The overall goal of this research is to prevent foodborne illness associated with human consumption of raw or partially-cooked molluscan shellfish. Effective post-harvest processing technologies that are recognized by the US FDA have had limited acceptance in the domestic industry due to high initial capital equipment costs and the economics of transporting and storing of shell oysters. This new project was developed to increase our knowledge and understanding on use of ozonated microbubbles in the depuration of Eastern oysters (Crassostrea virginica) as a potential post-harvest processing technology. While other projects continue to look at intervention technologies like high salinity relaying and biological agents for the control of naturally occurring Vibrios in oysters, this proposed project will research novel technologies such as micro- and nano-bubbles for delivery of chemical control agents (ozone) in the depuration and/or wet storage of live shellfish. <P>The specific objectives of the research program are: Determine the antimicrobial efficacy of ozonated microbubbles on shellfish indicator organisms, pathogenic Vibrio bacteria and norovirus using artificial seawater; Evaluate the pathogen reduction, animal health and oyster meat quality in pilot-scale depuration and/or wet storage using ozonated microbubbles in natural seawater; and Validate the pathogen reduction and processing parameters in pilot-scale depuration and/or wet storage of Eastern oysters using ozonated microbubbles in natural seawater. <br/>This project will increase our knowledge and understanding on use of ozonated microbubbles in the depuration of Eastern oysters (Crassostrea virginica) as a potential post-harvest processing technology.
Current post-harvest processing (PHP) technologies recognized by the US Food and Drug Administration (FDA) for control of naturally occurring Vibrios in shellfish are mild heat treatment (cool pasteurization), cryogenic individual quick freezing (IQF) with extended frozen storage, high hydrostatic pressure (HHP) processing, and low-dose gamma irradiation (NSSP, 2009). While these PHP technologies are known to reduce Vibrios to non-detectable levels (less than 30 MPN/gram) in shellfish, general acceptance in the domestic oyster industry has been limited due to high initial capital equipment costs and the economics in transporting and storing of shell oysters (RTI, 2011). Shellfish depuration, on the other hand, has been available to the industry for over a century and is recognized for significantly reducing the levels of shellfish-borne illnesses (NSSP, 2009). Depuration is a commercial processing strategy where shellfish are relayed to locations with higher salinity water or placed in tanks of clean seawater and allowed to purge themselves of contaminants in a period of days (Richards, 1988; Richards, 1991, Richards, 2010). Water supplied to depuration tanks is treated to remove microbial contaminates through use of chlorine, UV light or ozone. Depuration is intended to reduce relatively low levels of contaminates in shellfish and is not intended for highly contaminated products. PHP technologies such as high-salinity relaying, shellfish depuration and use of biological or chemical control agents offers industry the added incentive of maintaining the oyster alive. Recently, micro- and nano-bubbles (MNBs) technology has attracted great interest in biological sciences and the water treatment industries. The ability to control bubble size and its composition, i.e., oxygen or ozone, offers some unique applications in a wide range of industries including the potential for use in post-harvest processing of molluscan shellfish. For example, the Institute for Environmental Management Technology (IEMT) at the National Institute of Advanced Industrial Science and Technology (AIST) in Japan has shown that microbubbles (less than 50 micrometer) shrink by dissolution of gas as they rise in water and disappear completely before reaching the water surface (Cameron, 2005). Nanobubbles (less than 1 micrometer) are more stable than microbubbles and can exist in water for months containing an appropriate concentration of electrolytes (salt) (Takahashi, 2010). Working with a small company called REO Research Institute; scientists discovered that infusing MNBs with oxygen and ozone have some remarkable effects on living organisms. Japanese scientists found that feeding oxygen-nanobubble water to cows and chickens alleviated a number of health issues such as intestinal parasites, bacterial infection and lethargy. They found purging shell oysters in ozone-nanobubble water for a day cleansed the oysters of bacterial and viral contaminates (Cameron, 2005). In addition, the IEMT/AIST reported the first successful inactivation of norovirus by using microbubbles infused with concentrated oxygen containing 2 percent ozone (AIST, 2004).
Ozonated microbubbles will be generated using a oxygen-ozone micro-nano bubble water sterilizer supplied by Reo Laboratory Co., Ltd. in Japan. The microbubbles will be monitored for ozone content using the indigo method (Bader and Hoigne, 1981). Artificial seawater will be used to supply water of various salinities, ozone levels and time to determine antimicrobial efficacy on shellfish indicators (total coliforms, fecal coliforms), Vibrio vulnificus, Vibrio parahaemolyticus, and norovirus. Mixed cultures of total and fecal coliform bacteria (indicator organisms) and pure cultures of Vibrio vulnificus and Vibrio parahaemolyticus will be used to determine the efficacy of ozonated microbubbles in artificial seawater. In addition, the ability to inactivate norovirus will be determined with the assistance of Dr. Jaykas and staff in the USDA NIFA funded NoroCORE project. The test method for the evaluation of antimicrobial and virucidal efficacy will be the AOAC bacterial use-dilution test for evaluating liquid surface disinfectants (US EPA, 2010). Microbiological analysis will be performed on control and treated oysters using the Most Probable Number (MPN) methods in the FDA Bacteriological Analytical Manual (BAM) (Kaysner and DePaola, 2004). Modified Cellobiose-Polymyxin B Colistin (mCPC) agar will be used for selection of Vibrio vulnificus and Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar will be used for selection of Vibrio parahaemolyticus. Colonies that are 1-2 mm in diameter, yellow, opaque, round and flat on mCPC are considered positive for Vv, while colonies that are 2-3 mm in diameter, opaque, round and green or bluish on TCBS are considered positive for Vp. MPN scores will be compared to 0.1, 0.01 and 0.001, 3-tube MPN tables for enumeration (Kaysner and DePaola, 2004). Pilot-scale depuration (water treatment) and/or wet storage (shellstock treatment) will be carried out in natural seawater that has been filtered to remove particulates and treated with ozonated microbubbles to remove microbial contaminates. The pilot-scale studies will utilize natural seawater that has undergone filtration to remove particulate and organic compounds. Natural seawater will be treated with ozonated microbubbles to remove microbial contaminates (depuration) prior to deliver to the holding tanks containing live oysters. A second set of experiments will be performed with ozonated bubbles delivered directly to the holding tanks with live oysters (wet storage) to allow the oyster to filter seawater containing an effective dose (2 ppm) of ozonated microbubbles for elimination of bacteria contained inside the oyster. These studies will be performed independently and the affect on pathogen reduction, animal health and oyster meat quality will be determined. Based on results obtained in objective two, pilot-scale depuration and/or wet storage studies will be performed to validate the pathogen reduction and process parameters to establish the post-harvest processing requirements as given in the National Shellfish Sanitation Program Guide and Interstate Shellfish Sanitation Conference.