Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT 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. 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. Progress was made on all objectives with a major focus on the ensuring (1) the food safety of catfish, seafood and produce, and are under the National Program 108-Food Safety, Component I: Food Borne Contaminants, and (2) food quality improvement under the National Program 306-Quality and Utilization of Agricultural Products, Component I: Foods. 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. We continued to acquire and install critical research equipment and personnel trained. Our major achievement was substantial progress on the construction of the Northern Gulf Aquatic Food Research Center (NGAFRC) funded at $8 million from other federal and state sources. The NGAFRC will be constructed on an 8 acre site in Ocean Springs, Mississippi. Currently, architects have completed the final documents, construction bid documents are to be released in the summer of 2024, and construction of the first of three phases is expected to begin in the early Fall of 2024. The NGAFRC will greatly improve our research capabilities and program and our ability to attract and recruit researchers with national and international reputations. Our ongoing strong and productive partnership with the USDA-Agricultural Research Service (ARS) and the catfish aquaculture and processing industry continues to demonstrate major strides towards improving the safety and quality of the fillet and by-products. We have completed research improving our ability to test and identify harmful pathogens as part of catfish and other aquatic food safety, made progress on removal strategies for off-flavors in farmed freshwater fish, and developed a singulation system for catfish fillets handling and processing. All of these efforts are published in peer reviewed journals. As part of an ongoing effort to expand the impact of this research program, an MSU internal request for proposals was issued and funded by the Mississippi Center for Enhancing Utilization and Safety of Catfish and Other Aquatic Foods. The RPF requested research with emphasis on application component that addresses one of the following two objectives: 1). Detect, characterize, and eliminate the pathogens, toxins, chemicals, allergens, and biologics that are accumulated in catfish and other aquatic food products as affected by production environment and food processing and packaging methods. 2). Apply advanced scientific and engineering principles to enhance manufacturing efficiency, meat and protein yield, food quality, shelf-life storage stability and product innovations and add value to bycatch and byproducts for expanding markets and/or increasing profitability of the aquatic food industry. Last year, 2023, five projects were funded for a 12 month period starting July 1, 2023. Awards are intended to be seed grants for new and innovative research. Funds are intended to build capacity for success in peer- reviewed science in terms of refereed publications and external competitive grants. These five funded projects are 1). ⿿Effects of chitosan on pathogenic and spoilage microbiota impacting the pre-harvest productivity and post-harvest safety and shelf life of Mississippi raised catfish, 2.), "Evaluating the silage technology to add value to catfish processing plants' by-products, 3). ⿿Hyperspectral detection of off- flavor causing cyanobacteria in catfish aquaculture with unmanned aerial systems, 4.) ⿿Recycling Catfish Bone for Plastic Composite 3D Printing, and 5.) ⿿Effects of environmental stress and antimicrobials on the health, microbiome, and product shelf-life of channel and hybrid catfish. Published findings will be included in the next annual report. Recently, an additional seven projects were funded for a 12 month period starting July 1, 2024. Accomplishments from each of these seven awarded projects will be included in the next annual report. The seven funded projects are 1). ⿿Understanding the accumulation and transmission of microbial-derived toxins in catfish and associated risk factors, 2). ⿿Assessing Consumer Awareness and Preferences for Food Safety Enhanced Catfish Products, 3.) ⿿Automated Preparation Technology for Skewering Operations, 4.) ⿿Optimizing ingredient formulations and processing variables associated with the marination of catfish, 5.) ⿿Assessment of chemical residues in store-bought catfish from Mississippi groceries, 6.) ⿿Evaluating the silage technology to add value to catfish processing plants' by-products year 2, and 7.) ⿿A Comprehensive Culturomics Approach to Recover and Characterize Catfish Spoilage Bacteria. The published results from these funded projects will greatly enhance the research results made possible by this collaboration with USDA-ARS. ACCOMPLISHMENTS 01 Development of an assay to detect species-specific and pathogenic genes of Vibrio parahaemolyticus. Vibrio parahaemolyticus can cause severe gastroenteritis in humans who consume contaminated seafood. Traditionally, detecting this bacterium and its harmful genes requires complex laboratory tests. ARS researchers at Mississippi State University location, developed a simpler and faster method using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD) assay for field use. The new test identifies Vibrio parahaemolyticus in just 20 minutes and can detect tiny amounts of DNA and bacteria, matching the sensitivity of complex lab tests. Trials demonstrated the assay's effectiveness in identifying the bacterium in oysters, proving it to be a quick and sensitive tool for detecting harmful genes of Vibrio parahaemolyticus. 02 Off-flavors and innovative removal strategies for farmed freshwater fish. Farmed freshwater fish are vital for meeting global protein demands, but off-flavors limit their appeal and consumer acceptance. Identifying and eliminating these off-flavors is crucial for both consumer satisfaction and industry success. ARS researchers at Mississippi State University location, examined the mechanisms behind off-flavor formation in farmed freshwater fish and presents innovative methods for their removal in aquaculture and post-harvest stages. Common off-flavors in freshwater fish include fishy, grassy, earthy, mushroom, muddy, and metallic tastes. Key strategies for off-flavor removal include ozone treatment, cold plasma, molecular sieves, edible coatings, photocatalysis, and probiotics. The review summarizes how protein degradation, microbial action, and lipid oxidation contribute to these off-flavors. The fish exhibit eight primary off-flavors, with fishy, earthy, and metallic being the most prominent. Six innovative removal strategies show promise for effectively addressing these off- flavors. 03 Development of a singulation system for handling catfish fillets. Among the various catfish products, fillets are particularly common and typically stored chilled or frozen after initial processing. However, when these fillets are removed from storage for further processing, they often stick together and require manual separation. This manual effort is necessary for steps like breading and quick-freezing the individual fillets. Due to a shortage of labor and rising costs, there is a pressing need for automated methods to handle this task, which would help boost the profitability of the United States. catfish industry. ARS researchers at Mississippi State University location, introduced a new system that uses water buoyancy and underwater streams to automatically separate catfish fillets. Researchers have examined and fine-tuned several operational parameters to improve the system's efficiency and output. They have also established guidelines for continuous production, ensuring that the process remains effective and productive. The insights from this research are expected to pave the way for more advanced and efficient singulation technologies for catfish and other fish species.
Impacts
(N/A)
Publications
- Bela-Ong, D.B., Thompson, K., Kim, H.J., Park, S., Jung, T.S. 2023. CD4+ T lymphocyte responses to viruses and virus-relevant stimuli in teleost fish. Fish and Shellfish Immunology. 142:109007. https://doi.org/10.1016/j.fsi. 2023.109007.
- Park, S., Zhang, Y. 2024. Development of multienzyme isothermal rapid amplification (MIRA) combined with lateral-flow dipstick (LFD) assay to detect species-specific tlh and pathogenic trh and tdh genes of vibrio parahaemolyticus.. Pathogens. 13(1):57. https://doi.org/10.3390/ pathogens13010057.
- Zhou, Y., Zhang, Y., Hong, H., Luo, Y., Li, B., Tan, Y. 2023. Mastering the art of taming: reducing bitterness in fish by-products derived peptides. Food Research International. 173(1). Article 113241. https://doi. org/10.1016/j.foodres.2023.113241.
- Zhou, Y., Zhang, Y., Liang, J., Hong, H., Luo, Y., Li, B., Tan, Y. 2024. From formation to solutions: Off-flavors and innovative removal strategies for farmed freshwater fish. American Journal of Medicine. 144:104318. https://doi.org/10.1016/j.tifs.2023.104318.
- Jing, Y., Liu, W., Lu, Y., Lyu, J., Yang, X., Wu, D., Zhang, X. 2024. Development of a singulation system for handling catfish fillets. International Journal of Advanced Manufacturing Technology. 128:81-89. https://doi.org/10.1007/s00170-023-11867-3.
- Zhang, Y., Chang, S. 2023. Microbial Transglutaminase Cross-Linking Enhances the Textural and Rheological Properties of the Surimi-like Gels Made from Alkali-Extracted Protein Isolate from Catfish Byproducts and the Role of Disulfide Bonds in Gelling. Journal of Functional Foods. 12(10) :2029. https://doi.org/10.3390/foods12102029.
- Chen, R., Chang, S., Gillen, A.M., Chen, P., Zhang, B. 2024. Relationships between protein and other chemical composition and texture of tofu made from soybeans grown in different locations. Journal of Food Science. 89:1428-1441. https://doi.org/10.1111/1750-3841.16922.
- Liu, A., Phillips, K., Jia, J., Deng, P., Zhang, D., Chang, S., Lu, S. 2023. Development of a qPCR Detection Approach for Pathogenic Burkholderia cenocepacia Associated with Fresh Vegetables. Food Microbiology. 115:104333. https://doi.org/10.1016/j.fm.2023.104333.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT 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. 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. 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, and are under the National Program 108-Food Safety, Component I: Food Borne Contaminants. The third objective also has a focus on the food quality improvement under the National Program 306- Quality and Utilization of Agricultural Products, Component I: Foods. 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. Protease inhibitor including trypsin inhibitor in soybean and whey were studied as they could be used as an ingredient for enhancing fish products. In support of Objective 1, 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. In this period, we continued to acquire critical research equipment and have them installed and personnel trained. The major achievement was to complete the installation of an exhaust system for a wet chemistry laboratory. In addition, about $8 million were acquired from the state sources for building a Northern Gulf Aquatic Food Research Center (NGAFRC), for the first phase of building construction for the NGAFRC on a piece of 4-acre land, which was purchased last year. We have met with architects on planning laboratories to begin building in Spring of 2023. Currently, the seafood research laboratory is locating at near the sea level and subject to flood. We continued to foster our partnership with the USDA-Agricultural Research Service (ARS) and the catfish aquaculture and processing industry to improve the safety and quality of the fillet and by-products. We have completed a high hydrostatic pressure processing project investigating the inactivation of bacteria in oysters produced by an oyster aquaculture farm in Alabama by using hydrostatic pressure up to 600 mPa and time from one to five minutes. Data have been collected and analyzed, and a manuscript is being prepared for publication. In support of Objective 2, we continued to determine the mechanistic approach, by which certain pathogenic bacteria may be reduced in catfish, seafood and produce. We have successfully completed the experiments to enhance the detection of pathogenic Vibrio vulnificus in oysters by using an innovative recombinase-polymerase system in conjunction with a lateral flow dipstick assay. The method is more sensitive and rapid for screening than the FDA-approved methods for Vibrio analyses and will be very useful to oyster aquaculture and processing industries for controlling these pathogens. The method is more sensitive than traditional culture-gene probe method or the polymerase chain reaction (PCR) method. Results from our collaboration of the USDA-ARS laboratory in Delaware and the Texas A&M University, on testing the effectiveness of riboflavin would affect norovirus inactivation by x-ray irradiation has been written into a manuscript. Progress was made continuously on the development of low- level tolerance to antibiotic trimethoprim in Listeria monocytogenes after sublethal adaptation to quaternary ammonium compound (QAC). Using eight L. monocytogenes strains, researchers in the Department of Food Science, Nutrition, and Health Promotion at the Mississippi State University determined, the changes in short-range of minimum inhibition concentrations (MIC), growth rate, and survival for heterologous stress response to trimethoprim, after sublethal exposure to daily cycles of fixed or gradually increasing concentration of QAC. When adapted to daily cycles of fixed or gradually increasing sublethal concentrations of QAC, three main findings were found in eight L. monocytogenes strains against trimethoprim: (a) 3 of the 8 strains exhibited significant increases in short-range minimum inhibitory concentration (MIC) of trimethoprim by 1.7 to 2.5 fold in QAC-adapted subpopulations as compared to non-adapted cells; (b) 2 of the 8 strains exhibited significant increase in growth rate in trimethoprim by 1.4 to 4.8 fold in QAC-adapted subpopulations compared to non-adapted cells; and (c) 5 of the 8 strains yielded significantly higher survival by 1.3-to-3.1 log CFU/mL in trimethoprim in QAC-adapted subpopulations compared to the non-adapted control. However, for 3/8 strains of L. monocytogenes, there were no increases in the survival of QAC-adapted subpopulations compared to non-adapted control in trimethoprim. These findings suggest the potential formation of low-level trimethoprim tolerant subpopulations in some L. monocytogenes strains where QAC may be used widely. A refereed journal article on this subproject was recently published in a peer-reviewed journal of ⿿Foods⿿ this year. In support of Objective 3, progress was made on the optimization of the extraction of proteins from catfish by-product, which included heads and bones from the fillet processing industry. Researchers in the Experimental Seafood Processing Laboratory (ESPL) in the Coastal Research and Extension Center of the Mississippi State University, continued to develop innovative methods for improving the color and texture of the surimi-like gels using water washing and the use of soy whey containing protease inhibitors. In the meantime, soybean and soymilk that contain trypsin inhibitors as affected by cultivars and food processing technologies were investigated. Results have been analyzed and reported in three peer-reviewed refereed journals. A tenure-track faculty position on food mechanical engineering was established to engage with the catfish processing industry to assist the processors to enhance meat yield and processing efficiency. If more meat can be recovered, catfish processing economy will be enhanced. Researchers at the ESPL also have completed the making of fish balls using the meat of oversized catfish, which are considered as byproduct since they cannot be processed by the normal setting of the automated filleting machine. Potato starch was found to enhance firmness of the fish balls, whereas seaweed and bacterial gums were found to weaken the gels of the fish balls. An international collaboration with Taiwan⿿s National Kaohsiung University of Science and Technologies led to two joint publications focusing on the use of high- hydrostatic pressure processing to extend the shelf-life of clam and milkfish fillet during cold storage, and to enhance meat yield. The findings can be applied to oyster and catfish product processing in the near future. ACCOMPLISHMENTS 01 Reducing pathogenic bacteria in catfish, seafood and produce. In support of our objectives, ARS researchers in Stoneville, Mississippi, continued to determine the mechanistic approach, by which certain pathogenic bacteria may be reduced in catfish, seafood and produce. Our researchers have successfully submitted their work for publication in the Journal of Food Protection on enhancing the detection of pathogenic Vibrio vulnificus in oysters by using an innovative recombinase polymerase system in conjunction with a lateral flow dipstick assay. The method is more sensitive and rapid for screening than the FDA approved methods for Vibrio analyses and will be very useful to oyster aquaculture and processing industries for controlling these pathogens. The manuscript has been accepted for publication. Results from our researcher collaboration with the USDA-ARS laboratory in Delaware and the Texas A&M University, on testing the effectiveness of riboflavin would affect norovirus inactivation by x-ray irradiation has been written into a manuscript and submitted to the journal of Radiation Physics and Chemistry. Progress was made continuously on the development of low-level tolerance to antibiotic trimethoprim in Listeria monocytogenes after sublethal adaptation to quaternary ammonium compound (QAC). Using eight L. monocytogenes strains, researchers in the Department of Food Science, Nutrition, and Health Promotion at the Mississippi State University, determined the changes in short-range of minimum inhibition concentrations (MIC), growth rate, and survival for heterologous stress response to trimethoprim, after sublethal exposure to daily cycles of fixed or gradually increasing concentration of QAC. 02 Development of innovative technologies for preventing illness caused by pathogens and chemical residues in foods. In further support of our objectives, ARS researchers in Stoneville, Mississippi, have continued to develop innovative technologies for preventing illness that may be caused by pathogens and chemical residues in foods. Substantial progress was made to optimize the safety of aquaculture products through innovative processes for reducing microbiological, physical, and chemical hazards in seafood/aquaculture products. In this period, we continued to acquire critical research equipment and have them installed and personnel trained. The major achievement was to complete the installation of several pieces of equipment for processing fish and fermentation. A complete canning laboratory with heat penetration was purchased and will be installed as soon as space is available. To increase space for research, about $8 million were acquired from the state sources to construct the Northern Gulf Aquatic Food Research Center (NGAFRC). We have met with architects on planning laboratories to begin building in Fall of 2023. Currently, our seafood research laboratory is located near the sea level and subject to flooding risk from hurricane storm surge. 03 High hydrostatic pressure processing project. ARS researchers in Stoneville, Mississippi, continued to foster our partnership with the USDA-Agricultural Research Service (ARS) and the catfish aquaculture and processing industry to improve the safety and quality of the fillet and by-products. We have completed a high hydrostatic pressure processing project investigate the inactivation of bacteria in oysters produced by an oyster aquaculture farm in Alabama by using hydrostatic pressure up to 600 mPa and time from one to five minutes. Data have been collected and analyzed, and a manuscript is being prepared for publication. In addition, soymilk is being made by using ultrahigh temperature heating to inactivate pathogens and trypsin inhibitors, and to reduce precipitation residues in food packages. This research is being continued. 04 Development of processing technologies for value-added utilization of foods. ARS researchers in Stoneville, Mississippi, have continued to characterize food materials and develop novel processing technologies for value-added utilization of foods produced in Mississippi. We have collaborated with USDA-ARS Soybean Genetic Research Unit in Stoneville, Mississippi, and with the University of Missouri to plant soybeans with different glycinin and beta-conglycinin composition for making value- added soy foods. More than 25 selected soybean lines were planted and characterized. Results are being analyzed. In the meantime, soybean and soymilk that contain trypsin inhibitors as affected by cultivars and food processing technologies were investigated. Progress was made on the optimization of the extraction of proteins from catfish by-product, which included heads and bones from the fillet processing industry. We continued to develop innovative methods for improving the color and texture of the surimi-like gels using water washing and the use of soy whey containing protease inhibitors. Results have been analyzed and reported in three peer-reviewed refereed journals. A tenure-track faculty position on food.
Impacts
(N/A)
Publications
- Chang, S., Zhang, Y. 2022. Color and texture of surimi-like gels made of protein isolate extracted from catfish by-products are improved by washing and adding soy whey. Journal of Food Science. 87(7):3057⿿3070. https://doi. org/10.1111/1750-3841.16229.
- Park, S., Chang, S. 2022. Development of recombinase polymerase amplification combined with lateral flow dipstick assay to detect hemolysin gene of Vibrio vulnificus in oysters. Journal of Food Protection. 85(12):1716⿿1725. https://doi.org/10.4315/JFP-21-455.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT 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. 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. 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, and are under the National Program 108-Food Safety, Component I: Food Borne Contaminants. The third objective also has a focus on the food quality improvement under the National Program 306- Quality and Utilization of Agricultural Products, Component I: Foods. 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. Protease inhibitor including trypsin inhibitor in soybean and whey were studied as they could be used as an ingredient for enhancing fish products. In support of Objective 1, 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. In this period, we continued to acquire critical research equipment and have them installed and personnel trained. The major achievement was to complete the installation of an exhaust system for a wet chemistry laboratory. In addition, about $8 million were acquired from the state sources for building a Northern Gulf Aquatic Food Research Center (NGAFRC) for the first phase of building construction for the NGAFRC on a piece of 4-acre land purchased last year. We have met with architects on planning laboratories to begin building in Spring of 2023. Currently, the seafood research laboratory is locating at near the sea level and subject to flood. We continued to foster our partnership with the USDA-Agricultural Research Service (ARS) and the catfish aquaculture and processing industry to improve the safety and quality of the fillet and by-products. We have completed a high, hydrostatic pressure processing project investigate the inactivation of bacteria in oysters produced by an oyster aquaculture farm in Alabama by using hydrostatic pressure up to 600 mPa and timed from one to five minutes. Data have been collected and analyzed, and a manuscript is being prepared for publication. In support of Objective 2, we continued to determine the mechanistic approach by which certain pathogenic bacteria may be reduced in catfish, seafood and produce. We have successfully completed the experiments to enhance the detection of pathogenic Vibrio vulnificus in oysters by using an innovative recombinase-polymerase system in conjunction with a lateral flow dipstick assay. The method is more sensitive and rapid for screening than the FDA-approved methods for Vibrio analyses and will be very useful to oyster aquaculture and processing industries for controlling these pathogens. The method is more sensitive than traditional culture-gene probe method or the PCR method. Results from our collaboration with the USDA-ARS laboratory in Delaware and the Texas A&M University, on testing the effectiveness of riboflavin would affect norovirus inactivation by x- ray irradiation has been written into a manuscript. Progress was made continuously on the development of low-level tolerance to antibiotic trimethoprim in Listeria monocytogenes after sublethal adaptation to quaternary ammonium compound (QAC). Using eight L. monocytogenes strains, researchers in the Department of Food Science, Nutrition, and Health Promotion at the Mississippi State University, determined the changes in short-range of minimum inhibition concentrations (MIC), growth rate, and survival for heterologous stress response to trimethoprim, after sublethal exposure to daily cycles of fixed or gradually increasing concentration of QAC. When adapted to daily cycles of fixed or gradually increasing sublethal concentrations of QAC, three main findings were found in eight L. monocytogenes strains against trimethoprim: (a) 3 of the 8 strains exhibited significant increases in short-range minimum inhibitory concentration (MIC) of trimethoprim by 1.7 to 2.5 fold in QAC- adapted subpopulations as compared to non-adapted cells; (b) 2 of the 8 strains exhibited significant increase in growth rate in trimethoprim by 1.4 to 4.8 fold in QAC-adapted subpopulations compared to non-adapted cells; and (c) 5 of the 8 strains yielded significantly higher survival by 1.3-to-3.1 log CFU/mL in trimethoprim in QAC-adapted subpopulations compared to the non-adapted control. However, for 3/8 strains of L. monocytogenes, there were no increases in the survival of QAC-adapted subpopulations compared to non-adapted control in trimethoprim. These findings suggest the potential formation of low-level trimethoprim tolerant subpopulations in some L. monocytogenes strains where QAC may be used widely. A refereed journal article on this subproject was recently published in a peer-reviewed journal of ⿿Foods⿿ this year. In support of Objective 3, progress was made on the optimization of the extraction of proteins from catfish by-product, which included heads and bones from the fillet processing industry. Researchers in the Experimental Seafood Processing Laboratory (ESPL) in the Coastal Research and Extension Center of the Mississippi State University, continued to develop innovative methods for improving the color and texture of the surimi-like gels using water washing and the use of soy whey containing protease inhibitors. In the meantime, soybean and soymilk that contain trypsin inhibitors as affected by cultivars and food processing technologies were investigated. Results have been analyzed and reported in three peer-reviewed refereed journals. A tenure-track faculty position on food mechanical engineering was established to engage with the catfish processing industry to assist the processors to enhance meat yield and processing efficiency. If more meat can be recovered, catfish processing economy will be enhanced. Researchers at the ESPL also have completed the making of fish balls using the meat of oversized catfish, which are considered as byproduct since they cannot be processed by the normal setting of the automated filleting machine. Potato starch was found to enhance firmness of the fish balls, whereas seaweed and bacterial gums were found to weaken the gels of the fish balls. An international collaboration with Taiwan⿿s National Kaohsiung University of Science and Technologies led to two joint publications focusing on the use of high- hydrostatic pressure processing to extend the shelf-life of clam and milkfish fillet during cold storage, and to enhance meat yield. The findings can be applied to oyster and catfish product processing in the near future. ACCOMPLISHMENTS 01 Enhancing the safety and quality of oyster meat using high hydrostatic pressure processing (HPP). High hydrostatic pressure processing is one of the four FDA-approved post-harvest processing technology for reducing the risk of eating raw oysters. The effect of HPP processing technology on the oysters produced in the Northern Gulf region had not been comprehensively studied. The objective of this research was for ARS researchers in Stoneville, Mississippi, to search for the best HPP conditions for processing oysters over a wide range of pressures and processing time intervals. Our completed results have identified the range of the conditions that could facilitate shucking and to reduce pathogens and to enhance shucking efficiency as well as to increase meat yield. The results can be transferred to preserve the natural quality of oysters with improved safety and to enhance marketing raw oysters to the consumers. 02 Development of a rapid, sensitive, and equipment-free detection of Vibrio vulnificus in oysters. Vibrio vulnificus in oysters and in beach water is very hazardous to human health since it can cause the death of people with physical wound infected by this pathogen. The official method approved by FDA and NSSP (the National Shellfish Sanitation Program) for Vibrio vulnificus analysis uses culturing method and followed by gene hybridization, and would take 4-5 days to complete. ARS researchers' in Stoneville, Mississippi, objective was to develop a rapid, sensitive and equipment-free method for the detection of this pathogen. Our completed results confirmed that the test could be done in 30 min. This method will have broad applications in the seafood industries, and will be useful for government agencies for making decisions for beach closure, when this pathogen is detected in the beach water. 03 Optimizing efficiency of protein extraction from catfish by-products, containing heads and frames. Catfish by-products (skin, heads and bones) , which account for more than 200 million pounds each year and almost 40-50% of the total fish proteins, have been considered as a waste for a long time. ARS researchers' in Stoneville, Mississippi, objective was to enhance the recovery of proteins and improve its quality through understanding of the protein structures. Our results showed that protein product⿿s functional performance, such as color could be improved by repeated washing. In addition, the texture profile of the surimi-gels could be improved by the use of soy whey to control the protein degradation during cooking. The engineering data obtained will pave the foundation to contribute to making of successful fish products that are acceptable to the consumers. 04 Recent findings showed that such gradual exposure to sublethal concentrations of biocides (sanitizers) could co-select for bacterial cells that are tolerant to lethal concentrations of biocides. Therefore, ARS researchers' in Stoneville, Mississippi, objective was to understand the role of sublethal concentrations of biocides in the emergence of heterologous stress-response in L. monocytogenes and if it would lead to the antibiotic tolerance/resistance development. In this project, we tested three approaches for continuous exposure to sublethal concentrations of QAC against actively growing planktonic cells of L. monocytogenes and evaluated the subsequent changes in antibiotic susceptibility against trimethoprim by three different methods. Our findings showed that there was a development of low-level tolerance to trimethoprim in L. monocytogenes strains after exposure to sublethal concentrations of QAC. These findings are useful in identifying the predisposing conditions for slow emergence of fluoroquinolone-resistant strains of L. monocytogenes, which may create food safety risk. The findings also can contribute to the development of early detection methods for detecting Listeria monocytogenes in the future.
Impacts
(N/A)
Publications
- Kode, D., Nannapaneni, R., Chang, S. 2021. Low-level tolerance to antibiotic trimethoprim in QAC-adapted subpopulations of Listeria monocytogenes. Food and Agricultural Immunology. https://doi.org/10.3390/ foods10081800.
- Chang, S., Zhang, Y. 2022. Color and texture of surimi-like gels made of protein isolate extracted from catfish by-products are improved by washing and adding soy whey. Journal of Food Science. 10.1111/1750-3841.16229.
- Shang, S., Zhang, Y. 2021. Trypsin inhibitor activity, phenolic content and antioxidant capacity of soymilk as affected by grinding temperatures, heating methods and soybean varieties. LWT - Food Science and Technology. 153:112424. https://doi.org/10.1016/j.lwt.2021.112424.
- Chang, S., Yang, Y., Zhang, Y. 2022. Determination of protease inhibitors, glycinin and beta-conglycinin in soybeans and their relationships. Journal of Food Science. 87:1082-1095. https://doi.org/10.1111/1750-3841.16054.
- Lin, C., Lee, Y., Kung, H., Cheng, Q., Qu, T., Chang, S.K., Tsai, Y. 2021. Inactivation of microbial loads and retardation of quality loss in Asian hard clam (Meretrix lusoria) using high-hydrostatic-pressure processing during refrigerated storage. Food Control. 133(Part A):108583. https://doi. org/10.1016/j.foodcont.2021.108583.
- Tsai, Y., Kung, H., Lin, C., Hwang, C., Liu, S., Huang, C., Chang, S.K., Lee, Y. 2022. Combined effect of brine salting and high-hydrostatic- pressure processing to improve the microbial quality and physicochemical properties of milkfish fillet. International Journal of Food Properties. 25:872-884. https://doi.org/10.1080/10942912.2022.2066120.
- Chen, D.M., Moore, M., Willis, E.L., Kouba, A.J., Vance, C.K. 2022. The impact of time and environmental factors on the mitochondrial vesicle and subsequent motility of amphibian sperm. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. 268:111191. https://doi.org/10.1016/j.cbpa.2022.111191.
- Santos-Rivera, M., Woolums, A.R., Thoreson, M., Meyer, F., Vance, C.K. 2022. Bovine respiratory syncytial Virus (BRSV) infection detected in exhaled breath condensate of dairy calves by near-infrared aquaphotomics. Molecules. 27(2):549-562. https://doi.org/10.3390/molecules27020549.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT 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. 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. 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 all of which have a major focus on the ensuring the food safety of catfish, seafood and produce. The third objective also has a focus on the food quality improvement. 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. In support of Objective 1, significance progress was made to optimize the safety of aquaculture products through innovative processes for reducing microbiological, physical, and chemical hazards in seafood/aquaculture products. We continued to renovate our laboratories which included transferring and re-installing equipment from the main Mississippi State University campus to the Experimental Seafood Processing Laboratory. Achievements included installation of an exhaust system for a wet chemistry laboratory, acquisition of $3 million by the Mississippi Agricultural and Forestry Experimentation (MAFES) towards building a Northern Gulf Aquatic Food Research Center (NGAFRC), with an additional $3.8 million pending from the Mississippi Department of Marine Resources for the first phase of building construction for the NGAFRC on a piece of 4-acre land. We continued to foster our partnership with the USDA-Agricultural Research Service (ARS) and the catfish aquaculture and processing industry to improve the safety and quality of catfish fillet and by- products. A multi-million dollar research project proposal was prepared with a coalition of 15 scientists from MAFES, USDA-ARS, Auburn University and Louisiana State University. We continued to work on the effect of processing, selected chemicals and storage conditions on the spoilage bacteria growth and sensory quality of fresh catfish fillet for extending product shelf-life and demonstrated shelf-life of fillet could be extended for 2-4 days. We initiated a subproject to determine the effect of dry and wet steam on cleaning and sanitation of food processing equipment. Preliminary data showed certain combinations of stream pressure, hot water content and conveyor speed resulted in significant reductions in bacteria and food residues. Using steam for cleaning and sanitation does not require any chemicals, such as chlorine or acids, and would be very useful for improving safety and quality of fish products. Another project has been initiated to determine the effects of high hydrostatic pressure processing to inactivate bacteria in farmed oysters, preliminary data from this project is being analyzed. In support of Objective 2, we continued to study mechanisms by which certain pathogenic bacteria may be reduced in catfish, seafood and produce including new methods to improve the rapidity, sensitivity and specificity for detecting various pathogens in response to innovative treatments. We completed certification requirements by Food and Drug Administration (FDA) for our Vibrio analysis protocols and are waiting for a FDA visit (delayed due to Covid-19) to certify our laboratory. A novel recombinase-polyermase/dipstick assay was developed allowing more sensitive and rapid detection of pathogenic Vibrio vulnificus in oysters, and this technology is under review for a patent application. This technology will be very useful to oyster aquaculture and processing industries for controlling Vibrio, an important pathogen in shellfish. Further optimization of the method is planned. Significant progress was also made on the development of a rapid system for detecting pathogenic Burkholderia species from fresh vegetables and catfish. Primers-probe combinations have been designed for the detection of pathogenic strains which allow detection of as few as 10 bacterial cells in a sample. We also collaborated with a researchers at the USDA-ARS laboratory in Delaware, on testing how riboflavin, a free-radical initiator, would affect norovirus inactivation by x-ray irradiation. Progress was made on understanding the development of low-level tolerance to fluoroquinolone antibiotic ciprofloxacin in Listeria monocytogenes after sublethal adaptation to quaternary ammonium compound (QAC). Results suggest the potential formation of low-level ciprofloxacin-tolerant subpopulations in some L. monocytogenes strains when exposed to residual QAC concentrations and such cells if not inactivated might create food safety risk. In support of Objective 3, progress was made on the optimization of the extraction of proteins from catfish by-product, which included heads and bones from the fillet processing industry. Researchers in the Experimental Seafood Processing Laboratory in the Coastal Research and Extension Center of the Mississippi State University continued to investigate how the combinations of the effect of particle size and alkalinity of extraction water could increase the recovery of fish protein from the ground catfish by-products. This research sub-objective was co-supported by an USDA-NIFA competitive grant. Incorporation of whey protein into fish protein isolates improved properties or resulting protein gels. Over-sized catfish are a problem for the industry and have dramatically reduced value, and scientists in the Experimental Seafood Processing Laboratory also determined that adding starch improved the firmness and quality of value-added ⿿fish-ball⿿ products from the meat of oversized catfish. The technologies developed in this project can also be applied to the other types of fish, and will produce a significant effect towards the sustainability of US agriculture and aquaculture. A competitive grant was obtained from the USDA-NIFA-AFRI research programs for enhancing the emulsion stability of the soymilk beverage systems. An international collaboration with Taiwan⿿s National Kaohsiung University of Science and Technologies led to several publications focusing on the synthesis of butyrate esters of resveratrol, which is a bioactive compound present in grape and other fruits, for enhancing antioxidant capacity and health promotion. Optimizing efficiency of protein extraction from catfish by-products, containing heads and frames. Catfish by-products (skin, heads and bones), which account for more than 200 million pounds each year and almost 40- 50% of the total fish proteins, have been considered as a waste for a long time. Our objective was to enhance the recovery of proteins and improve its food functions through understanding of the protein structures. Our results showed that protein product⿿s functional performance was related to physical and chemical conditions of the extraction, which subsequently affects the protein molecular structures. Based on our finding of 30% mass recovery, more than 60 million pounds of the value-added protein product could be recovered for making various protein products, such as fish cake or fish sausage for human consumption. The engineering data obtained will pave the foundation for building a protein recovery plant to contribute to the vitality of the rural economy. ACCOMPLISHMENTS 01 Evaluation of dry and wet steam to reduce bacterial contamination on a continuously moving conveyor. Processing equipment cleaning and sanitation are very important to ensure that low bacterial contamination occurs during processing of catfish and other food products to ensure the safety and quality of the products. ARS reseachers in Stoneville, Mississippi, have the objective to use dry and wet steam to spread on the moving conveyer in a continuous manner to achieve the killing of the bacteria and removing catfish residues. A new Clean-In-Place (CIP) technology, Optima Steamer SE-II, was tested for its effectiveness for cleaning and sanitizing. Our preliminary results showed that by controlling the steam pressure, water content in the jet stream and moving speed, pathogens can be effectively reduced. The results will be confirmed by conducting more studies. If proven effective, the new steam system can be adopted by the catfish filleting company for improving quality and safety of the fillet products. 02 Enhancing the safety and quality of oyster meat using high hydrostatic pressure processing (HPP). High hydrostatic pressure processing is one of the four FDA-approved post-harvest processing technology for reducing the risk of eating raw oysters. The effect of HPP processing technology on the oysters produced in the Northern Gulf region has not been comprehensively studied. ARS researchers in Stoneville, Mississippi, have the objective to search for the best HPP conditions for processing oysters over a wide range of pressures and processing time intervals. The preliminary results have identified the range of the conditions that could facilitate shucking and to reduce pathogens. More studies are needed. If successfully completed, the oyster processing companies can adopt the processing conditions to preserve the natural quality of oysters with improved safety and to enhance marketing raw oysters to the consumers. 03 Development of a rapid, sensitive, and equipment-free detection of Vibrio vulnificus in oysters. Vibrio vulnificus in oysters and in beach water is very hazardous to human health since it can cause the death of people infected by this pathogen. The official method approved by FDA and NSSP (the National Shellfish Sanitation Program) for Vibrio vulnificus analysis uses culturing method and followed by gene hybridization, and would take 4-5 days to complete. ARS researchers in Stoneville, Mississippi, have the objective to develop a rapid, sensitive and equipment-free method for the detection of this pathogen. Our preliminary results are promising since detection can be accomplished in 30 min. An invention disclosure has been filed by MAFES at the Mississippi State University. If successfully patented, this method will have broad applications in the seafood industries, and will be useful for government agencies for making decisions for beach closure, when this pathogen is detected in the beach water. 04 Decreasing biofilm formation by planktonic cells of Listeria monocytogenes in the presence of sodium hypochlorite. Listeria monocytogenes is regularly exposed to different kind of stresses in the food processing environment. Some processing conditions can exert stress to this pathogen to enhance its persistence in harsh environmental conditions. Along with stress adaptation, biofilm formation by L. monocytogenes is another form of survival mechanism, which makes bacterial elimination from food contact surfaces a serious challenge. ARS researchers in Stoneville, Mississippi, have the objective to understand how sub-inhibitory chlorine in the form of sodium hypochlorite, a commonly used sanitizer, affected planktonic cells and its subsequent biofilm formation ability. The results showed that chlorine decreased L. monocytogenes biofilm formation on polystyrene surface at sub-inhibitory concentration levels. Such anti- biofilm effect was found to be associated with the reduced attachment on polystyrene surface. The research would allow the food processing industry to choose the correct type of equipment materials, which come in contact with foods to facilitate elimination of this pathogen with the aid of chlorine sanitation. 05 Understanding the level of tolerance to fluoroquinolone antibiotic ciprofloxacin in QAC-adapted subpopulations of Listeria monocytogenes. In the food processing environment, even though sanitizers and disinfectants are routinely used at 50-100 times greater than that of their minimum bactericidal concentration (MBC) to kill foodborne bacterial pathogens, both planktonic cells and biofilms cells of these pathogens present in the crevices may be frequently exposed to a gradient of concentrations of biocides in the processing environments. Recent findings showed that such gradual exposure to sublethal concentrations of biocides can co-select for bacterial cells that are tolerant to lethal concentrations of biocides. Therefore, ARS researchers in Stoneville, Mississippi, have the objective to understand the role of sublethal concentrations of biocides in the emergence of heterologous stress-response in L. monocytogenes and if it would lead to the antibiotic tolerance/resistance development. In this project, we tested three approaches for continuous exposure to sublethal concentrations of QAC against actively growing planktonic cells of L. monocytogenes and evaluated the subsequent changes in antibiotic susceptibility against ciprofloxacin by three different methods. Our findings showed that there is a development of low-level tolerance to ciprofloxacin in L. monocytogenes strains after exposure to sublethal concentrations of QAC. These findings are useful in identifying the predisposing conditions for slow emergence of fluoroquinolone-resistant strains of L. monocytogenes, which may create food safety risk.
Impacts
(N/A)
Publications
- Kode, D., Nannapaneni, R., Bansa, M., Chang, S.C., Cheng, W., Sharma, C.S., Kiess, A. 2021. Low-level tolerance to fluoroquinolone antibiotic ciprofloxacin in QAC-adapted subpopulations of Listeria monocytogenes. Microorganisms. 9(5):1052. https://doi.org/10.3390/microorganisms9051052.
- Tan, Y., Chang, S. 2021. Protein extraction pH and cross-linking affect physicochemical and textural properties of protein gels made from channel catfish by-products. Journal of the Science of Food and Agriculture. https://doi.org/10.1002/jsfa.11126.
- Bansal,, M., Dhowlaghar, N., Nannapaneni, R., Kode, D., Chang, S.C., Sharma, C.S., Mcdaniel, C., Kiess, A. 2020. Decreased biofilm formation by planktonic cells of Listeria monocytogenes in the presence of sodium hypochlorite. Food Microbiology. 96. Article 103714. https://doi.org/10. 1016/j.fm.2020.103714.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report 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. 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. All objectives were planned and completed by the ARS researchers 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, and are under the National Program 108-Food Safety, Component I: Food Borne Contaminants. The third objective also has a focus on the food quality improvement under the National Program 306-Quality and Utilization of Agricultural Products, Component I: Foods. 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. In support of Objective 1, 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. In this period, we continued to renovate our laboratories and install, re-calibrate the equipment, which was transferred to the Experimental Seafood Processing Laboratory from the main campus to build the food safety program. Mississippi State University researchers at Starkville, Mississippi, also continued to build close partnership with the catfish aquaculture and processing industry to improve the safety and quality of the fillet and by-products. Mississippi State University researchers at Starkville, Mississippi, have performed a comprehensive study on spoilage bacteria inhibition and sensory assessment of plastic film packed fresh catfish fillet (simulating grocery market shelf conditions) placed under light-emitting diode (LED) light as affected by treatments with chitosan and organic acids and Mississippi State University researchers at Starkville, Mississippi, also have completed a study on extension of the shelf life of fresh fillet in the box packed in ice to simulate the conditions during shipping and storage. The results showed under certain conditions, shelf-life of fillet could be extended for 2-4 days. This progress is very significant to the catfish industry. Mississippi State University researchers at Starkville, Mississippi, also conducted a study to develop more efficient x-ray irradiation conditions by optimizing accelerating voltage and using aluminum filter. Results showed that conditions under high accelerating voltage and the use of 1 mm aluminum filter to remove low energy rays could enhance killing efficiencies on pathogens such as Escherichia. coli O157:H7 and Vibrio species in pure cultures and in whole shell oysters. The results were published in the International Association of Food Protection⿿s annual conference and in a peer-reviewed journal of Food Control. In support of Objective 2, we continued to determine the mechanistic approach by which the certain pathogenic bacteria may be reduced in catfish, seafood and produce. Mississippi State University researchers at Starkville, Mississippi, continued to use Polymerase Chain Reaction analysis and to develop assays to improve the sensitivity and specificity for detecting various isolates in response to innovative treatments. Mississippi State University researchers at Starkville, Mississippi, have completed the requirements by Food and Drug Administraion for certification of our Vibrio analysis protocols and is waiting for FDA to complete the next site visit to certify our laboratory. Mississippi State University researchers at Starkville, Mississippi, have initiated a study to enhance the detection of pathogenic Vibrios (Vibrio parahemolyticus and Vibrio vulnificus) in oysters by using a novel combinase-polymerase method. If successful, the method will be more sensitive and rapid for screening than the FDA-approved methods for Vibrio analyses and will be very useful to oyster aquaculture and processing industries for controlling these pathogens. Significant progress was also made on the development of rapid detection systems for pathogenic Burkholderia spp. from fresh vegetables and catfish. Twenty-three pairs of PCR primers were designed and synthesized based on genome-wide comparison with the related bacterial genomes. A few pairs of PCR primers were successfully identified for detection of Burkholderia cenocepacia. Furthermore, a pair of qPCR primer and a probe were designed and synthesized. The pair of primers and the probe were further tested using different bacterial strains. The genomes of B. cenocepacia strains were GC rich (GC content in most of the gnome of these strains >66.8% that submitted to GenBank) and the high GC reduces the sensitivity of qPCR. To increase the sensitivity of q-PCR assay, q- PCR reaction system was modified by supplementing different concentrations of dimethyl sulfoxide, 1,3-propanediol (pro), trehalose (tre), betaine (BT) and increased DNA polymerase concentration. Comprehensive assays showed that the primer-probe system was able to detect as less as 10 bacterial cells in a sample. Multiple samples of fresh vegetables were collected from different locations or grocery stores. Standard procedures for bacterial isolation and Burkholderia identification were conducted. None of the isolates of Burkholderia spp. were recovered from the samples. In another subproject, previously we reported the adaptation of Salmonella to sublethal NaOCl and subsequent formation of stable rugose morphotype. Additional progress has been made on the quantification of the survival of this rugose morphotype in lethal concentrations of NaOCl. The biofilm architectures of this rugose morphotype induced by subinhibitory NaOCl using scanning and transmission electron microscopy have been characterized. The determination on the biofilm- and rugose-related gene expression in Salmonella typhimurium ATCC 14028 induced by sublethal NaOCl was published this year. The gene expression data indicated that the expression of biofilm regulator (csgD), curli (csgA, csgB, and csgC) and cellulose (bcsE) was significantly increased in rugose morphotype of S. typhimurium ATCC 14028 when induced by sequential exposure to sublethal concentrations of NaOCl. Also, structural differences in rugose and smooth morphotype planktonic and biofilms were characterized by scanning and transmission electron microscopy. At higher magnification, Scanning Electron Microscope micrographs revealed that a majority of rugose morphotype cells were interconnected with extracellular materials while smooth morphotype cells were separated from each other for both S. typhimurium ATCC 14028 and for S. Heidelberg ATCC 8326. In support of Objective 3, progress was made on the optimization of the extraction of proteins from catfish by-product, which included heads and bones from the fillet processing industry. Researchers in the Experimental Seafood Laboratory in the Coastal Research and Extension Center of the Mississippi State University continued to investigate how the combinations of the effect of particle size and alkalinity of extraction water could increase the recovery of fish protein from the ground catfish by-products. This research sub-objective was co-supported by an USDA-NIFA competitive grant. The whey proteins from the soy protein extraction that was rich in trypsin inhibitor activity did not show a significant effect on extraction yield and protein integrity. More than 30% of the proteins in the mixture of catfish heads and frames was recovered from the extraction at an optimal pH. The protein gel functional properties were analyzed, and the results showed alkalinity of the extraction water affected the gel functional properties and need to be further optimized. Scientists in the Experimental Seafood Laboratory also initiated a study on making value-added fish ball products from fresh fillet with the inclusion of starch and gums and the results were compared to commercial fish balls. Preliminary experiments showed that the texture of the fish balls was affected by starch or gum content. Based on last year⿿s data, a refereed publication was published in a top journal of Food Chemistry on the determination of gut microbiota and short chain fatty acid composition as affected by legume type and processing methods as assessed by simulated in vitro digestion assays. This study could serve as a foundation for producing value-added bean fiber prebiotic products for health improvement. In addition, significant progress was made to identify the type of probiotic bacteria that could grow well in soymilk yoghurt products, which may be used as a milk yoghurt substitute for people who are allergic to cow⿿s milk products. Accomplishments 01 Extension of shelf-life of fresh fillet in retail tray pack under light- emitting diode stored with refrigeration. Fresh fish is well known to have short shelf-life and the catfish industries have long wished to find ways to extend shelf-life of catfish fillet to enhance marketing. In this study, ARS researchers in Stoneville, Mississippi, used various non-phosphorus organic acids to treat catfish fillet and the spoilage organisms and food quality of the fillet were analyzed over a 10-day period. Results showed that control fillet without treatments had only 4 days of shelf-life. Organic acids could extend the shelf-life to 6 to 10 days. The results will be confirmed by conducting more studies. If proven effective and not damaging to flavor, the impact of extending days of shelf-life by using selected acids will be very large to the catfish industry. 02 Extension of shelf-life of catfish fillet buried in ice in shipping containers. Currently, the shelf-life of the fresh fillet is around 10- 11 days after filleting. Extending days of shelf-life would allow the transport of the fillet to farther locations to expand fillet market. Currently, fresh catfish fillet is marketed mostly in the Southeastern parts of the United States. In this study, ARS reseachers in Stoneville, Mississippi, will find treatments that could extend the shelf-life of fillet packed in ice in shipping boxes. Our results showed that under certain organic acids or natural bacteriocin treatments, the shelf-life could be extended by 2-4 days. If confirmed with additional research, this will make a very significant contribution for expanding fresh fillet market to most of the continental states of our nation. 03 Optimizing novel x-ray technologies to produce oysters with minimum pathogens while maintaining live fresh oysters for enhanced values. Pathogens such as Vibrio species can cause human illness and therefore, damage oyster aquaculture. Premium oysters are often eaten in their raw state, and if pathogens are present and not inactivated, can easily cause illness. X-ray is the only technology that can kill Vibrio without killing the oysters. ARS reseachers in Stoneville, Mississippi, concluded that that x-ray⿿s killing efficiency can be optimized to improve pathogen inactivation while maintaining the life of oysters for better marketing. Our research added new knowledge to the science and technology for the preservation of raw foods since the technologies can also be applied to other foods, such as fruits and vegetables that are consumed raw to protect the consumer health. 04 Studying the effect of the combination of the effect of particle size and alkalinity of extraction water on protein extraction and product functional properties. Fresh catfish by-products (skin, heads and bones) , accounting for 200 million pounds each year and almost 40% of the total fish proteins, have been considered as a waste for a long time. ARS reseachers in Stoneville, Mississippi, showed significant amount of proteins could be extracted. The extracted protein possessed functional properties for use to make fish protein gel products. Based on our finding of 30% mass recovery, more than 60 million pounds of the value- added protein product could be recovered for human consumption. The technologies can be used as a foundation for building a protein recovery plant to contribute to the rural economy. 05 Determining the biofilm- and rugose-related gene expression in Salmonella typhimurium ATCC 14028 induced by sublethal stress and structural differences by microscopy. In the food processing facility, Salmonella biofilm is regularly exposed to sublethal stresses. The tendency of S. typhimurium and S. Heidelberg rugose morphotypes to form denser biofilm under sublethal stress condition may increase their ability to persist in the food processing environment. The sequential exposure of sodium hypochlorite at sublethal concentrations modulated the rugose and biofilm related gene expression levels in S. typhimurium. The research is important to show mechanistic of resistance and has added significant knowledge in the scientific literature. Based on these findings by ARS researchers in Stoneville, Mississippi, food processors will develop a strong control protocol for cleaning and sanitation of food processing equipment and hard-to-reach contact surfaces to reduce biofilms of Salmonella and other foodborne bacterial pathogens. 06 Gut microbiota and short chain fatty acid composition were affected by legume type and processing methods as assessed by simulated in vitro digestion assays. In recent years, legumes are gaining considerable interest globally due to a positive association between the consumption of legume and a reduction in the risk of cardiovascular diseases, obesity, type-2 diabetes and cancers. The objectives of this project were to investigate the effect of processing methods on the digestibility of soybean and pinto bean, and to characterize how the undigested fiber two differently processed beans affected the gut microbiota and short chain fatty acid formation. ARS reseachers in Stoneville, Mississippi, showed that soluble fiber was better than insoluble fiber in giving benefits to the gut microbiota. Mississippi State University Scientists at Starkville, Mississippi, have added significant knowledge to the literature by publishing a refereed article this year. The new findings can be used as a foundation to develop the technology for producing novel food products for enhancing human health through modulation of gut microbiota.
Impacts
(N/A)
Publications
- Tan, Y., Gao, H., Chang, S., Bechtel, P.J., Mahmoud, B. 2019. Comparative studies on the yield and characteristics of myofibrillar proteins from catfish heads and frames extracted by two methods for making surimi-like protein gel products. Food Chemistry. 272:133-140.
- Wu, Y., Chang, S. 2020. The efficacy of X-ray doses on Vibrio vulnificus in pure culture and Vibrio parahaemolyticus in pure culture and inoculated farm-raised live oysters (Crassostrea virginica) with different acceleration voltages. Food Control. 115:107277.
- Chen, Y., Chang, S., Zhang, Y., Hsu, C., Nannapaneni, R. 2020. Gut microbiota and short chain fatty acid composition as affected by legume type and processing methods as assessed by simulated in vitro digestion assays. Food Chemistry. 312:126040.
- Shi, M., Tan, Y., Chang, S., Li, J., Maleki, S.J., Puppala, N. 2019. Peanut allergen reduction and functional property improvement by means of enzymatic hydrolysis and transglutaminase crosslinking. Food Chemistry.
- Bansal, M., Nannapaneni, R., Kode, D., Chang, S., Sharma, C., Mcdaniel, C., Kiess, A. 2019. Rugose morphotype in Salmonella Typhimurium and S. Heidelberg induced by sequential exposure to subinhibitory NaOCl aids in biofilm tolerance to lethal NaOCl on polystyrene and stainless steel surfaces. Frontiers in Microbiology. 10:2704.
- Tan, Y., Chang, S., Meng, S. 2019. Comparing the kinetics of the hydrolysis of by-product from channel catfish (Ictalurus punctatus) fillet processing by eight proteases. LWT - Food Science and Technology. 111:809- 820.
- Shi, M., Li, J., Chang, S., Maleki, S.J. 2019. Quantitative and kinetic analyses of peanut allergens as affected by food processing. Food Chemistry.
- Abeysundara, P., Dhowlaghar, N., Nannapaneni, R. 2019. Influence of cold stress on the survival of Listeria monocytogenes Bug60 and ScottA in lethal alkali, acid and oxidative stress. LWT - Food Science and Technology. 100:40-47.
- Dhowlaghar, N., Shen, Q., Nannapaneni, R., Schilling, W., Samala, A. 2019. Survival of acid stress adapted cells of Listeria monocytogenes serotypes 1/2a and 4b in commonly used disinfectants in broth and water models. LWT - Food Science and Technology. 109:201-206.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report 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. 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. All Objectives were planned and completed by the Agriculture Research Service (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, and are under the National Program 108-Food Safety, Component I: Food Borne Contaminants. The third objective also has a focus on the food quality improvement under the National Program 306-Quality and Utilization of Agricultural Products, Component I: Foods. 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. Under Objective 1, 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. In this period, we devoted a great deal of time to install and test the newly purchased 350 kV x-ray machine. In addition, we have prepared an environment chamber, which can maintain temperature and modified atmosphere during x-ray irradiation. In 2018, researchers at the Experimental Seafood Processing Laboratory of the Mississippi State University acquired a tailor-made X-ray irradiator (Kimtron 350kV), which was customized for food irradiation research. With this X-ray machine, the current (mA), irradiation filter and accelerating voltage can be optimized for the best pathogen inactivation and food preservation with an auto-dosing system. Progress was made to design and manufacture an environmental chamber, which can control the irradiation temperature and atmosphere of the samples during irradiation to study effect of temperature and oxygen replacement. To prove the impact of accelerating voltage on pathogen inactivation in pure culture, different energy levels of X-ray were applied on two different food pathogens (Escherichia coli O157:H7 and Vibrio parahaemolyticus) and determined the efficacy of X-ray of different accelerating voltage and filtration. Research showed that lower irradiation doses than conventional method was required to achieve the same killing efficacy, when proper acceleration voltage and aluminum filter were used. Under Objective 2, significant progress was made to understand the mechanisms influencing microbial survival of selected pathogens in seafood/aquaculture products. Significant progress was made on the development of rapid detection systems for pathogenic Burkholderia (B.) spp. from fresh vegetables and catfish. Twenty-three pairs of PCR primers were designed and synthesized based on genome-wide comparison with the related bacterial genomes. A few pairs of PCR primers were successfully identified for detection of B. cenocepacia. Furthermore, a pair of qPCR primer and a probe were designed and synthesized. The pair of primers and the probe were further tested using different bacterial strains. The genomes of B. cenocepacia strain are GC rich (GC content in most of the gnome of these strain >66.8% that submitted to GenBank) and high GC reduces the sensitivity of qPCR. To increase the sensitivity of q- PCR assay, q-PCR reaction system was modified by supplemented of different concentrations of dimethyl sulfoxide, 1,3-propanediol (pro), trehalose (tre), betaine (BT) and increased DNA polymerase content. Comprehensive assays showed that the primer-probe system can be able to detect as less as 10 bacterial cells from a sample. Significant progress was also made in the understanding of the formation of rugose morphotype in Salmonella (S.) Typhimurium and S. Heidelberg and its inactivation. Scientist in the Department of Food Science, Nutrition and Health Promotion and the Department of Poultry Science discovered that progressive and sequential exposure to increasing subinhibitory concentrations of NaOCl at 37°C led to the development of stable rugosity in S. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326. Then the stable behavior of rugose morphotype was confirmed by frequent subculturing of rugose Salmonella on tryptic soy agar and tryptic soy broth without sodium hypochlorite (NaOCl). These results showed that Salmonella rugose is a stable morphotype and did not revert to smooth morphotype after adaptation. However, there is a knowledge gap on the biofilm architecture of rugose morphotype and the influence of subinhibitory concentration of NaOCl on rugose biofilm formation ability. Also, there is a lack of information on the effects of lethal concentrations of NaOCl against Salmonella biofilms of rugose morphotype on different food-contact surfaces. The results showed that the rugose morphotype of S. Typhimuirum ATCC 14028 and S. Heidelberg ATCC 8326 produced dense biofilm on polystyrene surface as a function of increased formation of extracelluar polymeric compounds. The efficacy of dietary trans-cinnamaldehyde to combat bacterial infections of catfish was investigated by researchers in the Department of Basic Science at Mississippi State University and at the USDA-ARS in Starkville, Mississippi. The results showed the application of trans-cinnamaldehyde on catfish feed was effective in protecting fish from Edwardsiella ictaluri infection. Under Objective 3, progress was made on the optimization of the extraction of proteins from catfish by-product, which included heads and bones from the fillet processing industry. The effect of particle size and the use of the soy protein whey to inhibit the degradation of fish protein during extraction were studied. The kinetics of the hydrolysis of by-products from channel catfish fillet processing by eight proteases were compared and the most suitable enzymes and their concentrations were identified for preparing protein hydrolysates from the by product. In addition, progress was made on the quantitative and kinetic analyses of peanut allergens as affected by food processing, including frying, steaming, boiling, microwaving and roasting and published a paper in a high-quality journal. Furthermore, progress was made on the determination of gut microbiota and short chain fatty acid composition as affected by legume type and processing methods as assessed by simulated in vitro digestion assays. Accomplishments 01 Rapid detection systems for pathogenic Burkholderia species from fresh vegetables and catfish. Pathogenic Burkholderia are extremely rich in nature and some cause life-threatening cystic fibrosis. Unfortunately, sensitive and specific approaches are not available in the literature for identification and detection of the bacteria. Scientists in the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, and the USDA-ARS scientists in Auburn, Alabama, jointly worked on this investigation to develop rapid detection systems for pathogenic Burkholderia from fresh vegetables and catfish using genetic screening methods. The bacterial genetic makeups were analyzed for development of gene-based detection approaches. One genetic system has been developed specifically for detection of Burkholderia cenocepacia. This method is very sensitive and can detect as few as 10 bacterial cells in a food sample and can be adopted by the food industry easily. The sensitive gene-based technology can be used for detection of the bacteria to ensure fresh food safety. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi. 02 Formation of rugose morphotype in Salmonella species and its inactivation. Salmonella biofilms acts as a continuous source for cross- contamination in the food processing environments. Understanding the nature of the biofilms, its physical, chemistry and biological properties (smooth or rugose forms) are important to enable the industry to control the biofilms. In this study, a stable morphotype of Salmonella rugose was first induced by sequential exposure to subinhibitory concentrations of sodium hypochlorite, a sanitation agent. The developed rugose biofilms were characterized for their forming abilities on polystyrene and stainless steel surfaces. The Salmonella biofilms were treated with biocidal agents and the reduction in bacteria counts were analyzed. The biofilm reduction in bacteria counts for the rugose was lower than the smooth morphotype on polystyrene and stainless-steel surfaces. Under electron microscope, a denser biofilm in Rugose form was discovered to be more tolerant to the sanitation agent such as biocidal NaOCl concentrations commonly used in the food processing plants. The study generated valuable information for future development of intervention technologies to reduce biofilm deposition in the food processing plant. Reducing contamination of foods is important to consumers and the food industry. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi. 03 Efficacy of dietary trans-cinnamaldehyde to combat bacterial infections of catfish. Antimicrobial resistance can be developed in catfish production when synthetic antibiotics are used. The objective of this project was to identify potential intervention strategies using natural antimicrobials to reduce catfish mortality associated with bacterial infections. The antibacterial activity of trans-cinnamaldehyde, a natural phytochemical, was confirmed using in vivo and in vitro studies. Results from catfish challenge experiment demonstrated that incorporation of trans-cinnamaldehyde in catfish feed significantly lowered mortality associated with Edwardsiella (E.) ictaluri infection than those fed the control diet. It was also found that trans- cinnamaldehyde supplementation has the potential to modify the gut microbiota composition in a positive direction by increasing the abundance of beneficial bacteria. These findings suggest that trans- cinnamaldehyde could serve as a promising alternative to antibiotics for use in aquaculture. The information also has an impact on reducing the use of antibiotics in fish production. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi. 04 Developing novel x-ray technologies to enhance pathogen killing efficiencies. Foods consumed raw require processing methods that can preserve the raw texture and taste quality. Researchers in the Experimental Seafood Processing Laboratory of the Coastal Research and Extension Center have installed a new 350 kV x-ray machine for processing vegetables and oysters. The preliminary experiments using E. coli O157:H7 and Vibrio (V.) parahaemolyticus proved the research hypothesis that by varying the degree of acceleration energy (voltage) and the inclusion of an aluminum filter, much greater inactivation of these pathogens in liquid buffer occurred. The preliminary optimization effort showed less irradiation dose than that has been used in the past can achieve safety and also preserve better oyster quality. To the best of our knowledge, this is the first time the effects of different energy levels have been systematically compared on the efficacy of the radiation sterilization. The new technology developed has great application value since the irradiation industry can achieve a bactericidal effect several times higher than the literature record using conventional irradiation method. The novel method developed is of interest to the food industry and the scientific communities. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi. 05 Studying the effect of particle size of ground catfish by-product on protein extraction. Fresh catfish by-products, a mixture of heads and frames, contain protein that can be extracted to produce value-added protein product. Currently, more than 200 million pounds of by-product are produced each year, and if recovery rate is more than 20%, more than 40 million pounds of the protein product could be resulted from the recovery process. To improve the protein recovery rate, studying each step of the processing is important. The by-products were ground into various particle sizes, and, protein in the ground materials was extracted with varying ratios of solid to water, for varying periods of time and alkalinity for optimal protein recovery. The particle size distribution of catfish mince prepared by different grinding methods were verified. Results showed the mince with the smallest particle size that was produced in the laboratory gave the highest solid and protein recovery. Water-to-mince ratios studied had no effect on the protein recovery. The preliminary study revealed that alkalinity was an important factor to the protein recovery. More combinations of two factors (extraction time and extraction alkalinity) are being tested to optimize the protein extraction. The data obtained from the current study is needed for estimating commercial feasibility and will serve as a basis for further protein recovery using trypsin inhibitor for preventing degradation during extraction. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi. 06 Comparing the kinetics of the hydrolysis of catfish by-products by eight proteases. Value-added protein hydrolysates can be made from catfish by- products for the applications as flavoring materials or as a nutrition supplement. The objective of this study was to investigate the kinetics of the enzymatic hydrolysis of catfish by-products. Eight enzyme sources, including papain, ficin, bromelain, neutrase, alcalase, protamex, novo-proD and thermolysin were used for hydrolysis. Proteolytic activities of those proteases were examined at different hydrolysis temperature, proteases cost for reaching certain degree of hydrolysis was predicted. Degree of hydrolysis of the hydrolysates and hydrolysis kinetics were studied. Emulsifying and foaming property and stability of selected hydrolysates were evaluated. Results indicated that thermolysin had the highest activity at 70°C. Ficin was the most efficient in hydrolyzing the by-product. The hydrolysis curves fit an engineering kinetic model very well. This study provides important engineering information for choosing proteases and processing conditions for optimization of efficiency, yield and economy. This study will lead to value- added protein products from catfish by- products and will benefit farmers and the consumers as well. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi. 07 Quantitative and kinetic analyses of peanut allergens as affected by food processing. Peanut is a major crop in South Eastern region of the United States and its products and ingredients are widely used in the food industry. However, peanuts are a major allergenic food, which contains four major allergens with differences in allergenic potency. Peanuts are normally cooked by various ways before consumption, but the effect of each heat treatment method is unknown and is the target of this study. In this study, we characterized the reaction kinetics of peanut allergens as affected several major thermal processing methods with temperature and time intervals, which produced palatable peanut products. The reactivities of the peanut proteins to human plasma antibody in different protein fractions were analyzed. The relationships between thermal processing (time) and log transformed water- soluble/total extractable major allergen content were characterized. Results showed that among all the methods with optimal processing point for palatable texture of peanuts, frying appeared to be the best to reduce the immunoreactivity of Ara h 2, which is the most potent allergen in peanuts. The research provides new knowledge to the food industry and the consumers to select different types of processing for reducing the potential of peanut allergenicity. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi. 08 Gut microbiota and short chain fatty acids as affected by legume type and processing. Legumes are rich in dietary fibers (prebiotics) and have gained considerable interest globally due to their ability to reduce the risk of cardiovascular diseases, obesity, type-2 diabetes and cancer by consumption of beans. New knowledge on the variety of legume and how their fiber types affects gut health will benefit consumers. The effect of processing methods on the digestibility of soybeans and pinto beans were investigated and the effect of these beans⿿ fiber residues after digestion on the gut microbiota and short chain fatty acid formation were studied by genetic and gas chromatographic methods. Results showed significant differences in the microbiota and short chain fatty acid composition between the two legume types and among samples treated by autoclaving and germination. Short chain fatty acids, which have been reported to modulate diabetes and heart disease, were higher in soluble fiber treatment than that in the insoluble fractions. Bean fibers promoted the growth and activities of beneficial gut microflora. The soluble and insoluble digested residues functioned differently in shaping the gut microbial community. Soluble fibers have much more positive effect than the insoluble fibers. The new findings contributed to the new understanding of the potential gut health benefit of beans as affected by the consumption of different types of legumes and processing method. This study was planned and completed by scientists at the Mississippi State University in collaboration with ARS scientists in Stoneville, Mississippi.
Impacts
(N/A)
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report 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. 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. 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 �g/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 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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). 08 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.� 09 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.
Impacts
(N/A)
Publications
- 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.
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report 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. 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. All Objectives were planned and completed by the ARS scientists in Stoneville, Mississippi, in collaboration with the scientists in the Mississippi State University. Progress was made on all three objectives and their sub-objectives, all of which have a major focus on the ensuring the food safety of catfish, seafood and produce. Objective 1: 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. X-ray irradiation was used successfully to reduce bacterial pathogens and norovirus in oysters and seafood, such finfish products used in sushi products, and in selected freshly cut fruits. Various sanitation methods using acids, alkali, low-temperature and oxidative agents used in reducing pathogens such as Salmonella and Listeria. Progress was made on the understanding of the development of cross- resistance of the sub-lethal concentrations of the sanitation agents, and the use of antibiotics. This is important for future development of intervention methods for controlling pathogens in during food processing and storage. Objective 2: Significant progress was made to understand the mechanisms influencing microbial survival of selected pathogens in seafood/ aquaculture products. Progress has been made to identify the molecular differences between virulent (high-risk) and non-virulent (low-risk) pathogens. Novel detection methods were invented to detect pathogens in a rapid and less costly manner. The specific genetic factors, which were related to pathogen�s adhesive ability to attach on the surface of the equipment of food processing, were identified. Progress was made to develop the methods for detection of toxins that could be present in catfish under certain environmental conditions. Progress was made on the development of chemical polymers from agricultural waste (fiber from cotton stalk) and seafood waste (chitin from shrimp shells) to reduce pathogens and heavy metals in fish ponds. Objective 3: Through the collaboration with the USDA-ARS Southern Regional Research Center, progress was made on the extraction of useful proteins for making useful products from catfish by-products (such as heads, frames and skin) after filleting and from the invasive species of carps (silver carp) and the quality characteristics of the protein products were characterized. Progress was also made on the understanding of environment temperature and oxygen content of the water in the pond on the color of the fish fillet. This would lead to enhancement of the fillet quality for marketing. Accomplishments 01 Utilization of the invasive carp species for surimi products. Invasive carps are undesirable for the ecology of the Mississippi River and its tributary water. Utilization of the carp meat as a food protein source for surimi products will contribute to the elimination of carps from the river. ARS researchers in Stoneville, Mississippi, showed that the surimi made from carp meat is very firm. Different types of starch was used to reduce the firmness making the gel more palatable and whiter. The cooking loss of surimi gel containing 6% starch was significantly lower than the control without adding starch when modified starches were applied. Fish sauce was successfully produced from the frame and offal of silver carp by fermentation. The research provides technology for further improvement of the quality of food made from carp, and will allow the food industry to adopt the technology for productive use of this invasive species. 02 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. The results obtained by ARS researchers in Stoneville, Mississippi, showed that protein recoveries form heads, frames, and skins exceeded 50% of that in the raw materials. The by-product has little value and was considered as processing waste. Better laboratory scale extraction of gelatin from fish skins was developed. The findings were presented to the scientists and industry representatives in the annual meetings of the Institute of the Food Technologists. A national competitive grant proposal was resubmitted to USDA-NIFA (National Institute of Food and Agriculture) for consideration. The review was favorable and in the medium funding priority. A revised proposal, including mathematical prediction modelling for predicting product quality and more economic feasibility study was submitted again for consideration. The preliminary studies done on this project will lay the foundation for commercial production of protein products in the future. The impact is in terms of millions of dollars and creation of many jobs for Mississippi and the region. 03 Analysis of allergens in peanut varieties and assessment of effects of food processing on peanut allergens. Peanut allergy is one of the most common causes of food related death. Peanut allergy is typically life- long and the number of people with peanut allergies in developed countries, including the United States, appears to be increasing. Despite the seriousness of peanut allergy, to date, there is no cure for peanut allergy. Currently the only available treatment is complete peanut avoidance. However, avoiding peanuts in food product is difficult because of its ubiquitous use as an ingredient in processed foods. Thus, production of a peanut that has reduced levels of allergens will benefit consumers who suffer from peanut allergies. To this end, scientists at Mississippi State University have analyzed 122 peanut varieties and found that eight peanut varieties have reduced levels of major allergen. ARS researchers at the Mississippi State University, Mississippi, in collaboration with ARS researchers in New Orleans, Louisiana, also found that frying peanut seeds for eight minutes could reduce the level of a major allergen. This information can help growers to choose peanut varieties with reduced allergen levels and inform food industries about the useful peanut processing method for reducing allergen levels. The results will benefit consumers greatly. 04 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 will have increased levels of arsenic. Consumption of food with a high heavy metal would damage consumers� health. ARS researchers at Mississippi State University, Mississippi, in collaboration with ARS researchers located in Stuttgart, Arkansas, have identified the high and low arsenic rice germplasm. 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 have very minor effect on other minerals. The exciting results suggest that continuing the research may substantially contribute to reducing human arsenic exposure from rice consumption. 05 Reducing aflatoxin contamination in corn production using resistant lines. Corn is frequently infected by a soil fungal pathogen Aspergillus flavus in the pre-harvest and post-harvest stages. Infected corn by A. flavus can produce aflatoxins B1 and B2, and ingestion of corn contaminated with aflatoxins causes liver damages and suppresses the immune systems. One of the strategies in reducing aflatoxin contamination is to breed maize lines with resistance to the fungus. With the collaboration of scientists in USDA-ARS in Starkville, Mississippi, scientists in the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology at Mississippi State University have tested genes encoding small ribonucleic acid (RNA) molecules in resistant maize plants that may be involved in resistance to aflatoxin accumulation. This study facilitates our understanding of the molecular mechanisms governing maize resistance to the fungus and aflatoxin accumulation. The goals of this research are to reduce the loss to aflatoxin contamination of corn and eliminate an important food and feed safety problem. 06 Development of a rapid detection system for pathogenic Burkholderia spp. A certain species of pathogenic Burkholderia bacteria is associated with soil and water where vegetables and fish are grown and if transmitted to humans can caused illness. However, no method is available for detection of pathogenic Burkholderia from fresh vegetables and catfish. Isolated from Fresh vegetable and catfish, ARS researchers at Mississippi State University, Mississippi and ARS researchers in Auburn, Alabama, have isolated from vegetables and catfish 88 sequenced genomes or genome drafts of the Burkholderia strains in the National Center for Biotechnology Information GenBank to identify unique regions for the genes. They have identified one pair of gene primers that produces a unique gene product from the one specific Burkhoderia that is not in the rest of bacterial tested. From 45 samples collected from the local grocery stores, which included lettuce, cucumber, cabbage, celery, onion, and catfish, two specific bacterial isolates of B. contaminans were recovered from the samples sweet onion and celery stalk. In addition, some animal and human pathogens, such as Brucella species was recovered from a celery stalk. The results were significant for future development of a sensitive method for detecting pathogenic Burkhoderia that will lead to the reduction in human illness, particularly in consuming uncooked vegetables.
Impacts
(N/A)
Publications
- Soni, B., Hassan, E., Schilling, M.W., Mahmoud, B. 2016. Transparent bionanocomposite films based on chitosan and TEMPO-oxidized cellulose nanofibers with enhanced mechanical and barrier properties. Carbohydrate Polymers. doi:e10.1016.
- Lee, Y., Wang, C. 2017. Morphological change and decreasing transfer rate of biofilm-featured Listeria monocytogenes EGDe. Journal of Food Protection. 80:368-375.
- Abeysundara, P., Nannapaneni, R., Soni, K., Sharma, C.S., Mahmoud, B. 2016. Induction and stability of oxidative stress adaptation in Listeria monocytogenes EGD (Bug600) and F1057 in sublethal concentrations of H2O2 and NaOH. International Journal of Food Microbiology. 238:288-294.
- Shen, Q., Pandare, P., Soni, K., Nannapaneni, R., Mahmoud, B., Sharma, C.S. 2016. Influence of temperature on alkali stress adaptation in Listeria monocytogenes. Food Control. 62:74-80.
- Mahmoud, B.M., Nannapaneni, R., Chang, S.C., Wu, Y. 2016. Improving the safety and quality of raw tuna fillets by x-ray irradiation. Food Control. 60:569-574.
- Wu, Y.W., Chang, S.C., Nannapaneni, R., Coker, R., Haque, Z., Mahmoud, B.M. 2016. The efficacy of x-ray doses 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. 2017. Effect of x-ray treatments on Escherichia coli O157:H7, Listeria monocytogenes, Shigella flexneri, Salmonella enterica and inherent microbiota on whole mangoes. Letters in Applied Microbiology. 62(2):138-144.
- Salehi, S., Howe, K., Brooks, J.P., Lawrence, M., Bailey, H., Karsi, A. 2016. Identification of salmonella enterica serovar Kentucky genes involved in attachment to chicken skin. BMC Microbiology. 16:168.
- Tan, Y., Chang, S., Zhang, Y. 2017. Comparison of alpha-amylase, alpha- glucosidase and lipase inhibition activity of the phenolic substances in two black legumes of different genera. Food Chemistry. 214:259-268.
- Sah, S.K., Reddy, K.R., Li, J. 2016. Abscisic acid and abiotic stress tolerance in crop plants. Frontiers in Plant Science. 7(571):1-26.
- Wu, Y.M., Chang, S., Nannapaneni, R., Zhang, Y., Coker, R., Mahmoud, B. 2017. The effects of x-ray treatments on bioaccumulated murine norovirus-1 (MNV-1) and survivability, inherent microbiota, color, and firmness of Atlantic oysters (Crassostrea virginica) during storage at 5�C for 20 days. Food Control. 73:1189-1194.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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.
Impacts
(N/A)
Publications
- 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.
- 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.
- 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.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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. 09 Various edible proteins, including whey protein concentrate, bovine serum albumin and �-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.
Impacts
(N/A)
Publications
- 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.
- Narayanan, L.A., Edelmann, M. 2014. Ubiquitination as an efficient molecular strategy employed in salmonella infection. Frontiers in Immunology. 5:558.
- 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.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. The results indicated that more than 5-log colony forming unit (CFU) reduction of Salmonella enterica populations on tuna was achieved using 0. 5 kilogray(kGy) X-ray irradiation. Furthermore, 0.6 kGy X-ray reduced Salmonella to less than detectable level. Heating treatment of Listeria (L.) moncytogenes strains showed a diversity of heat tolerance. Exposing to sublethal heat would increase heat tolerance of the pathogen. Pocket size detection kits for detecting food borne are being validated against official methods. The low cost one-tube method was successfully adapted to 96-well microplate system. The next step is to conduct validation study for Vibrio vulnificus and Vibrio parahaemolyticus. A quantitative polymerase chain reaction (PCR) assays was developed and applied to differentiate genetic variants of Edwardsiella (E.) tarda for comparative evaluation of pathology, virulence and dissemination in channel catfish and hybrid catfish. Results showed E. piscicida may be more prevalent in catfish aquaculture than E. tarda. Results showed pegylated gold nanoparticles (AuNPs) could be used as an important platform for studying ligand interactions with AuNps. Researchers at Mississippi State University (MSU) have discovered new primers that can differentiate serotypes of Listeria pathogens. Development of molecular methods to distinguish high-risk serotypes and low-risk serotypes of Listeria would enable determination of the risk associated with the isolation of Listeria monocytogenes from seafood, including channel catfish. A Listeria monocytogenes surface anchor protein was found not to contribute to listerial collagen binding or cell adherence, but appeared to contribute to virulence and intracellular replication. The use of small molecules to block the attachment of LmF2365 to catfish fillet is being continued. Scientists at MSU showed a protein ImoH7858_0396 was a novel virulent factor of L. monocytogenes, and deletion of an alkylated DNA (AlkD)-like genetic determinant affected flagellar motility gene expression, biofilm formation, virulence and stress responses of L. moncytogenes. The physiological functions of deubiquitinating enzymes (DUBs) as potential drug targets in Salmonellosis in catfish and chicken were characterized. Chemical proteomics data showed that activities of host DUBs were regulated during Salmonella infection. Cheddar whey solution (2%, weight/volume) was subjected to nanoemulsification by high pressure homogenization at pressures. The results indicated samples treated with both chedder whey (CW) (subjected to 20,000 PSI of pressure) and chitosan exhibited the best results for storage of the catfish fillet. There was an almost six-fold reduction in oxidative degradation as a result of nano-emulsion coating containing chitosan. The stability and antioxidative activities of a nanoemulsion consisting of a whey protein concentrate and peanut oil were investigated. Based on total radical trapping potentials (TRAP) analyses, the antioxidative potential of the nanoemulsions were significantly enhanced by adding sulfobetaine 3-10, though further addition of Trition X-100 had no significant effect on antioxidative properties. Characterization of phenolic substances and antioxidant capacity of muscadine grape and quality of food grade soybeans have been initiated. Thirteen major varieties bred by ARS researchers at the Cochran Fruit Research Center in Mississippi were analyzed for juice quality and health promotion components. Results showed that black varieties had the highest phenolic substances. However, oxygen radical absorbance capacity was not associated the skin color. Food grade soybeans grown by the ARS researchers in Stoneville, MS, were analyzed for protein quality for making foods. Results showed some varieties have potential to be utilized for foods. Significant Activities that Support Special Target Populations: Large amount of catfish consumed in the United States is imported due to the low cost of the foreign products. The United States Department of Agriculture (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 aquaculture products in 2013 was about $7.5 billion. Mississippi catfish production ranks number one in the nation with $165 million value. The catfish production has been in decrease phase due to foreign competition and high cost of fish feed. Concomitantly, other agricultural products are increasing overtime with chicken and soybeans being the two largest crops. For the U.S. catfish growers to compete for the market share, it is important to conduct research for the development of safe, high quality domestic catfish products. 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. Mississippi State scientist has visited food industries in Mississippi to discuss how we may use our research to support the science community as well as the agricultural, aquaculture and food industry. Our current findings on the mechanisms for pathogens on formation and their control by X-ray, coating with antioxidants and other processing methods were presented in various meetings including the 2014 annual meeting of the Institute of Food Technologists in New Orleans, LA. Validation of the rapid pathogen detection system will lead to a low-cost product that can be used to enhance safety of catfish and seafood, fresh produce and poultry products to contribute to the marketability of these products produced in the United States. This research also will contribute to the expansion of the manufacturing sectors to utilize the abundant Mississippi agricultural products to produce value-added end products to enhance the economy of the food and agricultural communities. Accomplishments 01 Development of X-ray technology for reducing Salmonella pathogens. Researchers at Mississippi State University discovered X-ray at 0.5-0.6 kilogray (kGy) could effectively reduce Salmonella enterica in tuna fillet. Spoilage microorganisms are also significantly reduced. This is important since tuna is eaten raw and the flavor and texture of X- ray irradiated products can be preserved. 02 Development of rapid assays for detection of Vibrios species. Scientists at Mississippi State University (MSU) have improved the sensitivity of the rapid single tube (pocket size) assays for independent detection of Vibrio vulnificus and Vibrio parahaemolyticus strains of bacteria from seafood by converting it to a 96-well pocket size detection systems while maintaining the reliability, sensitivity, and rapidity. The test is still being validated by the Food and Drug Administration (FDA) laboratory in Alabama. One grant proposal has been submitted to National Oceanic and Atmospheric Administration for competitive funding. 03 Development and application of quantitative polymerase chain reaction (PCR) assays for genetic variants of Edwardsiella (E.) tarda. Mississippi State University (MSU) scientists have completed the study on the development and application of quantitative PCR assays for genetic variants of Edwardsiella (E.) tarda for comparative evaluation of pathology, virulence and dissemination in channel, blue and hybrid catfish. Successful primers and molecular methods have been developed to differentiate various types of Edwardsiella species. MSU Scientists, at the National Warmwater Aquaculture Center (NWAC) in Stoneville, MS, have found that E. piscicida is more prevalent than Edwardsiella (E.) tarda in catfish aquaculture in southeastern region of the USA. A journal article has been published, and two presentations were made in scientific meetings. 04 Characterization of attachment factors of Listeria (L.) monocytogenes. Scientists at Mississippi State University (MSU) have identified and characterized an attachment factor of L. monocytogenes. A Listeria monocytogenes surface anchor protein has been found not to contribute to listerial collagen binding or cell adherence, but appeared to contribute to virulence and intracellular replication. The resistance and subsequent adaptation of biofilm cells of foodborne bacterial pathogens to any antimicrobials by a frequent repetitive exposure is a great concern. An internalin-like protein that is involved in the adhesion and invasion of Listeria monocytogenes is also a novel virulence factor. A protein encoded by alkylated DNA (AlkD)-like protein is a bifunctional protein that represses flagellar motility and influnences stress responses through its DNA glycosylase activity. This result provides insides for biofilm formation and virulence of Listeria. 05 Role of physiological functions of deubiquinating enzymes in salmonellosis. Scientists at Mississippi State University (MSU) have identified potential drug targets in foodborne disease salmonellosis and developed a sample preparation method which can be used for tissue- based proteomics in animals to study catalytically active enzymes with important cellular functions. 06 Use of nanoemulsified cheddar whey (CW) and chitosan to reduce oxidative degradation of farm raised retailed cut catfish fillet. The results have potential to lead to the development of natural preservatives with remarkable antioxidative efficacy, that can significantly increase the shelf-life of farm raised retailed cut catfish fillet, without the undesirable health effects resulting from synthetic preservative agents.
Impacts
(N/A)
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. The results indicated that the 0.1 and 0.2 kGy X-ray treatments significantly reduced the Salmonella populations on tuna samples. Biomarkers on Vibrio vulnificus and Salmonella were discovered and optimized on its expression in 24 hours. Researchers at Mississippi State University (MSU) have been successful in developing polymerase chain reaction (PCR) primers that can differentiate serotypes of Salmonella. New primer sets are currently being tested to differentiate serotypes 1/ 2a and 3b. Identification and evaluation of attachment of Listeria monocytogenes on the surface of ready-to-eat products have been started. Scientists at MSU have screened 32 genes encoding surface proteins and lipases of the strain to find highly expressed genes on lettuce leaves. Results showed that transcription levels of five genes were significantly up-regulated on lettuce leaves. It was a hypothesized one of these genes involved in an attachment to vegetables and the gene was named Listeria cellulose- binding protein (LCP). Scientists at MSU constructed a LCP mutant (LCP) by deleting the LCP gene and this mutant lost its ability to attach on the surfaces of leafy vegetables and cantaloupe skin. Results strongly suggest that LCP plays an important role in an attachment to vegetables and fruits. Scientists at MSU investigated the effects of essential oils of thyme and oregano and their antimicrobial phenolic constituent carvacrol were evaluated for their ability to inhibit biofilm formation and inactivate preformed Salmonella biofilms. The presence of nonbiocidal concentrations of thyme oil, oregano oil, and phenolic carvacrol at 0.006 to 0.012% suppressed Salmonella spp. Scientists at MSU are using novel proteomic techniques to discover new biomarkers in chicken and catfish relevant in foodborne disease, such as chicken salmonellosis. ARS Scientists are also discovering new active enzymes (kinases and deubiquitinases) and have developed and optimized an animal tissue processing method, which is compatible with the chemical proteomics to uncover these enzygmes. Techniques to identify novel enzygmes through mass spectrometry have been developed and have discovered a gene in animal macrophage cells (UCHL5) involved in bacterial survival in macrophages. The quality attributes, descriptive sensory characteristics, and muscle proteomes (sarcoplasmic and myofibrillar) of normal and reddish channel catfish (Ictalurus punctatus) fillets were evaluated. Reddish fillets had greater redness and yellowness values and less lightness than normal fillets, but no differences existed in cooking loss and instrumental texture. The reddish fillets were more bitter, earthy, grassy, metallic, and oxidized, and had more off-flavor than normal fillets. Analyses of the sarcoplasmic proteome revealed higher abundance of hemoglobin (beta) in reddish fillets. Scientists at MSU identified the protein biomarkers responsible for redness in catfish fillets. Significant Activities that Support Special Target Populations: The United States Department of Agriculture (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. Expansion of markets through development of safe, high quality domestic catfish products will benefit United States catfish farmers. 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. Mississippi State scientist has visited food industries in Mississippi to discuss how we may use our research to support the science community as well as the agricultural, aquacultural and food industry. Our findings on the effect of temperature on Salmonella (S.) Typhimurium and S. enterica biofilm formation and their control by essential oils was presented at the International Food Technology 2012 meetings in Las Vegas, June 24-29, 2012. ARS Scientist have set up collaboration with University of Arkansas to identify changes in protein expression in poultry upon Salmonella infection by using different Salmonella serovars and mutants. Accomplishments 01 Development of single tube assays for detection of Vibrios species. Scientists at Mississippi State University (MSU) have developed single tube (pocket size) assays for independent detection of Vibrio vulnificus and Vibrio parahaemolyticus strains of bacteria from seafood by using the unique biochemical features found in each of these organisms. The test is still to be validated by the FDA laboratory in Alabama. At the conclusion of another segment of this project, ARS Scientists anticipate developing a multiplex quantitative polymerase chain reaction (PCR) assay for detection of all high-risk Listeria monocytogenes (LM) serotypes. ARS Scientists have already shown that multiplex PCR with primers from LMOF2365_0088 and LMOF2365_2059 will detect isolates in serotypes 1/2a, 1/2b, 1/2c, and 4b. In the currently proposed project, ARS Scientists will develop primer/probe combinations that will allow this assay to be quantitative. ARS Scientists also anticipate improving this assay to allow distinction of serotypes 3c, 3b, 4d, and 4e from the high-risk serotypes. 02 Development and application of quantitative polymerase chain reaction (PCR) assays for genetic variants of Edwardsiella tarda. Mississippi State University (MSU) scientists have begun the study on the development and application of quantitative PCR assays for genetic variants of Edwardsiella (E.) tarda for comparative evaluation of pathology, virulence and dissemination in channel, blue and hybrid catfish. ARS Scientists have developed and validated a set of PCR primers to distinguish between various phenotypically indistinguishable Edwardsiella's and have identified that of the three (E. tarda, E. piscicida and E. piscicida-like), E. piscicida is predominant in catfish aquaculture. A manuscript has been submitted to the Diseases of Aquatic Organisms and is currently under review. 03 Characterization of attachment factors of Listeria (L.) monocytogenes. Scientists at Mississippi State University (MSU) have identified and characterized an attachment factor of L. monocytogenes. This study helps understand how L. monocytogenes attaches on the surface of vegetables and fruits and the information may help to prevent the initial attachment. The resistance and subsequent adaptation of biofilm cells of foodborne bacterial pathogens to any antimicrobials by a frequent repetitive exposure is a great concern. This work shows that the essential oils of thyme, oregano and carvacrol show strong antimicrobial activity against Salmonella biofilm cells that are present on polystyrene or stainless steel coupon surfaces. Essential oils are made up of several antimicrobial constituents, so there is less chance of adaptive resistance and these compounds can serve as a hurdle response to bacterial cell adaptation. 04 Role of physiological functions of deubiquinating enzymes in salmonellosis. Scientists at Mississippi State Univeristy (MSU) have identified potential drug targets in foodborne disease salmonellosis and developed a sample preparation method which can be used for tissue- based proteomics in animals to study catalytically active enzymes with important cellular functions. 05 Factors related to development of red fillet in catfish. From the study on determining the relationships between environment factors, catfish, physiology, rigor mortis and meat quality, a method was developed to characterize the proteome of catfish fillets with red color defect.
Impacts
(N/A)
Publications
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. Research related to quantification, identification and control of pathogens in food products, in particular Listeria (L.) monocytogenes, is focused on developing strategies for understanding virulence factors and reducing L. monocytogenes in catfish, seafood, and other meat products. Virulent factors for L. monocytogenes have been identified as well as proteins responsible for attachment strength on reay-to-eat (RTE) foods. This information will be critical in developing tests and protocols required to control L. monocytogenes in catfish, seafood, and other RTE products. Rapid and sensitive tests for detection of Salmonella and Escherichia (E) coli 0157:H7, and were developed, whereas tests for detection of Vibrio vulnificus and Listeria, and botulism neurotoxin are being developed and validated. These assays assay will be useful for screening live/on farm and final products. Understanding of catfish fillet redness is being studied with the vision of helping develop industry guidelines to enhance filet color uniformity or predict filet color. Antimicrobials in the form of bacteriophages or naturally-derived were tested against pathogens in vitro and in seafood matrices with good results. Minimum inhibitory concentrations were found for each of the compounds or for those used in a mix. Ready-to-eat catfish products were developed. These could increase the offer of value-added catfish products in the market, expanding the industry competitiveness edge. These research projects will play a role in assuring safe, high-quality food products to consumers and will be important to maintaining viable aquaculture and seafood industries in the U.S. Significant Activities that Support Special Target Populations: Assuring safety and improving quality of domestic farm-raised catfish will benefit operators of small farms by expanding markets for catfish products. 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. Expansion of markets through development of safe, high quality domestic catfish products will benefit U.S. catfish farmers, a significant portion of which are classified as small farmers. Accomplishments 01 Determination of the genome sequence of a virulent serotype of Listeria monocytogenes. Listeria monocytogenes is a human pathogen but some strains and other species could be non-virulent. This work determined th genomic sequence and identified 58 virulence-specific genes for a virule strain of Listeria monocytogenes. This can potentially allow development of methods to distinguish pathogenic and nonpathogenic isolates. Polymerase chain reaction (PCR) assays based on eight of these virulence specific genes were tested on a panel of listerial isolates, and two of the genes showed good potential. The goal is development of a multiplex real-time PCR assay for simultaneous and rapid differentiation and quantification of high-risk and low-risk L. monocytogenes serovars from food, particularly aquaculture products. This assay will allow us to address important questions regarding persistence and replication of different listerial serotypes in food and in processing environments. 02 Novel genetic differences between pathogenic and nonpathogenic isolates. Discrimination between pathogenic and non-pathogenic Listeria (L.) monocytogenes with accurate diagnostic tools can help the industry (food and fish) better discern the safety of their products. A large number of food recalls occurs annually because of the possibility of being contaminated with L. monocytogenes, even though the strain may not be virulent. The identification of novel targets that could differentiate pathogenic and nonpathogenic isolates has taken on increased importance. Results show that one could discern between pathogenic and non-pathogeni strains by determing the presence or absence of certain genes. This coul lead to the development of tools to identify whether a product or animal carries a pathogenic strain or whether it is safe to eat even though it may carry Listeria monocytogenes. 03 Natural antimicrobials that can inhibit Vibrio (V.) vulnificus and V. parahaemolyticus in shucked oysters. Vibrio in oysters account for many foodborne outbreaks, are responsible for economic and social hardship to fishermen and others, and also responsible for increased health care cos This work looked at some natural antimicrobials that could be utilized inhibit Vibrio spp. growth and thus prevent foodborne illnesses. It was found that citric acid, grape seed extract and lactic acid solutions at fairly high concentrations could be used to suppress growth of these pathogens. These findings can serve to develop formulations to add to oysters to minimize foodborne illnesses when consumed. 04 Attachment of Listeria monocytogenes on the surface of ready-to-eat (RTE products. Understanding the mechanisms of attachment may help to elucidate the persistence of Listeria monocytogenes in ready-to-eat products and thus find ways to inhibit it by detaching it from the food not allowing attaching to the food. A gene was identified as responsible for attachment (at least in part) of Listeria monocytogenes in RTE foods like fresh fruits and vegetables. The binding was found to occur with cellulosic material in the fresh produce. This hypothesis was tested and validated. Thus, a system to prevent attachment to cellulosic material o to detach the organism can be designed. This can enhance the safety of fresh and fresh-cut fruits and vegetables, a category of foods responsib for a large number of foodborne outbreaks annually. 05 Enhanced functional properties of tannic acid after thermal hydrolysis. Tannic acid has been proven to possess antioxidant and antimicrobial properties thus being a powerful bioactive component. However, there cou be potential for enhancing its functional properties by manipulation of the compound per se or in food or other matrices. Thermal hydrolysis at high pressures of fresh tannic acid resulted in shorter chain but more powerful components produced: gallic acid and pyrogallol. This process could be utilized to enhance natural antimicrobial properties of tannic- acid containing foods or to enhance functional properties of tannic acid to be used as additives in food and feed. 06 Detection of Botulinum toxin contamination of catfish. Clostridium Botulinum Type E is associated with seafood including aquaculture produc It has been discovered that this organism is beginning to affect fish a the farm level since it produces a powerful toxin, Neurotoxin E. It is very difficult to separate the toxin from a complex matrix like fish. Preliminary results have shown progress in eliminating enzymes responsib for eliminating the toxin and thus make the detection method more sensitive and accurate. The development of this procedure will aid the catfish and other aquaculture/seafood industries tremendously as a diagnostics tool to react and treat prior to total loss. 07 Potassium acetate and lactate enhance quality of marinated catfish fille Shelf-life of fresh/iced catfish fillets and other fresh products is a problem in the industry. Development of systems that can extend shelf-li while adding value to these products can be beneficial to the industry. The treated fillets had a longer self-life and were preferred by consume (when fried) over untreated fillets. Thus, incorporation of these organi salts in catfish products not only enhances their quality but also provides for longer shelf-life and thus fewer losses. 08 A quick and easy test for identification of human pathogens in seafood a other products and the environment. Testing at the processing level for the presence of pathogens in the food is very tedious, time consuming an requires lots of training for the most part. Novel testing kits utilizin two-phase media with a mix of indicators, growth promoters and/or inhibitors have been developed or are being developed. These kits will allow processors and handlers of food to screen their production lots an their environments in relatively short times for the presence of pathoge and thus clear product for market in a rapid and reliable manner. 09 Prevention of food safety and food quality problems associated with larv nematodes in commercial catfish. The most prevalent population of turtl in commercial catfish ponds are the red-eared sliders and that they are frequently heavily infected with two nematodes, Serpinema sp. and Camallanus sp. which can also infect fish. Both Serpinema and Camallanu have been sequenced and work is underway to develop molecular probes to screen for their presence. This work will provide for a rapid screening tool for these nematodes and thus reduce fish losses at the farm.
Impacts
(N/A)
Publications
- Soni, K., Nannapaneni, R. 2010. Bacteriophage significantly reduces Listeria monocytogenes on raw salmon fillet tissue. Journal of Food Protection. 73:32-38.
- Donaldson, J.B., Nanduri, B., Pittman, J., Givaruangsawar, S., Burgess, S. C., Lawrence, M.L. 2011. Proteomic expression profiles of virulent and avirulent strains of Listeria monocytogenes isolated from macrophages. Journal of Proteomics. 74(10):1906-1917.
- Steel, C.L., Donaldson, J.R., Paul, D., Barnes, M.M., Burgess, S.C., Arick, T., Bridges, S.M., Lawrence, M.L. 2011. Genome sequesnce of lineage III Listeria monocytogenes strain HCC23. Journal of Bacteriology. 193:3679- 3680.
- Kim, T., Silva, J.L., Jung, Y. 2010. Enhanced functional properties of tannic acid after thermal hydrolysis. Food Chemistry. 126:116-120.
- Soni, K., Nannapaneni, R., Tasara, T. 2011. The contribution of transcriptomic and proteomic analysis in elucidating stress adaptation responses of Listeria monocytogenes. Foodborne Pathogens and Disease. 8:842-852.
- Bradley, E.M., Williams, B., Schilling, W., Coggins, P., Crist, C., Yoder, S.W. 2011. Effects of sodium lactate and acetic acid derivatives on the quality and sensory characteristics of hot-boned pork sausage patties. Meat Science. 88:145-150.
- Soni, K., Nannapaneni, R., Tasara, T. 2011. An overview of stress response proteomes in Listeria monocytogenes. Agriculture, Food and Analytical Bacteriology. 1:66-85.
- Kin, S., Schilling, W., Smith, B.S., Silva, J., Kim, T., Pham, A. 2011. Potassium acetate and potassium lactate enhance the microbiological and physical properties of marinated catfish fillets. Journal of Food Science. 76(4):5242-5250.
- Soni, K., Nannapaneni, R., Hagens, S. 2010. Reduction of Listeria monocytogenes on the surface of fresh channel catfish fillets by bacteriophage listex P100. Foodborne Pathogens and Disease. 7:427-434.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. Approach (from AD-416) These funds will be used for 1) Extramural research with Mississippi Center for Food Safety and 2) Aflatoxin research. Research related to control of pathogens in food products, in particular Listeria monocytogenes, is focused on developing strategies for reducing pathogens in catfish, seafood, and other meat products. New generally regarded as safe (GRAS) antimicrobials for control of bacterial pathogens in aquaculture products are being developed and tested for their effectiveness. Effects of irradiation on shelf-life extension and pathogen reduction are being determined for several foods, including catfish. A rapid, inexpensive antibody-based assay is being developed for detecting botulism neurotoxin in catfish. This assay will be useful for screening purposes. A luciferase reporter gene has been inserted into Salmonella enterica and selectively produces light in viable cells. This technique allows real-time detection of the bacteria at low levels and allows monitoring of bacterial progression under different environmental and chemical conditions. Training of catfish producers and processors was conducted to reduce potential biosecurity and food safety risks. These research projects will play a role in assuring safe, high-quality food products to consumers and will be important to maintaining viable aquaculture and seafood industries in the U.S. Accomplishments 01 Inactivation of Salmonella enetrica on cantaloupes and mangos by X-ray. Fruits including cantaloupe and mango are an essential part of healthy eating. The United States Department of Agriculture recommends that consumers include more fruit and vegetables in their diet to decrease th risk of cardiovascular disease and cancer. As produce consumption has increased, a significant increase in the number of foodborne disease outbreaks and illnesses associated with fresh produce has been reported. Many outbreaks, especially those caused by the bacterial pathogen Salmonella, have been associated with cantaloupe and mango. Mississippi State University scientists determined effects of X-ray on reduction of bacteria and shelf-life in cantaloupes and mangos inoculated with Salmonella. Salmonella was reduced more than 10,000,000 cells per gram cantaloupe and mango with 2.0 and 1.0 kGy X-ray doses. In addition the inherent microflora (other bacteria, yeasts, and molds) was reduced to less than 10 cells per gram. This research demonstrates X-ray irradiati is an effective method for reducing pathogens and microflora in cantaloupes and mangos and has important implication for food safety. 02 Inactivation of Salmonella, Escherichia (E.) coli O157: H7 and Shigella species on parsley and green peppers by X-ray. Several recent foodborne disease outbreaks associated with vegetables including parsley and peppe have been reported. Outbreaks have been associated with bacterial pathogens Salmonella, Escherichia coli O157:H7 and Shigella spp. Mississippi State University scientists studied effects of X-ray doses ( 1, 0.5, 1.0 and 1.5 kGy) on parsley and green peppers inoculated with Escherichia coli O157:H7, Salmonella enterica and Shigella flexneri. The effect of X-ray on microflora counts (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated parsley and green peppers was also evaluated. Pathogenic bacteria were nearly emlinated on green peppers and parsley with 1.0 and 1.5 kGy X-ray. Furthermore, treatment with X-ray significantly reduced the initial inherent microflora on parsley and green peppers and throughout refrigerated storage for 30 days. This research demonstrates significant benefit to consumers through use of X-ray to reduce pathogens and extend shelf-life in parsley and green peppers. 03 Reduction of Listeria monocytogenes on smoked salmon and catfish using X ray treatment. Listeria monocytogenes is a foodborne pathogen that caus severe disease in humans. Listeria monocytogenes is widely found in the environment and has been isolated from seafood products. L. monocytogene can easily be destroyed by cooking in contaminated seafood; however its presence in ready-to-eat seafood, particularly smoked fish, is a safety concern. Mississippi State University scientists determined the efficacy of X-ray irradiation on the inactivation of Listeria monocytogenes and shelf-life of smoked salmon and catfish. Smoked catfish and salmon fille were inoculated with a 3-strain mixture of Listeria monocytogenes and treated with 0.1, 0.5, 1.0 and 2.0 kGy X-ray. Un-irradiated and irradiated samples were stored at 5�C for 35 days and periodically teste for L. monocytogenes counts. X-ray of 1.0 kGy significantly reduced all bacteria, including L. monocytogenes. X-ray of 2.0 kGy extended shelf-li to 35 days in smoked salmon and 30 days in smoked catfish. This researc demonstrated X-ray irradiation can be used to improved safety and extend shelf-life of smoked fish and is beneficial to consumers. 04 Characterization of Campylobacter and E. coli in a real-time bioluminescent assay. Catfish, red meat and poultry processing fall und the authority of the United States Department of Agriculture Food Safety Inspection Service. The goal of Mississippi State University scientists to assist the food animal industry in meeting current and future federal safety regulations. As part of this effort we have established and test an in vitro real-time bioluminescent-based reporter system for monitorin Salmonella and Escherichia coli in food production and processing environments. This system is based on the light-producing gene (lux) whi is inserted into the bacterial chromosome causing emission of light and allows rapid, efficient detection of these pathogens. We demonstrated th system was effective for tracking Salmonella and E. coli on refrigerated raw catfish fillets, boneless chicken fillets, and ground beefs. Using t reporter system, we evaluated three antimicrobial compounds for their efficacy to inhibit growth of E. coli. We also initiated work to allow u to study the attachment of Salmonella to chicken skin. Our work demonstrates the utility of this reporter system for use in studying the foodborne pathogens. Understanding this mechanism will aid in characterizing persistence of Salmonella and E. coli, two common and important food pathogens, in production and processing environments. 05 Prevention of potential food quality problems associated with an emergin larval nematode in commercial catfish meat. Recently unidentified larva nematodes were found in the skin and muscle of commercial catfish, causi lesions that compromised the quality and appearance of the whole meat an fillet product. These parasites were tentatively identified as drucunculoids with aquatic turtles, fish and aquatic invertebrates as hosts in the life cycle of these parasites. The objectives of this project were to: confirm the host (turtle species) involved in the life cycle of this unknown larval nematode; identify the invertebrates that serve as intermediate hosts for the larval stages of this parasite, and develop polymerase chain reaction (PCR)-specific probes to detect the li stages of this parasite in the invertebrate hosts. The parasite was sequenced and found to be 98% homologous with the spiruroid Camillanus ( oxycephalus confirming that red-eared sliders in catfish ponds are infected with at least one nematode, S. trispinosum, which has the potential to infect catfish. Invertebrate collections were also done in additional ponds (n=5) with histories of poor performance, and no larval nematode stages were found in these ponds. We determined that red-ear slider turtles are infected with a nematode that also infects fish. We have now generated sequencing data both on this parasite and the unknown drucunculoid-like nematode which can be used in the development of PCR primers that will be more efficient and sensitive in the detection of other stages of these parasites in the fish and invertebrate hosts. This will provide the diagnostic tools necessary for the development of management programs to control/eradicate these invertebrates and interru the life cycle prior to infection, preventing the encystment of these larval stages in the catfish tissue. 06 Assessment, development and implementation of training materials for foo defense/safety and biosecurity issues within the catfish processing industry. Agriculture has been listed as a potential target for terrori in which the security of our food supply may be breached. Food defense a traceability plans are designed to evaluate all areas of a business and infrastructure including personnel, ingredients, supplies, transportatio processing, distribution, and product tracking. The objective of this research and subsequent training sessions was to promote awareness and educate catfish farmers and processors on the principles of food defense biosecurity and traceability of agricultural commodities and ingredients used in human food production. Training sessions were provided at a choi of two different dates and locations. Participants were asked a series o questions involving different areas of food defense, safety, biosecurity and traceability. Of those surveyed, 74% said they have a food defense plan; 48% had a working traceability plan; and 52% had a biosecurity pla for catfish. Additionally, a pre-and post-assessment were given to evaluate the effectiveness of the training sessions. Overall 68% of participants earned an equal or higher score on the post-assessment. Training will continue through seminars, workshops, supplementary materials, and checklists. While many larger and corporate structured enterprises in aquaculture were found to be more prepared on certain principles, there is a need to advance training and knowledge. With prop training and awareness, farmers and others throughout the chain are bett prepared to continue, and even increase, the security of our food supply 07 Use of acid and sweet whey as edible coating to control oxidative deterioration of catfish fillets. Retail value of U.S. farm grown catfi was estimated to be over $1.56 billion in 2005 and is an important contributor to the economies of numerous southern states including Mississippi. Unfortunately, oxidative degradation resulting in off-color and flavor development starts once any muscle foods are processed. Henc there is significant commercial interest in identifying food-grade antioxidants for use as coating agents. We determined that acid (cottage cheese) and sweet (Edam and Cheddar) whey could effectively and inexpensively be used in edible coatings to control the oxidative deterioration of catfish fillet. In addition we found that controlled he treatment (thermization) enhanced antioxidative capacity of whey based o measurement of protein degradation indicators in catfish fillets. Over storage period of 14 days (2�C), all treatments dramatically and favorab affected these indicators (e.g., average 66% reduction by the treatments in amines compared to uncoated control). Colorimetry indicated significant improvement in fillet color in all treatments with Cheddar whey being the best. Moisture loss during storage was reduced an averag of approximately 30% in all treatments with Cheddar whey again being the best. This was speculated to be due to greater amount of casein peptides and free amino acids in Cheddar whey. Data indicate that natural, nutritious, inexpensive and abundantly available whey, with or without thermization, can significantly enhance shelf quality and benefit the catfish industry and consumers. 08 The development of an ultra-sensitive analytical method to detect Botulinum toxin contamination of catfish. Visceral toxicosis of catfish (VTC) is a relatively new disease syndrome that affects food size catfis raised in commercial catfish ponds. Over the past ten years losses associated with this disease have been estimated to be in excess of one million pounds. Serum neutralization studies and endopep mass spectrometry indicate the disease is a form of botulism, caused by a tox produced by the bacterium Clostridium botulium. Development of a sensiti low cost, toxin free assay for measuring catfish toxin-specific antibod production will be useful for monitoring and managing this disease. We continued work to optimize extraction of Botulinum neurotoxin (BoNT) fro catfish serum, muscle and intestinal tract; improve sensitivity and quantitative detection capabilities for BoNT; and initiated development methods to study intact BoNT. Fish muscle/solvent ratios for BoNT extraction were optimized and the serum protein sample preparation was modified by filtration which increased BoNT detection via liquid chromatography-mass spectrometry/mass spectometry (LC-MS/MS). Applicatio of new technology and software allowed detection of the toxin at the 166 pg level. Work with BoNT is complicated by the toxicity of the intact protein. The model protein Proteinase K was selected and is being used t test technologies and develop protocols that can be used to study intact BoNT. This research will provide insights into monitoring and managing V in catfish. 09 New generally regarded as safe bacteriophage LISTEXTM P100 for reduction of Listeria monocytogenes in aquaculture products. Listeria monocytogen is a deadly foodborne pathogen. Freshwater and marine fish are sometimes inadvertently contaminated with very low levels of L. monocytogenes. Intervention/elimination strategies for L. monocytogenes in processed, r and smoked seafood products are currently inadequate. Recently the Food and Drug Administration and the United States Department of Agriculture approved a novel antimicrobial, bacteriophage Listex P100, which is commercially available and has generally regarded as safe (GRAS) status for the application on both raw and ready-to-eat food products to contro L. monocytogenes. Mississippi State University scientists� research results reveal a strong bactericidal activity of bacteriophage P100 phag against L. monocytogenes on raw salmon fillets. Previous studies demonstrate that the L. monocytogenes cells present in a biofilm matrix show greater resistance to antimicrobial agents than those present in planktonic conditions. Our work demonstrated the potential of phage treatment as an alternative strategy for the control of L. monocytogenes biofilms on the stainless steel surfaces. This technology has potential to improve control of L. monocytogenes in seafood processing environment 10 Identification and evaluation of attachment of Listeria monocytogenes on the surface of ready-to-eat products. Listeria (L.) monocytogenes is we known to possess a strong attachment and biofilm formation abilities. Th bacterium has unique ability to attach to various ready-to-eat (RTE) surfaces and processing equipment which makes it difficult to prevent th cross contamination during food processing. Mississippi State University scientists determined surface proteins and virulence factors of L. monocytogenes associated with attachment on vegetables. Activated listerial cells were inoculated on lettuce leaves and incubated at 4�C. tested 31 genes encoding surface proteins and 8 genes encoding virulence factors of L. monocytogenes using real-time PCR. The transcriptional levels of some surface proteins on the lettuce leaf were up-regulated after inoculation. However, the gene expression of virulence factors did not change. The results suggest that cell surface proteins and sigB (a k regulator of general stress) may be involved in attachment of Listeria t surfaces. This study will help us understand how L. monocytogenes surviv or grow on the surface of leafy vegetables. The information will help preventive strategies to reduce the contamination of L. monocytogenes on vegetables.
Impacts
(N/A)
Publications
- Bae, D., Crowley, M.R., Wang, C. 2011. Transcriptpome analysis of Listeria monocytogenes grown on a ready to eat meat matrix. Journal of Food Protection. 74:1104-1111.
- Wang, C., Chou, C., Tseng, C., Ge, X., Pinchuk, L.M. 2011. Early gene response of human brain endothelial cells to Listeria monocytogenes. Canadian Journal of Microbiology. 57:441-446.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. Approach (from AD-416) These funds will be used for 1) Extramural research with Mississippi Center for Food Safety and 2) Aflatoxin research. This report serves to document research conducted under Specific Cooperative Agreements 0500-00031-001-02S, 0500-00031-001-03S, and 0500- 00031-001-04 (Mississippi Center For Food Safety and Postharvest Technology) between the ARS and Mississippi State University. Research related to control of pathogens in food products, in particular Listeria (L.) monocytogenes, is focused on developing strategies for reducing L. monocytogenes in catfish, seafood, and other meat products. New generally regarded as safe (GRAS) antimicrobials for control of bacterial pathogens in ready-to-eat products are being developed and tested for their effectiveness. Studies on the genetic and environmental determinants of virulence of L. monocytogenes have demonstrated that both genotype and environment play a role in the virulence of this important food pathogen. This information will be critical in developing tests and protocols required to control L. monocytogenes in catfish, seafood, and other meat products. A rapid, inexpensive antibody-based assay is being developed for detecting botulism neurotoxin. This assay will be useful for screening purposes. A luciferase reporter gene has been inserted into Salmonella enterica and selectively produces light in viable cells. This technique allows real-time detection of the bacteria at low levels and allows monitoring of bacterial progression under different environmental and chemical conditions. Bacteria numbers and bioluminescence correlated well in all strains. These research projects will play a role in assuring safe, high-quality food products to consumers and will be important to maintaining viable aquaculture and seafood industries in the U.S. Significant Activities that Support Special Target Populations Assuring safety and improving quality of domestic farm-raised catfish will benefit operators of small farms by expanding markets for catfish products. 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 1,370 catfish farms in the United States, 515 farms (38% of the total) reported annual revenues of less than $25,000. Expansion of markets through development safe, high quality domestic catfish products will benefit U.S. catfish farmers, a significant portion of which are classified as small farmers. Accomplishments 01 Improving the Safety of Oysters and Shrimp by X-ray Irradiation. The lon term goal of this project is to improve the safety of seafood (oysters a shrimp) using X-ray technology for the seafood industry under approved regulatory status. We determined the efficacy of X-ray on the inactivati of pathogenic bacteria (E. coli O157: H7, Salmonella spp., Shigella spp. and Vibrio spp.) on seafood. Live, freshly harvested Atlantic oysters (Crassostrea virginica), and frozen, cooked and peeled ready to eat (RTE shrimp with tails were used in this study. A cocktail mixture of three strains of each pathogen: E. coli O157:H7 (C7927, EDL933 and 204P); Salmonella (S.) enterica (S. enteritidis, S. typhimurium and S. newport) and V. parahaemolyticus (ATCC 17802, 2006-002 and 2006-005) were used as inoculants of fresh oysters and thawed shrimp. Irradiation doses (0.1, 2, 0.3, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 kGy) were generated using the RS 2400 system. At each examined dose, two treated samples of oysters or shrimp were removed from the exposure chamber and bacterial populations were isolated and enumerated. More than one million cells of coli O157: H7, S. enterica, S. flexneri and Vibrio (V.) parahaemolyticu were reduced on RTE shrimp after treatment with 2-4 kGy X-ray. Also treatment with low dose (0.75 kGy) killed all inherent bacteria on RTE shrimp. X-ray treatments significantly reduced the population of Vibrio parahaemolyticus in oysters. Vibrio parahaemolyticus reduced by more tha one million cells per gram with 2.0 kGy X-ray doses in half shell oyster The same reduction was achieved with 5.0 kGy X-ray doses in whole shell oysters. X-ray treatment did not kill the oysters even with the highest dose (5.0 kGy). 02 New Generally Regarded as Safe (GRAS) Bacteriophage LISTEXTM P100 for Quantitative Reduction of Listeria (L.) monocytogenes Loads in Aquacultu Products/Seafood Products. The new 2008 farm bill legislation (PL 110-24 signed into law on June 18, 2008 extends Food Safety Inspection Service (FSIS�s) inspection jurisdiction to include domestic and imported farmraised catfish. This legislation may lead to new federal regulations on microbiological requirements across aquaculture products based on ris analysis data. Recent studies show that L. monocytogenes is prevalent in 23.5% of catfish obtained from various retail stores in the U.S. Also, 2 to 47% of fresh channel catfish fillets were contaminated with L. monocytogenes. Treatment with highly specific and effective bacteriophag that parasitize L. monocytogenes before or directly after filleting may an effective way of reducing L. monocytogenes. FDA recently approved the bacteriophage Listex P100 bacteriophage preparation for all raw and rea to-eat foods to control L. monocytogenes. The effectiveness of bacteriophage Listex P100 in reducing L. monocytogenes on fresh catfish fillet tissue was determined as a function of: (i) Listex P100 contact time; (ii) Listex P100 dose; (iii) storage temperature; and (iv) and storage duration. L. monocytogenes reduction was influenced by phage contact time and phage dose regardless of temperature. Treatments with phage P100 dose of 5�107 PFU/cm2 yielded a reduction of 1.5 to 2.3 log10 CFU/g in L. monocytogenes at all temperatures. A 30 min contact time wit phage P100 treatment was essential for reductions in L. monocytogenes. Phage P100 titer was stable on catfish tissue and overall reductions in monocytogenes counts were maintained over a 10-day shelf-life of fresh catfish fillet tissue. These findings illustrate the effectiveness of alternative GRAS antimicrobial bacteriophage Listex P100 in reducing L. monocytogenes from the surface of fresh catfish fillets. 03 Channel Catfish Immune Recognition of Botulium Neurotoxin E: Development of An Immunoassay to Detect Botulism Exposure in Channel Catfish. Visceral toxicosis of catfish (VTC) is a relatively new disease syndrome that affects food size catfish raised in commercial catfish ponds. Over the past ten years losses associated with this disease have been estimat to be in excess of one million pounds. Serum neutralization studies and endopep mass spectrometry indicate the disease is a form of botulism, caused by a toxin produced by the bacterium Clostridium botulium E. Development of a sensitive, low cost, toxin free assay for measuring catfish toxin-specific antibody production will be useful for monitoring and managing this disease. The purpose of this work was to map the recognition profile of the heavy chain of botulium neurotoxin E (BoNT/E) We have developed a solid-phase enzyme-linked immunosorbant assay (ELISA utilizing peptide sequences from BoNT/E using archived serum samples fro effected and non-effected catfish. Protein immune recognition dictates that certain rules must be followed, therefore readily available compute programs were employed for antibody epitope prediction. Results revelaed 5 candidate peptide sequences which play a role in parent molecule recognition and possible neutralization. We also chemically coupled a carrier molecule (bovine serum albumin) with the recognized sequences as well as unrelated, non reactive sequences for future immunization studie 04 Complete the Listeria (L.) monocytogenes HCC23 Genome and Identify Novel Genetic Differences Between Pathogenic and Nonpathogenic Isolates. As this project has progressed, the identification of novel targets that could differentiate pathogenic and nonpathogenic isolates has taken on increased importance. We previously demonstrated that three internalin genes, lmo2821 (inlJ), lmo2470, and inlC, are absent in a nonpathogenic monocytogenes serotype 4a isolate from healthy channel catfish (HCC23) b it is not known whether the absence of these genes in HCC23 contributes its nonpathogenic phenotype. We transferred all three genes into the HCC chromosome. The HCC23 gene insertions were evaluated using the cell lin Caco-2 to determine whether lmo2821, lmo2470, and inlC would confer increased ability of HCC23 to invade human colonic epithelium. Results showed that, as expected, pathogenic isolate F2365 had increased ability to invade compared to HCC23. Currently it appears that none of the thre internalin genes caused increased adherence, but experiments are still being completed. The HCC23 genome sequence was formatted and submitted GenBank. Ortholog analysis showed that L. monocytogenes and L. innocua share a �core genome� composed of >2300 genes. Importantly, ortholog analysis also allowed identification of �virulence specific� genes that are present in pathogenic isolates EGD-e and F2365 but are missing in HCC23 and L. innocua. In total, 58 virulence-specific genes were identified. Many of the virulence-specific genes encode hypothetical proteins or surface-predicted proteins that need further studies to determine their function. 05 A Global Analysis of Meat Matrix-mediated Gene Expression in Listeria (L monocytogenes. The central hypothesis is that L. monocytogenes present meat products can enhance its virulence and ability to survive under har environments. Microarray and quantitative real-time polymerase chain reaction (qRT-PCR) technologies were used to identify genes expressed differentially in F2365 (serovar 4b) L. monocytogenes grown at a high sa condition similar to conditions in ready to eat (RTE) meat products to those grown in standard medium. Thirteen and 31 genes were up-regulated and down-regulated in response to salt stress, respectively. Specificall genes related to glycine betaine/L-proline ABC transporters and ribosoma proteins were up-regulated. Genes associated with PTS system, its metabolic enzymes, transport and binding proteins involved in cellular membrane and the uptake of carbohydrates, and internalin D were down- regulated. This is the first study that reports a global gene expression of L. monocytogenes in response to osmotic stress. The data suggest that monocytogenes has ability to adapt to a high salt condition and enhance the bacterium�s ability to survive and grown in RTE that can potentially result in listeriosis outbreaks. The information that helps to understan Listeria�s behaviour under a high salt condition will benefit other scientists and the food processing industry. 06 Whey Based Edible Films-reduce Oxidative Degradation of Meats. In 2008, S. production of dry whey was more than 504 million kg and whey solids a used in various food systems. Whey can be categorized as sweet or sour based on ph (high pH referred to as sweet and low pH referred to as sour Thermization (heat treatment) of whey enhances production of Maillard reaction products (MRPs) that are highly antioxidative. We investigated the effect of thermizations at 70�C thermostability of fresh sweet whey protein concentrates (WPC). Short time thermization treatments (5 and 1 min), respectively, of sweet whey significantly enhanced thermostability across all pH levels and heat-exposure times. Effects of thermized whey as a barrier and antioxidative protection to pork loin (longissimus dors and cubed (tenderized) beef steak (semimembranosus) was determined. Fre cheddar whey was skimmed, pasteurized and batch thermized at 70�C and resulting WPCs were used to coat meat. Oxidative stability was determin using Thiobarbituric Acid Reactive Substance (TBARS) and peroxide value (PV) every 24 h up to 4 days of storage at 4�C. Colorimetric measuremen showed the mean of the color consistency index for L*, a* and b* scales sour whey powder (SWP) to be higher than other treatments. SWP performed better than other protein sources for reducing oxidative degradation, as reflected by an increase in carbonyls and decrease in sulfhydryls. Moisture barrier property of SWP based coating was the best and Carboxymethyl cellulose (CMC) significantly enhanced this property of Calcium caseinate (CC) and soy protein isolate (SPI). Data showed that S can effectively be used to extend quality and shelf life of meat. 07 Effects of Polyphosphates, Hydrostatic Pressure, Irradiation and Fumigation on Quality and Safety of Catfish and Other Meat Products. Changes in flavor compounds and sensory quality of food products were detected due to irradiation, pest prevention practices, animal harvestin methods, and product processing. Research on catfish filets marinated v vacuum tumbling and multi-needle injection indicated use of agglomerated phosphates optimized yields in vacuum tumbled catfish fillets, when compared to sodium tripolyphosphate, due to the presence of variable phosphate chain lengths and enhanced solubility. Optimal yields and quality of catfish filets produced with a particular agglomerated blend sodium phosphates applied through multi-needle injection appear to be associated with this blends high pH and ionic strength. A highly solubl phosphate blend with a high pH is optimal for injector systems used on catfish filets. We determined that the irradiation of ground beef was successful at eradicating Escherichia coli O157:H7 but did not negativel impact sensory quality, but hydrostatic pressure treatment did not eliminate Escherichia coli O157:H7 and negatively impacted the consumer acceptability of ground beef. Fumigation with methyl bromide only had minimal effects on the volatile composition of dry cured ham and did not result in formation of any unsafe chemicals in dry-cured ham. 08 Development of Real-time Bioluminescent Assays for Investigating Salmonella, Campylobacter and Escherichia (E.) coli in Foods of Animal Origins. The Mississippi broiler and catfish industries are vital to th state�s economy. Poultry has an economic impact of $6.5B and the value catfish production in Mississippi for 2006 was $273 million. Together these industries employ over 85,000 people statewide. With both poultry and catfish processing now under Food Safety Inspection Service (FSIS) inspection, increased regulatory burdens face the industries. Our long- range goal to help the food animal industry meet federal food safety regulations by developing in vitro real-time models for studying three organisms (Salmonella, E. coli, and Campylobacter) associated with human disease from foods of animal origin. Luminescence is a potentially very sensitive detection method to estimate bacterial cell density in vitro. We have shown insertion of a light-producing gene (lux), engineered to produce light only in a viable cell, provides real-time detection of Salmonella enterica strains at low levels under various environmental conditions. We have used this assay to monitor dose-dependent responses of Salmonella to three common antimicrobial compounds (AMCs) (bleach, acetic acid, and hydrogen peroxide). These results indicate that the ass is sensitive in monitoring affects of antimicrobial compounds on this pathogen. 09 Development of Testing and Identification of Listeria and L. monocytogen in Catfish and Its Environment. We have developed novel testing procedures for testing the presence of Listeria and L. monocytogenes in catfish and its environment (plant), for determining the strains to facilitate traceback and investigation, and we have developing promising antimicrobials and other procedures to enhance safety and quality of the product.
Impacts
(N/A)
Publications
- Haque, Z.U., Williams, J.B., Mikel, W.B. 2010. Influence of Whey Peptides on the Surface Activity of k-casein and �-lactoglobulin. International Journal of Dairy Technology. 63(2)190-196.
- Chen, B.Y., Pyla, R., Kim, T.J., Silva, J.L., Jung, Y.S. 2010. Antibiotic Resistance in Listeria Species Isolated from Catfish Fillets and Processing Environment. Letters in Applied Microbiology. 50:662-632.
- Kim, T.J., Weng, W.L., Silva, J.L., Marshall, D.A. 2010. Identification of Natural Antimicrobial Substances in Red Muscadine Juice against Enterobacter sakazakii. Journal of Food Science. 75:M150-M154.
- Chen, B.Y., Pyla, R., Kim, T.J., Silva, J.L., Jung, Y.U. 2010. Prevalence and Contamination Patterns of Listeria monocytogenes in Fresh Catfish Fillets and their Processing Plants. Food Microbiology. 27:645-652.
- Kim, T.J., Silva, J.L., Weng, W.L., Chen, W.W., Corbitt, M., Jung, Y.S. 2009. Inactivation of Enterobacter sakazakii by Water-soluble Muscadine Seed Extracts. International Journal of Food Microbiology. 129:3295-3299.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. Approach (from AD-416) These funds will be used for 1) Extramural research with Mississippi Center for Food Safety and 2) Aflatoxin research. Original start and term dates 5/14/93-5/13/98, then 05/14/98 - 05/13/03, then 05/14/03 - 05/13/08. Changing start and term dates from 5/14/03-5/13/08. Extending for an additional 5 years. klj Significant Activities that Support Special Target Populations Research related to control of pathogens in food products, in particular Listeria monocytogenes, is focused on developing strategies for reducing L. monocytogenes in catfish, seafood, and other meat products. New generally regarded as safe (GRAS) antimicrobials for control of bacterial pathogens in ready-to-eat products are being developed and tested for their effectiveness. Studies on the genetic and environmental determinants of virulence of L. monocytogenes have demonstrated that both genotype and environment play a role in the virulence of this important food pathogen. This information will be critical in developing tests and protocols required to control L. monocytogenes in catfish, seafood, and other meat products. A rapid, inexpensive antibody-based assay is being developed for detecting botulism neurotoxin. This assay will be useful for screening purposes. A luciferase reporter gene has been inserted into Salmonella enterica and selectively produces light in viable cells. This technique allows real-time detection of the bacteria at low levels and allows monitoring of bacterial progression under different environmental and chemical conditions. Bacteria numbers and bioluminescence correlated well in all strains. These research projects will play a role in assuring safe, high-quality food products to consumers and will be important to maintaining viable aquaculture and seafood industries in the U.S. Significant Activities that Support Special Target Populations Assuring safety and improving quality of domestic farm-raised catfish will benefit operators of small farms by expanding markets for catfish products. 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 1,370 catfish farms in the United States, 515 farms (38% of the total) reported annual revenues of less than $25,000. Expansion of markets through development safe, high quality domestic catfish products will benefit U.S. catfish farmers, a significant portion of which are classified as small farmers.
Impacts
(N/A)
Publications
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. Approach (from AD-416) These funds will be used for 1) Extramural research with Mississippi Center for Food Safety and 2) Aflatoxin research. Original start and term dates 5/14/93-5/13/98, then 05/14/98 - 05/13/03, then 05/14/03 - 05/13/08. Significant Activities that Support Special Target Populations Research related to control of pathogens in food products, in particular Listeria monocytogenes, is focused on developing strategies for reducing L. monocytogenes in catfish, seafood, and other meat products. New generally regarded as safe (GRAS) antimicrobials for control of bacterial pathogens in ready-to-eat products are being developed and tested for their effectiveness. Studies on the genetic and environmental determinants of virulence of L. monocytogenes have demonstrated that both genotype and environment play a role in the virulence of this important food pathogen. This information will be critical in developing tests and protocols required to control L. monocytogenes in catfish, seafood, and other meat products. A rapid, inexpensive antibody-based assay is being developed for detecting botulism neurotoxin. This assay will be useful for screening purposes. A luciferase reporter gene has been inserted into Salmonella enterica and selectively produces light in viable cells. This technique allows real-time detection of the bacteria at low levels and allows monitoring of bacterial progression under different environmental and chemical conditions. Bacteria numbers and bioluminescence correlated well in all strains. These research projects will play a role in assuring safe, high-quality food products to consumers and will be important to maintaining viable aquaculture and seafood industries in the U.S. Publications Attila Karsi, Kevin Howe, Tasha B. Kirkpatrick, Robert W. Wills, R. Hartford Bailey, and Mark L. Lawrence. 2008. Development of bioluminescent Salmonella strains for use in food safety. BioMed Central- Microbiology 2008, 8:10. Battula, V., Schilling, M.W., Vizzier-Thaxton, Y., Behrends, J.B., Williams, J.B., Schmidt, T.B. 2008. The effects of low atmosphere stunning and deboning time on broiler breast meat quality. Poultry Science. 87:1202-1210. Bohoua, G. L. and Haque, Z.U. 2007. Surface activity of surfactants and dairy proteins. Milchwissenschaft. 62(4):394-397. Chen, B.Y., Kim, T.J., Jung, Y.S. and Silva, J.L. 2008. Attachment strength of Listeria monocytogenes and its internalin negative mutants. Food Biophysics (Accepted 5-08, # FOBI120R1, Available online: http://www. springerlink.com/content/j21464621762nxu1/). Significant Activities that Support Special Target Populations Assuring safety and improving quality of domestic farm-raised catfish will benefit operators of small farms by expanding markets for catfish products. 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 1,370 catfish farms in the United States, 515 farms (38% of the total) reported annual revenues of less than $25,000. Expansion of markets through development safe, high quality domestic catfish products will benefit U.S. catfish farmers, a significant portion of which are classified as small farmers.
Impacts
(N/A)
Publications
- Kim, T., Weng, W.L., Stojanovic, J., Lu, Y., Jung, Y.S., Silva, J.L. 2008. Antimicrobial Effect of Water-Soluble Muscadine Seed Extracts on Escherichia coli O157:H7. Journal of Food Protection. 71:1465-1468.
- Liu, D., Lawrence, M.L., Ainsworth, A.J. 2008. A Novel PCR Assay for Listeria welshimeri Targeting Transcriptional Regulator Gene lwe1801. Journal of Rapid Methods and Automation in Microbiology. 16:152-161.
- Mujihid, S., Pechan, T., Wang, C. 2007. Improved Solubilization of Surface Proteins from Listeria monocytogenes for Two-dimensional Gel Electrophoresis. Electrophoresis. 28:3998-4007.
- Pham, A.J., Schilling, M.W., Yoon, Y., Kamadia, V.V., Marshall, D.L. 2008. Characterization of Fish Sauce Aroma Impact Compounds Using GC-MS, SPME- Osme-GCO, and Stevens' Power Law Exponents. Journal of Food Science. 73(4) :C268-C274.
- Attila, K., Howe, K., Kirkpatrick, T.B., Wills, R.W., Hartford-Bailey, R., Lawrence, M.L. 2008. Development of Bioluminescent Salmonella Strains for Use in Food Safety. BioMed Central-Microbiology. 8:10.
- Chen, B.Y., Kim, T.J., Jung, Y.S., Silva, J.L. 2008. Attachment Strength of Listeria monocytogenes and Its Internalin Negative Mutants. Food Biophysics. 3:329-332.
- Liu, D., Lawrence, M.L., Ainsworth, A.J., Austin, F.W. 2007. Toward an Improved Laboratory Definition of Listeria monocytogenes Virulence. International Journal of Food Microbiology. 118(2):101-15.
- Battula, V., Schilling, M.W., Vizzier-Thaxton, Y., Behrends, J.B., Williams, J.B., Schmidt, T.B. 2008. The Effects of Low Atmosphere Stunning and Deboning Time on Broiler Breast Meat Quality. Poultry Science. 87:1202- 1210.
- Gandy, A.E., Schilling, M.W., Coggins, P.C., White, C.W., Yoon, Y., Kamadia, V.V. 2008. The Effect of Pasteurization Temperature on Consumer Acceptability, Sensory Characteristics, Volatile Compound Composition, and Shelf-Life of Fluid Milk. Journal of Dairy Science. 91:1769-1777.
- Kim, T.Y., Jung, S., Silva, J.L., Danviriyakul, S. 2007. Detection and Rapid Purification of Internalin B as A Protein Marker in Listeria monocytogenes. Food Biotechnology. 21:161-168.
- Liu, D., Lawrence, M.L., Pinchuk, L.M., Ainsworth, A.J., Austin, F.W. 2007. Characteristics of Cell-mediated, Anti-listerial Immunity Induced by A Naturally Avirulent Listeria monocytogenes Serotype 4a Strain HCC23. Archives Of Microbiology. 188(3):251-6.
- Liu, D., Lawrence, M.L., Austin, F.W., Ainsworth, A.J. 2007. A Multiplex PCR for Species- and Virulence-specific Determination of Listeria monocytogenes. Journal of Microbiological Methods. 71(2):133-140.
- Liu, D. 2008. Epidemiology. In Liu, D. (ed) Handbook of Listeria monocytogenes, Pages 27-59. CRC Press, Florida.
- Liu, D., Lawrence, M.L., Ainsworth, A.J., Austin, F.W. 2008. Genotypic Identification. In Liu, D. (ed) Handbook of Listeria monocytogenes, Pages 169-201. CRC Press, Florida.
- Roche, S.M., Velge, P., Liu, D. 2008. Virulence Determination. In Liu, D. (ed) Handbook of Listeria monocytogenes, Pages 241-270. CRC Press, Florida.
- Liu, D. (ed). 2008. Handbook of Listeria monocytogenes. CRC Press, Boca Raton, Florida.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report 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. Approach (from AD-416) These funds will be used for 1) Extramural research with Mississippi Center for Food Safety and 2) Aflatoxin research. Original start and term dates 5/14/93-5/13/98, then 05/14/98 - 05/13/03, then 05/14/03 - 05/13/08. Accomplishments Effects of freezing and duration of refrigeration prior to freezing on levels of Vibrio vulnificus in oyster meat. Pathogenic strains of Vibrio vulnificus are natural inhabitants of estuarine environments world wide and can be transmitted to humans through consumption of raw shellfish which flourish in the same estuaries. Until recently, most post harvest processes for preserving and extending shelf-life of in-shell and shucked oysters have centered on refrigeration, but research to find other processes to eliminate this pathogen has expanded. Recent information gained through validation studies of freezing (individually quick frozen (IQF)) (Schwarz, 2003) has indicated variability in success of freezing depending on storage conditions prior to freezing. Cryogenic freezing successfully reduced Vibrio vulnificus numbers, with an adjusted gross mean (AGM) of 87,000 MPN/g oyster meat, to < 30 MPN/g oyster meat in 7 days and to non-detectable levels (<3 MPN/g oyster meat) in 35 days. However, there was no difference in reduction of Vibrio vulnificus in oysters refrigerated for different times up to 4 days prior to freezing, indicating Vibrio vulnificus did not become more cold tolerant or more process resistant after refrigeration. Knowledge gained from this study will provide specific information for industry partners interested in processing using IQF freezing technology. Establishing specific guidelines for transport and storage of oysters deemed for IQF processing will maximize the effect of freezing on vibrio reduction in half shell oysters. This research directly addresses National Program 108 � Food Safety, Component 1.2 � Pathogens, Toxins and Chemical Contaminansts � Postharvest; Problem Statements 1.2.4 Processing Intervention Strategies and 1.2.6 Safety and Health. Genome sequencing of L. monocytogenes serotype 4a strain HCC23. Understanding genetic factors associated with virulence in L. monocytogenes will allow better detection and control of virulent strains. Sequence differences between L. monocytogenes strains HCC23 (non- virulent) and serotype 4b strain F2365 (virulent) were mapped and approximately 1,500 single nucleotide polymorphisms were resolved in HCC23. High throughput resequencing was conducted to resolve additional sequence differences. The total genome size of HCC23 was 2,943,866 bp in length. There were 74,923 high confidence mutations between F2365 and HCC23, indicating the two genomes are highly divergent. This indicates several genes are altered in HCC23 compared to virulent strain F2365, and several others are missing. When the genome of 4a strain HCC23 is completed, comparison of the genome of this avirulent strain with the F2365 genome will allow identification of novel virulence genes. Understanding genetic factors affecting virulence of L. monocytogenes will be important to improve food safety in seafood and other foods. This research directly addresses National Program 108 � Food Safety, Component 1.2 � Pathogens, Toxins and Chemical Contaminansts � Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Characterize genes that show genetic changes between virulent and avirulent L. monocytogenes. L. monocytogenes is a foodborne bacterial pathogen that employs a number of virulence-associated proteins (e.g., ActA and internalins) that enable penetration and movement within host cells. While actA and many internalin genes were examined previously by DNA arrays, they have not been fully characterized by PCR. Thus, oligonucleotide primers were designed from L. monocytogenes EGD-e actA and internalin genes (ie, lmo0327, lmo0331, lmo2396, lmo2445 and lmo2470) and used in PCR for examination of 29 L. monocytogenes and 5 other Listeria strains. L. monocytogenes serotypes demonstrated notable variations in their actA and internalin genes, with high virulence serotypes possessing more internalins than low virulence serotypes. Understanding genetic factors affecting virulence of L. monocytogenes will be important to improve food safety in seafood and other foods. This research directly addresses National Program 108 � Food Safety, Component 1.2 � Pathogens, Toxins and Chemical Contaminansts � Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Isolation of virulent and avirulent L. monocytogenes from the murine macrophage cell line J774.1. Understanding genetic factors associated with virulence in L. monocytogenes will allow better detection and control of virulent strains. Identifying differences in protein expression between virulent and avirulent strains will allow a better understanding of genetic determinants of virulence. However, studying of protein expression requires large amount of protein. By increasing incubation time and altering the infection procedure, we have increased the amount of bacteria harvested from J774 cells to enable us to harvest sufficient bacterial proteins to conduct high throughput proteomics of L. monocytogenes proteins. The differences in protein expression between virulent and avirulent strains at different time points may explain the difference in virulence. Understanding genetic factors affecting virulence of L. monocytogenes will be important to improve food safety in seafood and other foods. This research directly addresses National Program 108 � Food Safety, Component 1.2 � Pathogens, Toxins and Chemical Contaminansts � Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Development of a multiplex PCR for species- and virulence-specific determination of L. monocytogenes. L. monocytogenes is composed of multiple serotypes/strains showing varied virulence potential. As a means to implement effective control and prevention measures for L. monocytogenes, it is necessary to identify virulent, disease-causing strains from avirulent, nonpathogenic strains that are relatively harmless. After examination of a collection of 29 L. monocytogenes and 5 strains from the other Listeria species by PCR and Southern blot, a multiplex PCR was developed that incorporates genes (inlA, inlC, and inlJ) potentially related to virulence in L. monocytogenes gene. The species identity of the 29 L. monocytogenes strains was verified through the amplification of a 800 bp fragment with the inlA gene primers, and the virulence potential of these strains was ascertained by the formation of 517 bp and/or 238 bp fragments with the inlC and inlJ gene primers, respectively. Use of the multiplex PCR targeting inlA, inlC, and inlJ genes facilitated rapid, simultaneous confirmation of L. monocytogenes species identity and virulence potential. Rapid, accurate identification of L. monocytogenes species and potential for virulence has important implications for controlling this important food pathogen. This research directly addresses National Program 108 � Food Safety, Component 1.2 � Pathogens, Toxins and Chemical Contaminansts � Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Evaluation of acid, alkali and salt tolerance among Listeria species. Being ubiquitously present in the natural environment, Listeria species (especially L. monocytogenes) are renowned for their ability to withstand external pH, temperature, and osmotic stresses, and to survive a variety of food manufacturing processes. Therefore, a detailed understanding of non-monocytogenes Listeria species in terms of their biochemical and molecular features is vital to the development of novel diagnostic procedures for improved discrimination of pathogenic from non-pathogenic Listeria species. We determined effect of extreme pH and salt concentrations on survival of 16 Listeria strains (with three each of L. grayi, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri, and one L. monocytogenes strain). There were significant differences among Listeria species in their ability to survive salt concentrations and pH. We are currently assessing if the differnces in tolerance of Listeria species and strains to salt concentration and pH are associated with their virulence. Understanding relationships between virulence and environmental tolerance in Listeria species will provide insights into virulence of this important pathogen and potential methods for controlling it. This research directly addresses National Program 108 � Food Safety, Component 1.2 � Pathogens, Toxins and Chemical Contaminansts � Postharvest; Problem Statements 1.2.5 Omics, 1.2.6 Safety and Health, and 1.2.8 Pathogenecity. Determine effects of chemical compounds, diffusion method, and cooking method on masking of off-flavor compounds in fish. The incidence of �off- flavor� remains a major issue for the catfish farming industry and other farmed seafood. Off-flavor in catfish is typically due to uptake of algal derived compounds which impart a objectionable flavor and odor to the fillet. Methods for reducing or masking off-flavor compounds would benefit the catfish farming industry. We determined effects of chemical compounds, diffusion methods, and cooking methods for reducing or masking off-flavor from 2-methylisoborneol (MIB) in processed channel catfish. There were no differences among diffusion methods (dipping, tumbling, injecting). Acetic acid (1-2%) reduced off-flavor odor, but induced a sour flavor. Citric or lactic acid at 1%, 3 ppm ozone for 10-30 min, 0. 5% hydrogen peroxide for 20 min or, sodium bicarbonate at 1-7% were not effective for masking or reducing MIB. Sodas like 7-up� and Sprite� were shown to decrease off-flavor while adding a pleasant sweet flavor to the catfish fillet. Frying and marinating prior to baking were found to be effective in decreasing sensory off-flavor. GC/MS analysis showed that MIB was reduced by acetic acid and cooking but not by the soda treatments. Reduction or masking of off-flavor due to MIB appears to be possible and could greatly benefit the catfish industry by allowing sale of catfish that would otherwise have poor consumer acceptance. This research directly addresses National Program 106- Aquaculture; Component 9 Quality, Safety, and Variety of Aquaculture Products for Consumers. Determine effects of sampling site on level of off-flavor compounds in catfish flesh. Off-flavor compounds may not be consistent throughout catfish fillets, and therefore, the location on the fillet from which a sample is taken may influence if a sample is determined to be off-flavor. We evaluated effects of sampling location on off-flavor assessment in catfish fillets. Muscle in the peritoneal cavity contained 3-5X higher amount of geosmin than other parts. Other sites on the fillet showed differences in concentration of off-flavor, with lean areas showing less off-flavor than fatty areas. This suggests that fatty areas of the fillet, such as the muscle in the peritoneal cavity, should be sampled when screening for off-flavor catfish to insure a rigorous testing and minimize the incidence of truly off-flavor fish being classified as on- flavor. This research directly addresses National Program 106- Aquaculture; Component 9 Quality, Safety, and Variety of Aquaculture Products for Consumers. Significant Activities that Support Special Target Populations Assuring safety and improving quality of domestic farm-raised catfish will benefit operators of small farms by expanding markets for catfish products. 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 1,370 catfish farms in the United States, 515 farms (38% of the total) reported annual revenues of less than $25,000. Expansion of markets through development of safe, high quality domestic catfish products will benefit U.S. catfish farmers, a significant portion of which are classified as small farmers. Technology Transfer Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 15 Number of Newspaper Articles,Presentations for NonScience Audiences: 6
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Progress 10/01/05 to 09/30/06
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Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This research directly addresses components of National Program 108 Food Safety, National Program 306 Quality and Utilization of Agricultural Products, and National Program 106 Aquaculture. United States consumers spending on seafood products is currently over $55 billion annually, however, much of this demand is met through foreign imports. The catfish farming industry is the largest segment of U.S. aquaculture with about 600 million pounds of catfish being produced annually. Increasing production and consumption of catfish and other domestically produced seafood will benefit U.S. producers, processors, and consumers. Assurance of safe, high quality products will play a crucial role in expanding domestic production and consumption of catfish and other seafood products. Development
and evaluation of technologies that allow identification and reduction of food borne microbial pathogens, spoilage bacteria, physical hazards, and chemical contamination in seafood; and education of processors and consumers on seafood safety are crucial to assuring seafood safety, improving product quality, and developing new products. This research directly impacts and benefits producers, processors, and consumers of catfish and other seafood products. The research will assure consumers receive safe, high quality seafood products, resulting in growth in the industries involved in production and processing of these products. We have addressed issues associated with seafood safety and quality through a multidisciplinary program focused on developing and evaluating technologies for improving seafood safety and quality. The overall goal of this project is to develop technologies useful for assuring safety and improving quality of catfish and seafood products. Specific areas of
research have been identified through interactions with industry representatives, extension service personnel, and researchers in other disciplines and states. 2. List by year the currently approved milestones (indicators of research progress) This research project has not been through the OSQR process; therefore, a Project Plan with approved milestones has not been developed at this time. Primary objectives for the project and tentative milestones for FY 2007, FY 2008, and FY 2009 are listed in section 3b. 4a List the single most significant research accomplishment during FY 2006. This accomplishment is related to National Program 108 Food Safety. Listeria monocytogenes is common intracellular bacterium that can cause listerosis in humans, a potentially serious illness that accounts for a significant portion of human foodborne diseases. The virulence of L. monocytogenes varies depending on lineage, with some serotypes causing listerosis in humans while other serotypes do not. An
avirulent strain of L. monocytogenes (strain HCC23) was evaluated for its ability to stimulate an immune response and provide protection against subsequent infection with virulent Listeria in mice. Mice infected with strain HCC23 exhibited protection against subsequent infection with virulent L. monocytogenes (strain EGD) and L. ivanovii (strain ATCC 19119). This research has increased knowledge of factors determining virulence of this important food borne pathogen and demonstrated that avirulent L. monocytogenes may have potential as a vaccine against virulent Listeria. 4b List other significant research accomplishment(s), if any. This accomplishment is related to National Program 108 Food Safety. The virulence of L. monocytogenes, a common intracellular bacterium that can cause listerosis in humans, varies depending on genetic lineage. Genetic relationships among L. monocytogenes isolates (36 strains from catfish, 35 strains from seafood, and 37 strains from humans) were
determined. Results suggest detected genetic differences in L. monocytogenes isolates can be used to predict their potential for causing listerosis in humans. L. monocytogenes isolates from catfish and seafood pose a low threat of causing illness in humans. This accomplishment is related to National Program 108 Food Safety. Microbrial flora present in raw oysters can pose human health issues and reduce shelf life of product. X-ray radiation was evaluated as a technique for reducing the bacterium Vibrio vulnificus, a potential human pathogen, and other bacterial species associated with spoilage in live, in-shell and shucked oysters. An x-ray exposure of 0.75 kGy reduced V. vulnificus to undectable levels and extended shelf life of oysters by greater than 1 week by reducing spoilage bacteria. X-ray radiation has potential to reduce pathogen and increase shelf life in raw oysters. This accomplishment is related to National Program 108 Food Safety. Attachment strength of microbes
influences ability to clean food contact surfaces, and has implications for sanitation of seafood processing equipment and microbial contamination of seafood. A method was developed to quantify attachment strength of common food borne bacterial pathogens. This method allows rapid determination of attachment strength of bacterial pathogens on food contact surfaces. Information on attachment strength of pathogens will be useful in developing and evaluating sanitation of processing equipment and better understanding the transmission of food borne pathogens. 4c List significant activities that support special target populations. Assuring safety and improving quality of farm-raised catfish will benefit operators of small farms by expanding markets for catfish products. 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 1,370 catfish farms in the United States, 515 farms (38% of the total) reported
annual revenues of less than $25,000. Expansion of markets through development safe, high quality catfish products will benefit catfish farmers, a significant portion of which are classified as small farmers. 5. Describe the major accomplishments to date and their predicted or actual impact. The focus of this project is to assure safety and improve quality of catfish and other seafood products, accomplishments relate to National Program 108 Food Safety, National Program 306 Quality and Utilization of Agricultural Products, and National Program 106 Aquaculture. Customers benefiting from the research include producers, processors, and consumers of catfish and seafood products. Specifically, effects of x-ray irradiation, ozonation, UV irradiation, modified atmospheric conditions, hot water pasteurization, and various solutions have been studied to determine effects on food borne pathogens and spoilage bacteria in catfish and other seafood. Specific antimicrobials and methodologies
have been developed and are currently being used by catfish processors. Factors associated with virulence of Listeria monocytogenes, a food borne pathogen, have been identified and tests to differentiate between virulent and avirulent Listeria monocytogenes have been developed. Increased shelf-life and safety of catfish have been achieved. Information and technologies have been shared with other scientists, catfish and seafood processors, and consumers. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The benefits of this project will be continued development of technologies and techniques to improve catfish and seafood quality and safety. A better understanding of virulence and control of Listeria, an important food borne pathogen, has been achieved
and will lead to safer catfish and seafood products. Processors of catfish and other seafood have inquired about commercial application of technologies being developed and evaluated for improving shelf life of products. Information on current research and seafood safety education has been provided to customers through workshops, meetings, and site visits to processing plants. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Dickson, J.S., Marshall, D.L. 2006. Food microbiology and safety: Basic requirements. In Handbook of Food Science, Technology and Engineering, 4 Volume Set. Hui, Y.H., E. Castell-Perez, L.M. Cunha, I. Guerrero Legarreta, H.H. Liang, Y.M. Lo, D.L. Marshall, W.K. Nip, F. Shadidi, F. Sherkat, R.J. Winger, and K.L. Yam (Eds.). CRC Press, Taylor & Francis Group Boca Raton, FL, Volume IV Food Technology and Food Processing,
Chapter 184, pp. 184-1-184-8. Duran, G.M., Marshall, D.L. 2006. Cleaning a processing plant. In Handbook of Food Science, Technology and Engineering, 4 Volume Set. Hui, Y.H., E. Castell-Perez, L.M. Cunha, I. Guerrero Legarreta, H.H. Liang, Y. M. Lo, D.L. Marshall, W.K. Nip, F. Shadidi, F. Sherkat, R.J. Winger, and K.L. Yam (Eds.). CRC Press, Taylor & Francis Group, Boca Raton, FL, Volume IV Food Technology and Food Processing, Chapter 196, pp. 196-1- 196-7. Marshall, D.L., Dickson, J.S. 2006. Contaminants. In Handbook of Food Science, Technology and Engineering, 4 Volume Set. Hui, Y.H., E. Castell- Perez, L.M. Cunha, I. Guerrero Legarreta, H.H. Liang, Y.M. Lo, D.L. Marshall, W.K. Nip, F. Shadidi, F. Sherkat, R.J. Winger, and K.L. Yam (Eds.). CRC Press, Taylor & Francis Group, Boca Raton, FL, Volume IV Food Technology and Food Processing, Chapter 194, pp. 194-1-194-10.
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