<OL> <LI> To develop real-time PCR methodology for the rapid determination of the total viable bacterial population on seafood<LI> To assess the extent to which protozoa are able to harbor and protect L. monocytogenes from sanitizing agents<LI> To determine the minimum growth temperature of Bacillus cereus isolates derived from seafood and to determine the extent of toxin production at such minimum growth temperatures<LI> Evaluation of extracts of fungal-processed phytochemical extracts and extracts of edible mushrooms as antimicrobials against L. monocytogenes on seafoods including studies on the development of synergistic combinations of food-grade and GRAS antimicrobials at minimal doses against this organism <LI> To improve control measures for foodborne microbial pathogens in seafood by incorporating liposomal-encapsulated antimicrobials in seafoods to enhance antimicrobial efficacy <LI> To clarify how the omega 3 fatty acid stearidonic acid derived from seafood is metabolized by hepatocytes as well as determining the activity of stearidonic acid on cholesterol metabolism <LI> To optimize the liposomal encapsulation of omega 3 fatty acids derived from seafood for addition to a wide variety of food products for the purpose of significantly increasing the publics health <LI> To develop methodology to prevent lipid oxidation of proteins extracted from fish muscle by treatment with cations and organic acids.
NON-TECHNICAL SUMMARY: PCR presently detects both live and dead bacteria. The development of PCR methodology for quantifying only viable bacteria will allow the rapid assessment of the quality of seafood. Documenting the numbers of spore forming toxin bacteria on seafood will delineate the extent of such a problem with seafood. The development of new methods for inhibiting the refrigerated growth and destroying bacterial pathogens on seafood will enhance the safety of seafood. Refrigerated seafood keeps for only 7-9 days. By applying liposomal-encapsulated antimicrobials to seafoods, microbial pathogens will be reduced and the refrigerated storage life of such seafoods will be enhanced. The development of technology to encapsulate omega 3 fatty acids in stabilized liposomes will allow their incorporation into many foods to reduce arthrosclerosis. There is a significant amount of underutilized fish species from which protein can be extracted and used for a variety of food products. The development of technology to prevent the lipid oxidation of such extracted protein has the potential to expand the sources of seafood derived protein for human consumption and to enhance profitability to the seafood industry. The purposes are to enhance the quality and safety of seafood, reduce arthrosclerosis, and increase the utilization of under utilized fish species.
APPROACH: We will develop real time PCR methodology for rapid quantitation of solely live bacteria on seafood tissue, 2. determine the ability of Listeria monocytogenes and Listeria innocua to grow within protozoa and evaluate the sensitivity of these two organisms to sanitizers during intracellular protozoan growth, 3. assess the risk derived from the occurrence of spore forming toxin producing bacteria on seafood, 4. assess the effectiveness of newly developed phytochemical antimicrobial agents for enhancing the safety of seafoods, 5. enhance the control of pathogenic bacteria on seafood and extend refrigerated storage life of seafood by applying liposomes containing antimicrobials, 6. determine the effect of specific omega 3 fatty acids derived from fish tissue on cholesterol metabolism, 7. assess and optimize the stability of liposomes laden with omega 3 fatty acids for incorporation into a wide variety of food products, 8. reduce the oxidizability of membrane lipids of extracted fish proteins by treatment in Situ,
PROGRESS: 2005/08 TO 2008/08<BR>
OUTPUTS: Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The use of 10 ìg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 ìg/ml. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 100,000 CFU/ml at 4, 20, and 37oC yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70C to a minority of the mixed population compared to membrane damage. When inoculated into water, the numbers of L. monocytogenes gradually decreased by 3.8 log, however, when co-inoculated with the protozoan A. castellanii the total cell numbers of L. monocytogenes remained constant over 27 days, indicating that the presence of A. casellanii greatly enhanced the survival of L. monocytogenes in water. Clonal and synergistic oregano phenolic extracts were effective in inouculated fish fillets, where at 800 ppm of oregano clonal extracts reduced the growth of Listeria monocytogenes by 3-4 logs compared to controls. Soybean and cranberry fermented extracts were similarly effective as phenolic phytochemical hurdles in seafood,. This alternative strategy to use phytochemicals from botanical processing by dietary fungi such as Rhizopus oligosporus and Lentinus edodes are promising and food-grade with low flavor backgrounds. Further we have combined fungal processed phenolic phytochemicals from soybean and fungal extracts from edible mushrooms such as shiitake (Lentinus edodes) and have developed combinations that have the potential to be inhibitory against seafood pathogens at lower doses.Isolates of Bacillus cereus isolated from seafood were tested for antibiotic resistance. Publications <BR> PARTICIPANTS: A total of 3 postdoctoral scientists, 8 graduate students,and 4 undergraduates worked on this project. <BR> TARGET AUDIENCES: The seafood Indusdtry, FDA, USDA,and researchers at other universities will find these result of interest and value. <BR> <BR>
IMPACT: 2005/08 TO 2008/08<BR>
Results will allow rapid molecular enumeration of total bacteria on seafood. Enhanced preservation and safety of seafood can be achieved by application of results.
PROGRESS: 2006/08/15 TO 2007/08/15<BR>
At 37C the MPN of of E. coli O157:H7 (strain ATCC 43895) in fish silage at pH 3.5 was reduced from 10 billion cells per ml to less than 1/ml after 18 hrs of incubation). At 20C, 6 days were required to reduce the MPN below 1/ml. At 4C, 36 days of incubation were required to reduce the MPN below 1/ml with viability of less than 1 MPN/ml still detected after 39 hr but not at 42 hr. We have developed GRAS phenolic synergies of oregano and cranberry to control V. parahaemolyticus in seafood. We have isolated clonal herbs and that are specific for inhibiting Listeria monocytogenes. After optimization of the parameters, we have been able to use DGGE with environmental shrimp processing plant samples to obtain a view of microbial diversity over time in floor drains. These gels show approximately 18-25 bands. In order to confirm these results, the 16S rDNA sequences were amplified, cloned and sequenced to confirm the degree of environmental diversity. A total of 23 S. putrefaciens strains were isolated from various sources of the tilapia fish farm on different dates. The results indicate that strains of S. putrefaciens were found to constitute up to 70% of the bacterial flora in the water from the same tilapia tank at different sampling dates. Based on RAPD analysis the 23 strains of S. putrefaciens could be broken down into seven separate composite RAPD types with the use of the 3 random primers. This indicated that strains of S. putrefaciens from the tilapia farm were genotypically heterogeneous but with several common clones predominating. The mols % GC of the different RAPD types ranged from 45.5 to 48.9%. Oil-in-water emulsions containing cationic droplets stabilized by lecithin-chitosan membranes were produced using a two-stage process. A primary emulsion containing anionic lecithin-coated droplets was prepared by homogenizing oil and emulsifier solution using a high pressure valve homogenizer (5 wt% corn oil, 1 wt% lecithin, 100 mM acetic acid, pH 3.0). A secondary emulsion containing cationic lecithin:chitosan-coated droplets was formed by diluting the primary emulsion with an aqueous chitosan solution (1 wt% corn oil, 0.2 wt% lecithin, 100 mM acetic acid and 0.036 wt% chitosan. Our results showed that the secondary emulsions had better stability to droplet aggregation during thermal processing (30 to 90C for 30 minutes), freeze-thaw cycling (10C for 22 hours/30C for 2 hours) and high calcium chloride contents (500mM calcium chloride) and exhibited less lipid oxidation (peroxide formation), than primary emulsions. Studies on the reduction of lipid oxidation in fish muscle protein Extracts have involved acid and/or alkali solubilization is a recent method developed to separate muscle proteins with good functional properties. Citric acid and calcium chloride were more effective in reducing sensitivity to oxidation when added to disrupted whole muscle tissue, rather than when added to disrupted pre-washed tissue, where the soluble constituents have been largely removed. Citric acid and calcium chloride were found to provide an effective means of inhibiting lipid oxidation in muscle proteins extracted from minced cod tissue.
IMPACT: 2006/08/15 TO 2007/08/15<BR>
Enhance the bacteriological safety of seafood, reduce the oxidatin of solubilized fish muscle protein, and reduce the oxidation of omega-3 fatty acids to be added to foods by their encapsulation and hence achieve reduction of artherosclerosis.