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ARS Home » Southeast Area » Stoneville, Mississippi » Warmwater Aquaculture Research Unit » Research » Research Project #144153

Research Project: Umbrella Project for Food Safety

Location: Warmwater Aquaculture Research Unit

2009 Annual Report

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.

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

Progress Report
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.

1. Improving the Safety of Oysters and Shrimp by X-ray Irradiation. The long-term goal of this project is to improve the safety of seafood (oysters and shrimp) using X-ray technology for the seafood industry under approved regulatory status. We determined the efficacy of X-ray on the inactivation 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); 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, 0.2, 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 E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus 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 than one million cells per gram with 2.0 kGy X-ray doses in half shell oysters. 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).

2. New GRAS Bacteriophage LISTEXTM P100 for Quantitative Reduction of Listeria monocytogenes Loads in Aquaculture Products/Seafood Products. The new 2008 farm bill legislation (PL 110-246) signed into law on June 18, 2008 extends food Safety Inspection Service's (FSIS’s) inspection jurisdiction to include domestic and imported farm-raised catfish. This legislation may lead to new federal regulations on microbiological requirements across aquaculture products based on risk 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, 25 to 47% of fresh channel catfish fillets were contaminated with L. monocytogenes. Treatment with highly specific and effective bacteriophages that parasitize L. monocytogenes before or directly after filleting may be an effective way of reducing L. monocytogenes. FDA recently approved the bacteriophage Listex P100 bacteriophage preparation for all raw and ready-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 with phage P100 treatment was essential for reductions in L. monocytogenes. Phage P100 titer was stable on catfish tissue and overall reductions in L. monocytogenes counts were maintained over a 10-day shelf-life of fresh catfish fillet tissue. These findings illustrate the effectiveness of alternative GRAS (generally recognized as safe) antimicrobial bacteriophage Listex P100 in reducing L. monocytogenes from the surface of fresh catfish fillets.

3. 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 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 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 from effected and non-effected catfish. Protein immune recognition dictates that certain rules must be followed, therefore readily available computer programs were employed for antibody epitope prediction. Results revelaed 4-5 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 studies.

4. Complete the 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 L. monocytogenes serotype 4a isolate from healthy channel catfish (HCC23) but it is not known whether the absence of these genes in HCC23 contributes to its nonpathogenic phenotype. We transferred all three genes into the HCC23 chromosome The HCC23 gene insertions were evaluated using the cell line 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 three internalin genes caused increased adherence, but experiments are still being completed. The HCC23 genome sequence was formatted and submitted to 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.

5. A Global Analysis of Meat Matrix-mediated Gene Expression in Listeria monocytogenes. The central hypothesis is that L. monocytogenes present in meat products can enhance its virulence and ability to survive under harsh environments. We used microarray and quantitative real-time polymerase chain reaction (qRT-PCR) technologies to identify genes expressed differentially in F2365 (serovar 4b) L. monocytogenes grown at a high salt 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. Specifically, genes related to glycine betaine/L-proline ABC transporters and ribosomal proteins were up-regulated. Genes associated with phosphtransferase (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 L. monocytogenes has ability to adapt to a high salt condition and enhances the bacterium’s ability to survive and grown in RTE that can potentially result in listeriosis outbreaks. The information that helps to understand Listeria’s behaviour under a high salt condition will benefit other scientists and the food processing industry.

6. Whey Based Edible Films-reduce Oxidative Degradation of Meats. In 2008, the U.S. production of dry whey was more than 504 million kg and protein rich whey solids are being extensively used in various food systems. Thermostability (TS) is an important functionality that impacts potential utilization of whey solids. Thermization presumably enhances the production of Maillard reaction products (MRPs) that are highly antioxidative. Whey can be categorized as sweet or sour based on ph (high pH referred to as sweet and low pH referred to as sour). We investigated the effect of varied levels of pre-heat treatments, or thermizations at 70 degrees Celsius, of fresh sweet whey (Edam and Cheddar) on the thermostability (TS) of whey protein concentrates (WPCs). Our data demonstrated that short time thermization treatments for 5 and 10 min, respectively, of Edam and Cheddar whey significantly enhanced TS across all pH levels and heat-exposure times studied. We investigated the efficacy of thermized Cheddar whey in providing a barrier and antioxidative protection to pork loin (longissimus dorsi) and cubed (tenderized) beef steak (semimembranosus). Fresh Cheddar whey from the Mississippi State University (MSU) Dairy Plant was skimmed, pasteurized and batch thermized at 70°C. Coating solutions for the meats were made using WPC as the main constituent. Oxidative stability was determined using TBARS and peroxide value (PV) every 24 h up to 4 days of storage at 4°C. Data clearly showed that the relatively easy and safe thermization step significantly enhanced antioxidative properties of Cheddar whey protein concentrate used as a coating solution on pork loin and cubed beef steak. Sour whey powder (SWP) based edible coatings, with and without CM-cellulose (CMC), were used to reduce oxidative degradation of cut beef steak stored at 2°C for 5 days. Comparison was with calcium caseinate (CC) and soy protein isolate (SPI).

7. Effects of Polyphosphates, Hydrostatic Pressure, Irradiation and Fumigation on Quality and Safety of Catfish and Other Meat Products. In our food safety research projects, we have been able to detect changes in flavor compounds and sensory quality of food products due to irradiation, pest prevention practices, animal harvesting methods, and product processing. Research on catfish filets marinated via 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 of sodium phosphates applied through multi-needle injection appeared to be associated with this blends high pH and ionic strength. A highly soluble 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 negatively impact sensory quality. In addition, this research revealed that the hydrostatic pressure treatment that was used did not eliminate Escherichia coli O157:H7 and negatively impacted the consumer acceptability of the ground beef. Fumigation of hams with methyl bromide only had minimal effects on the volatile composition of dry cured ham and did not cause the formation of any unsafe chemicals in the dry-cured ham. Research on catfish filets marinated via 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.

8. Development of Real-time Bioluminescent Assays for Investigating Salmonella, Campylobacter and E. coli in Foods of Animal Origins. The Mississippi broiler and catfish industries are vital to the state’s economy. Poultry has an economic impact of $6.5B and the value of 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 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 assay is sensitive in monitoring affects of antimicrobial compounds on this pathogen.

9. Development of Testing and Identification of Listeria and L. monocytogenes 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.

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.