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Virginia Sea Grant funded research

Use of High Hydrostatic Pressure (HHP) Treatments to Eliminate Vegetative Pathogens in Fresh Crab Meat and Spores and Spore Forming Pathogens in Fresh Crab Meat and Shrimp

Michael L. Jahncke and George Flick

Objectives of this project are to: 1) establish survival curves and D values for three individual strains of L. monocytogenes in pure cultures at pressures of 310, 340, 420, 440, and 590 MPa at 21° and 45°C; 2) establish survival curves and D values for a mixture of three strains of L. monocytogenes inoculated in fresh crab meat at pressures of 45, 000, 50,000, 60,000, 65,000 and 85, 000 psi at 21° and 45°C; 3) establish survival curves and D values for a mixture of three strains of L. monocytogenes inoculated in fresh crab meat treated with Nisin (500 or 1000 IU/g), or ALTATM 2341 (0.1 or 1%), or sodium lactate (1M) at pressures of 45, 000, 50,000, 60,000, 65,000 and 85, 000 psi at 21° and 45°C; 4) determine the effect of HHP (with and without added Nisin, or ALTATM 2341 or sodium lactate) treatments on aerobic plate counts (APC) and fecal coliform (FC) numbers in fresh crab meat; 5) select the most effective treatment combinations, and determine the effect of HHP treatment, by itself, or in combination with Nisin or ALTATM 2341 or sodium lactate on the sensory characteristics of fresh crab meat using triangle tests and consumer acceptance sensory studies; and 6) determine the effect of HHP treatment, by itself, or in combination with Nisin or ALTATM 2341 or sodium lactate on shelf life of fresh crab meat.

The proposed analytical methodology is based on the FDA Bacteriological Analytical Manual (FDA/BAM, 1995). Three different strains of L. monocytogenes (Scott A, V7, D43) will be evaluated. Both pure cultures and inoculated fresh crab meat will be evaluated. Strains will be maintained on tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) slants at refrigeration temperatures. Specific HHP processing parameters with and without Nisin or ALTATM 2341 or sodium lactate additions will be established to achieve a 6-7 log reduction in pure cultures and in inoculated crab meat. Pure cultures of single strains will be treated at temperatures of 21° and 45°C. Specific time/pressure processing parameters will be established to achieve a 6-7 log reduction. Samples will be stored in a refrigerator (4°C) before and after treatment. The L. monocytogenes cultures will be prepared as described above. Crab meat samples (25-50 g) will be inoculated with three strains to obtain 107 CFU/g. The inoculated crab meat will be mixed by hand for 1 min and then stomached for 2 min to ensure that the cells are uniformly distributed throughout the meat. (Note: The appropriate concentrations of Nisin, or ALTATM 2341 or sodium lactate will also be added and mixed into the crab meat). Portions (25-50 g) of fresh crab meat will be placed into pouches and vacuum-sealed. Duplicate pouches will be placed and heat-sealed in a s25-Jan-2008ore and after treatment. The physical properties of varying HHP times, temperatures, and pressures will be determined for cooked crab meat and shrimp. The effects of the various treatments on physical properties will be defined by objective and subjective tests to determine processes that have minimal effects on produce quality and acceptability. Once acceptable HHP processes are identified, crab meat and shrimp will be processed and the surviving microorganisms isolated using standard (e.g., Nutrient Agar) and resuscitation (BHI Media, Chopped Meat Media) media. The recovered microorganisms will be identified by genus and species (where possible) using the Sherlock Microbial Identification System. A heat shock procedure (80°C for 10 min) will be applied to the processed products to promote spore germination. Spores of two B. cereus serovars will be subjected to varying HHP pressures, times, temperatures, with and without chemical additives (carbon dioxide, Nisin, ALTA). The spores will be harvested after 80 – 90% sporulation after growth in Nutrient Broth and Nutrient Agar. The spores will be washed and suspended at a density of 107/ml in 20 ml of the germination media. The spores will be processed to determine the lethality of the various processes. Recovery of the viable spores will be accomplished using McIlvaine citrate phosphate buffer with a heat treatment and subsequent growth on Nutrient Agar. Crab meat and shrimp will be inoculated with 102 and 104 organisms/g of one B. cereus serovars. The products will be stored at varying temperatures and times (and chemicals if they have a positive effect) to determine the ability of the B. cereus to grow competitively with the normal product microflora. The samples will be evaluated for acceptance by odor only since they will contain a human pathogen.

Beneficiaries of this research include the seafood processing industry that is under constant pressure to increase the safety of their ready-to-eat products; regulators who are seeking improved post-processing methods to increase food safety, non-government organizations that are demanding higher food safety standards; and citizens who are concerned about the safety of the food. The research results will be of special interest to processors of crab meat, shrimp, crawfish, hot smoked fish and other similar products. The USDA and FDA have released their L. monocytogenes risk assessment for RTE foods. Cooked RTE products are identified as high-risk food products (CFSAN/ FSIS, 2001). Thus, the FDA will focus their current and future inspection activities on high-risk RTE food products, such as fresh crab meat. Several crab processing companies in Virginia have been placed under Consent Decrees by the FDA due to the presence of L. monocytogenes in fresh crab meat products. Information from this project will provide additional relevant data to help refine the USDA and FDA risk assessment on L. monocytogenes for RTE fresh crab meat. This research will determine the ability of pathogenic microbial spores to survive various HHP treatments and the effect HHP treatments have on either increasing or decreasing the potential for food-borne poisoning. There is the possibility HHP process could compromise food safety since HHP processes would destroy vegetative microorganisms thereby permitting the growth of pathogenic spore forming microorganisms. Industry is also reluctant to consider purchasing HHP equipment since the cost is high and the economic/food safety benefits questionable.