On-line screening and identification of free radical scavenging compounds in Angelica dahurica fermented with Eurotium cristatum using an HPLC-PDA-Triple-TOF-MS/MS-ABTS system.

Eurotium cristatum, a beneficial fungus isolated from Fuzhuan tea, was used to ferment Angelica dahurica for the first time. The antioxidant capacities of the extracts before and after fermentation were compared using ABTS, DPPH and FRAP assays. The results showed that the antioxidant capacities of the extracts acquired using organic solvents were greater after fermentation. Moreover, based on a comparison of the HPLC chromatograms, the chemical composition of Angelica dahurica changed substantially during fermentation. To further understand the changes in its antioxidant constituents, an on-line HPLC-PDA-Triple-TOF-MS/MS-ABTS system was employed. Twelve antioxidants belonging to three different classes were detected and identified, and their antioxidant capacities were preliminarily evaluated. The results indicated that the substances produced during the fermentation of Eurotium cristatum played important roles in enhancing the antioxidant capacity.

Effect of water activity and temperature on the growth of Eurotium species isolated from animal feeds / Efecto de la actividad del agua y la temperatura en el crecimiento de especies de Eurotium aisladas de alimentos para animales

Background. Xerophilic fungi represent a serious problem due to their ability to grow at low water activities causing the spoiling of low and intermediate moisture foods, stored goods and animal feeds, with the consequent economic losses. Aims. The combined effect of water activity and temperature of four Eurotium species isolated from animal feeds was investigated. Methods. Eurotium amstelodami, Eurotium chevalieri, Eurotium repens and Eurotium rubrum were grown at 5, 15, 25, 37 and 45°C on malt extract agar adjusted with glycerol in the range 0.710-0.993 of water activities. Results. The cardinal model proposed by Rosso and Robinson (2001) was applied to fit growth data, with the variable water activity at fixed temperatures, obtaining three cardinal water activities (awmin, awmax, awopt) and the specific growth rate at the optimum aw (μopt). A probabilistic model was also applied to define the interface between growth and no-growth. The cardinal model provided an adequate estimation of the optimal aw to grow and the maximum growth rate. The probabilistic model showed a good performance to fit growth/no-growth cases in the predicted range. Conclusions. The results presented here could be applied to predict Eurotium species growth in animal feeds (AU)

Antecedentes. Los hongos xerófilos son un problema importante debido a su capacidad de crecer a bajas actividades del agua, lo que causa el deterioro de alimentos a humedades bajas e intermedias, de materias primas almacenadas y de piensos para animales, con las consecuentes pérdidas económicas. Objetivos. Se llevó a cabo un estudio sobre el efecto de los factores ambientales (temperatura y actividad del agua) sobre el crecimiento de cuatro especies pertenecientes al género Eurotium aisladas de piensos para animales. Métodos. Se estudió el crecimiento de Eurotium amstelodami, Eurotium chevalieri, Eurotium repens y Eurotium rubrum a valores de actividad de agua en el rango 0,710-0,993 en el medio de cultivo agar extracto de malta modificado con glicerol, y valores de temperatura de 5, 15, 25, 37 y 45°C. Resultados. El modelo cardinal propuesto por Rosso y Robinson (2001) se aplicó para realizar el ajuste de datos con la actividad del agua como variable a una temperatura fija; se obtuvieron tres valores cardinales de actividad del agua (awmin, awmax, awopt) y la tasa de crecimiento específico en el valor óptimo de aw (μopt). También se aplicó un modelo probabilístico para definir la interfase entre crecimiento y no crecimiento. El modelo cardinal presentó una adecuada estimación del awopt y la máxima velocidad de crecimiento. El modelo probabilístico fue adecuado para el ajuste de los casos de crecimiento/falta de crecimiento en el rango previsto. Conclusiones. Los resultados presentados en este artículo pueden aplicarse para pronosticar el crecimiento de especies de Eurotium en piensos para animales (AU)

Effect of water activity and temperature on the growth of Eurotium species isolated from animal feeds.

BACKGROUND: Xerophilic fungi represent a serious problem due to their ability to grow at low water activities causing the spoiling of low and intermediate moisture foods, stored goods and animal feeds, with the consequent economic losses. AIMS: The combined effect of water activity and temperature of four Eurotium species isolated from animal feeds was investigated. METHODS: Eurotium amstelodami, Eurotium chevalieri, Eurotium repens and Eurotium rubrum were grown at 5, 15, 25, 37 and 45°C on malt extract agar adjusted with glycerol in the range 0.710-0.993 of water activities. RESULTS: The cardinal model proposed by Rosso and Robinson (2001) was applied to fit growth data, with the variable water activity at fixed temperatures, obtaining three cardinal water activities (awmin, awmax, awopt) and the specific growth rate at the optimum aw (µopt). A probabilistic model was also applied to define the interface between growth and no-growth. The cardinal model provided an adequate estimation of the optimal aw to grow and the maximum growth rate. The probabilistic model showed a good performance to fit growth/no-growth cases in the predicted range. CONCLUSIONS: The results presented here could be applied to predict Eurotium species growth in animal feeds.

Quantifying the effect of water activity and storage temperature on single spore lag times of three moulds isolated from spoiled bakery products.

The inhibitory effect of water activity (aw) and storage temperature on single spore lag times of Aspergillus niger, Eurotium repens (Aspergillus pseudoglaucus) and Penicillium corylophilum strains isolated from spoiled bakery products, was quantified. A full factorial design was set up for each strain. Data were collected at levels of aw varying from 0.80 to 0.98 and temperature from 15 to 35°C. Experiments were performed on malt agar, at pH5.5. When growth was observed, ca 20 individual growth kinetics per condition were recorded up to 35days. Radius of the colony vs time was then fitted with the Buchanan primary model. For each experimental condition, a lag time variability was observed, it was characterized by its mean, standard deviation (sd) and 5th percentile, after a Normal distribution fit. As the environmental conditions became stressful (e.g. storage temperature and aw lower), mean and sd of single spore lag time distribution increased, indicating longer lag times and higher variability. The relationship between mean and sd followed a monotonous but not linear pattern, identical whatever the species. Next, secondary models were deployed to estimate the cardinal values (minimal, optimal and maximal temperatures, minimal water activity where no growth is observed anymore) for the three species. That enabled to confirm the observation made based on raw data analysis: concerning the temperature effect, A. niger behaviour was significantly different from E. repens and P. corylophilum: Topt of 37.4°C (standard deviation 1.4°C) instead of 27.1°C (1.4°C) and 25.2°C (1.2°C), respectively. Concerning the aw effect, from the three mould species, E. repens was the species able to grow at the lowest aw (awmin estimated to 0.74 (0.02)). Finally, results obtained with single spores were compared to findings from a previous study carried out at the population level (Dagnas et al., 2014). For short lag times (≤5days), there was no difference between lag time of the population (ca 2000 spores inoculated in one spot) and mean (nor 5th percentile) of single spore lag time distribution. In contrast, when lag time was longer, i.e. under more stressful conditions, there was a discrepancy between individual and population lag times (population lag times shorter than 5th percentiles of single spore lag time distribution), confirming a stochastic process. Finally, the temperature cardinal values estimated with single spores were found to be similar to those obtained at the population level, whatever the species. All these findings will be used to describe better mould spore lag time variability and then to predict more accurately bakery product shelf-life.

Identification and quantification of antifungal compounds produced by lactic acid bacteria and propionibacteria.

Fungal growth in bakery products represents the most frequent cause of spoilage and leads to economic losses for industrials and consumers. Bacteria, such as lactic acid bacteria and propionibacteria, are commonly known to play an active role in preservation of fermented food, producing a large range of antifungal metabolites. In a previous study (Le Lay et al., 2016), an extensive screening performed both in vitro and in situ allowed for the selection of bacteria exhibiting an antifungal activity. In the present study, active supernatants against Penicillium corylophilum and Aspergillus niger were analyzed to identify and quantify the antifungal compounds associated with the observed activity. Supernatant treatments (pH neutralization, heating and addition of proteinase K) suggested that organic acids played the most important role in the antifungal activity of each tested supernatant. Different methods (HPLC, mass spectrometry, colorimetric and enzymatic assays) were then applied to analyze the supernatants and it was shown that the main antifungal compounds corresponded to lactic, acetic and propionic acids, ethanol and hydrogen peroxide, as well as other compounds present at low levels such as phenyllactic, hydroxyphenyllactic, azelaic and caproic acids. Based on these results, various combinations of the identified compounds were used to evaluate their effect on conidial germination and fungal growth of P. corylophilum and Eurotium repens. Some combinations presented the same activity than the bacterial culture supernatant thus confirming the involvement of the identified molecules in the antifungal activity. The obtained results suggested that acetic acid was mainly responsible for the antifungal activity against P. corylophilum and played an important role in E. repens inhibition.

Modelling of the growth/no growth interface of Wallemia sebi and Eurotium herbariorum as a function of pH, aw and ethanol concentration.

High sugar products (sugar content > 50%) are generally considered to be stable against all forms of microbial spoilage during a prolonged shelf life of several months. However, one specific subgroup of micro-organisms, the xerophilic moulds, can develop quite fast on the surface of food products with a reduced water activity (< 0.85). The chance whether these xerophilic moulds are able to grow on the food product depends on the combination of intrinsic factors (e.g., water activity and pH) and the storage conditions (e.g., temperature). This study examines the development of growth/no growth models for the xerophilic moulds Wallemia sebi and Eurotium herbariorum in a sugar rich broth. Growth/no growth models are predictive models that are designed to give a prediction about the probability of growth of a spoilage micro-organism under a specific set of environmental conditions. In this research, a water activity between 0.75 and 0.90, a pH between 5.0 and 6.2, an ethanol concentration between 0% and 5% (g EtOH/g H2O) and their interactions were tested. The inoculated media were stored at 22 °C (± 1 °C) during a prolonged test period (up to 120 days). The obtained models were also validated in a chocolate-based food product (ganache). The resulting growth/no growth models show that the growth of W. sebi and E. herbariorum can be inhibited for a prolonged time (> 3 months) if an ethanol concentration of 5% on the water phase is present in the food product, irrespective of water activity values between 0.89 and 0.755. The necessary amount of ethanol for shorter shelf lives can be calculated with the models that were built. Although the models have not been validated thoroughly in actual food products, the preliminary results that were obtained by testing the model on a ganache indicated that the models are capable of delivering safe predictions.

Modeling growth of three bakery product spoilage molds as a function of water activity, temperature and pH.

The objective of this study was to quantify the effect of water activity, pH and storage temperature on the growth of Eurotium repens, Aspergillus niger and Penicillium corylophilum, isolated from spoiled bakery products. Moreover, the behaviors of these three mold species were compared to assess whether a general modeling framework may be set and re-used in future research on bakery spoilage molds. The mold growth was modeled by building two distinct Gamma-type secondary models: one on the lag time for growth and another one on the radial growth rate. A set of 428 experimental growth curves was generated. The effect of temperature (15-35 °C), water activity (0.80-0.98) and pH (3-7) was assessed. Results showed that it was not possible to apply the same set of secondary model equations to the three mold species given that the growth rate varied significantly with the factors pH and water activity. In contrast, the temperature effect on both growth rate and lag time of the three mold species was described by the same equation. The equation structure and model parameter values of the Gamma models were also compared per mold species to assess whether a relationship between lag time and growth rate existed. There was no correlation between the two growth responses for E. repens, but a slight one for A. niger and P. corylophilum. These findings will help in determining bakery product shelf-life and guiding future work in the predictive mycology field.

Taxonomic revision of Eurotium and transfer of species to Aspergillus.

Aspergillus section Aspergillus contains economically important, xerophilic fungi that are widely distributed in nature and the human environment and are known for their ability to grow on substrates with low water activity. The taxa were revised based on sequence data from four loci, PCR fingerprinting, micro- and macromorphology, and physiology. The number of taxa was reduced to 17 species, all of which can be distinguished with sequence data from either the caM or RPB2 locus. The original description of A. proliferans was supplemented by a description of its teleomorph. This species seems to be relatively common and often has been confused with A. glaucus. In addition, green sporulating isolates of A. niveoglaucus isolated from food and several other substrates are indistinguishable in phenotype from A. glaucus. A dichotomous key based on ascospore size and ornamentation and the ability to grow at specific combinations of temperature and water activity is provided for identification of species. In response to recent changes in the botanical code, we transferred the Eurotium species to Aspergillus and selected one name for each species.

The mycobiota of three dry-cured meat products from Slovenia.

The surface mycobiota of three types of Slovenian dry-cured meat products were isolated from a total of 75 items of product that were sampled periodically during the drying/ripening stage of processing. The predominant filamentous fungal genus isolated was Penicillium. Eurotium spp., Aspergillus versicolor and Cladosporium spp. were isolated from only two of the products. Eight Penicillium species were identified. Penicillium nordicum was recovered frequently. Penicillium nalgiovense was recovered less frequently, from one product only (a salami), while a yet-to-be described species Penicillium "milanense" was isolated from 21 items. The other penicillia were rarely isolated. Of the isolated and identified species, those that can produce mycotoxins are: A. versicolor, Penicillium brevicompactum, Penicillium chrysogenum, P. nordicum, and Penicillium polonicum. Their growth on dry-cured meat products is undesirable.

Effect of reduced water activity and reduced matric potential on the germination of xerophilic and non-xerophilic fungi.

Reduction in water activity (a(w)) is used as a microbiological hurdle to prevent food spoilage. To minimize the levels of salt and sugar, which are commonly used to reduce a(w), the potential of food structure as a microbiological hurdle needs to be assessed. The concept of matric potential (Psi(m)) is used to measure the effect of food structure on water movement. This study reports the effect of reduced a(w) and reduced Psi(m) on the germination of xerophilic fungi (represented by Eurotium herbariorum) and non-xerophilic fungi (represented by Aspergillus niger) on model glycerol agar media. Germination curves were plotted with the percentage of germinated spores against time. The germination time (t(G)), which is defined as the time at which 50% of the total viable spores have germinated, was estimated using the Gompertz model. Total viable spores was defined as those spores that were able to germinate under the optimum a(w) and Psi(m) conditions for each species, i.e. 0.95 a(w) and 2.5% agar for E. herbariorum and 0.98 a(w) and 2.5% agar for A. niger. As a(w) decreased from 0.90 to 0.85 a(w), t(G) increased significantly for both the xerophilic fungi and non-xerophilic species at equivalent matric potential values. When matric potential was reduced from -12 kPa (2.5% agar) to -38 kPa (12.5% agar), t(G) of A. niger was significantly extended at 0.90 a(w); however, t(G) remained the same for A. niger at 0.85 a(w), and for E. herbariorum at 0.80, 0.85 and 0.90 a(w). This study demonstrated that the germination time for non-xerophilic and xerophilic fungi was extended by reduced a(w), however the effect of reduced Psi(m) was limited.

[Species composition of food-spoiling mycelial fungi].

We investigated the composition of the microflora that spoils foodstuffs (the surface of hard cheeses and sausages) at agribusiness factories. Mycelial fungi, mostly ascomycetes of the order Eurotiales belonging to the genus Penicillium play the main role in spoiling food. Most representatives of these fungi are mesophiles and possess the capacity for utilizing nutrient substrates in surface and submerged cultures.

Moulds and ochratoxin A on surfaces of artisanal and industrial dry sausages.

The use of moulds as a seasoning for sausage can have both desirable and undesirable consequences. The desirable consequences are the creation of a successful product that appeals to consumers. The undesirable consequences are due to the growth of undesirable moulds that produce highly toxic secondary metabolites referred to as mycotoxins. The aim of the paper was to investigate the presence of moulds producing ochratoxin A (OTA) on the surface of sausages from northern Italy. A total of 757 mould strains were isolated from sausage casings. The most frequently identified species were Penicillium nalgiovense, Penicillium oxalicum, Eurotium amstelodami, Penicillium olsonii, Penicillium chrysogenum, Penicillium verrucosum, Penicillium viridicatum, and Eupenicillium crustaceum. Aspergillus ochraceus was detected in only one production lot. Approximately 45% of these samples were positive for the presence of OTA. On the casings of the investigated sausages, the lowest and highest OTA values were 3 and 18 microg/kg, respectively. The OTA concentration was reduced to below the limit of detection (LOD) by brushing and washing the sausages prior to sale. From these data it appears that the presence of OTA on the surface of sausage (on the casings) is not indicative of any health risk for human consumption of sausage, since OTA was not identified inside the dry meat.

Inactivation of food spoilage fungi by ultra violet (UVC) irradiation.

The effect of ultraviolet irradiation (254 nm, UVC) on Aspergillus flavus, Aspergillus niger, Penicillium corylophilum and Eurotium rubrum was investigated using three different exposure techniques. Survival was determined for spores suspended in liquid medium after 1, 2 and 3 min UVC exposure at 4644 J/m(2)/min. The same UVC dose was applied to spores on the surface of agar plates for 5, 10, 15, 30, 60 and 120 s. Spores of A. niger were dried onto a membrane filter, then exposed to UVC treatment. In the liquid medium, treatments from 1-3 min significantly (P<0.001) reduce the number of viable spores. On the surface of agar plates, after a 15 s exposure, a 80-99% reduction of viable spores was observed for all species except A. niger, for which the reduction was only 62%. For spores dried onto filter membranes, a 3.5 log(10) reduction was achieved for A. niger after 180 s exposure. These observations suggest that UVC irradiation can effectively inactivate spores of A. flavus, P. corylophilum, E. rubrum and A. niger but the efficacy of UVC radiation against fungal spores varies significantly according to methods of exposure to the irradiation, and among genera.