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Production and world consumption of spices are constantly increasing. Although the antimicrobial properties of some spices are well documented, their use in the agri-food industry is also responsible for microbial contamination and spoilage. Bacterial spores introduced by spices can withstand different preparation processes, particularly thermal treatments, leading to food alterations during storage. This review brings together data from the literature about the prevalence and concentrations of spore-forming bacteria in all commercially available spices. The sporeformers found in spices belong mainly to the genera Bacillus and Clostridium. Such contaminations are very common and sometimes reach high levels, as in pepper and turmeric. Bacillus licheniformis and Bacillus cereus are the most frequently detected species. Studying the harvesting, processing, and storage procedures for spices provides elements to explain why high prevalence and concentrations are observed. Spices are mostly produced in developing countries on small farms using traditional production methods. Spices become contaminated by bacterial spores in two main ways: by contact with soil during harvesting or drying, as for pepper, or by cross-contamination during the water-cooking step, as for turmeric. From these observations, we propose some recommendations. Different methods that can be used to eliminate bacterial spores from spices are presented indicating their efficiency and the limitations of their use.

This study reports the prevalence of sporeforming bacteria and contamination levels of a variety of powdered dairy products from cows', ewes', goats' and mares’ milk produced in France. The concentration of mesophilic spores, thermophilic spores and highly heat-resistant thermophilic spores was assessed in 61 dairy powders. Thermophilic spore concentration was highly variable between powders, likely due to the different manufacturing processes used for transforming milk into dairy powders. The different stages of processing, particularly heat treatment close to 55 °C had a strong impact on selection of thermophilic bacteria contaminating dairy powders. For some products, thermophilic spore counts were as high as 5.89 log TSC g−1. Of the 313 thermophilic isolates selected, 93.3% belonged to the species Geobacillus stearothermophilus (43.5%), Bacillus licheniformis (30.0%) and Anoxybacillus flavithermus (19.8%). These results confirm the presence of the three majority species reported worldwide.

Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges from 4.7 to 7.4, and temperature from 10°C to 37°C for B. weihenstephanensis and from 18°C to 59°C for B. licheniformis. Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits, was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat-treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C during 12 min or at 95°C during 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite they both led to a tenfold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth by 1.2 fold and lowered the proportion of spores able to germinate and outgrow by 1.4 fold. Interestingly, we showed by a proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 compared to those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH. Importance Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sub-lethal heat-treatment. Using flow cytometry we were able to follow at a single cell level the changes in the physiological states of heat-stressed spores of Bacillus sp. and to discriminate between dormant spores, germinated spores and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth, and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sub-lethal heat-treatment, a common situation in food processing and a concern for food preservation and safety.

Spore-forming bacteria are implicated in cases of food spoilage or food poisoning. In their sporulated form, they are resistant to physical and chemical treatments applied in the food industry and can persist throughout the food chain. The sporulation leads to an increase in the concentration of resistant forms in final products or food processing equipment. In order to identify sporulation environments in the food industry, it is necessary to be able to predict bacterial sporulation according to environmental factors. As sporulation occurs after bacterial growth, a kinetic model of growth-sporulation was used to describe the evolution of vegetative cells and spores through time. The effects of temperature, pH and water activity on the growth and the sporulation abilities of Bacillus subtilis BSB1 were modelled. The values of the growth boundaries were used as inputs to predict these effects. The good description of the sporulation kinetics by growth parameters suggests that the impact of the studied environmental factors is the same on both physiological process. Suboptimal conditions for growth delay the appearance of the first spores, and spores appear more synchronously in suboptimal conditions for growth. The developed model was also applicable to describe the growth and sporulation curves in changing temperature and pH conditions over time.

Bacterial spores are formed within vegetative cells as thick-walled bodies resistant to physical and chemical treatments which allow the persistence and dissemination of the bacterial species. Spore-forming bacteria are natural contaminants of food raw materials and sporulation can occur in many environments from farm to fork. In order to predict spore formation over time, we developed a model that describes both the kinetics of growth and the differentiation of vegetative cells into spores. The model includes a classical growth model with the addition of only two sporulation-specific parameters: the probability of each vegetative cell to sporulate, and the time needed to form a spore once the cell is committed to sporulation. The growth-sporulation model was evaluated using the spore-forming, Gram positive bacterium,Bacillus subtilisand the biological meaning of the sporulation-specific parameters was validated using a derivative strain that produces the green fluorescent protein as a marker of sporulation initiation. The model accurately describes the growth and the sporulation kinetics in different environmental conditions and further provides valuable, physiological information on the temporal abilities of vegetative cells to differentiate into spores.ImportanceThe growth-sporulation model we developed accurately describes growth and sporulation kinetics. It describes the progressive transition from vegetative cells to spores with sporulation parameters which are meaningful and relevant to the sporulation process. The first parameter is the mean time required for a vegetative cell to differentiate into a spore (i.e. the duration of the sporulation process). The second sporulation parameter is the probability of each vegetative cell forming a spore over time. This parameter assesses how efficient the sporulation process is, how fast vegetative cells sporulate and how synchronous the bacterial population is for sporulation. The model constitutes a very interesting tool to describe the growth and the sporulation kinetics in different environmental conditions and it provides qualitative information on the sporulation of a bacterial population over time.

International audience; In foodstuffs, physico-chemical interactions and/or physical constraints between spores, inhibitors and food components may exist. Thus, the objective of this study was to investigate such interactions using a model emulsion as a microbial medium in order to improve bacterial spore control with better knowledge of the interactions in the formulation.Emulsions were prepared with hexadecane mixed with nutrient broth using sonication and were stabilized by Tween 80 and Span 80. The hexadecane ratio was either 35% (v/v) or 50% (v/v) and each emulsion was studied in the presence of organic acid (acetic, lactic or hexanoic) at two pH levels (5.5 and 6). Self-diffusion coefficients of emulsion components and the organic acids were measured by Pulsed Field Gradient-Nuclear Magnetic Resonance (PFG-NMR). The inhibition effect on the spore germination and cell growth of Bacillus weihenstephanensis KBAB4 was characterized by the measure of the probability of growth using the most probable number methodology, and the measure of the time taken for the cells to germinate and grow using a single cell Bioscreen® method and using flow cytometry. The inhibition of spore germination and growth in the model emulsion depended on the dispersed phase volume fraction and the pH value. The effect of the dispersed phase volume fraction was due to a combination of (i) the lipophilicity of the biocide, hexanoic acid, that may have had an impact on the distribution of organic acid between hexadecane and the aqueous phases and (ii) the antimicrobial activity of the emulsifier Tween 80 detected at the acidic pH value. The interface phenomena seemed to have a major influence. Future work will focus on the exploration of these phenomena at the interface.

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis ) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

Dehydrated raw materials used to produce bread are known to be highly contaminated with spore-forming bacteria. Once incremented in the bread, spores will germinate and exponential growth of amylase producing species such as Bacillus amyloliquefaciens may cause ropy bread spoilage in Mediterranean countries and huge economical costs. The aim of this study was to perform challenge test studies to compare growth experimental counts in bread and in silico growth predictions for various bread recipes. B. amyloliquefaciens ISPA-S109.3 was selected after previous characterization underlining its resistance to the baking process and high spoilage potential. Challenge tests were performed according to standardized methods with ISPA-S109.3 spore inoculation (4log spore/g) in the ingredient mixture. Home bread-making machine was used to bake the 4 bread recipes, i.e. wheat bran bread (WBB) and white wheat bread +/- bioingredient yielding various intrinsic parameters (aw: 0.93-0.96 and pH: 4.67-5.87). Bread incubation was performed at 20, 25 and 30°C +/- 0.01. Artificially contaminated pan bread and controls were prepared in three replicates. Distinction between ISPA-S109.3 and naturally contaminated Bacillus was performed using Rep PCR fingerprinting. The enumeration of ISPA-S109.3 was performed to determine growth kinetics and mathematical modeling using Sym'Previus. Based on fitted experimental growth kinetics, growth rate in WBB at 30°C was determined (µmax= 0.415+/-0.013h-1) and optimal growth rate calculated (µopt=1.61+/-0.048h-1) to further predict growth for the 5 tested conditions. Comparisons between experimental kinetics and growth predictions underlined that challenge test data were comprised into the 90% confidence interval of the predictions. These results highlight that major impact on growth was due to pH, aw and temperature while the impact of food matrix quantified by the µopt was sufficient, even in the case of sourdough. This study received funding from FP7-222-654-2 DREAM project and data related to strain and behavior diversity were incremented in Sym'Previus.

International audience; Heat resistance of spores is affected by many factors such as temperature, pH, water activity (aw) and others. Previous studies have reported that free fatty acids can affect the germination and growth of bacterial spores. In this study, we investigated the influence of free fatty acids in heating medium or in recovery medium on the heat resistance of spores of Bacillus cereus NTCC 11145 and Clostridium sporogenes Pasteur 79.3. Four free fatty acids were studied: palmitic, palmitoleic, stearic and oleic acid. During thermal treatments, the impact of these FFA in heating media was generally low, but the presence of free fatty acids in the recovery medium highly decreases bacterial spore apparent heat resistance, particularly with unsaturated fatty acids. A mathematical model was developed to describe and quantify the influence of free fatty acids in recovery media on the D-values. The z'FFA parameter values which quantify the impact of free fatty acids were determined. The variation of this parameter value according to the free fatty acid type was compared with MIC value variation given in the literature. The model enables the decrease in D-values in the presence of free fatty acids to be estimated. The high concentrations of free fatty acids in liver or canned duck may explain the microbial stability with low sterilization values applied.

Thermophilic spore forming bacteria present in the farm environment are found at low concentrations in raw milk. They are resistant to pasteurization treatments applied in industry and can develop during the manufacturing process of dairy powders and be found in high concentrations. In order to evaluate the contamination of dairy powders produced in France, an enumeration and an evaluation of the species diversity of thermophilic spores was carried out on 61 samples collected from industrials allowed to highlight a great diversity of spore contaminants. It appears that A. flavithermus, G. stearothermophilus and B. licheniformis are the most present thermophilic spore-forming bacterial species in dairy powders. These prevalence depend on the type of dairy powder studied. A phenotypic diversity regarding the enzymatic activity has shown with a spoilage potential for A. flavithermus, G. stearothermophilus and more particularly for B. licheniformis.As a result, contaminated powders used as ingredients in food formulations are possible sources of spoilage. A detailed analysis of the manufacturing process of dairy powders, associated with the growth capacities of thermophilic spore-forming bacteria, allowed to evaluate the steps of the manufacturing process of the powders favouring the development, the formation of biofilms and spores of the studied species. Finally, the estimations of the capacity of biofilm formation by A. flavithermus and G. stearothermophilus on the one hand and of their resistance to the cleaning treatments on the other hand revealed that the A. flavithermus species is the most resistant to the alkaline treatments whereas the resistances to the acid treatments seem to be similar for both species. The results obtained during this thesis have allowed to evaluate the growth capacities of thermophilic spore forming bacteria in the dairy powder industry and to understand the levers allowing to better control their development and their elimination., Les bactéries sporulées thermophiles présentes dans l’environnement de la ferme, se retrouvent en faibles concentrations dans le lait cru. Elles résistent aux traitements de pasteurisation appliqués en industrie et peuvent se développer au cours des procédés de fabrication des poudres laitières pour s’y retrouver en fortes concentrations. Afin d’évaluer la contamination des poudres laitières produites en France, un dénombrement et une évaluation de la diversité des espèces des spores thermophiles mené sur 61 échantillons recueillis auprès d’industriels a permis de mettre en évidence une grande diversité de contaminants sporulés. Il apparait que A. flavithermus, G. stearothermophilus et B. licheniformis sont les espèces bactériennes sporulées thermophiles les plus présentes dans les poudres laitières. Ces prévalences dépendent du type de poudre laitière étudiées. Une diversité phénotypique est observée lors de l’analyse des activités enzymatiques avec un potentiel d’altération observée pour A. flavithermus, G. stearothermophilus et plus particulièrement pour B. licheniformis.Il en résulte que les poudres contaminées utilisées comme ingrédients dans les formulations alimentaires sont de possibles sources d’altération. Une analyse détaillée du procédé de fabrication des poudres laitières, associée aux capacités de croissance des bactéries sporulées thermophiles a permis d’évaluer les étapes du procédé de fabrication des poudres susceptibles de favoriser le développement des espèces étudiées, de la formation de biofilms et de spores. Enfin les estimations d’une part de la capacité de formations de biofilms par A. flavithermus et par G. stearothermophilus et d’autre part de leurs résistances aux traitements de nettoyage en place ont révélé que l’espèce A. flavithermus est la plus résistante aux traitements alcalins alors que les résistances aux traitements acides semblent similaires pour les deux espèces. Les résultats obtenus au cours cette thèse ont permis d’évaluer les capacités de croissance des bactéries sporulées thermophiles dans les industries des poudres laitières et d’appréhender des leviers permettant de mieux maitriser leurs développements et leurs éliminations.