A sublethal dose of ethanol (5%, vol/vol), acid (HCl, pH 4.5 to 5.0), H 2 O 2 (500 ppm), or NaCl (7%, wt/vol) was added to a Listeria monocytogenes culture at the exponential phase, and the cells were allowed to grow for 1 h. Exponential-phase cells also were heat shocked at 45؇C for 1 h. The stress-adapted cells were then subjected to the following factors at the indicated lethal levels-NaCl (25%, wt/vol), ethanol (17.5%, vol/vol), hydrogen peroxide (0.1%, wt/vol), acid (pH 3.5), and starvation in 0.1 M phosphate buffer at pH 7.0 (up to 300 h). Viable counts of the pathogen, after the treatment, were determined on Trypticase soy agar-yeast extract, and survivor plots were constructed. The area (h ⅐ log 10 CFU/ml) between the control and treatment curves was calculated to represent the protective effect resulting from adaptation to the sublethal stress factor. Adaptation to pH 4.5 to 5.0 or 5% ethanol significantly (P < 0.05) increased the resistance of L. monocytogenes to lethal doses of acid, ethanol, and H 2 O 2 . Adaptation to ethanol significantly (P < 0.05) increased the resistance to 25% NaCl. When L. monocytogenes was adapted to 500 ppm of H 2 O 2 , 7% NaCl, or heat, resistance of the pathogen to 1% hydrogen peroxide increased significantly (P < 0.05). Heat shock significantly (P < 0.05) increased the resistance to ethanol and NaCl. Therefore, the occurrence of stress protection after adaptation of L. monocytogenes to environmental stresses depends on the type of stress encountered and the lethal factor applied. This "stress hardening" should be considered when current food processing technologies are modified or new ones are developed.
Changes in the thermotolerance of Listeria monocytogenes at different stages of growth and in the presence of stress factors were studied. Stresses investigated include starvation and treatment with hydrogen peroxide, ethanol, and low pH. Stressed cells were suspended in phosphate buffer (0.1 M, pH 7.0) and their thermotolerance was determined by measuring D56°C-value using the capillary tube method. D56°C remained constant (1.0 min) during the exponential phase of growth and then increased to a maximum of 8.6 min during the late exponential and early stationary phases. Cells of L. monocytogenes were harvested when in the exponential phase and suspended in phosphate buffer at pH 7.0. The cells were then starved by incubating the suspension for up to 163 h at 30°C. D56°-values increased during starvation and reached a maximum of 13.6 min. Ethanol (0.5 to 12%, vol/vol), acid (pH 4.0 to 7.0), or H2O2 (27 to 500 ppm) was added to L. monocytogenes cultures in the exponential phase and the cells were allowed to grow for 1 h (one to two doublings of the control treatment). All treatments caused increases in the thermotolerance of the pathogen. Maximum tolerance was observed in cells exposed to 4 to 8% ethanol, pH 4.5, and 500 ppm H2O2; the corresponding averages for D56°C-values were 4.1, 8.8, and 2.9 min, respectively.
Resistance of biological indicators for sterilization in pharma and food applications is often determined via fraction negative experiments. The ISO 11138 standards prescribe the Holcomb-Spearman-Karber procedure (HSKP) for calculating D value using these methods. However, HSKP imposes limitations on experimental designs. While ISO 11138 requires inactivation kinetics to not significantly deviate from first order, the HSKP does not provide a step to check such departure, or its extent. Maximum likelihood estimation (MLE) is an alternative approach for D value determination from fraction negative experiments. Compared to HSKP, it allows researchers to tailor the experimental design to practical needs, potentially reducing overall sample size in the process. This work presents a development of a 2004 published worksheet for MLE calculation, providing tighter confidence intervals and an improved way for Improbability calculation. Ways to exploit the design flexibility of MLE approach are presented, including simulated experiments and examples on how to optimize experimental design according to various situational needs, also in cases where expected D values are of the order of one or a few seconds as is commonly encountered in the sterilization of aseptic fillers for food. Detection of deviation from first-order kinetics using the enhanced Improbability methods is discussed; one of the examples provided by ISO 11138-1 for D value determination by HSKP contains data that deviate significantly from first order.
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