Relationships between the inhibitory efficacy and physicochemical properties of six organic acids and monolaurin against Bacillus weihenstephanensis KBAB4 growth in liquid medium.

Organic acids are widely used in foodstuffs to inhibit pathogen and spoiler growth. In this study, six organic acids (acetic, lactic, propionic, phenyllactic, caprylic, and lauric acid) and monolaurin were selected based on their physicochemical properties: their molecular structure (carbon chain length), their lipophilicity (logP), and their ability to dissociate in a liquid environment (pKa). The relation between these physicochemical properties and the inhibitory efficacy against B. weihenstephanensis KBAB4 growth was evaluated. After assessing the active form of these compounds against the strain (undissociated, dissociated or both forms), their MIC values were estimated in nutrient broth at pH 6.0 and 5.5 using two models (Lambert & Pearson, 2000; Luong, 1985). The use of two models highlighted the mode of action of an antibacterial compound in its environment, thanks to the additional estimation of the curve shape α or the Non-Inhibitory Concentration (NIC). The undissociated form of the tested acids is responsible for growth inhibition, except for lauric acid and monolaurin. Moreover, long-carbon chain acids have lower estimated MICs, compared to short-chain acids. Thus, the inhibitory efficacy of organic acids is strongly related to their carbon chain length and lipophilicity. Lipophilicity is the main mechanism of action of a membrane-active compound, it can be favored by long chain structure or high pKa in an acid environment like food.

Impact of sodium chloride and carbon dioxide on conidial germination and radial growth of Penicillium camemberti.

Penicillium camemberti is a domesticated species adapted to the dairy environment, which is used as adjunct cultures to ripen soft cheeses. A recent population genomics analysis on P. camemberti revealed that P. camemberti is a clonal lineage with two varieties almost identical genetically but with contrasting phenotypes in terms of growth, color, mycotoxin production and inhibition of contaminants. P. camemberti variety camemberti is found on Camembert and Brie cheeses, and P. camemberti variety caseifulvum is mainly found on other cheeses like Saint-Marcellin and Rigotte de Condrieu. This study aimed to evaluate the impact of water activity (aw) reduced by sodium chloride (NaCl) and the increase of carbon dioxide (CO2) partial pressure, on conidial germination and growth of two varieties of P. camemberti: var. Camemberti and var. Caseifulvum. Mathematical models were used to describe the responses of P. camemberti strains to both abiotic factors. The results showed that these genetically distant strains had similar responses to increase in NaCl and CO2 partial pressure. The estimated cardinal values were very close between the strains although all estimated cardinal values were significantly different (Likelihood ratio tests, pvalue = 0.05%). These results suggest that intraspecific variability could be more exacerbated during fungal growth compared with conidial germination, especially in terms of macroscopic morphology. Indeed, var. Caseifulvum seemed to be more sensitive to an increase of CO2 partial pressure, as shown by the fungal morphology, with the occurrence of irregular outgrowths, while the morphology of var. Camemberti remains circular. These data could make it possible to improve the control of fungal development as a function of salt and carbon dioxide partial pressure. These abiotic factors could serve as technological barriers to prevent spoilage and increase the shelf life of cheeses. The present data will allow more precise predictions of fungal proliferation as a function of salt and carbon dioxide partial pressure, which are significant technological hurdles in cheese production.

Impact of carbon dioxide on the radial growth of fungi isolated from dairy environment.

In dairy industry, filamentous fungi are used as adjunct cultures in fermented products for their technological properties but they could also be responsible for food spoilage and mycotoxin production. The consumer demands about free-preservative products has increased in recent years and lead to develop alternative methods for food preservation. Modified Atmosphere Packaging (MAP) can inhibit fungal growth and therefore increase the food product shelf-life. This study aimed to evaluate radial growth as a function of CO2 and more particularly carbonic acid for fourteen adjuncts and/or fungal spoiler isolated from dairy products or dairy environment by using predictive mycology tools. The impact of the different chemical species linked to CO2 (notably carbonic acid) were study because it was reported previously that undissociated carbonic acid impacted bacterial growth and bicarbonates ions were involved in modifications of physiological process of fungal cells. A significant diversity in the responses of selected strains was observed. Mucor circinelloides had the fastest growth rates (µ > 11 mm. day-1) while Bisifusarium domesticum, Cladosporium herbarum and Penicillium bialowiezense had the slowest growth rates (µ < 1 mm. day-1). Independently of the medium pH, the majority of strains were sensitive to total carbonic acid. In this case, it was not possible to conclude if CO2 active form was gaseous or aqueous so modeling were performed as a function of CO2 percentage. Only Geotrichum candidum and M. circinelloides strains were sensitive to undissociated carbonic acid. Among the fourteen strains, P. bialowiezense was the less sensitive strain to CO2, no growth was observed at 50% of CO2 only for this strain. M. lanceolatus was the less sensitive strain to CO2, the CO250 which reduce the growth rates by 50% was estimated at 138% of CO2. Low CO2 percentage improved the growth of Penicillium expansum, Penicillium roqueforti and Paecilomyces niveus. Mathematical models (without and with optimum) were suggested to describe the impact of CO2 percentage or undissociated carbonic acid concentration on fungal growth rate.

Synergistic interaction between pH and NaCl in the limits of germination and outgrowth of Clostridium sporogenes and Group I Clostridium botulinum vegetative cells and spores after heat treatment.

Group I Clostridium botulinum and Clostridium sporogenes are physiologically and genetically closely related. Both are widely distributed in the environment and can cause foodborne botulism. In this work, a physiological study was conducted with 37 isolates from spoiled canned food and five referenced strains of C. sporogenes (three isolates) and Group I C. botulinum (two isolates). Growth limits of vegetative cells were established as a function of pH and NaCl concentration in PYG modified medium (PYGm) at 30 °C for 48 days. The heat resistance of the spores was studied for 2 min and 10 min at 102 °C and 110 °C. This physiological study (pH, NaCl growth limits and heat resistance) allowed the selection of 14 isolates of C. sporogenes (twelve isolates) and Group I C. botulinum (two isolates) representative of the diversity found. This panel of 14 selected isolates (11 isolated from spoiled canned food and three reference strains), were whole genome sequenced, but no association of physiological and genetic characteristics could be detected. Finally, we studied the ability of spores to germinate and grow from 5 isolates (four C. sporogenes and one Group I C. botulinum), under stress conditions generated by pH and NaCl following a low intensity heat treatment. The accumulation of these 3 stresses creates synergies that will strongly reduce the probability of spore growth in pH and salt conditions where they usually proliferate. The effect is progressive as the conditions become drastic: the number of decimal reduction observed increases translating a probability of growth which decreases. This study provides a better understanding of the behaviour of C. sporogenes and Group I C. botulinum isolates and shows how the combination of pH, NaCl and heat treatment can help prevent or minimise foodborne botulism outbreaks.

Impact of water activity on the radial growth of fungi in a dairy environment.

Filamentous fungi are used in the dairy industry as adjunct cultures in fermented products, but can also lead to food spoilage, waste and economic losses. The control of filamentous fungi with abiotic factors contributes to longer food shelf life and prevention of fungal spoilage. One of the main abiotic factors for controlling fungal growth in foods is water activity (aw). This study aimed to evaluate radial growth as a function of aw for sixteen fungal adjuncts and/or spoilers isolated from dairy products or a dairy environment. Glycerol (a non-ionic compound) and sodium chloride (NaCl, an ionic compound) were used to adjust the aw of culture media. This study showed significant diversity in the responses of the tested fungal strains as a function of medium aw. The growth response of Penicillium bialowiezense and Sporendonema casei was binary, with no clear decrease of growth rate until the growth limit, when the aw was reduced. For the strains of Bisifusarium domesticum, Mucor circinelloides and Penicillium camemberti, a decrease of aw had the same impact on radial growth rate regardless of the aw belonging to their growth range. For the strains of Aspergillus flavus, Cladosporium herbarum, Geotrichum candidum, Mucor lanceolatus, Penicillium expansum, Penicillium fuscoglaucum, Penicillium nalgiovense, Paecilomyces niveus, Penicillium roqueforti, Penicillium solitum and Scopulariopsis asperula, the impact of a decrease in aw was more pronounced at high aw than at low aw. A mathematical model was suggested to describe this impact on the radial growth rate. For all tested species, radial growth was more sensitive to NaCl than glycerol. The ionic strength of NaCl mainly explains the difference in the effects of the two solutes.

A Bayesian Approach to Describe and Simulate the pH Evolution of Fresh Meat Products Depending on the Preservation Conditions.

Measuring the pH of meat products during storage represents an efficient way to monitor microbial spoilage, since pH is often linked to the growth of several spoilage-associated microorganisms under different conditions. The present work aimed to develop a modelling approach to describe and simulate the pH evolution of fresh meat products, depending on the preservation conditions. The measurement of pH on fresh poultry sausages, made with several lactate formulations and packed under three modified atmospheres (MAP), from several industrial production batches, was used as case-study. A hierarchical Bayesian approach was developed to better adjust kinetic models while handling a low number of measurement points. The pH changes were described as a two-phase evolution, with a first decreasing phase followed by a stabilisation phase. This stabilisation likely took place around the 13th day of storage, under all the considered lactate and MAP conditions. The effects of lactate and MAP on pH previously observed were confirmed herein: (i) lactate addition notably slowed down acidification, regardless of the packaging, whereas (ii) the 50%CO2-50%N2 MAP accelerated the acidification phase. The Bayesian modelling workflow-and the script-could be used for further model adaptation for the pH of other food products and/or other preservation strategies.

Effect of abiotic factors and culture media on the growth of cheese-associated Nectriaceae species.

Nectriaceae species have been described in various natural environments or as plant or human pathogens. Within this family, the Bisifusarium domesticum species is of particular interest for food mycologists as it is used for technological functions in various cheese productions. Moreover, it has only been isolated from the cheese environment so far and, until recently, was the only Nectriaceae species described in this food product. Recently, four novel cheese-associated Nectriaceae species have been described, including two associated to the Bisifusarium genus and two to a new genus, Longinectria gen. nov.. These observations raise questions concerning the potential adaptation of these species to the cheese environment. In this context, this study first focused on determining the impact of abiotic factors on the growth of isolates belonging to the five cheese-associated species (i.e. B. allantoides sp. nov., B. domesticum, B. penicilloides sp. nov., L. lagenoides gen. nov. sp. nov. and L. verticilliforme gen. nov. sp. nov.) but also included phylogenetically close species. To do so, fungal growth kinetics in liquid medium (Potato Dextrose Broth) were determined by laser nephelometry at different temperatures, pH and water activities using NaCl as a depressor. Growth modeling was then performed to estimate cardinal values for each abiotic factor. Secondly, fungal growth was also evaluated on Potato Dextrose Agar (synthetic medium), cheese agar (cheese-mimicking medium) and Raclette de Savoie cheese (actual cheese). Our results clearly highlighted physiological differences in growth characteristics between the studied cheese-associated Nectriaceae spp. and the "non-cheese" species which could suggest, for the former, an adaptation to this food matrix. Indeed, regarding the impact of the tested abiotic factors, statistical analyses confirmed this dichotomy, with for example the lowest optimal temperatures estimated for the cheese-associated species (Topt 19.1-23.1 °C) while the other Bisifusarium species exhibited the highest optimal temperatures (Topt 26.1-36.2 °C). As for the impact of growth media, radial growth measurements highlighted that B. domesticum was the least affected species for growth on Raclette de Savoie and even grew faster on cheese agar than on synthetic medium confirming its strong adaptation to the cheese environment.

The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) provides new possibilities for optimizing processes to manage risk of C. sporogenes spoilage.

Clostridium sporogenes has been widely used as a surrogate for proteolytic C. botulinum for validating thermal processes in low-acid cans. To limit the intensity of heat treatments, industrials must use other ways of control as an association of acidic and saline environment after a low heat treatment. The probability of growth of pH (7-4.4), sodium chloride concentration (0-11%) and heat treatment (80°C-10 min; 100°C-1.5 min and 5.2 min) were studied on C. sporogenes PA 3679 spores and vegetative cells. Vegetative cells or heat-treated spores were inoculated in PYGm broth at 30 °C for 48 days in anaerobic conditions. Vegetative cells growth (pH 4.6-pH 4.5; 7%-8% NaCl) range is larger than the spore one (pH 5.2-pH 5.0; 6%-7% NaCl). Spores germination and outgrowth rage is decreased if the spores are heat-treated at 100 °C for 1.5 min (pH 5.5-5.3; 4%-5% NaCl) and 5.2 min (pH 5.7-5.3; 4%-5% NaCl). The C. sporogenes PA 3679 spores germination and outgrowth is impacted by their physiological state. The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) opening new possibilities for optimizing food formulation processes to manage the risks of C. sporogenes spoilage.

Application of a path-modelling approach for deciphering causality relationships between microbiota, volatile organic compounds and off-odour profiles during meat spoilage.

Microbiological spoilage of meat is considered as a process which involves mainly bacterial metabolism leading to degradation of meat sensory qualities. Studying spoilage requires the collection of different types of experimental data encompassing microbiological, physicochemical and sensorial measurements. Within this framework, the objective herein was to carry out a multiblock path modelling workflow to decipher causality relationships between different types of spoilage-related responses: composition of microbiota, volatilome and off-odour profiles. Analyses were performed with the Path-ComDim approach on a large-scale dataset collected on fresh turkey sausages. This approach enabled to quantify the importance of causality relationships determined a priori between each type of responses as well as to identify important responses involved in spoilage, then to validate causality assumptions. Results were very promising: the data integration confirmed and quantified the causality between data blocks, exhibiting the dynamical nature of spoilage, mainly characterized by the evolution of off-odour profiles caused by the production of volatile organic compounds such as ethanol or ethyl acetate. This production was possibly associated with several bacterial species like Lactococcus piscium, Leuconostoc gelidum, Psychrobacter sp. or Latilactobacillus fuchuensis. Likewise, the production of acetoin and diacetyl in meat spoilage was highlighted. The Path-ComDim approach illustrated here with meat spoilage can be applied to other large-scale and heterogeneous datasets associated with pathway scenarios and represents a promising key tool for deciphering causality in complex biological phenomena.

Effects of temperature, pH and water activity on the growth and the sporulation abilities of Bacillus subtilis BSB1.

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.

Spoilage of fresh turkey and pork sausages: Influence of potassium lactate and modified atmosphere packaging.

Fresh poultry and pork meat products represent highly perishable products which are susceptible to spoil within a few days after production. Lactate addition and modified atmosphere packaging are common preservation strategies used to overcome spoilage. This study aimed to identify the effects of these strategies and their possible interactions on spoilage indicators simultaneously on fresh pork and turkey sausages. Ten batches of raw meat (turkey or pork) sausages were industrially produced with different lactate concentrations (0, 1 or 2% w/w in turkey and 0, 0.57 and 1.13% w/w in pork), packed under different gas mixtures (air, MAP1: 70% O2 - 30% CO2 and MAP2: 50% CO2 - 50% N2) and chill stored during 22 days. Spoilage responses including enumeration of total aerobic mesophilic and lactic acid bacteria, measurement of pH and colour, evaluation of visual defects and off-odour, were monitored. Effects of lactate and modified atmosphere packaging (MAP) as well as random effect of the batch variability were studied using a mixed effect model. Despite initial batch variability, significant effects of lactate and gas packaging were observed but in a different way in turkey and pork. Our results suggest that for fresh turkey sausages, the gas mixture enriched in oxygen enhanced off-odour perception and sausage discolouration from red to dark grey / brown colour. Unlike turkey sausages, in pork sausages, lactate did not significantly influence the monitored spoilage responses, whereas MAP (70% O2-30% CO2) reduced the off-odour perception. The developed model could be useful to estimate the effect of preservation strategies on spoilage occurrence while considering industrial batch variability.

Spoilage of Chilled Fresh Meat Products during Storage: A Quantitative Analysis of Literature Data.

A literature search was performed on spoilage of fresh meat products by combining keyword query, text mining and expert elicitation. From the 258 collected studies, a quantitative analysis was first performed to identify the methods which are the most used to evaluate spoilage beside the preservation strategies suggested. In a second step focusing on a subset of 24 publications providing quantitative data on spoilage occurrence time, associations between spoilage occurrence time of meat products and specific spoilage indicators were investigated. The analysis especially focused on factors well represented in the 24 publications, i.e., gas packaging (O2 and CO2) and storage temperature. Relationships between spoilage occurrence and several microbiological indicators were also sought. The results point out possible advantages of removing dioxygen in packaging to delay spoilage occurrence, whereas, in the presence of dioxygen, the carbon dioxide proportion in the gas mixtures was shown to influence spoilage occurrence. The collected data clearly reveal a potentially protective role of lactic acid bacteria. Besides, while a spoilage role could be attributed to Pseudomonas spp., the growth of mesophilic aerobic microbes, Brochothrix spp. and Enterobacteriaceae seemed independent of spoilage occurrence time.

Large-scale multivariate dataset on the characterization of microbiota diversity, microbial growth dynamics, metabolic spoilage volatilome and sensorial profiles of two industrially produced meat products subjected to changes in lactate concentration and packaging atmosphere.

Data in this article provide detailed information on the diversity of bacterial communities present on 576 samples of raw pork or poultry sausages produced industrially in 2017. Bacterial growth dynamics and diversity were monitored throughout the refrigerated storage period to estimate the impact of packaging atmosphere and the use of potassium lactate as chemical preservative. The data include several types of analysis aiming at providing a comprehensive microbial ecology of spoilage during storage and how the process parameters do influence this phenomenon. The analysis includes: the gas content in packaging, pH, chromametric measurements, plate counts (total mesophilic aerobic flora and lactic acid bacteria), sensorial properties of the products, meta-metabolomic quantification of volatile organic compounds and bacterial community metagenetic analysis. Bacterial diversity was monitored using two types of amplicon sequencing (16S rRNA and GyrB encoding genes) at different time points for the different conditions (576 samples for gyrB and 436 samples for 16S rDNA). Sequencing data were generated by using Illumina MiSeq. The sequencing data have been deposited in the bioproject PRJNA522361. Samples accession numbers vary from SAMN10964863 to SAMN10965438 for gyrB amplicon and from SAMN10970131 to SAMN10970566 for 16S.

Differentiation of Vegetative Cells into Spores: a Kinetic Model Applied to Bacillus subtilis.

Spore-forming bacteria are natural contaminants of food raw materials, and sporulation can occur in many environments from farm to fork. In order to characterize and 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 is based on a classical growth model and enables description of the kinetics of sporulation with the addition of three parameters specific to sporulation. Two parameters are related to the probability of each vegetative cell to commit to sporulation and to form a spore, and the last one is related to the time needed to form a spore once the cell is committed to sporulation. The goodness of fit of this growth-sporulation model was assessed using growth-sporulation kinetics at various temperatures in laboratory medium or in whey for Bacillus subtilis, Bacillus cereus, and Bacillus licheniformis The model accurately describes the kinetics in these different conditions, with a mean error lower than 0.78 log10 CFU/ml for the growth and 1.08 log10 CFU/ml for the sporulation. The biological meaning of the parameters was validated with a derivative strain of Bacillus subtilis 168 which produces green fluorescent protein at the initiation of sporulation. This model provides physiological information on the spore formation and on the temporal abilities of vegetative cells to differentiate into spores and reveals the heterogeneity of spore formation during and after growth.IMPORTANCE The growth-sporulation model describes the progressive transition from vegetative cells to spores with sporulation parameters describing the sporulation potential of each vegetative cell. Consequently, the model constitutes an interesting tool to assess the sporulation potential of a bacterial population over time with accurate parameters such as the time needed to obtain one resistant spore and the probability of sporulation. Further, this model can be used to assess these data under various environmental conditions in order to better identify the conditions favorable for sporulation regarding the time to obtain the first spore and/or the concentrations of spores which could be reached during a food process.

Dispersed phase volume fraction, weak acids and Tween 80 in a model emulsion: Effect on the germination and growth of Bacillus weihenstephanensis KBAB4 spores.

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.

Modeling the Effect of Modified Atmospheres on Conidial Germination of Fungi from Dairy Foods.

Modified atmosphere packaging (MAP) is commonly applied to extend food shelf-life. Despite growth of a wide variety of fungal contaminants has been previously studied in relation to modified-atmospheres, few studies aimed at quantifying the effects of dioxygen (O2) and carbon dioxide (CO2) partial pressures on conidial germination in solid agar medium. In the present study, an original culture method was developed, allowing microscopic monitoring of conidial germination under modified-atmospheres in static conditions. An asymmetric model was utilized to describe germination kinetics of Paecilomyces niveus, Mucor lanceolatus, Penicillium brevicompactum, Penicillium expansum, and Penicillium roquefoti, using two main parameters, i.e., median germination time (τ) and maximum germination percentage (Pmax ). These two parameters were subsequently modeled as a function of O2 partial pressure ranging from 0 to 21% and CO2 partial pressure ranging from 0.03 to 70% (8 tested levels for both O2 and CO2). Modified atmospheres with residual O2 or CO2 partial pressures below 1% and up to 70%, respectively, were not sufficient to totally inhibit conidial germination,. However, O2 levels < 1% or CO2 levels > 20% significantly increased τ and/or reduced Pmax , depending on the fungal species. Overall, the present method and results are of interest for predictive mycology applied to fungal spoilage of MAP food products.

Modelling the effect of water activity reduction by sodium chloride or glycerol on conidial germination and radial growth of filamentous fungi encountered in dairy foods.

Water activity (aw) is one of the most influential abiotic factors affecting fungal development in foods. The effects of aw reduction on conidial germination and radial growth are generally studied by supplementing culture medium with the non-ionic solute glycerol despite food aw can also depend on the concentration of ionic solutes such as sodium chloride (NaCl). The present study aimed at modelling and comparing the effects of aw, either modified using NaCl or glycerol, on radial growth and/or conidial germination parameters for five fungal species occurring in the dairy environment. The estimated cardinal values were then used for growth prediction and compared to growth kinetics observed on commercial fresh cheese. Overall, as compared to glycerol, NaCl significantly increased the fungistatic effect resulting from aw reduction by extending latency and/or reducing radial growth rates of Paecilomyces niveus, Penicillium brevicompactum, Penicillium expansum and Penicillium roqueforti but not of Mucor lanceolatus. Besides, NaCl significantly reduced aw range for conidial germination and delayed median germination time of P. expansum but not of P. roqueforti. Despite these observations, cardinal aw values obtained on glycerol-medium yielded similar predictions of radial growth and germination time in commercial fresh cheese as those obtained with NaCl. Thus, it indicates that, for the studied species and aw range used for model validation, the use of NaCl instead of glycerol as a aw depressor had only limited impact for fungal behavior prediction.

Walking dead: Permeabilization of heat-treated Geobacillus stearothermophilus ATCC 12980 spores under growth-preventing conditions.

Although heat treatment is probably the oldest and the most common method used to inactivate spores in food processes, the specific mechanism of heat killing of spores is still not fully understood. The purpose of this study is to investigate the evolution of the permeabilization and the viability of heat-treated spores during storage under growth-preventing conditions. Geobacillus stearothermophilus spores were heat-treated under various conditions of temperature and pH, and then stored under conditions of temperature and pH that prevent growth. Spore survival was evaluated by count plating immediately after heat treatment, and then during storage over a period of months. Flow cytometry analyses were performed to investigate the Syto 9 permeability of heat-treated spores. Sub-lethally heat-treated spores of G. stearothermophilus were physically committed to permeabilization after heat treatment. However, prolonged heat treatment may abolish the spore permeabilization and block heat-treated spores in the refractive state. However, viability loss and permeabilization during heat treatment seem to be two different mechanisms that occur independently, and the loss of permeabilization properties takes place at a much slower rate than spore killing. Under growth-preventing conditions, viable heat-treated spores presumably lose their viability due to the permeabilization phenomena, which makes them more susceptible to the action of adverse conditions precluding growth.

Knowledge of the physiology of spore-forming bacteria can explain the origin of spores in the food environment.

Spore-forming bacteria are able to grow under a wide range of environmental conditions, to form biofilms and to differentiate into resistant forms: spores. This resistant form allows their dissemination in the environment; consequently, they may contaminate raw materials. Sporulation can occur all along the food chain, in raw materials, but also in food processes, leading to an increase in food contamination. However, the problem of sporulation during food processing is poorly addressed and sporulation niches are difficult to identify from the farm to the fork. Sporulation is a survival strategy. Some environmental factors are required to trigger this differentiation process and others act by modulating it. The efficiency of sporulation is the result of the combined effects of these two types of factors on vegetative cell metabolism. This paper aims to explain and help identify sporulation niches in the food chain, based on features of spore-former physiology.

Temperature, water activity and pH during conidia production affect the physiological state and germination time of Penicillium species.

Conidial germination and mycelial growth are generally studied with conidia produced under optimal conditions to increase conidial yield. Nonetheless, the physiological state of such conidia most likely differs from those involved in spoilage of naturally contaminated food. The present study aimed at investigating the impact of temperature, pH and water activity (aw) during production of conidia on the germination parameters and compatible solutes of conidia of Penicillium roqueforti and Penicillium expansum. Low temperature (5°C) and reduced aw (0.900 aw) during sporulation significantly reduced conidial germination times whereas the pH of the sporulation medium only had a slight effect at the tested values (2.5, 8.0). Conidia of P. roqueforti produced at 5°C germinated up to 45h earlier than those produced at 20°C. Conidia of P. roqueforti and P. expansum produced at 0.900 aw germinated respectively up to 8h and 3h earlier than conidia produced at 0.980 aw. Furthermore, trehalose and mannitol assessments suggested that earlier germination might be related to delayed conidial maturation even though no ultra-structural modifications were observed by transmission electron microscopy. Taken together, these results highlight the importance of considering environmental conditions during sporulation in mycological studies. The physiological state of fungal conidia should be taken into account to design challenge tests or predictive mycology studies. This knowledge may also be of interest to improve the germination capacity of fungal cultures commonly used in fermented foods.