.Z. 2015. Efficacy of sweet whey containing final dips in reducing protein oxidation in retail-cut cubed beefsteak. Annals Food Science and Technology. 16(1):85-89.

Zhang, X., Haque, Z.Z. 2015. Generation and stabilization of whey-based monodisperse naoemulsions using ultra-high pressure homogenization and small amphipathic co-emulsifier combinations. Journal of Agricultural and Food Chemistry. 63(45):10070-10077.

Soni, B., Hassan, E., Mahmoud, B. 2015. Chemical isolation and characterization of different cellulose nanofibers from cotton stalks. Carbohydrate Polymers. 134:581-589.

Soni, B., Hassan, E., Schilling, W., Mahmoud, B. 2016. Transparent bionanocomposite films based on chitosan and tempo-oxidized cellulose nanofibers with enhanced mechanical and barrier properties. Carbohydrate Polymers. 151:779-789.

Reddy, S., Akgul, A., Karsi, A., Hossam, A., Wills, R., Lawrence, M. 2015. The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication. Microbial Pathogenesis. 92:19-25.

Wu, Y., Chang, S., Nannapaneni, R., Coker, R., Haque, Z., Mahmoud, B. 2015. The efficacy of X-ray does on murine norovirus-1 (MNV-1) in pure culture, half-shell oyster, salmon sushi, and tuna salad. Food Control. 64:77-80.

Mahmoud, B., Nannapaneni, R., Chang, S., Coker, R. 2016. Effect of X-ray treatments on Escherichia coli 0157:H7, Listeria monocytogenes, Shigella flexneri, Salmonella enterica and inherent microbiota on whole mangoes. Letters in Applied Microbiology. 62:138-44.

Qian, S., Kamlesh, S., Ramakrishna, N. 2015. Stability of sublethal acid stress adaptaion and induced cross protection against lauric arginate in Listeria monocytogenes. International Journal of Food Microbiology. 203:49-54.

Sulagna, S., Nitin, D., Amanda, L., Ramakrishna, N., Chander, S., Barakat, M. 2015. Transmission electron microscopy study of Listeria monocytogenes serotype 1/2a cells exposed to sublethal heat stress and carvacrol. Journal of the Mississippi Academy of Sciences. 60:300-304.

Qian, S., Pooja, P., Kamlesh, S., Ramakrishna, N. 2016. Influence of temperature on alkali stress adaptation in Listeria monocytogenes. Food Control. 62:74-80.