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.

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.

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.

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.

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.

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).

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.

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.

A model for the effect of pH on the growth of chalk yeasts.

Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera were isolated from spoiled packaged sliced bread. These chalk yeasts were characterized by a wide range of pH for which growth was almost optimum. Thus, the curve growth vs pH exhibited plateau and sharp profiles close to the minimum and the maximum pH. This study described a chalk yeast model (CYM) for the effect of pH derived from a new germination model for fungi (Dantigny, P., Nanguy, S., P.-M., Judet-Correia, D., and Bensoussan, M. 2011, International Journal of Food Microbiology, 146, 176-181). The CYM is asymmetric, versatile, based on parameters with biological significance, and compatible with the gamma concept. The CYM was compared to the cardinal pH model (CPM) which is widely used to describe the effect of pH on microbial growth. The CYM exhibited RMSE values two fold less than those obtained with the CPM for H. burtonii, and S. fibuligera for which plateaus were clearly observed. For P. anomala, the plateau was less obvious, but the RMSE value obtained with the CYM was similar to that found with the CPM. The CYM could extend its use to represent the effect of pH on mold growth.

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.

Effect of inoculum size and water activity on the time to visible growth of Penicillium chrysogenum colony.

In order to assess the effect of the inoculum size on the time to visible growth for Penicillium chrysogenum, the correlation described by González et al. (González, H.H.L., Resnik, S.L., Vaamonde, G., 1987. Influence of inoculum size on growth rate and lag phase of fungi isolate from Argentine corn. International Journal of Food Microbiology 4, 111-117) was compared to the model introduced by Gougouli et al. (Gougouli, M., Kalantzi, K., Beletsiotis, E., Koutsoumanis, K.P., 2011. Development and application of predictive models for fungal growth as tools to improve quality control in yogurt production. Food Microbiology 28, 1453-1462). Based on the regression coefficient, the latter model performed better than the former one to fit the data obtained for P. chrysogenum grown on Potato Dextrose Agar at 25 °C. Inoculum sizes in the range 10(1)-10(5) spores were tested at 0.930, 0.950, 0.970, and 0.995 aw. By extrapolation of the straight line, the model of Gougouli et al. (2011) provided accurate estimations of the time to visible growth for a single spore inoculum, tvg (N=1). In order to avoid experiments at reduced water activities, the influence of water activity on the model parameters, and on the ratio tvg (N=1) over the germination time was assessed.

Effect of storage conditions (relative humidity, duration, and temperature) on the germination time of Aspergillus carbonarius and Penicillium chrysogenum.

Fungal conidia are disseminated, often in the air, for a certain period of time, prior to contaminating food products. The objective of this study was to examine the effects of the relative humidity, RH (%), time (day), and temperature (°C) during this period of time, called "storage", on the germination time, τ (h), of Aspergillus carbonarius and Penicillium chrysogenum. A Doehlert design was used in the range, 20-100% RH, 2-28 days, and 5-25 °C. As compared to un-stored conidia, the germination time of conidia stored at 60% RH, 15 days, 5 °C was increased by 23 and 28%, for A. carbonarius and P. chrysogenum, respectively. Stored conidia exhibited a minimum τ value at 60% RH, and 100% RH for A. carbonarius and P. chrysogenum, respectively. For these species, τ was minimum for 2 days of storage. The effect of temperature was RH dependent for A. carbonarius. The germination time of stored conidia was clearly greater than that of fresh conidia obtained in the laboratory. This result should be taken into account to determine the mould free shelf-life of food products.

A new model for germination of fungi.

The objective of this study was to design a germination model dedicated to fungi. The percentage of germinated spores, P(%), depended on the maximum percentage of germination P(max) (%), the germination time, τ (h) and a design parameter, d (-) according to : [formula in text]. The model was capable to fit satisfactorily either apparent symmetric and asymmetric shapes of germination curves. The accuracy of τ determined by using the logistic or the present model was at least twice that obtained by the Gompertz equation. In contrast to the logistic model, the new model is by essence asymmetric. Therefore, its use is consistent with skewed distributions of the individual germination times that were observed experimentally in many cases.

Impact of water activity of diverse media on spore germination of Aspergillus and Penicillium species.

The effects of water activity (a(w)) of diverse media i/ culture medium for sporogenesis, a(w sp) ii/ liquid spore suspension medium, a(w su) and iii/ medium for germination, a(w ge), on the germination time t(G) of Aspergillus carbonarius, Aspergillus flavus, Penicillium chrysogenum and Penicillium expansum were assessed according to a screening matrix at 0.95 and 0.99 a(w). It was shown that i/ reduced t(G)s were obtained at 0.95 a(w sp) except for P. expansum ii/ a significant effect of a(w su) on t(G) was demonstrated for A. carbonarius, P. chrysogenum and P. expansum iii/ the most important factor for controlling the germination time was the medium for germination except for A. carbonarius (a(w su)). In accordance with the fact that fungal spores can swell as soon as they are suspended in an aqueous solution it is recommended to re-suspend fungal spores in a solution at the same water activity as that of subsequent germination studies.

Validation of a predictive model for the growth of Botrytis cinerea and Penicillium expansum on grape berries.

The objective of this study was to develop and to validate a model for predicting the combined effect of temperature and a(w) on the radial growth rate, mu, of Botrytis cinerea and Penicillium expansum on grape berries. The proposed strategy was based on the gamma-concept developed previously [Zwietering, M.H., Wijtzes, T., de Wit, J.C., van't Riet, K. 1992. A decision support system for prediction of the microbial spoilage in foods. Journal of Food Protection. 12, 973-979]: mu=mu(opt).gamma(T).gamma(a(w)), where the gamma functions were cardinal models with inflection (CMI), mu(opt) the radial growth rate on grape berries. Firstly, the cardinal temperatures and a(w)'s were estimated independently from experiments carried out on Potato Dextrose Agar. Secondly, the gamma concept was validated i/ on a synthetic grape juice medium (SGJ) and ii/ on a grape juice agar (GJA). Accuracy and bias factors were closer to 1 with the latter analogue, thus suggesting that GJA should be preferred to SGJ. Thirdly, an experimental protocol taken into account the isotropic nature of fungal growth was developed for estimating mu(opt) on grape berries. This study demonstrated that CMI's can be validated on agri-food products over a wide range of temperature and a(w) using the described methodology.

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.

Use of a logistic model to assess spoilage by Byssochlamys fulva in clarified apple juice.

The percentage P (%) of spoiled bottles (n=40) of clarified apple juice due to Byssochlamys fulva, was modeled by using a logistic model: P = P(max)/1 + exp (k(tau-t)) where P(max) (%) the maximum percentage of spoiled bottles, k (h(-1)) a slope parameter and tau (h) the time for P=P(max)/2. Bottles of pasteurized apple juice were inoculated with B. fulva IOC 4518 ascospores for low and high initial loads, 4.8+/-2.3 ascospores/100mL and 19.3+/-4.6 ascospores/100mL respectively and incubated at 21 degrees C and 30 degrees C. P(max) was not significantly different from 100% except for a low initial load at 21 degrees C. Model parameters were estimated with a good accuracy, RMSE in the range 3.89-7.50. Then the model was used to determine the time for 10% bottles spoiled, t(10%). This time was greater at low initial loads, 57.4 and 104 h at 30 and 21 degrees C respectively, than at high initial loads 23.9 and 75.1h at 30 and 21 degrees C respectively. This study demonstrated that even at a very low initial contamination, clarified apple juice can be easily spoiled by B. fulva highlighting the importance of controlling critical control steps of fruit juice processing (i.e., fruit washing, juice filtration and pasteurization).

Preparation of fungal conidia impacts their susceptibility to inactivation by ethanol vapours.

A common protocol employed for the preparation of conidia employs flooding a fungal colony grown on semi-solid media under optimum conditions with an aqueous solution. In contrast, conidia produced in a natural environment are usually not hydrated when disseminated in air and can be produced under water stress. In order to simulate the latter conditions, cultures were grown at different water activities and conidia were dry-harvested on the lid by turning the dishes upside-down then gently tapping the bottom of the box. This study aimed at assessing the effect of the preparation of fungal conidia on their inactivation by ethanol vapours. Firstly ethanol vapours (either 0.30 or 0.45 kPa) were applied to conidia obtained from the standardised protocol and to dry-harvested conidia for some species of Penicillium. While all dry-harvested conidia remained viable after 24 h of treatment, about 1.0, 3.5 and 2.5 log(10) reductions were observed for hydrated conidia of Penicillium chrysogenum, Penicillium digitatum and Penicillium italicum respectively. Secondly ethanol vapours (0.67 kPa) were applied to dry-harvested conidia obtained from cultures grown at 0.99 a(w) and at reduced water activities. For all species, the susceptibility to ethanol vapours of conidia obtained at 0.99 a(w) was significantly greater than that of conidia obtained at reduced water activities. Conidia produced in a natural environment under non-optimal conditions would be much more resistant to ethanol vapours than those produced in the laboratory. This phenomenon may be due to a reduced intracellular water activity of dry-harvested conidia.