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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** 2020 Annual Report **Objectives**
                                

Research will address methods to determine the presence of pathogens in catfish/catfish products and to maximize elimination methods.  Detection techniques will be developed to aid in processing and packaging operations, which should further enhance product safety.  Specifically the new objectives are: 1)Optimize safety of aquaculture products through innovative processes for reducing microbiological, physical and chemical hazards in seafood/aquaculture products.  2)Determine the mechanisms influencing microbial survival of selected pathogens in seafood/aquaculture products.  3)Optimize the market value of seafood/aquaculture products through enhanced food safety and quality. **Approach**
                                

Catfish.  Determine optimum rates of microbial reduction through innovative processing in catfish products including evaluation of consumer acceptance.  Determine viable methods of hazard reduction (smoking, acidulants, antimicrobials, etc) in catfish products during processing and storage.  Determine the methods by which these methods reduce hazards within the products evaluated.  Enhance the physical safety of catfish fillets with innovative analysis technology.

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

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

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

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

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

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

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

Seafood/Produce.  Evaluate consumer acceptance of products enhanced through various processing methods.   Preparation techniques, ingredient inclusion, packaging and storage methods will be evaluated at various time frames and inclusion rates to determine specie specific parameters limitations.  Analyze economics of various market potentials. **Progress Report**
                                

All objectives were planned and completed by the ARS 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**
                                

1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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. **Review Publications**
                            *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.*
                                *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. https://doi.org/10.1016/j.foodchem.2019.126040.*
                                *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. https://doi.org/10.1016/j.foodchem.2019.125186.*
                                *Shi, M., Li, J., Chang, S., Maleki, S.J. 2019. Quantitative and kinetic analyses of peanut allergens as affected by food processing. Food Chemistry. https://doi.org/10.1016/j.fochx.2019.100004.*
                                *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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