Research Project: Umbrella Project for Food Safety

Location: Warmwater Aquaculture Research Unit

2018 Annual Report

Objectives
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.

Approach
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.

Progress Report
All objectives were planned and completed by the ARS scientists in Stoneville, Mississippi, in collaboration with the scientists at the Mississippi State University. Progress was made on all objectives and their sub-objectives, all of which have a major focus on the ensuring the food safety of catfish, seafood and produce. Production, processing and distribution of fish, seafood and produce are very diverse and extensive, and the system is vulnerable to the introduction of contaminants through the environment, natural processes and the delivery system. Significant progress was made to optimize the safety of aquaculture products through innovative processes for reducing microbiological, physical and chemical hazards in seafood/aquaculture products. During catfish processing, residues such as water runoff, muscle protein extract and mucus present on the skin surface may promote the persistence of Salmonella and Listeria (L.) monocytogenes in the processing environment. Salmonella and L. monocytogenes strains were found to be able to grow and form biofilms in the concentration as low as 15 µg/mL of the catfish mucus extract. The growth rate and biofilm formation by these foodborne bacterial pathogens increased with increasing concentration and temperature. No major differences were found among their strains that were tested for their ability to form biofilm in catfish mucus extract. Biofilm production of Salmonella Blockley on buna-N rubber was less than stainless steel, polyethylene and polyurethane surfaces in catfish mucus extract. Disinfectants containing a mixture of quaternary ammonium compound with hydrogen peroxide, or peroxyacetic acid with hydrogen peroxide and octanoic acid were effective at reducing of biofilm cells on the stainless-steel surface to a non-detectable level. The result from this biofilm study is important for future development of intervention methods for controlling pathogens in food processing and storage. Significant progress was made to understand the mechanisms influencing microbial survival of selected pathogens in seafood/aquaculture products. Progress was made on the detection of protein biomarkers for high-risk Listeria monocytogenes. Total protein from three L. monocytogenes strains Lm-33007, Lm-33014, and Lm-33077 was extracted in three replicates and fractionated by molecular weight. Twelve protein fractions of each replicate were digested with trypsin and resolved using high-performance liquid chromatography and mass spectrometer (HPLC-MS). Mass spectra were matched against peptide databases from each respective genome. All spectra that mapped to either of the other two representative lineage strains were removed. Candidate fractions were further screened against all other Lineage I, II and III proteins available on NCBI (National Center for Biotechnology Information) database. Six unique proteins from Lineage III (ST33077_2218, ST33077_2323, ST33077_2770, ST33077_1897, ST33077_1926 and ST33077_1129) were cloned and expressed. Expression was optimized, and recombinant proteins were purified using histidine tags. Four were purified (ST33077-1897, ST 33077-2270, ST 33077- 1926 and ST 33077-1129). The identity of the cloned proteins was confirmed via LC-MS analysis. Rabbit polyclonal serum was prepared against these four proteins. Progress was also made towards commercialization of a rapid detection kit for pathogen detection. Various possible formulations were conceptualized based on the original formula described in a patent. A final formula was modified based on the concept of having a commercial kit that could be mailed/shipped to the users and could be used in an easy and reliable manner. The modified formulation now needs to be validated. Progress was made on the optimization of the extraction of collagen from catfish skin, which is a by-product from the fillet processing industry. Hydrochloric acid extraction with the addition of a homogenization process at pH 2.4 was found to be able to extract 60% of the skin protein for food and cosmetic uses. Pepsin-aided extraction, which degraded the protein structure of the collagen made the gels was softer than the collagen gels extracted by acetic or HCl extraction. Meanwhile, fish muscle protein has been extracted from silver carp and the quality characteristics of the protein products were characterized with the enhancement of the properties by addition of starches. Six types of starches were tested, and some were found to be more suitable than others for modifying the texture of the carp protein gels. Fish sauce was made from carp bone frames and viscera, and the sensory properties of the fish sauce were compared with the commercial fish sauce.

Accomplishments
1. Utilization of the by-product (heads and bones) from the invasive carp species for fish sauce. Through a cooperative agreement with ARS scientists at Stoneville, Mississippi, researchers at the Mississippi State University, Coastal Research and Extension Center and the Department of Food Science, Nutrition, and Health Promotion showed that fish sauce was successfully produced from the frame and viscera of silver carps by fermentation. The preliminary results from sensory evaluation showed that carp fish sauce, a non-conventional fish sauce, may be used to replace a certain level of commercial fish sauce made from the sea fish sources. More study is needed. If successful, the research will provide technology for utilization of the by-product of the invasive carp species after filleting. Utilization of carps will help eliminate the invasive carps from the Mississippi River and its tributaries.

2. Value-added utilization of catfish by-product from the fillet processing industries. In the fillet industry, approximately 55% of the catfish are the by-product, that amount to be more one-hundred million pounds per year in the state of Mississippi alone. The by-product contains significant amount of proteins. The by-products need to be processed or disposed, otherwise will cause environmental problem. Through a cooperative agreement with ARS scientists at Stoneville, Mississippi, the results obtained from the scientists at the Mississippi State University, Department of Food Science, Nutrition, and Health Promotion and Coastal Research and Extension Center showed that protein recoveries form heads, frames, and skins exceeded 50% of that in the raw materials. Based on this preliminary result, a highly competitive research grant was obtained from USDA-NIFA-AFRI program for undertaking more extensive studies for developing value-added products from catfish by-products (such as heads, bone frames and skin) after filleting and to determine the commercial feasibility of the extraction processes. This grant will help the researchers at the Coastal Research and Extension Center to improve the extraction of proteins from the by-product and for determining commercial feasibility of the extraction process. Successful research will yield a large economic impact in terms of millions of dollars and creation of many jobs for Mississippi and the region.

3. Extraction of the collagen protein from the catfish skin. Through a cooperative agreement with ARS scientists at Stoneville, Mississippi, researchers at Mississippi State Univerisity, Coastal Research and Extension Center, successfully recovered approximately 60% yields of the skin protein by an acid extraction in conjunction with a homogenization process. The extracted collagen from catfish may be used for food gelation and cosmetic applications to replace pork skin collagen, which is undesirable to the Muslim and Jewish populations.

4. Detection of protein biomarkers for high-risk Listeria (L.) monocytogenes. L. monocytogenes strains vary in virulence, but protein-based methods for differentiating strains do not exist. Through a cooperative agreement with ARS scientists at Stoneville, Mississippi, scientists at the Mississippi State University College of Veterinary Medicine and the Institute for Genomics, Biotechnology and Biocomputing discovered protein biomarkers unique for the three major genetic lineages of L. monocytogenes. Four of these biomarkers for L. monocytogenes Lineage III were cloned and purified. Identification of protein biomarkers for differentiating L. monocytogenes strains could allow rapid, low-technology methods for differentiating strains in the field and in processing plants, thus lowering the cost for detection.

5. Protecting catfish processing surfaces from biofilms of foodborne bacterial pathogens. Salmonella and Listeria monocytogenes (L.) are widely distributed in the catfish and poultry processing environments. When the normal free-floating cells of these pathogens attach to the processing equipment surface, they persist as biofilms, which can survive under harsh environmental conditions. Biofilms once formed, remain as a continuous source of cross contamination of food products in the processing environments. Through a cooperative agreement with ARS scientists at Stoneville, Mississippi, scientists at Mississippi State University in Starkville, Mississippi, have determined the role of catfish mucus extract in biofilm formation by Salmonella and L. monocytogenes in catfish processing environments. All food-equipment contact surfaces, including stainless steel, polyethylene, polyurethane and buna-n rubber supported varying levels of biofilm formation by Salmonella and L. monocytogenes when low concentrations of catfish mucus extract was present. A mixture of commercial disinfectants containing quaternary ammonium compound with hydrogen peroxide, or peroxyacetic acid containing hydrogen peroxide and octanoic acid was far more effective than single compound disinfectants for the removal of these biofilms on catfish processing equipment surfaces.

6. Impact of florfenicol use on the development of antimicrobial resistance in catfish intestinal microbiome. Florfenicol is extensively used as an antibiotic on catfish farms, which may lead to antimicrobial resistant pathogens, a global health concern that causes an estimated $20 billion in health care costs each year. Understanding the effects of antimicrobials on the gastrointestinal tract is warranted due to the importance of gut bacteria for animal health and tracking the source of antibiotic resistance. Therefore, our objective was to identify the impact of florfenicol treatment on the intestinal microbiota in catfish. Scientists at the Mississippi State University in Starkville, Mississippi, and ARS scientist at Stoneville, Mississippi, investigated the microbial changes in catfish intestine after florfenicol feeding. Results from 16S rRNA taxonomic analysis revealed shift in community composition following florfenicol-fed. Among these changes, relative abundance of Enterobacteriaceae increased following florfenicol application, whereas the relative abundance of Aeromonas and Plesiomonas decreased. These findings are expected to elucidate the risks associated with antibiotic selection, use, and development of best practices for prudent use of antibiotic in fish production.

7. Development of rapid detection systems for pathogenic Burkholderia species in fresh vegetables and catfish. Since Burkholderia is extremely ubiquitous in soil and nature and some species can cause respiratory complications for cystic fibrosis patients, it is important to develop rapid, sensitive and specific approaches for the identification and detection of pathogenic Burkholderia. Scientists at the Mississippi State University in Starkville, Mississippi, have analyzed bacterial genetic makeups for the development of qPCR-based detection of Burkholderia cenocepacia. This method is able to find as few as 15 bacterial cells from a food sample. Additional 20 pairs of primers are being evaluated for other bacteria. In 2017, the presence of Burkholderia contaminans in sweet onion and celery was confirmed. The sensitive qPCR-based system can be used for the detection of this bacteria to ensure food safety, particularly for the safety of fresh vegetables that are eaten raw (uncooked).

8. Conversion of a 96-well detection kit into a one-tube, commercially viable detection kit for Vibrio species. Through a cooperative agreement with ARS scientists at Stoneville, Mississippi, scientists at Mississippi State University Food Science, Nutrition and Health Promotion department, along with a commercial partner, have developed a concept for a commercially viable, simple one-tube test kit for detection of Vibrio in food (molluscan shellfish) and water samples. This will lead to an easy, reliable method for testing water and food samples for the presence of Vibrio species, sometimes known as “flesh-eating bacteria.”

9. Developing a method to effectively remove arsenic from rice bran, and reducing arsenic content in rice through genetic and food processing research. Arsenic is a toxic heavy metal that tends to accumulate in rice. Fish and rice grown in arsenic contaminated water would have increased levels of arsenic. Consumption of food with a high heavy metal would damage consumers’ health. Through a cooperative agreement with ARS scientists at Stoneville, Mississippi, Mississippi State University researchers located in Mississippi State, Mississippi, in collaboration with USDA-ARS scientists located in Stuttgart, Arkansas, have identified the high and low arsenic rice germplasms. Excellent germplasm lines were collected worldwide and grown in Starkville, Mississippi. The arsenic contents of selected key germplasm lines were detected and verified. Crosses were made for mapping population construction. A novel procedure to reduce rice arsenic content was established. The method can substantially reduce arsenic and lead content in cooked rice, but does not significantly affect other minerals. The exciting results suggest that this research may substantially contribute to reducing human arsenic exposure from rice consumption.

Review Publications
Abdelhamed, H., Tekedar, H., Ozdemir, O., Hsu, C., Arick, M., Karsi, A., Lawrence, M. 2018. Complete genome sequence of multidrug-resistant Edwardsiella ictaluri strain MS-17-156. Genome Announcements. http://doi.org/10.1128/genomeA.00477-18.
Reichley, S.R., Ware, C., Khoo, L.H., Greenway, T.E., Wise, D.J., Bosworth, B.G., Lawrence, M.L., Griffin, M.J. 2017. Comparative susceptibility of channel catfish, Ictalurus punctatus; blue catfish, Ictalurus furcatus; and channel female Blue male Hybrid catfish to Edwardsiella piscicida, Edwardsiella tarda, and Edwardsiella anguillarum. Journal of the World Aquaculture Society. 49(1):197-204.
Reddy, S., Turaga, G., Abdelhamed, H., Banes, M.M., Wills, R.W., Lawrence, M.L. 2017. Listeria monocytogenes PdeE, a phosphodiesterase that contributes to virulence and has hydrolytic activity against cyclic mononucleotides and cyclic dinucleotides. Microbial Pathogenesis. 110:399-408.
Dhowlaghar, N., Abeysundara, P., Nannapaneni, R., Schilling, N.W., Chang, S., Cheng, W.H., Sharma, C.S. 2017. Biofilm formation by Salmonella spp. in catfish mucus extract under industrial conditions. Food Microbiology. 70(4):172-180.
Dhowlaghar, N., Abeysundara, P., Nannapaneni, R., Schilling, M.W., Chang, S., Cheng, W.H., Sharma, C.S. 2017. Growth and biofilm formation by Listeria monocytogenes in catfish mucus extract on four food-contact surfaces at 22°C and 10°C and their reduction by commercial disinfectants. Journal of Food Protection. 81(1):59-67.
Dhowlaghar, N., Bansal, M., Schilling, M.W., Nannapaneni, R. 2018. Scanning electron microscopy of Salmonella biofilms on various food-contact surfaces in catfish mucus. Food Microbiology. 74(9):143-150.
Obe, T., Nannapaneni, R., Sharma, C.S., Kiess, A. 2018. Homologous stress adaptation, antibiotic resistance, and biofilm forming ability of Salmonella enterica serovar Heidelberg (ATCC8326) on different food-contact surfaces following exposure to sub-lethal chlorine concentrations. Poultry Science. 97(3):951-961.
Akgul, A., Al-Janabi, N., Das, B., Lawrence, M., Karsi, A. 2018. Small molecules targeting LapB protein prevent Listeria attachment to catfish muscle. PLoS One. 12(12):1-10.
Ross, M.K., Lee, J.H., Hou, X., Kummari, E., Borazjani, A., Edelmann, M.J. 2017. Endocannabinoid hydrolases in avian HD11 macrophages identified by chemoproteomics: inactivation by small molecule inhibitors and pathogen-induced downregulation of their activity. Molecular and Cellular Biochemistry. http://doi.org/10.1007/s11010-017-3237-0.
Abdelhamed, H., Ozdemir, O., Tekedar, H., Arick, M., Hsu, C., Karsi, A., Lawrence, M. 2018. Complete genome sequence of multidrug-resistant plesiomonas shigelloides strain MS-17-188. Genome Announcements. http://doi.org/10.1128/genomeA/00387-18.