Response of <i>Listeria monocytogenes</i> to high hydrostatic pressure or freeze-thaw cycles following exposure to selected environmental stresses
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The purpose of this investigation was to examine the viability of Listeria monocytogenes Scott A NADC 2045 that endured selected environmental stresses and were then subsequently exposed to freeze-thaw cycles or high hydrostatic pressure. The environmental stresses investigated in relation to freeze-thaw cycle survival include acid shock (HCl, pH 4.0-6.0), alkali shock (NaOH, pH 8.0-11.0), ethanol shock (2.0% -0.5%), oxidative shock (H2O2, 50-500ppm), and acid adaptation. All shock stresses were applied to exponential phase cells whereas non-stressed exponential phase cells served as a control. Freeze-thaw cycles involved freezing at -18yC for 24 h and thawing at 30yC for 7 min. Injury evaluation for all freeze-thaw treatments were performed by comparing colony counts of the pathogen on tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) to counts on modified oxford agar (MOX). All samples were serially diluted (10-fold) in Buffered Peptone Water (BPW) and surface-plated on appropriate agar media. Inoculated agar plates were incubated at 35yC and bacterial colonies were counted at 72 h. Starvation of washed stationary phase cells in physiological saline (0.85% (w/v) NaCl) over 12 days was examined at 2-day intervals for viability and resistance to high hydrostatic pressure. Starvation preparation involved the static growth of L. monocytogenes in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) and washing these cells twice in 0.85% NaCl. Cells were then suspended in fresh physiological saline and held at 25yC during the starvation period. Pressurization at 400 MPa from 1 to 75 s was achieved using the Food Lab High-Pressure Food Processor (Stansted Fluid Power Ltd, Essex, U.K.). Viability of pressurized L. monocytogenes was examined after serial dilution in 0.1% peptone and plating on TSAYE followed by incubation of 35yC for 48 h. Control cultures were non-starved stationary phase cells. Results for the freeze-thaw cycles and environmental stresses indicate no statistically significance difference in freeze resistance or injury of the stressed pathogen compared to the control. When examining controls, there was a decrease in viability after 1 cycle of 0.5 log CFU/ml and 0.74 log CFU/ml after 4 cycles. This decrease occurred irrespective of any prior environmental stress tested. No statistically significant freeze-thaw injury was found among cells that endured prior stresses or in freeze-thaw treated cells compared to control cells. For starved L. monocytogenes, approximately a 2 log CFU/ml decrease was seen in viability after 2 days of starvation. Viability remained stable for the remaining 10 days. Maximum D-values (at 400 MPa) of 19.88 s, 18.6 s and 18.5 s were observed after 8, 6, and 10 days of starvation, respectively. D-value (at 400 MPa) of the control was 11.85 s. Overall significance of freeze-thaw results for the food industry is that freeze-thaw resistance of L. monocytogenes does not seem to be affected by certain prior environmental stresses on this pathogen. Reductions after 4 freeze-thaw cycles in the controls were 0.74 Log CFU/ml, which represented a significant decrease in viability. Based on the results of the present study, the exposure of L. monocytogenes Scott A to certain environmental stresses does not increase the resistance of this organism to freeze-thaw cycles. Also, starved L. monocytogenes cells developed a higher resistance to high pressure processing compared to non-starved cells. The increased high pressure resistance of starved L. monocytogenes should be considered when aiming to design safe food processing protocols involving high hydrostatic pressure technology.