Influence of Sporulation Temperature on Germination and Growth of B. weihenstephanensis Strains in Specific Nutrients and in an Extended Shelf-Life Refrigerated Matrix Under Commercial Pasteurization and Storage Conditions

Abstract

Extended shelf-life (ESL) refrigerated ready-to-eat foods are thermally pasteurized to ensure food safety and stability. However, surviving psychrotrophic Bacillus cereus spores can still pose a challenge. Studies predicting their behavior often overlook sporulation conditions. This study investigated the effect of sporulation temperature on germination of three Bacillus weihenstephanensis strains in specific nutrients (inosine and/or amino acids) with or without prior heat activation (80 °C, 10 min). Sporulation temperature variably affected germination, with stronger effects in moderately responsive strains and nutrients. Heat activation strongly stimulated germination, particularly in nutrients with poorer responses, mitigating differences induced by sporulation temperature. The influence of sporulation temperature on germination and growth in an ESL matrix at refrigeration temperatures (4 °C or 8 °C) in vacuum packaging after heat activation or commercial pasteurization (90 °C, 10 min) was also studied. The latter treatment increased germination rates of surviving spores; however, some strains suffered damage and lost viability upon germination at 4 °C but recovered and grew at 8 °C. These findings highlight the need to account for variability in spore recovery and outgrowth during quantitative risk assessments for psychrotrophic B. cereus in ESL foods.

Keywords: Bacillus weihenstephanensis; bacterial spores; extended shelf-life refrigerated foods; germination; heat resistance; sporulation temperature.

Figure 1

Vegetative cells (dark cells) and spores (bright cells) of B. weihenstephanensis WSBC 10204 observed under phase-contrast microscopy during the sporulation process.

Figure 2

Counts (log CFU/mL) of germinated spores of strains (A) WSBC 10202, (B) WSBC 10204, and (C) SC produced at T_min (blue), T_opt (green), or T_max (red) without and with applying a prior heat treatment (80 °C, 10 min or 90 °C, 10 min) in an ESL matrix after 24 h of incubation at 25 °C. Values in the figures correspond to averages and standard deviations calculated from three biological replicates. Different lowercase letters indicate statistically significant differences (p ≤ 0.05) among spores produced at different sporulation temperatures within each treatment and strain. Different capital letters indicate statistically significant differences (p ≤ 0.05) among treatments within each strain and sporulation temperature. N.D.: not determined; germination was not tested because heat treatment caused >1.5 log reductions.

Figure 3

Inactivation (expressed as log (N₀/N_t)) of (A) WSBC 10202, (B) WSBC 10204, and (C) SC spores produced at T_min (blue), T_opt (green), or T_max (red) by heat treatment at 90 °C for 10 min in the ESL matrix. The dotted line indicates the limit of quantification (300 CFU/mL). Values in the figures correspond to averages and standard deviations calculated from three biological replicates. Different lowercase letters indicate statistically significant differences (p ≤ 0.05) among spores produced at different sporulation temperatures within each strain.

Figure 4

Plate counts (log CFU/mL) of germinated spores of WSBC 10202 produced at T_opt (●) and WSBC 10204 sporulated at T_min (■), T_opt (■), or T_max. (■) in the ESL matrix after heat treatment at (A) 80 °C for 10 min or (B) 90 °C for 10 min during storage at 4 °C in vacuum packaging. Values in the figures correspond to averages and standard deviations calculated from three biological replicates. The dotted line indicates the limit of quantification (300 CFU/mL).

Figure 5

Growth kinetics of WSBC 10202 spores produced at T_opt (■) and WSBC 10204 sporulated at T_min (■), T_opt (■), or T_max. (■) in an ESL matrix after heat treatment at (A) 80 °C for 10 min or (B,C) 90 °C for 10 min during storage at (A,B) 4 °C or (C) 8 °C in vacuum packaging. The dotted line indicates the limit of detection (200 CFU/mL).