Effects of heating and recovery media pH on the heat resistance of Alicyclobacillus acidoterrestris Ad 746 spores.

AIMS: Alicyclobacillus acidoterrestris is a sporulating, acidophilic bacterial species which spoils acidic beverages such as fruit juices. This work aims to quantify the heat resistance of A. acidoterrestris spores and their recovery potential as a function of heating and recovery media pH. METHODS AND RESULTS: The heat treatments were carried out with the strain of A. acidoterrestris Ad 746 in Bacillus acidoterrestris thermophilic medium. The pH of the heating medium from pH 7 to pH 2 nonsignificantly reduced the heat resistance. However, the pH levels of the recovery media strongly affected the apparent heat resistance of this strain. The maximum heat resistance was found when the pH was 4·70 and decreased when the pH decreased to pH 2·8, close to the minimum growth pH and when the recovery medium pH increased to pH 5·3. CONCLUSION: The heating medium pH has a slight effect on the spore heat resistances of this acidophilic species. However, the pH of the recovery media strongly affected the apparent heat resistance of this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained parameters quantifying the heat resistance of A. acidoterrestris spores are tools to optimize the heat treatments and to control its development.

Comparison of three Bacillus amyloliquefaciens strains growth behaviour and evaluation of the spoilage risk during bread shelf-life.

This study aims at the characterisation of growth behaviour of three strains of Bacillus amyloliquefaciens, isolated from ropy bread (ATCC8473), wheat grain (ISPA-S109.3) and semolina (ISPA-N9.1) to estimate rope spoilage risk in pan bread during shelf-life using the Sym'Previus tool. Cardinal values and growth/no growth boundaries were determined in broth, while artificial spore inoculations were performed in dough for various pan bread recipes to compare experimental counts with in silico growth simulations. Finally, two storage scenarios were tested to determine the probability to reach a spoilage threshold during bread shelf-life. Similarly to the safety criteria fixed for Listeria monocytogenes contamination in foodstuff complying with EC regulation, a potential rope spoilage threshold was arbitrary fixed at 5 log CFU/g for B. amyloliquefaciens. This study further underlines a higher rope spoilage potential of the ISPA strains as compared to the ATCC strain, thus emphasizing the interest to characterise both wild strains and reference strain to account for biological variability. In conclusion, this study showed that available decision making tools which are largely recognized to predict behaviour of pathogenic strains, shall also be used with spoilage strains to help maintain food quality and extend shelf-life.

Sensitivity of Bacillus weihenstephanensis to acidic changes of the medium is not dependant on physiological state.

This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance of vegetative cells of Bacillus weihenstephanensis. The psychrotolerant strain KBAB4 was cultured until the mid-exponentially phase (i) in BHI, (ii) in BHI supplemented with 2.5% salt or (iii) in BHI acidified at pH 5.5 with HCl. The growing cells were subsequently inactivated in lethal acid conditions ranging from 4.45 to 4.70. Based on statistical criteria, a primary mixed-Weibull model was used to fit the acid inactivation kinetics. The acid resistance was enhanced for acid-adapted cells and decreased for salt-adapted cells. The secondary modelling of the bacterial resistance allowed the quantification of the change in pH leading to a ten folds variation of the bacterial resistance, i.e. cells sensitivity (zpH). This sensitivity was not significantly affected whatever the preliminary mild exposure and the presence of sub-populations with different acid resistances. These results highlighted that pre-incubation conditions influence bacterial acid resistance without affecting the sensitivity to acidic modifications, with a 10 fold reduction of Bacillus acid resistance observed for a reduction of 0.37 pH unit. Quantification of such adaptive stress response might be instrumental in quantitative risk assessment more particularly in food formulation, particularly for low-acid minimally processed foods.

Predicting heat process efficiency in thermal processes when bacterial inactivation is not log-linear.

The food industry widely uses the F-value which considers microbial log-linear inactivation, while microbial heat inactivation may result in a non-log-linear inactivation pattern due to genetic or phenotypical heterogeneity. This may yield discrepancies in predicting microbial heat inactivation under dynamic conditions of heat treatment. In this paper, we suggest the calculation of the equivalent time of heat treatment at a given temperature to overcome these constraints. To validate our proposal, the heat inactivation of Bacillus pumilus, showing non-log-linear behavior, was predicted for 4 different heat inactivation profiles and bacterial enumeration was performed to determine whether prediction errors were acceptable. When the proportion of residuals in an acceptable zone from 1 log (fail safe) to 0.5 log (fail dangerous) was greater or equal to 70%, the model was considered as acceptable for predictions of the tested data. The new approach gave four different temperature profiles, with 96, 85, 85 and 100% of the residuals in the acceptable zone, indicating satisfactory prediction. Thus the proposed practical alternative to simulate microbial heat inactivation kinetics is able to extend the F-value to non-log-linear inactivation patterns.

Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour.