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Simple Summary: Ochratoxin A (OTA), a nephrotoxic mycotoxin categorized as a possible carcinogenic agent, is a current problem in the cheese manufacturing industry. The main OTA producers are Penicillium nordicum and Penicillium verrucosum. Therefore, we aimed to investigate different cheese ripening phases to hinder OTA production. We elaborated cheese analogues, inoculated both fungal species, and performed two different ripening phases, at 10 and 15 °C. Despite the fact that both were able to grow, they did not express genes related to OTA production, and no OTA was detected during two weeks of ripening. Therefore, we show that, when the first phases of ripening are maintained at <15 °C, the possibility of OTA production is dramatically minimized. This information is of utmost importance for industries to enhance the safety of their cheese products, which positively impacts human health. A lack of control of the technological abiotic parameters apparent during cheese manufacture, including temperature and relative humidity, results in this dairy product being prone to mold contamination. Sometimes, inoculant molds are used to obtain the characteristic sensory properties of this type of product. However, during the maturation process, some unwanted molds can colonize the ripening cheese and produce mycotoxins. Mycotoxigenic molds such as Penicillium nordicum and Penicillium verrucosum can colonize ripened cheeses, contaminating them with ochratoxin A (OTA), a nephrotoxic 2B toxin. Thus, the presence of OTA in cheeses could represent a hazard to consumers' health. This study has evaluated the growth and OTA production of P. nordicum and P. verrucosum on a cheese analogue under simulated ripening conditions of 10 and 15 °C and 0.96 water activity (aw). Ecophysiological, molecular, and analytical tools assessed the mold growth, gene expression, and OTA production under these environmental conditions. Both species were able to effectively colonize the cheese under these ripening conditions. However, neither species expressed the otapks and otanps biosynthetic genes or produced phenotypic OTA. Therefore, these results suggest a relatively low risk of exposure to OTA for consumers of this type of cheese product. The conditions used were thus appropriate for cheese ripening to minimize the potential for contamination with such mycotoxins. An appropriate adjustment of the technological ripening parameters during such cheese manufacture could contribute to OTA-free cheeses. [ABSTRACT FROM AUTHOR]

The current study aimed to detect fungi contamination in agricultural mushroom samples Agaricus bisporus which was taken from some areas of Baqubah district in Diyala governorate. It was shown that all the samples were contaminated 100%, total colony formation ranged 3.264×10-3 to 5.596 ×10-3, the results recorded 253 fungal isolates. The Canaan region recorded the highest number of fungal isolates, which was 84 isolates, followed by the regions of Buhruz, Khan Bani Saad and Baqubah, which were 66, 54 and 49 fungal isolates, in that order. The results of the study also showed that the fungal isolates that were isolated from the white agricultural fungus belonged to 3 fungal genera, called by Aspergillus, Rhizopus and Penicilium. The genus Aspergillus was superior in the number of species belonging to it since 6 species were recorded at a rate of 75% compared to the genus Rhizopus for which one species was recordedonly Rhizopus spp. The genus Penicillium recorded one species with a rate of 12.5 % each. The total species that were isolated and diagnosed were 8 fungal species: Aspergillus niger, A. parasiticus, A. nidulans, A. tamarii, A. fumigatus, A.ochraceus, Rizopus spp. and Penicillium verrucosum. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is a nephrotoxin that contaminates grains in storage. Moisture and temperature sensors give delayed responses due to their slow kinetic movement within the silo. This study examines if CO 2 production could predict OTA contamination and identify storage conditions exceeding the maximum limit (5 μg/kg). The impact of water activity levels (0.70–0.90 a w), temperatures (15 and 20 °C), and storage duration on (a) Penicillium verrucosum population, (b)CO 2 respiration rates (RR), and (c)ochratoxins concentrations in stored wheat was investigated. 96 samples were analysed for ochratoxins with LCMS-MS. RR was >7 times higher at wetter conditions than at drier a w levels. A positive correlation between CO 2 , OTA, OTB, and OTα was observed at the wettest conditions. OTA exceeded the limit at >0.80 a w (16% moisture content) with RR > 0.01 mg CO 2 kg−1 h−1. The knowledge of the RR of stored grain would alert grain farmers/managers to improve grain storage management. • CO 2 respiration rates will increase as the water activity (a w) of grains increases. • a w influences Penicillium populations and OTA, OTB, and OTα concentrations in wheat. • CO 2 can be used to predict ochratoxin contamination in very wet grains. • Monitoring changes in CO 2 and abiotic factors will improve grain storage management. [ABSTRACT FROM AUTHOR]

The study is directed to establish the minimizing effects of Syzygium aromaticum, Ocimum sanctum, and Cananga odorata essential oils on the growth and ochratoxin A (OTA) level of Aspergillus ochraceus and Penicillium verrucosum in maize grains. S. aromaticum essential oil (SAEO), O. sanctum essential oil (OSEO), and C. odorata essential oil (COEO) were extracted by hydro-distillation technique, and a total of 50, 44, and 48 chemical constituents were identified by gas chromatography-mass spectrometry (GC-MS), respectively. The SAEO and OSEO belong to the chemotype of eugenol, whereas, COEO was found to be the chemotype of thymol, limonene, and a-ylangene. The antifungal activity of essential oils (EOs) was determined by the micro-well dilution technique. The SAEO showed superior antifungal activity compared to OSEO, COEO, and synthetic antifungal agent nystatin, and its minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values against A. ochraceous and P. verrucosum were noticed as 1251 ± 42.32 and 1878 ± 28.47 µg/mL, and 0815 ± 22.69 and 1146 ± 51.19 µg/mL, respectively. The antifungal mechanism of EOs was unveiled by assessing the intracellular reactive oxygen species (ROS), ergosterol content, and membrane integrity. The antifungal investigations found that EOs caused fungal mortality by increasing the intracellular ROS, depleting ergosterol synthesis, and distracting membrane integrity. Finally, antifungal and antimycotoxin activity of EOs was demonstrated in maize grains. The SAEO, OSEO, and COEO have reduced the complete fungal growth and OTA level of A. ochraceous and P. verrucosum correspondingly at 2500 and 2500, 3500 and 2500, and 3500 and 3500 µg/g in maize. The EOs could act as natural antifungal agents; protect foodstuffs from fungal infection and mycotoxins during storage. [ABSTRACT FROM AUTHOR]

The present study examined the influence of primary food components on the antifungal activity of the essential oil of Origanum vulgare, carvacrol, thymol, eugenol, and trans-cinnamaldehyde against Penicillium verrucosum and Aspergillus westerdijkiae. The MIC was determined in food model media enriched with proteins (1, 5, or 10%), carbohydrates (1, 4, or 6%), or oil (1, 5, or 10%). Proteins increased the antifungal activity of O. vulgare essential oil, carvacrol, thymol, and eugenol, whereas the effect of trans-cinnamaldehyde decreased with increasing protein content. The presence of carbohydrates diminished the inhibitory effect of the natural preservatives on A. westerdijkiae; for P. verrucosum, their inhibitory effect increased with carbohydrates. Only the antifungal activity of trans-cinnamaldehyde did not depend on the carbohydrate content. The presence of oil had the strongest influence. At a concentration of 1% oil, the antifungal activity decreased significantly, and at 10% oil, almost no inhibition was observed. To investigate the effect of the antifungal agents on the morphology of the target molds, they were grown on malt extract agar containing carvacrol and trans-cinnamaldehyde and were examined by scanning electron microscopy. The hyphae, conidiophores, vesicles, and phialides were severely altered and deformed, and spore formation was clearly suppressed. The antifungal activity of natural preservatives is influenced by the food matrix. Proteins increase the impact of O. vulgare EO, carvacrol, thymol, and eugenol. Carbohydrates diminish the inhibition of natural preservatives on A. westerdijkiae. In the presence of oil, natural inhibitors lose their antifungal effect. Carvacrol and trans-cinnamaldehyde lead to distorted hyphae and loss of sporulation. [ABSTRACT FROM AUTHOR]

The present study examines the influence of the natural preservatives carvacrol, eugenol, trans-cinnamaldehyde and the essential oil (EO) Origanum vulgare on ochratoxin A (OTA) production and the mycelial growth of two food-related moulds, Penicillium verrucosum and Aspergillus westerdijkiae, by broth macro-dilution assay for 21 days. With the addition of ½ minimum inhibitory concentration (MIC) carvacrol, eugenol and O. vulgare EO, the mycelial dry weight of both moulds decreased significantly over the whole incubation period of 7, 14 and 21 days. Trans-cinnamaldehyde slightly stimulated the growth of A. westerdijkiae and P. verrucosum at 14 and 21 days of incubation. Growth inhibition did not accompany inhibition of OTA production. Although the growth of both moulds was inhibited after the addition of ½ MIC carvacrol, eugenol and O. vulgare EO, the OTA production of the strong mycotoxin producer A. westerdijkiae was stimulated. Only trans-cinnamaldehyde inhibited the production of OTA with the addition of ½ MIC. P. verrucosum produced significantly less OTA than A. westerdijkiae, and its mycotoxin production was almost completely inhibited by the addition of ½ MIC of the natural preservatives. [ABSTRACT FROM AUTHOR]

Ecological conditions during ripening of dry-cured meat products favour the growth of an uncontrolled mould population that could suppose a risk of ochratoxin A (OTA) production. In this work the influence of water activity (a w), temperature and substrate composition on fungal growth and OTA production by Penicillium nordicum and Penicillium verrucosum isolated from dry-cured meat products have been studied. In addition, the relative risk of OTA presence on dry-cured meat products has been evaluated using the Risk Ranger software. Fungal growth was observed in the range of 0.99–0.90 a w and 15–25 °C being mainly temperature-dependent. P. nordicum and P. verrucosum were able to produce OTA in every substrate in these ranges of a w and temperature. The production of OTA by P. verrucosum was mainly influenced by temperature and media composition. However, P. nordicum it is affected mainly by substrate or temperature depending on the strain studied. Both species produce a large amount of OTA on dry-cured ham and on dry-cured fermented sausage "salchichón" in environmental conditions usually found throughout the ripening of these products. The Risk Ranger software reveals that the relative risk of OTA on dry-cured meat products is 75%. Thus, control measures during dry-cured meat products processing to prevent OTA risk should be established. Unlabelled Image [ABSTRACT FROM AUTHOR]

The aims of this study were to evaluate the antifungal activity of the bioactive compound allyl isothiocyanate (AITC) against Aspergillus flavus (8111 ISPA) aflatoxins (AFs) producer and Penicillium verrucosum (D-01847 VTT) ochratoxin A (OTA) producer on corn, barley, and wheat. The experiments were carried out initially in a simulated silo system for laboratory scale composed of glass jars (1 L). Barley and wheat were contaminated with P. verrucosum and corn with A. flavus. The cereals were treated with a hydroxyethylcellulose gel disk to which 500 µL/L of AITC were added; the silo system was closed and incubated for 30 days at 21 °C. After that, simulated silos of 100 L capacity were used. Barley, wheat, and corn were contaminated under the same conditions as the previous trial and treated with disks with 5 mL of AITC, closed and incubated for 90 days at 21 °C. In both cases, the control test did not receive any antifungal treatment. The growth of the inoculated fungi and the reduction in the formation of AFs and OTA were determined. In the lab scale silo system, complete inhibition of fungal growth at 30 days has been observed. In corn, the reduction of aflatoxin B1 (AFB1) was 98.5%. In the 100 L plastic drums, a significant reduction in the growth of A. flavus was observed, as well as the OTA formation in wheat (99.5%) and barley (92.0%). [ABSTRACT FROM AUTHOR]

Abstract Ochratoxigenic penicillia can grow on the cured meat products surface during their ripening. Ingredients added to cured meat products throughout their processing, and specifically of dry-fermented sausages, consist of a potential strategy to prevent or minimise the hazard associated with ochratoxin A (OTA) in these products. The aim of this work was to evaluate the effect of different concentrations of NaCl, KCl and sucrose in a conducive medium on the growth and OTA production by two Penicillium verrucosum strains. In general, there were no statistical differences between their growth rates. Although OTA production was not completely avoided in the presence of KCl and sucrose, it was reduced by comparison with NaCl. The concentration of the ingredients also affected the OTA production by P. verrucosum. These results would mean that the replacement of NaCl with KCl would imply the production of potentially safer cured meat products in terms of the OTA presence, which is of great importance for the current trends to reduce the NaCl levels in this kind of products. Furthermore, the addition of sucrose should be considered one of the approaches of the hurdle technology for minimising OTA accumulation in cured meat products. Highlights • The role of cured meat ingredients on mould growth and OTA production was evaluated. • Two ochratoxigenic Penicillium verrucosum strains were investigated. • Type and amount of cured meat ingredients influenced growth and OTA accumulation. • Large differences between the tested two P. verrucosum strains were not found. • Formulation changes might provide safer cured meats in terms of the OTA presence. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) can cause toxicogenic effects in humans and animals when contaminated food products are consumed. Oat (Avena sativa), like any other cereal grain, can be contaminated with OTA when storage conditions are favorable for fungal growth and toxin production. South Dakota is among the leading oat-producing states in the United States. It is therefore important to determine the frequency of occurrence of the primary OTA-producing fungal species on oat grains produced in the state. In this study, we evaluated oat grain samples from South Dakota for the incidence of Penicillium verrucosum, the major ochratoxigenic fungus in temperate regions. Kernels from 12 oat cultivars grown at multiple locations in South Dakota from 2014 to 2016 (15 location-year combinations) were plated on dichloran yeast extract sucrose glycerol agar medium. P. verrucosum was detected on 0.30, 0.19, and 0.05% of the kernels tested in 2014, 2015, and 2016, respectively. Overall, 22 of the 360 evaluated samples had kernels contaminated with P. verrucosum. The fungal incidence of the contaminated samples ranged from 1 to 16%, and the majority of those samples originated from one location. All samples from 2014 and 2015 (n=240), except two, had no detectable levels of OTA. The concentration of OTA was well under the maximum limit recommended by the European Union for the two samples with detectable levels of OTA. [ABSTRACT FROM AUTHOR]

This study deals with the efficacy of Origanum onites, Salvia officinalis, and Mentha piperita essential oils (EOs) and their mixture (1:1:1, v/v/v) against ochratoxigenic P. verrucosum D-99756 and modelling the effects of EOs on fungal growth using modified Gomperz and logistic models. O. onites, S. officinalis, and M. piperita EOs were used at their minimal lethal concentration levels at 65, 125, and 250 μL/mL, respectively, which were determined in our preliminary studies. Inactivation of fungi sporulation by EO treatments was higher than 50-60% within 48 h, while 78.50-81.41% lethality was observed at the end of the 168 h for all of the tested EOs. The most effectiveness time for lethal effect was found at 84 h for O. onites and S. officinalis, while it was 168 h for mix EOs (p < 0.05). The modified Gompertz model successfully produced better fits than the logistic model as evidenced by the root mean square error, the modelling efficiency, and accuracy efficiency. In conclusion, data confirmed the potential use of plant and spice EOs for their fungistatic and fungicidal effects in food systems. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effect of synthetic detergent Merix (Henkel, Kruševac, Serbia), and its particular components-ethoxylated oleyl-cetyl alcohol and sodium tripolyphosphate on the growth and metabolic activity of Penicillium verrucosum. During 19 days of fungal cultivation in Czapek-Dox liquid medium supplemented with or without 0.5% pollutants, the following parameters were observed: pH, the total biomass dry weight, the quantity of free and total organic acids and proteolytic activity. The detergent caused a slight stimulatory (2.41%) effect whereas sodium tripolyphosphate and ethoxylated oleyl-cetyl alcohol provided a slight inhibitory action (0.59 and 2.75%, respectively) on the fungal biomass. The pollutants decreased pH values of the media and the quantity of free organic acids. In contrast, they enhanced the quantity of total organic acids. Proteolytic activity remained nearly unchanged (95.8%) in the presence of detergent and reduced to 80.1% in sodium tripolyphosphate-supplemented medium. In contrast, the enzyme activity sharply increased (260.8%) with ethoxylated oleyl-cetyl alcohol. The obtained results indicate the potential of P. verrucosum in bioremediation of environment contaminated with synthetic detergent taken in high concentration. [ABSTRACT FROM AUTHOR]

This study deals with the efficacy ofOriganum onites, Salvia officinalis, andMentha piperitaessential oils (EOs) and their mixture (1:1:1, v/v/v) against ochratoxigenic P. verrucosum D-99756 and modelling the effects of EOs on fungal growth using modified Gomperz and logistic models. O. onites, S. officinalis, and M. piperita EOs were used at their minimal lethal concentration levels at 65, 125, and 250 µL/mL, respectively, which were determined in our preliminary studies. Inactivation of fungi sporulation by EO treatments was higher than 50–60% within 48 h, while 78.50–81.41% lethality was observed at the end of the 168 h for all of the tested EOs. The most effectiveness time for lethal effect was found at 84 h for O. onites and S. officinalis, while it was 168 h for mix EOs (p< 0.05). The modified Gompertz model successfully produced better fits than the logistic model as evidenced by the root mean square error, the modelling efficiency, and accuracy efficiency. In conclusion, data confirmed the potential use of plant and spice EOs for their fungistatic and fungicidal effects in food systems. [ABSTRACT FROM AUTHOR]

Penicillium verrucosum is a fungus that can produce ochratoxin A and citrinin, two structurally related nephrotoxic mycotoxins. P. verrucosum usually occurs on wheat but can occasionally also be found in NaCl rich habitats such as salted cheeses or olives, indicating that this fungus can adapt to different environments. The ratio of ochratoxin A to citrinin produced by P. verrucosum is shifted to one of either mycotoxin at the expense of the other dependent on the environmental conditions. High NaCl concentrations shift secondary metabolite biosynthesis towards ochratoxin A production. P. verrucosum copes with NaCl stress by increased ochratoxin A biosynthesis, ensuring chloride homeostasis. Ochratoxin A carries chlorine in its molecule and can excrete chlorine from the cell. It was further shown that the regulation of ochratoxin A by high NaCl conditions is mediated by the HOG MAP kinase signal transduction pathway. Here it is shown that high oxidative stress conditions, evoked for example by increasing concentrations of Cu 2 + cations in the growth medium, shift secondary metabolite biosynthesis of P. verrucosum from ochratoxin A to citrinin. The production of citrinin normalizes the oxidative status of the fungal cell under oxidative stress conditions leading to an adaptation to these environmental conditions and protects against increased oxidative stress caused by increased Cu 2 + concentrations. Moreover citrinin also protects against light of short wavelength, which may also increase the oxidative status of the environment. The biosynthesis of citrinin is apparently regulated by a cAMP/PKA signaling pathway, because increasing amounts of external cAMP reduce citrinin biosynthesis in a concentration dependent manner. These conditions lead to the cross-regulation of the ochratoxin A/citrinin secondary metabolite pair and support the adaptation of P. verrucosum to different environments. [ABSTRACT FROM AUTHOR]

Abstract: In this study the differentially expressed protein population of Penicillium verrucosum grown either in the dark or under light with a wavelength of 450nm has been analyzed. Light of short wavelength led to oxidative stress in the fungal cell; under this condition the mycotoxin biosynthesis revealed a mutual shift from ochratoxin A to citrinin. Using a proteomic approach combining an optimized protein extraction method with 2-dimensional SDS-PAGE followed by HPLC-ESI-TOF-MS/MS mass spectrometric analysis, initially 56 significantly differential proteins (light vs. dark) were detected comprising proteins of a broad range of isoelectric points and molecular masses. In total, 46 proteins could be identified further by database query, most of these proteins are assumed to be involved in response to stress (e.g. antioxidative proteins, heat shock proteins) and general metabolic processes (e.g. glycolysis, ATP supply). Proteome analyses are necessary to unravel the regulation of secondary metabolite biosynthesis at a translational level. This may enable identification of proteins which are involved in mycotoxin biosynthesis, adaption processes or even stress compensation mechanisms. This study depicts the first proteome analysis of P. verrucosum. [Copyright &y& Elsevier]

Cereals are among the most vulnerable substrates to mycotoxin contamination. One of the major fungal species colonising the ecosystem of stored grain isPenicillium verrucosumbecause of its ability to produce ochratoxin A (OTA). Knowledge of favourable environmental conditions promoting fungal growth and mycotoxin biosynthesis seems to be a crucial step towards preventing mycotoxin formation in stored grain. The aim of the study was to examine the effect of temperature and different cereal substrates (wheat, triticale, rye, barley, maize, rice) on fungal growth and OTA and citrinin (CIT) production by a mycotoxin-producingP. verrucosumstrain at one moisture content. The correlation between fungal growth and mycotoxin biosynthesis was also investigated. For this purpose, cereal substrates moistened, autoclaved and inoculated withP. verrucosumspores were stored at 10°C, 20°C and 30°C for 40 days. After the storage period, the ergosterol (ERG) content as a fungal growth biomarker and OTA and CIT levels were determined using HPLC. Fungal growth was observed on all cereals at each temperature. Maximum ERG levels were found at 30°C and the values ranged from 401 to 3280 mg kg−1of grain. Among tested cereals, the highest ergosterol content was found on rice at all applied temperatures. Accumulation of OTA and CIT was detected in samples stored at 20°C and 30°C, while optimal conditions for the production of both mycotoxins were observed at 20°C. The maximum concentration of OTA (31.9 ± 4.3 (SD) μg kg−1) and CIT (74.8 ± 5.7 μg kg−1) were found on rice. The ability to biosynthesise OTA and CIT at 10°C was not detected. Analysis of variance showed that single factors, temperature and cereal matrices and their interactions had significant effects on ERG, OTA and CIT levels. Optimal conditions for fungus growth did not coincide with the ones for mycotoxin formation. [ABSTRACT FROM AUTHOR]

Growth of undesirable moulds is one of the major causes of defects in modern cheese production, and it is necessary to discover natural antifungal substances that are non-toxic to humans. Saffron spice has been successfully used as a new ingredient in some Italian or Spanish cheeses, but transference of safranal (the main compound responsible for saffron aroma) during cheesemaking has not yet been studied. In this study, the distribution of safranal in cheese, whey and requesón (Spanish whey cheese) and the antifungal potential of whey fractions against the mould Penicillium verrucosum were studied. To this end, a duplicate fabrication of cheeses was performed with raw ewe's milk with the addition of 40 μg safranal.kg −1 or control milk (no safranal addition). Safranal was more concentrated in cheese and requesón (78.91 and 76.95 μg.kg −1, respectively) than in whey and requesón whey (35.19 and 34.64 μg.kg −1, respectively). These liquid fractions showed good antifungal properties and inhibited mould with a safranal concentration of around 35 μg.kg −1, while a solution of 80 μg.kg −1 was required for a standard safranal solution to obtain a similar inhibition. These results reveal that whey has a synergistic effect with safranal in preventing cheese spoilage by moulds, which encourages making full use of this problematic by-product. [ABSTRACT FROM AUTHOR]

Abstract: Mycotoxin biosynthesis in Penicillium verrucosum is modulated by different molecular regulation mechanisms. One important mechanism is the HOG1 (high osmolarity glycerol) MAP kinase signaling pathway. In a comparative analysis three different P. verrucosum strains were selected from six strains with different ability to produce the mycotoxins ochratoxin and citrinin. The fungal strains were grown on laboratory medium supplemented with different concentrations of NaCl. It could be shown that there exists an interrelationship between the growth rate, the level of HOG phosphorylation and the mycotoxin biosynthesis under the respective growth condition. The weak to non ochratoxin producing P. verrucosum strain, BFE875, showed only a poor growth rate but the strongest HOG1 phosphorylation; the strong ochratoxin and citrinin producing strain BFE575 showed a reasonable HOG1 phosphorylation with an average growth rate; and the strong ochratoxin and weak citrinin producing strain BFE495 showed only a poor phosphorylation but the highest growth rate in 7days of incubation at 25°C. The magnitude of phosphorylation of the HOG1 protein seems to be inversely correlated with the degree of adaption of the fungus to hyperosmotic growth conditions. [Copyright &y& Elsevier]

Penicillium verrucosum, P. nordicum and Aspergillus carbonarius are three important ochratoxin A producing species. P. verrucosum is in addition able to produce citrinin. It has been shown earlier that P. nordicum is adapted to NaCl rich environments like salt rich dry cured foods or even salines. In this organism, the biosynthesis of ochratoxin A plays an adaptive role in this habitat. P. verrucosum generally can be found on cereals, but occasionally also on salt rich dry cured foods. In contrast A. carbonarius usually cannot be found in NaCl rich environments, but it occurs in another environment with high concentration of solutes, e.g., in sugar rich substrates like grapes and grape juices. Usually osmotic challenging conditions activate the HOG MAP kinase signal cascade, which in turn activates various osmo-regulated genes. In the current analysis, it could be demonstrated that in case of P. nordicum and P. verrucosum the NaCl induced production of ochratoxin A is correlated to the phosphorylation status of the HOG MAP kinase. Just the opposite was true for A. carbonarius. In this case, also higher amounts of NaCl in the medium lead to an increased phosphorylation status of HOG, but no increase in ochratoxin biosynthesis was observed. In contrast to the Penicillia, higher NaCl concentrations lead to a rapid cessation of growth by A. carbonarius. High glucose concentrations have much less impact on growth and the phosphorylation of HOG. [ABSTRACT FROM AUTHOR]

BACKGROUND: The use of aromatic plants and their extracts with antimicrobial properties may be compromised in the case of cheese, as some type of fungal starter is needed during its production. Penicillium verrucosum is considered a common cheese spoiler. The aim of this study was to evaluate the innovative use of certain aromatic plants as natural cheese covers in order to prevent mycotoxicogenic fungal growth ( P. verrucosum). A collection of 12 essential oils (EOs) was obtained from various aromatic plants by solvent-free microwave extraction technology, and volatile characterisation of the EOs was carried out by gas chromatography/mass spectrometry. RESULTS: The most effective EOs against P. verrucosum were obtained from Anethum graveolens, Hyssopus officinalis and Chamaemelum nobile, yielding 50% inhibition of fungal growth at concentration values lower than 0.02 µL mL−1. All EOs showed high volatile heterogeneity, with α-phellandrene, pinocamphone, isopinocamphone, α-pinene, camphene, 1,8-cineole, carvacrol and trans-anethole being found to be statistically significant in the antifungal model. CONCLUSION: The use of these aromatic plants as natural covers on cheese can satisfactorily inhibit the growth of some mycotoxicogenic fungal spoilers. Among the volatile compounds present, α- and β-phellandrene were confirmed as the most relevant in the inhibition. © 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Gallic acid (3,4,5-trihydroxybenzoic acid) was produced by microbial biotransformation of coffee pulp tannins byPenicillium verrucosum. Gallic acid production was optimized using response surface methodology (RSM) based on central composite rotatable design. Process parameters such as pH, moisture, and fermentation period were considered for optimization. Among the various fungi isolated from coffee by-products,Penicillium verrucosumproduced 35.23 µg/g of gallic acid on coffee pulp as sole carbon source in solid-state fermentation. The optimum values of the parameters obtained from the RSM were pH 3.32, moisture 58.40%, and fermentation period of 96 hr. Gallic acid production with an increase of 4.6-fold was achieved upon optimization of the process parameters. The results optimized could be translated to 1-kg tray fermentation. High-performance liquid chromatography (HPLC) analysis and spectral studies such as mass spectroscopy (MS) and1H-nuclear magnetic resonance (NMR) confirmed that the bioactive compound isolated was gallic acid. Thus, coffee pulp, which is available in enormous quantity, could be used for the production of value-added products that can find avenues in food, pharmaceutical, and chemical industries. [ABSTRACT FROM AUTHOR]

Mycotoxins are natural, secondary metabolites produced by fungi on agricultural commodities in the field and during storage under a wide range of climatic conditions. The ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by several species of Aspergillus and Penicillium. In this study, the influence of carbon and nitrogen sources on ochratoxigenic Penicillium species was assessed. The ochratoxigenic Penicillium species were isolated from poultry feed samples of Andhra Pradesh, India. The isolated ochratoxigenic Penicillium species were identified, screened and characterised as OTA producers by high performance thin layer chromatography (HPTLC) and confirmed by high performance liquid chromatography (HPLC). This experiment was carried out using Czapak yeast Autolysate (CYA) medium with different carbon (C) and nitrogen (N) sources at pH 6.5 and incubated at 25 ± 2°C under dark condition. Maximum OTA production was recorded in the presence of D-glucose followed by D-galactose and D-lactose as carbon sources. Similarly, the maximum amount of OTA production was observed in thiourea followed by potassium nitrate as nitrogen source. However, OTA production, final pH of the medium, and mycelial yield and OTA production of both the species of Penicillium varied with C and N present in the medium. The kinetics of the both species of Penicillium was observed for 30 days at an interval of three days. The maximum amount of OTA was detected by 12 and 15 days incubation periods for Penicillium nordicum and Penicillium verrucosum, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Penicillium.nordicum is an ochratoxin A producing filamentous fungus, which is adapted to sodium chloride and protein rich food environments like certain cheeses or dry cured meats. Penicillium.verrucosum usually occurs on cereals but can also be isolated from brined olives. It could be shown that sodium chloride has a profound influence on the regulation of ochratoxin A biosynthesis in both Penicillium species. High amounts of ochratoxin A are produced by P. nordicum over a wide concentration range of NaCl (5–100 g/l) with a weak optimum at about 20 g/l after growth on YES medium. P. verrucosum shifts secondary metabolite biosynthesis after growth on YES medium from citrinin at low to ochratoxin at elevated NaCl concentrations. The ochratoxin A biosynthesis of P. nordicum is accompanied by an induction of the otapksPN gene, the gene of the ochratoxin A polyketide synthase. A mutant strain unable to produce ochratoxin showed a drastic growth reduction under high NaCl conditions. Determination of the dry weight and the chloride content in the mycelium of the P. nordicum wild type strain and a non-ochratoxin A producing mutant strain showed a much higher increase of both parameters in the mutant compared to the wild type. These results suggest, that the constant biosynthesis and excretion of ochratoxin A, which itself contains a chloride atom, ensures a partial chloride homeostasis in the fungal cell. This mechanism may support the adaptation of ochratoxin A producing Penicillia to NaCl rich foods. [Copyright &y& Elsevier]

Abstract: In Spain, low ochratoxin A (OTA) levels have been detected in several pork products but there is no information published about the fungi involved in this OTA contamination. It is well known that P. verrucosum is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe where levels of OTA generally seem to be lower or not detected. Much less information is available about citrinin (CIT) and CIT producing species in cereals and their by products. The aim of this study was to determine, identify and characterize the occurrence of potential OTA and CIT producing Penicillium spp. from mixed feeds and raw materials purchased in the Spanish market and used as feedstuffs. A total of 155 Penicillium spp. isolates belonging to 34 species were analyzed in order to know if they are able to produce OTA and/or CIT. From these isolates, 11 P. verrucosum which were characterized by RAPD analyses, produced OTA. Fourteen isolates were CIT producers, 10 isolates of P. verrucosum and 4 of P. citrinum. Although the occurrence and abundance of OTA and CIT Penicillium producing species have been low in our study, our results confirm the potential risk of OTA and CIT production in feeds if stored improperly. Our results also confirm the occurrence of P. verrucosum in South European countries and that it is the only OTA producing Penicillium species in these substrates. [Copyright &y& Elsevier]

Abstract: In Spain, low ochratoxin A (OTA) levels have been detected in wheat and different wheat products but no information has been published about the fungi involved in this OTA contamination. Some species of the genera Penicillium and Aspergillus are known to form OTA but few of them are known to contaminate foods with this mycotoxin. Penicillium verrucosum, an important OTA producer typical of temperate and cold climates, is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe, where levels of OTA generally seem to be lower or is not detected. The aim of this study was to determine, identify and characterize the occurrence of potential OTA-producing Aspergillus spp. and Penicillium spp. from retail wheat flours purchased in the Spanish market and used for human consumption. A total of 105 Aspergillus isolates were analyzed in order to know whether they are able to produce OTA and/or citrinin (CIT). None of these isolates were able to produce these mycotoxins. However, 17 suspected P. verrucosum isolates were recovered and confirmed by RAPD analyses. Eleven isolates were OTA producers and 14 isolates produced CIT. Our results confirm the potential risk of OTA and CIT production in wheat flours if stored improperly and the occurrence of P. verrucosum in South European countries. This was the only species able to produce these mycotoxins. [Copyright &y& Elsevier]

Systematic expression analysis of mycotoxin biosynthesis genes by real-time PCR and microarray was carried out to examine the relationship between growth and general expression patterns in relation to single environmental factors such as temperature, water activity ( aw) and pH and water activity × temperature interactions. For single parameters, one major peak of expression occurred close to optimum growth conditions. However, a second minor peak was observed under suboptimal growth conditions, when intermediate environmental stress was imposed on Aspergillus parasiticus ( afl genes), Penicillium verrucosum ( ota genes) and Fusarium culmorum ( tri genes). This expression profile pattern was more pronounced in relation to changes in temperature and aw than to pH. In a two-factorial experimental design with temperature × aw regimes, again two peaks of expression were observed for cluster genes after microarray analysis, one close to those giving optimal growth and one under imposed stress conditions. Interestingly, when the activity of single genes of the microarray data were plotted in relation to the two parameters, again a two-peak expression profile became obvious independently for both parameters. Expression of the mycotoxin biosynthesis genes was followed exactly by phenotypic mycotoxin production. This expression profile appears to be generic across the mycotoxigenic fungi examined. [ABSTRACT FROM AUTHOR]

Summary: A Brazilian strain of Penicillium verrucosum was cultivated under different conditions in a two-step process, in order to verify the influence of nutrients, and of time periods of pre-fermentative and fermentative steps on the biosynthesis of metabolites. Extracellular and intracellular extracts were obtained from each culture in the four different production media used. Chemical profiles of the extracts were obtained by HPLC. Extract trypanocidal activities against trypomastigote forms of Trypanosoma cruzi were evaluated. The time period of incubation in the pre-fermentative and fermentative media, as well as the different nutrients tested, qualitatively and quantitatively modified the production of secondary metabolites by P. verrucosum, and the extract trypanocidal activities. [Copyright &y& Elsevier]

Ochratoxin A (OTA) is a secondary metabolite produced by several species of Aspergillus and Penicillium ; among them Aspergillus ochraceus and Penicillium verrucosum are two ochratoxigenic species capable of growing in different climates and thus contamination of food crops with OTA can occur worldwide. OTA can be found in a wide range of foods such as cereals, coffee, cocoa, spices, beer, wine, dried vine fruit, grapes and meat products. OTA is toxic to animals, it presents neurotoxic, immunotoxic and nephrotoxic effects. It has been implicated in a human kidney disorder known as Balkan Endemic Nephropathy. This review focuses on the ecophysiology of ochratoxin-producing Aspergillus ochraceus and Penicillium verrucosum , the effect of environmental factors on their germination, mycelial growth, and OTA production. Knowledge of environmental conditions required for sucessive stages of fungal development represent the first step towards preventing mycotoxin formation. Predictive models for different stages of fungal development are presented, which allow prediction of the time before spoilage as a function of the abiotic factors. Finally, the implications of these studies in management of barley, coffee and grapes are described. This can help to identify the critical control points in their production, storage and distribution processes. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to study the combined effects of water activity (a w, 0.75–0.99), temperature (10–30 °C) and isolate on spore germination and mycelial growth of ochratoxigenic Penicillium verrucosum isolates on barley meal extract agar (BMEA). Lag phases prior to germination (h), germination rates (h− 1) and growth rates (mm day− 1) were obtained at each set of conditions and for all the isolates. Analysis of variance showed a significant influence of the abiotic factors assayed on fungal development. Minimum a w levels found for germination and mycelial growth were 0.80 and 0.85, respectively, while maximum germination and growth rates were obtained at 0.95–0.99 a w level. Although maximum germination was obtained at 20 °C, significant differences were not found between growth rates at 20 and 30 °C. Data were modelled by MLR regressions and response surface models were obtained. Spore germination and mycelial growth of ochratoxigenic P. verrucosum isolates are significantly affected by water activity and temperature. Moreover, different isolates may have slightly different response to environmental factors. Predictive models obtained may allow a rough prediction of germination/growth as a function of the storage temperature and moisture content of barley grains. [Copyright &y& Elsevier]

j.c. frisvad, f. lund and s. elmholt. 2004.To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals,Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP).A total of 321 isolates ofP. verrucosumwere isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185P. verrucosumisolates, thus 84% of the isolates may represent different genets ofP. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual speciesP. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection ofP. verrucosumshowed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone.Among 184 ochratoxin A-producingPenicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the speciesP. verrucosumis phenotypically distinct and valid.AFLP fingerprinting ofPenicillium verrucosumindicates that genetic recombination takes place in this fungus. [ABSTRACT FROM AUTHOR]

Abstract: Studies were conducted to examine the effect of water activity (aw, 0.97, 0.95, 0.93), pH (4.5, 6.0) and preservative type and concentration (potassium sorbate (KS), calcium propionate (CaP), 300 and 3000 ppm) on colonisation and ochratoxin A (OTA) production by three strains of Penicillium verrucosum on bread analogues. At 25 °C, colony extension from a point source was similar for all three strains at the different environmental treatments examined, with optimum growth at 0.97 and 0.96 aw and pH 6. Temporal OTA production over 36 days varied significantly with strain, pH and aw level. Maximum OTA was produced at approximately 28–36 days for two of the strains with an optimum at 0.93 aw at pH 6.0. One strain produced 75–80 μg g−1 analogue under these conditions. However, at pH 4.5, optimum OTA was produced at 0.95 aw after a similar time period on bread analogues, with maximum amounts of about 25–35 μg g−1 bread. KS and CaP incorporation inhibited growth completely at pH 4.5 and 3000 ppm at all three aw levels. However, at suboptimal concentration, little growth inhibition occurred. At pH 6, even with 3000 ppm, growth of all three strains occurred. Where effective inhibition of growth was achieved (3000 ppm/pH 4.5, all aw levels), no OTA was detected. However, when 300 ppm was used, up to 7 μg g−1 was detected in bread analogues at pH 4.5. At pH 6, OTA was produced at all treatment conditions by all three strains, although preservative-treated bread had less present. Statistical analysis showed that many of the one-, two- and three-way interactions had a significant effect on growth and OTA production. These results suggest that there is a significant risk from OTA contamination if lower concentrations of existing preservatives are used in wheat-based bakery products contaminated by spoilage mycotoxigenic moulds such as P. verrucosum. [Copyright &y& Elsevier]

f. lund and j.c. frisvad. 2003. The aims of this study were to isolate and identify ochratoxin A (OTA) producing fungi in cereals containing OTA and to determine the best selective and indicative medium for recovery of OTA producing fungi. Seventy-six wheat, barley and rye samples from Europe containing OTA and 17 samples without OTA were investigated using three different media, dichloran yeast sucrose agar (DYSG), dichloran rose bengal yeast extract sucrose agar (DRYES) and dichloran 18% glycerol agar (DG18). Hundred kernels were plated on each medium and the kind and number of fungal OTA producers were recorded as percentage of infestation. Penicillium verrucosum was the sole OTA producer found in cereals. The average percentage of infestation of P. verrucosum counts was recorded as 28·3% on DYSG, 10·3% on DRYES and 9·9% on DG18 on the OTA containing samples and 0·8% on DYSG, 0·4% on DRYES and 0·6% on DG18 for the samples without OTA. Penicillium verrucosum was the sole OTA producer in European cereals. Determination of P. verrucosum infestation and infection was best detected on DYSG after 7 days at 20°C. The percentage of infestation of P. verrucosum found on DYSG and OTA content in cereals were correlated. More than 7% infestation of P. verrucosum indicated OTA contamination. The developed method could be used as a cereal quality control. [ABSTRACT FROM AUTHOR]

Contamination of maize and barley grain during storage by the toxigenic fungi Aspergillus flavus (A. flavus) and Penicillium Verrucosum (P. verrucosum) is both an economic and a public health problem, especially in less industrialized countries. Peracetic acid (PA) is a compound used for the disinfection of food and food contact surfaces. Unlike other disinfectants, it leaves no toxic residues and its decomposition products (CH 3 COOH, O 2 and H 2 O) are environmentally friendly. In order to apply PA to preserve maize and barley grain during storage, first, the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) against both fungi were determined in a 96-well sterile microplates. Then, the antifungal activity of the volatile PA was determined by depositing it on filter paper and Hydroxyethylcellulose (HEC) gels, which were placed in airtight 1L flasks together with PDA and maize and barley grain inoculated with both fungi. The MFC in liquid medium of A. flavus and P. verrucosum was 93.8 and 187.5 mg/L respectively. However, PA doses of 300 and 200 mg/L on HEC gels were required in order to observe significant antifungal and antimycotoxigenic activity in maize and barley grain inoculated with these fungi. [Display omitted] • Volatile peracetic acid presents antifungal activity against A. flavus and P. verrucosum. • Hydroxyethylcellulose gels with 300 mg/L of peracetic acid preserve stored maize and barley grains. • The mentioned gels inhibit the synthesis of aflatoxin B 1 and ochratoxin A in stored grain. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is one of the worldwide most important mycotoxins in terms of health and agroeconomic consequences. With the aim to promote the use of phytochemicals as alternatives to synthetic fungicides, the effect of hydroxycinnamic acids on the fungal growth and OTA yield by two major OTA-producing species was investigated. After a first step dedicated to the definition of most suitable culture conditions, the impact of 0.5 mM ferulic (FER), p-coumaric (COUM), caffeic and chlorogenic acids was evaluated on Aspergillus westerdijkiae and Penicillium verrucosum. Whereas no fungal growth reduction was observed regardless of the phenolic acid and fungal isolate, our results demonstrated the capacity of FER and COUM to inhibit OTA production. The most efficient compound was FER that led to a 70% reduction of OTA yielded by P. verrucosum and, although not statistically significant, a 35% inhibition of OTA produced by A. westerdijkiae. To further investigate the bioactivity of FER and COUM, their metabolic fate was characterized in fungal broths. The capacity of P. verrucosum to metabolize FER and COUM through a C2-clivage type degradation was demonstrated. Overall, our data support the potential use of FER to prevent OTA contamination and reduce the use of synthetic pesticides. [ABSTRACT FROM AUTHOR]

The present study evaluated the efficacy of allyl isothiocyanate (AITC) in avoiding the growth of Penicillium verrucosum and consequent ochratoxin A (OTA) production in barley during storage for 90 d. Environmental humidity was controlled using saturated salt solutions and moisture content (MC) of the grain was analyzed. Moreover, the residual concentration of AITC on stored barley was also examined. Samples with 20.6% of MC presented the highest absorption of AITC with levels ranging from 75 to 4 mg/kg at day 1 and 90, respectively. The population of P. verrucosum was significantly reduced after 24 h of AITC exposure. After 90 d, the non-treated control group reached a fungal population of 8.3 log CFU/g and 1.5 mg/kg of OTA. On the other hand, AITC at 50 μL/L was able to reduce the fungal population as well as the production of OTA to levels that were below the limit of detection, independently of the MC and the time of exposure. In conclusion, the AITC was absorbed by the barley and released gradually, inhibiting the growth of P. verrucosum and the production of mycotoxin. Therefore, AITC could be used as a fungicide to prolong the shelf life of the barley improving its safety. • AITC avoid the Penicillium verrucosum growth in barley stored for 90 days. • Fumigation of AITC could be used to improve barley safety. • 50 μL/L of AITC avoid Ochratoxin A production regardless of grain moisture content. [ABSTRACT FROM AUTHOR]