Impact of the physiological state of fungal spores on their inactivation by active chlorine and hydrogen peroxide.

This study aimed at assessing the impact of the physiological state of fungal spores on inactivation by sodium hypochlorite, 0.1% and 0.2% active chlorine, and 3% hydrogen peroxide. In this context, two physiological states were compared for 4 fungal species (5 strains). The first physiological state corresponded to fungal spores produced at 0.99 aw and harvested using an aqueous solution (laboratory conditions), while the second one corresponded to fungal spores produced under a moderate water stress (0.95 aw) and dry-harvested (mechanical harvesting without use of any water, mimicking food plant conditions). Aspergillus flavus "food plant" conidia were more resistant to all tested fungicide molecules than the "laboratory" ones. The same phenomenon was observed for Penicillium commune UBOCC-A-116003 conidia treated with hydrogen peroxide. However, this isolate did not exhibit any inactivation difference between "laboratory" and "food plant" conidia treated with sodium hypochlorite. Similarly, the physiological state of Cladosporium cladosporioides conidia did not impact the efficacy of the tested biocides. P. commune UBOCC-A-112059 "food plant" and "laboratory" conidia were more resistant to hydrogen peroxide and sodium hypochlorite, respectively. As for Mucor circinelloides, "laboratory" spores were more resistant to all disinfectant than the "food plant" ones. Noteworthy, regardless of the physiological state, all M. circinelloides and C. cladosporioides conidia were inactivated for 5 min treatment at 0.2% active chlorine and for 2.5 min treatment at 0.1% active chlorine, while the conidia of all the other species remained viable for these treatments. The obtained data indicate that the efficacy of disinfectant molecules depends not only on the encountered fungal species and its intraspecific diversity but also on the spore physiological state.

Production and migration of ochratoxin A and citrinin in Comté cheese by an isolate of Penicillium verrucosum selected among Penicillium spp. mycotoxin producers in YES medium.

Moldy food products that are not subject to pathogenic bacterial contamination could be trimmed by consumers to remove fungal mycelium before consumption. However, prior to giving such recommendations to consumers, it is necessary to evaluate potential mycotoxin migration in these products. This study aimed at quantifying citrinin (CIT) and ochratoxin A (OTA) accumulation and migration in a French semi-hard Comté cheese after artificial inoculation with a CIT- and OTA-producing Penicillium verrucosum strain. At 8 °C, CIT and OTA production started after 14 days and 28 days incubation, respectively; while at 20 °C, both mycotoxins were produced from day 7. At 20 °C, maximum CIT concentration, about 50000 ng/g, was 20 fold that at 8 °C. Regardless of temperature, maximum OTA concentration was about 4000 ng/g cheese. Maximum concentrations were obtained in the upper part of the cheese, but depending on incubation time, mycotoxins were detected up to 1.6 cm in depth. As long as only white mycelium developed on the cheese surface, trimming can be acceptable, but a blue mold color (due to fungal sporulation) was associated with the accumulation of significant amounts of mycotoxins so the product should be discarded.

Lag time for germination of Penicillium chrysogenum conidia is induced by temperature shifts.

In the environment, fungal conidia are subject to transient conditions. In particular, temperature is varying according to day/night periods. All predictive models for germination assume that fungal spores can adapt instantaneously to changes of temperature. The only study that supports this assumption (Gougouli and Koutsoumanis, 2012, Modelling germination of fungal spores at constant and fluctuating temperature conditions. International Journal of Food Microbiology, 152: 153-161) was carried out on Penicillium expansum and Aspergillus niger conidia that, in most cases, already produced germ tubes. In contrast, the present study focuses on temperature shifts applied during the first stages of germination (i.e., before the apparition of the germ tubes). Firstly, germination times were determined in steady state conditions at 10, 15, 20 and 25 °C. Secondly, temperature shifts (e.g., up-shifts and down-shifts) were applied at 1/4, 1/2, and 3/4 of germination times, with 5, 10 and 15 °C magnitudes. Experiments were carried out in triplicate on Penicillium chrysogenum conidia on Potato Dextrose Agar medium according to a full factorial design. Statistical analysis of the results clearly demonstrated that the assumption of instantaneous adaptation of the conidia should be rejected. Temperature shifts during germination led to an induced lag time or an extended germination time as compared to the experiments conducted ay steady state. The induced lag time was maximized when the amplitude of the shift was equal to 10 °C. Interaction between the instant and the direction of the shift was highlighted. A negative lag time was observed for a 15 °C down-shift applied at 1/4 of the germination time. This result suggested that at optimal temperature the rate of germination decreased with time, and that the variation of this rate with time depended on temperature.

Impact of temperature application and concentration of commercial sanitizers on inactivation of food-plant fungal spores.

This study aimed at quantifying the impact of the concentration of four commercial sanitizers and temperature on mold spores inactivation. The sanitizers were based on the following fungicide molecules, ethanol (ARVO 21 SR), active chlorine (ARVO CLM 600), hydrogen peroxide (Nocolyse Food) and triamine (P3 Topax 960). Food plant spores were produced under a moderate water stress, 0.95 aw and dry-harvested to simulate airborne spores responsible for contamination in the food industry. First, Aspergillus flavus, Cladosporium cladosporioides, Mucor circinelloides, and two Penicillium commune isolates were tested against the sanitizers at 20 °C and at a concentration recommended by the manufacturers. Overall, A. flavus was the less resistant species. Second the effects of concentration and temperature were assessed on the most resistant species, i.e., P. commune UBOCC-A-116003 (ARVO 21 SR and P3 Topax 960), P. commune UBOCC-A-112059 (ARVO CLM 600), and M. circinelloides (Nocolyse Food). With the exception of ARVO 21 SR, the observed inactivation kinetics were downward concave. The time necessary to obtain 4 log reduction, t4D, was estimated by means of the Weibull model. At 20 °C and at the recommended concentration by the manufacturers, t4D (min) for the most resistant strains were equal to 2.14 (ARVO 21 SR), 7.35 (ARVO CLM 600), 39.3 (Nocolyse Food) and 82.8 (P3 Topax 960). T4D was increased at lower concentrations and temperatures. These effects were more pronounced for ARVO 21 SR, t4D were about 10 fold and 20 fold the above reported value, 2.14 min, at 8 °C and by diluting the sanitizer by a 10:8 factor, respectively. The least effect of temperature, 3 fold, was shown for ARVO CLM 600, while concentration of P3 Topax 960 had no significant effect on t4D within the recommended utilization range.

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure.

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high-pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure-induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure-induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high-pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level.

Impact of intraspecific variability and physiological state on Penicillium commune inactivation by 70% ethanol.

The aim of this study was to assess the impact of the physiological state and intraspecific variability on the efficacy of 70% ethanol to inactivate conidia of Penicillium commune, used as a representative species of dairy product contaminants. Four physiological states were obtained by modifying the water activity during the production of conidia (0.995 and 0.950) and the harvesting conditions (hydrated and non-hydrated). These conditions were applied to four different P. commune strains isolated from contaminated dairy products. Five minutes exposure to 70% ethanol at ambient temperature allowed total inactivation of conidia (>4 log10) regardless of the physiological state or the strain. For 1 min exposure, regardless of the strains, only dry-harvested conidia produced at aw 0.950 exhibited survivors. Survival after 2 min exposure was observed for this physiological state for P. commune UBOCC-A-116003 only. For this strain, the impact of the physiological state was greater than 1.54 log10 between dry-harvested conidia produced at aw 0.950 that exhibited survivors after 1 min treatment and the 3 other kinds of conidia that were all inactivated. For 1 min exposure, by comparing the more resistant strain to the three other strains, the impact of the intraspecific variability was 2.35 log10. These results demonstrated that the physiological state of the conidia, the representativeness of the tested species and strains should be taken into account to assess the efficacy of disinfectants in dairies.

Production and migration of patulin in Penicillium expansum molded apples during cold and ambient storage.

The ability of three Penicillium expansum isolates to produce patulin was first evaluated in YES medium after incubation at 25 °C to select a high patulin producer. Then, a spore suspension of the selected P. expansum 3.78 strain was inoculated onto the surface of Golden delicious apples and incubated at 8 or 20 °C until the mold lesion reached a diameter of 1, 2 or 3 cm. For each lesion size, patulin was quantified from apple samples cut into 1 cm depthwise fractions and widthwise sized cylinders. Maximum patulin concentration, about 80,000 ng/g apple, was obtained at 8 °C for the center and surface sample of the 3 cm diameter lesion. Patulin was systematically found at the highest concentration in the lesions, but still quantified up to one centimeter next to the lesion. Patulin concentrations were not significantly different between the 8 and 20 °C incubation temperature, except for the 3 cm large lesions. Based on these findings, and for lesions less than or equal to 3 cm in diameter, we recommend to consumers to cut off at least 1 cm around and below the mold spot to limit patulin exposure. Apples should also be stored at cool temperatures, below 8 °C, to delay lesion development.

The logarithmic transformation should be avoided for stabilising the variance of mould growth rate.

Radial growth rate, micro (mm d(-1)) was evaluated by plotting the radius of the colony, r (mm) versus time (d) for Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Mucor racemosus, Rhizopus oryzae and Trichoderma harzianum at different T and a(w). For each of the 12 data sets, an analysis of variance of the raw growth rate data was performed. It was observed from the P-values that all square-root transformed values of micro were non-significant at the significance level alpha=0.05, whereas for untransformed values of micro, three of the 12 values were significant and for logarithmically transformed micro, nine of the 12 values were significant at the significance level. Therefore the logarithmic transformation that is used for bacteria should be avoided for these moulds except for A. flavus. In contrast the square-root transformation appeared suitable for stabilising the variance of mould growth rate in all cases.

Inactivation of conidia of Penicillium chrysogenum, P. digitatum and P. italicum by ethanol solutions and vapours.

A fractional factorial design, 2(5-1) experiments, was used for assessing the influence of 5 factors: water activity, aw [0.7, 0.9], temperature, T ( degrees C) [10, 30], mode of application, A [liquid, vapour], ethanol concentration, E (% w/w) [5, 10] and time, t (d) [1, 4] on the inactivation of spores of Penicillium chrysogenum, P. digitatum and P. italicum. Survival was determined by germination at optimal conditions within 3d. The experimental response was log (N 0/Nt), where N 0 and Nt (spore ml(-1)) the concentrations of viable spores at t=0 and t respectively. By a decreasing order of sensitivity to ethanol, moulds were ranked as followed: P. digitatum, P. italicum and P. chrysogenum. A greater inactivation for P. digitatum, P. italicum, that were the most sensitive moulds to ethanol, was obtained by applying vapour rather than ethanol solution. The order of significance of the main factors depended upon the mould. The key factor for explaining inactivation of P. chrysogenum was water activity. But, temperature was the main factor for explaining inactivation of P. digitatum and P. italicum. In the more drastic conditions, (i.e., 0.7 aw, 30 degrees C, 10% w/w ethanol), all spores were inactivated by applying liquid solution for 4d.

Distributions of the growth rate of the germ tubes and germination time of Penicillium chrysogenum conidia depend on water activity.

The effects of water activities for sporulation (a(wsp)) and germination (a(wge)) on the distributions of the growth rate of the germ tubes (mu) and the germination time (t(G)) of Penicillium chrysogenum conidia were determined by monitoring the length of the same germ tubes throughout the experiments automatically. No relationship between the individual t(G)'s and mu's could be established. Irrespective of the water activity for germination, mu was greater and t(G) was less for conidia produced at 0.95a(wsp) than that at 0.99a(wsp). At 0.99 a(wge) the mean and the standard deviation of t(G) were smaller than those obtained at 0.95a(wge). At 0.99a(wge), normal distributions for mu and t(G) were exhibited, but not at 0.95a(wge). The cumulative frequencies were used to reconstruct the germination curves. Great differences in the percentage of spores capable of germination (P(G)) and in the mean germination times between conidia produced at 0.95a(wsp) and at 0.99a(wsp) were clearly exhibited at 0.95a(wge), thus demonstrating the paramount influence of sporulation conditions on germination kinetics.

Mould germination: data treatment and modelling.

The objectives of this study were i/ to examine germination data sets over a range of environmental conditions (water activity, temperature) for eight food spoilage moulds, ii/ to compare the ability of the Gompertz equation and logistic function to fit the experimental plots, iii/ to simulate germination by assessing various distributions of the latent period for germination amongst a population of spores. Data sets (percentage germination, P (%), versus time, t) of Aspergillus carbonarius, Aspergillus ochraceus, Fusarium verticillioides, Fusarium proliferatum, Gibberella zeae, Mucor racemosus, Penicillium chrysogenum and Penicillium verrucosum were analysed. No correlation, or relationship between the mean percentage [mean (P)] and the variance [var (P)] was found. Therefore no transformation of the germination data was required. Experimental data were fitted by using the Gompertz equation P = A exp (-exp [mu(m) e/A (delta - t) + 1]) and the logistic function P = Pmax/(1 + exp (k (tau - t))). Based on the residual mean square error (RMSE), no model performed better than the other one. However, model parameters were generally determined more precisely with the logistic model than with the Gompertz one. The time course of fungal spore germination curves was simulated assuming different distributions of the latent period for germination, lag, amongst a population of spores. The growth rate of germ tubes was calculated by means of the relationship: lag x rate = k. For normal Gaussian distributions, germination curves were symmetrical with respect to the inflection point and should be modelled with the logistic function. Skewed distributions were capable of simulating an asymmetric germination curve that was fitted by the Gompertz model. Future studies should be conducted for assessing whether the distributions assumed in this paper are in accordance with the experimental distributions that are still unknown.

Modelling the effect of temperature, pH, water activity, and organic acids on the germination time of Penicillium camemberti and Penicillium roqueforti conidia.

In this study, the influence of environmental factors on the germination time of Penicillium camemberti and Penicillium roqueforti conidia was evaluated. To do so, the effects of i/temperature, pH, water activity, and ii/organic acids were determined using models based on i/cardinal values, and ii/minimum inhibitory concentration (MIC) respectively. Cardinal values for germination of conidia were not observed to be species dependent. Minimum temperatures were estimated to be below the freezing point, with an optimum of 26.9°C, and a maximum of 33.5°C. For both species, minimal and optimal aw values were found to be 0.83 and 0.99, respectively, while for pH these values corresponded to 2.9, and 5.6. MIC values could not be determined for lactic acid because conidia of both species germinated in up to 1M concentrations, the highest concentration tested. At pH5.6, P. camemberti (MIC=0.197M) was more sensitive to propionic acid than P. roqueforti (MIC=0.796M).

Modeling the heat inactivation of foodborne pathogens in milk powder: High relevance of the substrate water activity.

Due to the ability of foodborne pathogens to survive in low moisture foods, the decontamination of these products is an important issue in food hygiene. Up to now, such decontamination has mostly been achieved through empirical methods. The intention of this work is to establish a more rational use of heat treatment cycles. The effects of thermal treatment cycles on the inactivation of dried Salmonella Typhimurium, Salmonella Senftenberg, Cronobacter sakazakii and Escherichia coli were assessed. Bacteria were mixed with whole milk powder and dried down to different water activity levels (0.11, 0.25, 0.44 and 0.58). The rate of inactivated bacteria was determined after thermal treatment at 85°C, 90°C, 95°C and 100°C, from 0s to 180s in closed vessels, in order to maintain aw during treatment. In a first step, logarithmic bacterial inactivation was fitted by means of a classical loglinear model in which temperature and aw have a significant effect (p<0.05). DT,aw values were estimated for each T, aw condition and the results clearly showed that aw is a major parameter in the thermal decontamination of dried foods, a lower aw involving greater thermal resistance. In a second step, Bigelow's law was used to determine zT, a classical parameter relative to temperature, and yaw values, a new parameter relative to aw resistance. The values obtained for zT and yaw showed that the bacterium most resistant to temperature variations is Salmonella Typhimurium, while the one most resistant to aw variations is Escherichia coli. These data will help design decontamination protocols or processes in closed batches for low moisture foods.

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.

Standardisation of methods for assessing mould germination: a workshop report.

The first workshop on predictive mycology was held in Marseille, France, 2--4 February 2005 under the auspices of the French Microbiological society. The purpose of the workshop was to list the different techniques and definitions used by scientists for assessing mould germination and to evaluate the influence of the different techniques on the experimental results. Recommendations were made when a large consensus was obtained. In order to facilitate the study of germination, alternative methods to microscopic examination were examined.

Active packaging with antifungal activities.

There have been many reviews concerned with antimicrobial food packaging, and with the use of antifungal compounds, but none provided an exhaustive picture of the applications of active packaging to control fungal spoilage. Very recently, many studies have been done in these fields, therefore it is timely to review this topic. This article examines the effects of essential oils, preservatives, natural products, chemical fungicides, nanoparticles coated to different films, and chitosan in vitro on the growth of moulds, but also in vivo on the mould free shelf-life of bread, cheese, and fresh fruits and vegetables. A short section is also dedicated to yeasts. All the applications are described from a microbiological point of view, and these were sorted depending on the name of the species. Methods and results obtained are discussed. Essential oils and preservatives were ranked by increased efficacy on mould growth. For all the tested molecules, Penicillium species were shown more sensitive than Aspergillus species. However, comparison between the results was difficult because it appeared that the efficiency of active packaging depended greatly on the environmental factors of food such as water activity, pH, temperature, NaCl concentration, the nature, the size, and the mode of application of the films, in addition to the fact that the amount of released antifungal compounds was not constant with time.

Basis of predictive mycology.

For over 20 years, predictive microbiology focused on food-pathogenic bacteria. Few studies concerned modelling fungal development. On one hand, most of food mycologists are not familiar with modelling techniques; on the other hand, people involved in modelling are developing tools dedicated to bacteria. Therefore, there is a tendency to extend the use of models that were developed for bacteria to moulds. However, some mould specificities should be taken into account. The use of specific models for predicting germination and growth of fungi was advocated previously []. This paper provides a short review of fungal modelling studies.

Modelling the effect of ethanol on growth rate of food spoilage moulds.

The effect of ethanol (E) on the radial growth rate (mu) of food spoilage moulds (Aspergillus candidus, Aspergillus flavus, Aspergillus niger, Cladosporium cladosporioides, Eurotium herbariorum, Mucor circinelloides, Mucor racemosus, Paecilomyces variotii, Penicillium chrysogenum, Penicillium digitatum, Rhizopus oryzae and Trichoderma harzianum) was assessed in Potato Dextrose Agar (PDA) medium at a(w) 0.99, 25 degrees C. In order to model this effect, the Monod type equation described previously by Houtsma et al. (Houtsma, P.C., Kusters, B.J.M., de Wit, J.C., Rombouts, F.M., Zwietering, M.H., 1994. Modelling growth rates of Listeria monocytogenes as a function of lactate concentration. Int. J. Food. Microbiol. 24, 113-123.) was re-parameterised: mu = mu(opt)[K(E(max)-E)/K E(max)-2KE+E(max)E]; E(max) (%, wt/wt): ethanol concentration at which no growth occurs, K (%, wt/wt): ethanol concentration at which mu = mu(opt)/2, mu(opt) (mm day(-1)): growth rate at 0% ethanol. The model was capable of describing curves, mu vs. E, with either a concave shape (KE(max)/2) with a good accuracy (root mean square error (RMSE) < or = 0.136) with the notable exception of R. oryzae and T. harzianum. After growth rate data were square-root transformed to stabilise the variance, E(max) was estimated in the range 3% to 5% for all moulds with the exception of T. harzianum (E(max) 2.14%) and P. variotii (E(max) 6.43%). Ethanol would appear an effective additional barrier to inhibit fungal growth in food products and would represent an interesting alternative to the use of preservatives.

Modeling the effect of ethanol vapor on the germination time of Penicillium chrysogenum.

The influence of ethanol vapor on germination of Penicillium chrysogenum was determined on yeast nitrogen base plus glucose agar medium at 25 degrees C. Ethanol vapors were generated by 0 to 6% (wt/wt) ethanol solutions at the bottom of hermetically closed petri dishes. The logistic equation was used to describe the data as the percentage of germination versus time and to estimate the germination time. The effect of ethanol concentration on germination time was described by a new reparameterized equation, resulting in an estimated limiting ethanol concentration of 4.3%. Up to 3% ethanol, all spores germinated, and the germination time increased with increasing ethanol concentration. At 3.5 and 4%, some spores formed abnormal germ tubes and others were inhibited at the swelling stage. The inhibiting effect of ethanol was reversible under these experimental conditions.

Validation of a predictive model for the growth of chalk yeasts on bread.

The present study focused on the effects of temperature, T, and water activity, aw, on the growth of Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera on Sabouraud Agar Medium. Cardinal values were estimated by means of cardinal models with inflection. All the yeasts were xerophilic, and they exhibited growth at 0.85 aw. The combined effects of T, aw, and pH on the growth of these species were described by the gamma-concept and validated on bread in the range of 15-25 °C, 0.91-0.97 aw, and pH 4.6-6.8. The optimum growth rates on bread were 2.88, 0.259, and 1.06 mm/day for H. burtonii, P. anomala, and S. fibuligera, respectively. The optimal growth rate of S. fibuligera on bread was about 2 fold that obtained on Sabouraud. Due to reproduction by budding, P. anomala exhibited low growth on Sabouraud and bread. However, this species is of major concern in the baker's industry because of the production of ethyl acetate in bread.