Hydrodynamic and Hydrostatic Pressure Inactivation of Bacteriophage as a Model System in Food Processing

Approach: a. Hydrodynamic pressure (HDP) treatment and enumeration Previously identified bacteriophage will be inoculated on the surface of poultry meat, beef steaks, and beef hot dogs purchased at a local grocery store. These bacteriophage would have been identified as appropriate surrogates for norovirus. Bacteriophage will be cultivated and purified according to standard laboratory practices. Bacteriophage at high (107 PFU/ml) and low concentrations (104 PFU/ml) will be surface inoculated on specific locations of the hot dog. Inoculated hot dogs will be doubled packaged, first in a stomacher bag that will be folded, and secondly in a multi-layer barrier with built in bone-guard protection. The outer bag will be evacuated of air and sealed. Hot dogs that are packaged but not placed in the stainless steel container for HDP treatment will be considered control treatments. Packaged hot dogs will be placed in a stainless steel shock wave container with a capacity to hold 54 L. The container will be filled with water. A binary explosive (100 g) will be placed 30.5 cm above the packaged hot dogs. The stainless steel lid will be placed on this container and locked. The explosive will then be detonated. Microbiological analysis. Pouches containing hot dogs will be removed from the container and then aseptically opened. Hot dogs will be cut with a sterile instrument to isolate the inoculated portion. This portion will be placed in a filtered stomacher bag with 0.1 percent peptone and pummeled for 1 min. After pummeling, one ml of filtrate or filtrate diluted in 0.1 percent peptone will be mixed with 1 ml of host bacteria at ca. 108 CFU/ml and allowed to adsorb for 15 min at room temperature. The phage bacteria mixture will then be combined with 3 ml of 0.75 percent tryptic soy agar supplemented with 5mM MgSO4 (TSAM)and poured over a petri plate (15 x 100 mm) containing 1.5 percent TSAM. Plates will be allowed to solidify, and then incubated for 12-24 h at 37 deg C. Plaques will be counted and compared to control to determine if HDP treatment has reduced bacteriophage titers on hot dogs. Results will be analyzed using Statistical Analysis Software (SAS, Inc.) in appropriate statistical design to determine if significant differences result between HDP treated samples and control (non-HDP treated samples). <P>b. High pressure processing (HPP) and enumeration Poultry meat, beef steaks and beef hot dogs will be inoculated with bacteriophage as described in section (a). These samples will be placed in a sterile bone guard bag which will be heat sealed. Each inoculated meat sample will be placed in an individual pressure cell within the high pressure unit. Samples will be treated with hydrostatic pressure at various pressure levels (50 MPa to 400 MPa) for various time periods. After the elapsed time, an individual pressure cell will be isolated from the pressure unit and the pressure will be released. Samples will be removed from pressure cell, and aseptically removed from plastic boneguard bags. Bacteriophage titers from meat samples will be determined using the procedure described in section (a).