Progress 09/15/02 to 09/14/06
Outputs
Axenic cultures of two strains of Morganella morganii were exposed to high hydrostatic pressure (HHP) treatments of 200, 300, 350, 400 and 500 MPa. The cultures showed resistance to HHP treatments of 200, 300 and 350 MPa but showed a significant log reduction in growth at pressures of 400 and 500 MPa (eight log reduction and 90 percent inactivation of the M. morganii histidine decarboxylase). Given the results of the culture tests, three species of fish, bluefish, yellow fin tuna and Spanish mackerel were studied to determine the efficacy of HHP treatment to inactivate M. morganii in fish fillets. Fish were captured live in the Atlantic Ocean, iced, and transported to North Carolina State University and Virginia Tech for processing. Fillets from each species were inoculated with Morganella morganii, which is a known histidine producer. Both inoculated and uninoculated fillets were exposed to high hydrostatic pressures of 400 and 500 MPa for five minutes. Control fillets
were also inoculated but not exposed to HHP. Fillets were stored for up to five days at 10 C. In all three species of uninoculated fish, applying HHP of 400 MPa for 5 minutes reduced the overall microbial population by 2.2 to 4.6 logs after the fish had been stored for 5 days. In the fillets inoculated with the Morganella morganii (1,000,000 cells per gram) HHP treatment resulted in a reduction, after five days of storage, to 100 cells per gram or less. However, HHP processing changed the appearance of the fillets, resulting in a somewhat "cooked" appearance. To determine the effect of HHP processing on the marketability of the fish, bluefish and mackerel fillets were HHP processed dry, in water and in both a 0.9 and 3.5 percent saline solution and then presented to a 30-member sensory evaluation panel. The panel members were asked to visually compare the four different processing media used and to indicate if they would consider purchasing the fish. An untreated raw fillet was
included in the sensory panel evaluation as a control. Fresh raw mackerel was selected by 28 of 30 participants as acceptable for purchase; 22 participants said they would purchase or consider purchasing the mackerel that had been HHP-treated in a 3.5 percent saline solution. The other HHP treatments (dry, sterile water and 0.9 percent saline solution) were not as favorably received by the panel. Bluefish is a soft-tissue fish that proved not to be very acceptable to a 30-member panel even in its raw state. Given that bluefish was not acceptable raw, the fact that the panel found bluefish questionable to unacceptable no matter how it was processed indicates the panel results are not of any benefit in trying to determine if processing bluefish in liquid would help preserve its appearance. In summary, it appears that HHP treatment at 400 MPa for 5 minutes can significantly reduce scombrotoxin food poisoning in the fish fillets tested, but the resulting product may not be acceptable to
the general public, implying that it may be necessary to market it through food-services or knowledgeable chefs, as opposed to sold in retail stores.
Impacts
Histamine is one of the leading causes of illness from the consumption of seafood. This research shows that high hydrostatic pressure (HHP) processing of fillets of Spanish mackerel, yellow fin tuna and bluefish reduces histamines to within acceptable limits. In all three species of fish, applying HHP of 400 MPa for 5 minutes reduced the overall microbial population by 2.2 to 4.6 logs after the fish had been stored for 5 days. When the fish were spiked with the addition of 1,000,000 cells of stationary phase Morganella morganii and then high pressure processed and stored for five days, the Morganella morganii was reduced to 100 cells per gram or less. Appearance of high hydrostatic pressure treated fillets is altered, however the resulting product, while not acceptable for retail marketing, is acceptable for institutional use, where the majority of seafood is sold.
Publications
- Flick, Jr., G. J. 2006. ISO 22000:2005 Food Safety System Integrates HACCP. Global Aquaculture Advocate. 9(3):28-30.
- Flick, Jr., G. J. 2005. Scombrotoxins Part I: Problematic Compounds for Procedure, Processors. Global Aquaculture Advocate. 8(4):33-34.
- Flick, Jr. G. J. 2005. Scombrotoxins Part II: Prevalence in Seafood. Global Aquaculture Advocate. 8(5):32-34.
- Flick, Jr. G. J. 2006. Scombrotoxins Part III: Effect of Processing Operations on Product Safety. Global Aquaculture Advocate. 9(1):26, 28.
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Progress 10/01/04 to 09/30/05
Outputs
Fresh fish samples, tuna and mahi-mahi, were collected from North Carolina (NC) commercial vessels within 12 hr of capture. The fish were filleted prior to transport to Virginia Tech (VT) for high hydrostatic pressure processing. Processed samples were returned for microbiological and sensory evaluations. Replicate samples were kept frozen at VT to determine effect of high pressures on histidine decarboxylase activities. Replicates will be collected during spring of 2006. The total aerobic plate counts (TSA incubated at 37 C for 48 hr) were reported in August 2005. A total of 31 bacterial isolates were recovered from mahimahi treated at 400 MPa for 5 min. Twenty five isolates were identified by either the BBL Crystal identification or the BioMerieux API 20E system. Twenty of these isolates were Gram positive and 5 were Gram negative. Tuna samples yielded nine isolates from the 400 MPa at a 5 min treatment, eight of which were identified. Seven of these isolates were
Gram negative and 1 was Gram positive. The only isolate determined to be a histamine producer via Nivens media was a Gram negative bacteria isolated from mahimahi. A reduced number of samples have been obtained due to scheduling difficulties between fishing seasons, inclement weather, availability of high pressure unit and availability of student labor. A second tuna sample is being planned currently. The highest pressure treatments (400 MPa for 5 min) seems adequate to reduce fish fillet microbial populations to less than ten CFU per gram, however the sensory attributes are reduced. The literature suggests that Gram negative bacteria are primarily responsible for histamine production in scombroid fish. The literature is, however, concentrated on tuna species and may not represent scrombroid fish entirely. Future research activities will determine if the next tuna sample also produces a majority of Gram negative isolates which is in contrast to the mahimahi samples. Samples were taken
from mahimahi and stored in ice and transported to VT for pressure treatments at 200, 300, and 400 MPa for 2 or 5 min. Samples from all treatment combinations were evaluated both raw and cooked. Raw samples were placed in containers marked with 3 digit codes and held for 15 min before the evaluation. Cooked samples were placed in aluminum containers and covered with aluminum foil and coded. The samples were cooked at 177 C for 25 min in a preheated conventional oven and then served to panel members. Six panelists were trained in evaluating raw and cooked mahimahi and tuna during a 1 hr session. All panelists evaluated all samples once (each treatment twice). The effect of pressure treatments was only detected in raw samples. The samples had a cooked appearance and the cooked color was more intense in the thinner portions of the fish. Cooking effectively masked the color changes caused by the pressure treatments. Quality changes in cooked samples are solely related to storage time
(spoilage). Pressures up to 300 MPa decreased the quality of the raw samples, irrespective of application time. Treatmens of 400 MPa did not alter the samples significantly greater than the 300 MPa treatment.
Impacts
Results have shown that pressure treatments are not observed in cooked samples as in the raw. Cultures of Morganella morganii subjected to 300 and 400 MPA for 30, 60, and 120 seconds exhibited a loss of viability which appears as a first-order process, with a linear decrease in the log of colony forming units. Cells of M. morganii are highly sensitive to hydrostatic pressure when in the log phase of growth. A fully-grown culture of 1,000,000,000 cells was reduced to less than 10 cells in one minute at 400MPa. This research clearly demonstrates that some bacteria recognized as producing high histidine decarboxylase enzymes can be reduced or eliminated through pressurization treatments. High pressure has promise as an operational procedure capable of providing increased food safety to scombroid and scombroid like fish species through the reduction of selected bacteria species.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Because of the intense hurricane season of 2004, only two fishing trips could be conducted in Virginia. Bluefish were collected off the eastern coast of the Chesapeake Bay. A researcher accompanied the fishermen on the boat to ensure proper use of the temperature data recorders. Temperature data was recorded from the time the fish were caught until placed on ice and lowered to 40 degrees F or below. Fish were held below 40 degrees F until reaching Virginia Tech for microbial and histamine analysis. Microbial testing indicated bacterial loads averaged slightly less than 4 log. No histamine producing isolates have been identified from either fishing trip at this time. No samples of fish tested from either fishing trip have exhibited histamine concentrations higher than 3ppm. Previous fishing trips have yielded histamine concentrations from 0 to 15ppm, as well as identified species of histamine-producing bacteria. Histamine levels of 50ppm have been determined as the
level at which putrescine and cadaverine should subsequently be tested, but has not been needed at this point in the research. The correlation of time and temperature to the amount of histamine-forming bacteria and histamine in fish will be used to establish safe handling practices for industry. Additional fishing trips will be conducted in the spring and summer of 2005. Preliminary enzymatic studies using radiometric methods have been conducted to produce standard curves for the amount of histidine decarboxylase present in samples. A new extraction procedure adapted from Segner and Bohm (1994) is currently being investigated as an alternative method for isolation of histamine decarboxylase. Fish samples are homogenized in a 1:2 ratio with an extraction buffer solution. This homogenization buffer consists of 20mM potassium diphosphate (10mM diphasic, 10mM monophasic), 1mM EDTA, and 1mM mercaptoethanol. This technique has been used to obtain active histamine decarboxylase from fresh
fish tissue, which was confirmed using spectrophotometric methods. The extraction method is currently being used on fish that have undergone high-pressure treatments. The products of this extraction technique are presently awaiting analysis.
Impacts
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.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
The 35 liter high pressure processing equipment from Avure Technologies arrived at the Virginia Tech Food Science Building in May 2003. Preliminary experiments on the effect of high pressure processing, HHP, on survival of five histidine decarboxylating microorganisms have been accomplished. The five organisms were found to have very similar death curves when treated under the same high-pressure conditions. The effects of HHP on the injury of the native microflora of scrombiod fish were also examined. Microbial analysis of both injured and uninjured cells was conducted on pressure treated samples of raw yellowfin tuna. Preliminary results indicate that both injury and death increase as pressure is increased. The effect of HHP on the physical properties of raw yellowfin tuna was also examined to determine pressure and time combinations that would minimally change the sensory attributes of the product. Texture was analyzed using Kramer Shear Cell test on an Instron, and
color was measured using a Mintola colorimeter. Higher pressures were found to significantly affect both the color and texture of the product. A chiller for the HHP machine has been received and will be used to determine if lower processing temperature can be used to increase pressure treatments while minimally changing the physical properties of the product. The results from the culture survival studies and the physical property analysis will be used to determine optimal study parameters including times, pressures, temperature and number of cycles. Currently, researchers are fulfilling the requirements of the university Radiation Safety Office needed to begin enzyme assays. A change in personnel required that recertification and training occur before radiolabeled material could be used for the study. Dr. W. G. Niehaus has been consulted to provide assistance with the radiometric enzyme assay. Background information has been obtained to identify temperature and pH ranges that will be
used in enzyme assay optimization studies. The next steps in the study will be to determine the parameters to be used in the enzymatic assay and identify HHP treatments to be used throughout the study. Treatments will need to be both effective at the control of biogenic amine product as well as produce a product that will be acceptable to the consumer.
Impacts
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.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
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.
Impacts
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.
Publications
- No publications reported this period
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