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<ol> <li>Determine the effect(s) of varying high hydrostatic pressures, times, and cycles on bacterial produced enzymes of natural origin in two commercially caught fish species capable of producing scombrotoxin food poisoning.
<li>Determine the optimum combination of high hydrostatic presures, times, and cycles to produce fish filets and steaks having microbial populations less than 10 colony forming units/g (CFU/g) and the effects of the processes on consumer acceptability.
<li>Identify the genera and species of bacteria surviving high hydrostatic pressure treatments obtained from naturally contaminated fish.
<li>Determine the effect(s) of varying high hydrostatic pressures and times on enzyme activity produced by bacteria selected for their ability to produce active decarboxylase enzymes inoculated in the sterile tissue of five commercially caught fish species.
<li>Determine the potential for scombrotoxin food poisoning from biogenic amines produced by bacterial enzymes remaining on fish stored at varying temperatures after high hydrostatic processing to either kill or reduce bacterial populations.
<li>Determine the effect(s) of varying high hydrostatic pressures and times on the in vitro production of histamine and cadaverine from purified proginitor compounds and commercially obtained enzymes or enzymes purified from relevant bacteria.
<li>Implement an outreach program for stakeholders consisting of educational material development, scientific publications, and participation in professional and trade association meetings. </ol>
<ol> <li>Fish filets and steaks will be obtained 4 times each of 2 yrs from commercial fishermen in MD, VA and NC, processed under commercial conditions, and stored at 0, 5, and 15 C for 0, 3, 6, 9, 12 and 15 days or until unfit for human consumption. Decarboxylase activities on histidine, ornithine, and lysine will be determined by enzyme extraction and enzyme assays.
<li>Fish filets and steaks will be obtained as described for Obj 1, vacuum packed and subjected to 200, 400, and 500 MPa for 5, 10 and 15 min. at 18C. Samples will be subjected to 1, 2 and 3 cycles to determine effect of multiple cycles on vegetative microbial survival. Microbial samples will be obtained by suspending 20 g of tissue in 180 ml of 0.1% Peptone Broth, samples will be placed in a sterile Stomacher bag and homogenized for 1 min, serial dilutions made, and color measurements, sensory analysis, and statistical analysis performed. Data will be used to produce fish samples for Obj 4 and 5. If resistant microorganisms are encountered, Obj 3 will be implemented.
<li>Procedures as for Obj 2; after incubation for 48 and 168 hrs and alternative processes are unable to produce sterile fish tissue, resistant microorganisms will be identified. Sample colonies will be picked and regrown on agar from which they were isolated. Once isolates have been obtained and confirmed that a mixed colony is not present, microorganisms will be identified using Sherlock Microbial Identification System.
<li>Fish filets and steaks will be obtained as for Obj 1, and tissue samples will be inoculated with microorganisms that produce active decarboxylase enzymes. Samples will be stored at 5 and 10 C for 0, 50, 10, and 25 days and analyzed for quantitative histamine content using monoclonal antibody analysis. Growth of microorganisms will be monitored. Microbial suspensions will be subjected to 200, 300, 400 and 500 MPa for 5, 10 and 15 min at 18 C and 1, 2 and 3 cycles to determine resistance to varying pressures and cycles. Samples will be assayed for histidine, lysine, and ornithine decarboxylase.
<li>Procedures as Obj 4 except samples will be stored for 48 and 72 hrs and subjected to high hydrostatic pressure process to either destroy or inactivate the inoculating microorganisms. Samples will be incubated at 0, 3, 6 and 10 C for 0, 3, 6, 9, 12 and 15 days or until spoiled or inedible. Samples will be analyzed for histamine content; study will be replicated 6 times/yr.
<li>Commercially available enzymes (Histidine decarboxylase [non-pyridoxal phosphate] from Lactobacillus 30a [Sigma H3266, crude powder], histidine decarboxylase [pyridoxal phosphate] from clostridium perfringens [Sigma H8250, crude powder], lysine decarboxylase from Bacterium cadaveris [Sigma L0882, partially purified powder]) will be used in the assays. Decarboxylases will be purified from these powders if preliminary studies show promise. Histidine decarboxylase will be purified from cells of Morganella morganii.
<li>Results will be disseminated through presentations at natl and intnatl scientific and regional fishery meetings, refereed and trade journal articles, Cooperative Extension and Sea Grant publications, and websites. </ol>
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Scombrotoxin is a common source of illness in the US associated with the comsumption of certain finfish species. This research will answer whether high hydrostatic pressures provide protection against or increased vulnerability to scombrotoxin food poisoning thereby enabling food processors to know the safety or hazard presented through the application of high hydrostatic pressure processing.
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A research associate has been employed to direct this project and perform the required chemical and biological analyses to satisfy the project objectives. A meeting of the three cooperators (Virginia Tech, University of Maryland, and North Carolina State University)will be held in January, 2003 to coordinate project activities. The high hydrostatic pressure machine has been ordered and should arrive in March, 2003.
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Processors should be able to produce fish having an extended shelf life with biogenic amine concentrations that do not present a health risk. Also, a high hydrostatic pressure laboratory will be established that will be available to accommodate other food research activities.