Depuration tanks were set up at the MSU Experimental Seafood Processing Lab (ESPL) to control an environment for freshly harvested oysters from the Gulf of Mexico. These tanks were used to manipulate salinity and temperature to see if the effects could eliminate or reduce the harmful bacteriaV.v. in oysters. Most of the items needed for constructing a depuration tank can be purchased at a local hardware store with the exception of a few specialty items such as UV sterilizers, canister filters, chilling units, and salinity meters. These specialty items were purchased from a reputable aquaculture supply house.

Following harvest (within 24 hr), live oysters, Crassostrea virginica, were washed and placed into 2 live well tanks with a circulating water system equipped with UV sterilization tube. Water salinity varied with each set of experiments i.e. 12, 16, or 20 ppt sea salt. The temperature of both tanks was maintained at 78-800F.  Following acclimation of the oysters to their new environment, approximately 12 hours, the control tank continued to be maintained at the original temperature and the experimental tank temperature was reduced to 50-520 F using a circulating water chiller. Oysters were tested initially and after 7 and 14 days for aerobic plate counts (APC) andV.v.counts. Initial V.v. most probable numbers (MPN) ranged from an adjusted gross mean (AGM) of 14,300 to 111,667 MPN/gram oyster meat. At 12 and 16 ppt salinity water, the MPN was reduced by 2 log10 in the chiller tank and 1 log10 in the ambient tank. At the 20 ppt water salinity, there was a 3 log10 reduction in the ambient tank and again a 2 log10 reduction in the chiller tank. Further experiments conducted over the winter 2006-2007 determined that when initial V.v. numbers were <1000, rapid chilling at 16 ppt saline, for 7-14 days, was effective in reducing numbers to non-detectable levels (<3 MPN/g oyster meat).

Objectives

1. (a) Determine the most effective depuration conditions related to temperature, salinity, and time to reduce Vibrio vulnificus to <30 mpn/g oyster meat  (b) Perform a validation study for PHT processing using the most effective combination of temperature, salinity and time of process.
2. Provide demonstration workshops to processors and dealers on the PHT depuration method and results of research.
3. Perform a cost estimate on implementation of the "rapid-chill" post harvest process.

Methodology

Completion Report: 8/14/07

Immediately after harvest, oysters will be transported to the Experimental Seafood Processing Laboratory (ESPL) in Pascagoula, Mississippi, in insulated coolers and placed into a specially designed tank within 12 hours of their harvest. Water temperature at harvest will be recorded. The tank will circulate and filter water enriched with Sea Salt at varying salinity levels (5, 10, 15, and 20 ppt). Water quality tests will be performed daily to insure water quality throughout the depuration time.

A cooling system connected to the filtration system in the tank will allow temperature manipulation. The oysters will be placed in three depuration systems at ambient temperature (70-80 F) and then the temperature will be lowered in two of the tanks to reach a target temperature at harvest. The lower temperature tank will simulate winter water temperatures. Four bins of 50 oysters will be place in each of three depuration tanks. Two bins will be used to determine the mortality rate over time and the other two will be tested to determine the level of V.v. over time. The level of V.v. will be enumerated using a three tube MPN procedure followed by a standard chemical profile conformation method. Three depuration tanks will be used simultaneously at differing temperatures and salinity. The ESPL has already built one depuration tank and we have completed two preliminary studies to determine the feasibility of potential success for this project. Based on the preliminary studies, we anticipate a 5 log reduction of V.v. to occur in something less than 7 days.

A V.v. study will be conducted to determine a statistical validation of this process to classify this method as a post harvest process (PHT) according to the USFD/ISSC guidelines to reduce V.v. to <30 MPN/g oyster meat. Data will be collected on ten processed samples obtained on each of three different processing days for a total of 30 samples. Samples should be randomly samples throughout any one day. All samples on a processing day must come from the same lot of shellfish and be determined to have an adjusted geometric mean of MPN of 100,00 per gram of greater. Lower initial levels, when used, will only validate for those initial load levels. Standard MPN methods endorsed by the ISSC  will be used for the enumeration of V.v. Testing for processed samples will be a single dilution five-tube MPN inoculated with 0.1 g of shellfish per tube. For the process to be validated, no more than three samples out of 30 may fail. Failure is indicated by more than two out of five MPN tubes in any sample being positive. The validation will fail if any one sample has all five MPN tubes positive.

Rationale

Gulf Coast oysters, Crassostrea virginica, live in shallow estuarine waters with normal depths of 8 to 25 feet. Oysters filter feed plankton and other particles, including the pathogenic bacteria Vibrio vulnificus, by slightly opening their shell and pumping water through at a rate up to five liters per hour. V. vulnificus, a natural inhabitant of estuarine environments, is orally transferred to humans through the consumption of raw oysters. The objective of the study was to determine the effect of placing live oysters into a depuration system and adjusting the water temperature to simulate winter water temperatures. Varying temperatures and salinities were evaluated. Mortality rate of the oysters and levels of V. vulnificus will be monitored.

This experiment will attempted to establish a post harvest treatment, simulating natural environmental changes that occur from summer months (when V.v. numbers are high) to winter months (when V.v. numbers are low). A successful "rapid chill: depuration system" that effectively reduces the amount of V. vulnificus in live oysters while maintaining the quality of the oysters, would provide industry an inexpensive and effective treatment process and would provide consumers a safer oyster product. Oyster processors, especially small processors, would benefit from the low cost of implementation and increase in sales to local markets and especially to states requiring post harvest processed oysters within their markets.

Results

The rapid chill depuration system could be an additional method of postharvest processing for raw oyster products.  At present there are four methods which are approved for use in the processing of raw oyster products by the U.S. Food and Drug Administration (FDA), namely: heat-cool pasteurization (HCP), high-pressure processing (HPP), individual quick freezing (IQF), and irradiation. In 2007, there are nine commercial processing plants located in Mississippi, Louisiana, Florida, Texas and Washington which are currently using HCP, HHP and IQF processing systems for the rapidly growing and strictly regulated domestic markets of raw oyster products (http://msstate.edu/dept/crec/owmr.html).