C. Patrick Dunne

Sensitivity of spores of Bacillus subtilis and Clostridium sporogenes PA 3679 to combinations of high hydrostatic pressure and other processing parameters

The combined effects of pressure, temperature, pH and the presence of nisin or sucrose laurate on the survival of spores of Bacillus subtilis 168 and Clostridium sporogenes PA 3679 were investigated. Spore populations of PA 3679 were reduced by 2.5-log10 when exposed to 404 megapascals (MPa) at 25°C, pH 4.0 for 30 min, but the same treatment at pH 7.0 resulted in a <0.5-log10 reduction in spore counts. Pressurization of B. subtilis spores at 70°C, pH 6.0 or 7.0 for 15 min at 404 MPa resulted in a 5-log10 reduction as compared to a <0.5-log10 reduction for the same pressurization treatment at 25°C. For the inactivation of spores of B. subtilis and PA 3679, the addition of nisin to the plating medium appeared to be synergistic in some instances when combined with pressurization at elevated temperatures and reduced pH. B. subtilis 168 was resistant to 0.1% sucrose laurate, but when combined at ≤6.0 pH with a 15-min treatment of 404 MPa at 45°C, a dramatic synergistic effect eliminated spore suspensions of 1×106/ml.

Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat.

Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (<1.0%), 392 megaPascals (MPa) at 45 degrees C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.

Spectrofluorimetric assessment of bacterial cell membrane damage by pulsed electric field

A rapid fluorescence staining technique was used to assess cell membrane damage and ensuing injury and death caused by pulsed electric field (PEF) treatment. Cell suspensions of Lactobacillus leichmannii ATCC 4797, Listeria monocytogenes Scott A and Escherichia coli O157:H7 ATCC 35150 were subjected to PEF for145.6 μs at field strengths of 5–20 kV/cm. Immediately after PEF treatment, cells were stained with propidium iodide (PI), and changes in fluorescence intensity were measured with a spectrofluorimeter. Increase in field strength decreased the count of survivors and proportionally increased the fluorescence intensity, confirming that cell inactivation by PEF is caused by membrane damage. Cells of E. coli O157:H7 were incubated with or without EDTA before exposure to PEF, but similar inactivation was observed, regardless of the EDTA pre-treatment. Increase in the fluorescence intensity, however, was appreciable in the EDTA-PEF-treated cells. The fluorescence staining technique, therefore, revealed membrane-related injury when EDTA pre-treated cells were PEF-treated. In conclusion, the fluorescence staining technique can be used to assess membrane damage associated with PEF treatments and is potentially useful in determining the relative sensitivity of microorganisms to PEF or monitoring the efficacy of such treatments.

Pulsed Electric Field Alters Molecular Chaperone Expression and Sensitizes Listeria monocytogenes to Heat

ABSTRACT Pulsed electric field (PEF)-resistant and PEF-sensitive Listeria monocytogenes strains were sublethally treated with electric pulses at 15 kV/cm for 29 μs and held at 25°C for 5 to 30 min prior to protein extraction. The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting. After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A). The levels of GroEL and DnaJ increased back to the basal expression level within 30 min. A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment. Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578. Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 μs) and heat (55°C, 10 min) were applied in sequence. When PEF and heat treatments were applied separately, the populations of L. monocytogenes Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml. Cells treated first with PEF and incubated at 25°C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively. The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates. It is concluded that PEF sensitized L. monocytogenes to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level.

Evaluation of Batch and Semicontinuous Application of High Hydrostatic Pressure on Foodborne Pathogens in Salsa

The effects of high hydrostatic pressure (HPP; 545 MPa) on strains of Escherichia coli O157:H7, Listeria monocytogenes, enterotoxigenic Staphylococcus aureus, and nonpathogenic microorganisms were studied in tomato-based salsa. Products were evaluated for the survival of the inoculated pathogens following HPP treatment and after storage at 4 degrees C and 21 to 23 degrees C for up to 2 months. Inoculated samples without HPP treatment, stored under the same conditions, were also evaluated to determine the effects of the acid environment of salsa on the survival of inoculated strains. None of the inoculated pathogens were detected in the HPP-treated samples for all treatments throughout the storage period. Inoculated pathogens were detected in the non-HPP-treated samples stored at 4 degrees C after 1 month, with L. monocytogenes showing the highest level of survivors. In the non-HPP-treated samples stored at 21 to 23 degrees C, E. coli and S. aureus were not detected after 1 week, but L. monocytogenes was detected in low levels. Studies with nonpathogenic strains of the pathogens were conducted at Oregon State University using HPP treatments in a semicontinuous production system. The nonpathogenic microorganisms (E. coli, Listeria innocua, Listeria welshimeri, and nonenterotoxigenic S. aureus) were inoculated together into a feeder tank containing 100 liters of salsa. Microbiological results of samples collected before HPP treatment and from the aseptic filler were similar to those obtained for the pathogenic strains. No survivors were detected in any of the HPP-treated samples.

Pressure-Assisted Thermal Sterilization Validation

This chapters discusses the efforts by the US Army food industry academic consortium research to develop validation protocols and demonstrate efficacy of a pressure-assisted thermal sterilization process (PATS) for the production of a commercially sterile ambient stable, low-acid mashed potato product. Studies include qualification of the equipment, product, and package and process performance. Under the specified conditions of the validation study, it was concluded that the PATS process is capable of eliminating six log10 of heat- and pressure-resistant C. botulinum spores/145 g from the deliberately contaminated packs of mashed potatoes. Subsequently the consortium submitted a filing of a mashed potato product treated by PATS with FDA. FDA issued a letter of no objection to the consortium by 2009.