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ARS Home Southeast Area Stoneville, Mississippi Warmwater Aquaculture Research Unit Research





- Bosworth, Brian
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- Food Safety, (Animal and Plant Products) (108)

Research Project: Umbrella Project for Food Safety **Location:
                            Warmwater Aquaculture Research Unit** 2019 Annual Report **Objectives**
                                

Research will address methods to determine the presence of pathogens in catfish/catfish products and to maximize elimination methods.  Detection techniques will be developed to aid in processing and packaging operations, which should further enhance product safety.  Specifically the new objectives are: 1)Optimize safety of aquaculture products through innovative processes for reducing microbiological, physical and chemical hazards in seafood/aquaculture products.  2)Determine the mechanisms influencing microbial survival of selected pathogens in seafood/aquaculture products.  3)Optimize the market value of seafood/aquaculture products through enhanced food safety and quality. **Approach**
                                

Catfish.  Determine optimum rates of microbial reduction through innovative processing in catfish products including evaluation of consumer acceptance.  Determine viable methods of hazard reduction (smoking, acidulants, antimicrobials, etc) in catfish products during processing and storage.  Determine the methods by which these methods reduce hazards within the products evaluated.  Enhance the physical safety of catfish fillets with innovative analysis technology.

Seafood/Produce.  Determine the efficacy of IQF freezing, irradiation, and high pressure processing and other technologies on the safety and quality of oysters, shrimp and produce.  

Objective 2:  Catfish/ Seafood/Produce.  Determine the mechanistic approach by which the certain pathogenic bacteria may be reduced in aquatic species.  Utilize PCR analysis and other assays to determine the sensitivity and specificity of various isolates in response to innovative treatments.

Objective 3:   Catfish.  Enhance product value through innovative smoking and further processing of catfish fillets.  Value-added analysis will compared products to commodity value for product enhancement addition.  Evaluate value-added products to address potential food safety issues. 

Seafood/Produce.  Evaluate consumer acceptance of products enhanced through various processing methods.   Preparation techniques, ingredient inclusion, packaging and storage methods will be evaluated at various time frames and inclusion rates to determine specie specific parameters limitations.  Analyze economics of various market potentials.
Catfish.  Determine optimum rates of microbial reduction through innovative processing in catfish products including evaluation of consumer acceptance.  Determine viable methods of hazard reduction (smoking, acidulants, antimicrobials, etc) in catfish products during processing and storage.  Determine the methods by which these methods reduce hazards within the products evaluated.  Enhance the physical safety of catfish fillets with innovative analysis technology.

Seafood/Produce.  Determine the efficacy of IQF freezing, irradiation, and high pressure processing and other technologies on the safety and quality of oysters, shrimp and produce.  

Objective 2:  Catfish/ Seafood/Produce.  Determine the mechanistic approach by which the certain pathogenic bacteria may be reduced in aquatic species.  Utilize PCR analysis and other assays to determine the sensitivity and specificity of various isolates in response to innovative treatments.

Objective 3:   Catfish.  Enhance product value through innovative smoking and further processing of catfish fillets.  Value-added analysis will compared products to commodity value for product enhancement addition.  Evaluate value-added products to address potential food safety issues. 

Seafood/Produce.  Evaluate consumer acceptance of products enhanced through various processing methods.   Preparation techniques, ingredient inclusion, packaging and storage methods will be evaluated at various time frames and inclusion rates to determine specie specific parameters limitations.  Analyze economics of various market potentials. **Progress Report**
                                

All Objectives were planned and completed by the 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**
                                

1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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. 7. 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. 8. 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.


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