Aspergillus Carbonarius Growth Research Articles

Biocontrol activities of yeasts or lactic acid bacteria isolated from Robusta coffee against Aspergillus carbonarius growth and ochratoxin A production in vitro

Biocontrol Agents (BCAs) can be an eco-friendly alternative to fungicides to reduce the contamination with mycotoxigenic fungi on coffee. In the present study, different strains of bacteria and yeasts were isolated from Ivorian Robusta coffee. Their ability to reduce fungal growth and Ochratoxin A (OTA) production during their confrontation against Aspergillus carbonarius was screened on solid media. Some strains were able to reduce growth and OTA production by 85 % and 90 % and were molecularly identified as two yeasts, Rhodosporidiobolus ruineniae and Meyerozyma caribbica. Subsequent tests on liquid media with A. carbonarius or solely with OTA revealed adhesion of R. ruineniae to the mycelium of A. carbonarius through Scanning Electron Microscopy, and an OTA adsorption efficiency of 50 %. For M. caribbica potential degradation of OTA after 24 h incubation was observed. Both yeasts could be potential BCAs good candidates for Ivorian Robusta coffee protection against A. carbonarius and OTA contamination.

Plant extracts as biocontrol agents against Aspergillus carbonarius growth and ochratoxin A production in grapes

Aspergillus carbonarius (Bainier) Thom. is an important pathogen and ochratoxin A (OTA) producer in grapes that can be controlled by adopting sustainable approaches. Here we evaluate the application of natural plant extracts as an alternative to synthetic fungicides to reduce OTA contamination and to prevent infection of grapes by two isolates of A. carbonarius. In a preliminary screening, natural extracts of chestnut flower, cistus, eucalyptus, fennel, and orange peel were evaluated for their antifungal and anti-mycotoxigenic efficiency in a grape-based medium at concentrations of 10 and 20 mg/mL. Cistus and orange peel extracts demonstrated the best antifungal activity at both concentrations. Although the eucalyptus extract demonstrated no significant effect on Aspergillus vegetative growth, it significantly reduced OTA by up to 85.75 % at 10 mg/mL compared to the control. Chestnut flower, cistus, eucalyptus, and orange peel extracts were then tested at the lowest concentration (10 mg/mL) for their antifungal activity in artificially inoculated grape berries. The cistus and orange peel extracts demonstrated the greatest antifungal activity and significantly reduced mold symptoms in grapes. Moreover, all tested natural extracts were able to reduce OTA content in grape berries (17.7 ± 8.3 % - 82.3 ± 3.85 % inhibition), although not always significantly. Eucalyptus extract was particularly efficient, inhibiting OTA production by both strains of A. carbonarius by up to >80 % with no effects on fungal growth. The use of natural eucalyptus extract represents a feasible strategy to reduce OTA formation without disrupting fungal growth, apparently maintaining the natural microbial balance, while cistus and orange peel extracts appear promising as inhibitors of A. carbonarius mycelial growth. Our findings suggest that plant extracts may be useful sources of bioactive chemicals for preventing A. carbonarius contamination and OTA production. Nonetheless, it will be necessary to evaluate their effect on the organoleptic properties of the grapes.

Inhibition of Aspergillus carbonarius growth and Ochratoxin A production using lactic acid bacteria cultivated in an optimized medium

The Gram-positive bacteria lactic acid bacteria (LAB) are used in the food industry but are also known for inhibiting certain food spoilage microorganisms, especially fungi. Sources of nitrogen (N) for culture media are generally organic and expensive. Many attempts have been made to formulate economical culture media with alternative N sources obtained from agricultural and industrial byproducts. This study describes the design and optimization of an inexpensive culture medium for Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) MZ809351 strain B31. The culture medium was optimized using statistical experimental designs to identify the factors with the most significant effects on biomass concentration to reduce the overall cost, aiming to obtain a biomass concentration similar to that obtained with the reference LAB culture medium (de Man, Rogosa and Sharpe; MRS). Sodium acetate and magnesium sulfate were the most significant factors (p < 0.005), and their contents were reduced by 22 % and 40 %, respectively, without affecting biomass concentration. Malt germ extract (MGE) was used as an alternative nitrogen source to replace meat extract (ME) and proteose peptone (PP). Through these experiments, the composition of a culture medium that is less expensive than MRS broth was defined, which produced a biomass concentration (3.8 g/L) similar to that obtained with MRS medium. The inhibitory effects of two LAB strains isolated from the Ivory Coast and Mexico on the growth and production of ochratoxin A (OTA) in an ochratoxigenic fungus was tested. The minimum cellular concentration of the LAB to prevent the development of Aspergillus carbonarius Ac 089 and the production of OTA was determined in a model assay in Petri dishes. The conditions to inhibit the germination of A. carbonarius Ac 089 and the production of OTA were found. Using the optimized medium and a ratio of 2 × 104 LAB/spore (1 × 108 CFU/mL) strain B7 (L. plantarum MZ809351) and 2 × 103 LAB/spore (1 × 107 CFU/mL) strain B31 (L. plantarum MN922335) completely inhibited the growth of the fungus. A ratio of 2 × 105 LAB/spore (1 × 109 CFU/mL) was required to inhibit OTA production with strains B7 and B31. This study indicates the potential of cultivating LAB in an optimized and inexpensive culture medium for use as a biological control agent against ochratoxigenic fungi in food.

Combined metabolome and transcriptome analysis reveal the mechanism of eugenol inhibition of Aspergillus carbonarius growth in table grapes (Vitis vinifera L.)

During storage, Aspergillus carbonarius (A. carbonarius) can easily infect grape berries, resulting in a pronounced decline in nutritional value and substantial economic loss for the grape industry. Characterised by broad-spectrum antibacterial activity, eugenol is proven to significantly inhibit A. carbonarius and ochratoxin A (OTA) in vitro. In this study, the potential mechanism of eugenol against A. carbonarius in grapes (‘Kyoho’) was evaluated using integrative transcriptomic and metabolomics analyses. After eugenol treatment at 50 mM, the inhibition of OTA was reduced by 100%, despite a 56.2% inhibition of A. carbonarius. In the meantime, mycelial growth was completely inhibited by 100 mM eugenol in grape berries. The application of eugenol to grapes stimulated the activity of several enzymes involved in disease resistance, namely catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), chitinase (CHI), β-1,3-glucanase (GLU), cinnamate-4-hydroxylase (C4H), phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL) and glutathione (GSH) content. In addition, the contents of abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) in eugenol-treated grapes were higher after A. carbonarius inoculation. Combined transcriptomic and metabolomic analysis revealed that in phenylpropane biosynthesis, there were a variety of differentially expressed metabolites (DEMs) and differentially expressed genes (DEGs), and the plant hormone signalling pathway changed significantly. Among these, the levels of 47 polyphenol metabolites significantly increased in eugenol-treated grape berries compared to noneugenol-treated berries. Meanwhile, we investigated the transcript levels of 39 genes in 6 phytohormones signalling in response to eugenol-treated grape berries followed by A. carbonarius inoculation. These results suggest that eugenol positively improved the disease resistance of grapes and might be potentially beneficial for the prevention and treatment of A. carbonarius-caused disease.

Transcriptomic analysis reveals the inhibition mechanism of pulsed light on fungal growth and ochratoxin A biosynthesis in Aspergillus carbonarius

Pulsed light (PL) technology has a good effect on the control of fungi in postharvest fruit. In this present work, PL inhibited the growth of Aspergillus carbonarius in a dose-dependent manner, the mycelial growth decreased by 4.83 %, 13.91 % and 30.01 % at a fluence of 4.5 J·cm−2 (PL5), 9 J·cm−2 (PL10) and 13.5 J·cm2 (PL15), respectively. When inoculated with PL15 treated A. carbonarius, the scab diameter of the pears, ergosterol and OTA content was reduced by 23.2 %, 27.9 % and 80.7 % after 7 days, respectively. The third-generation sequencing technique was applied to study the transcriptome response of A. carbonarius treated with PL. Compared with the blank control, a total number of 268 and 963 differentially expressed genes (DEGs) were discovered in the group of PL10 and PL15, respectively. To be specific, a large amount of DEGs involved in DNA metabolism were up-regulated, while most of DEGs related to cell integrity, energy and glucose metabolism, ochratoxin A (OTA) biosynthesis and transport were down-regulated. In addition, the stress response of A. carbonarius was imbalanced, including up-regulation of Catalase and PEX12 and down-regulation of taurine and subtaurine metabolism, alcohol dehydrogenase and glutathione metabolism. Meanwhile, the results of transmission electron microscopy, mycelium cellular leakage and DNA electrophoresis indicated that PL15 treatment caused mitochondrial swelling, the destroyed cell membrane permeability and imbalance of DNA metabolism. The expression of P450 and Hal involved in OTA biosynthesis pathway were down-regulated in PL treated samples detected by qRT-PCR. In conclusion, this study reveals the molecular mechanism of pulsed light on inhibiting the growth, development and toxin production of A. carbonarius.

Antifungal and Cytotoxic Activity of Diterpenes and Bisnorsesquiterpenoides from the Latex of Euphorbia resinifera Berg.

Euphorbia resinifera latex has been extensively utilized in traditional medicine due to its range of bioactivities. Chromatographic separations on silica gel of ethanol extract of E. resinifera latex led to the development of a new procedure for isolating resiniferatoxin (4) via dried E. resinifera latex and the identification of nine compounds. Among these, catechol (7), protocatechuic acid (8) and 3,4-dihydroxyphenylacetic acid (9), known phenolic compounds, were identified for the first time in E. resinifera latex. Herein we investigated the effects of major compounds of the latex of E. resinifera on the yeast Saccharomyces cerevisiae, on the growth of Aspergillus carbonarius, a widespread fungal contaminant, and on the breast cancer cell line MCF7 as well as on MCF10A normal breast cells. 12-deoxyphorbol-13-isobutyrate-20-acetate (2) had an inhibiting effect on the growth of A. carbonarius, and 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) showed a negative effect on yeast cell growth and also a cytotoxic effect on breast cancer cell line MCF7, but not on MCF10A cells. Deglucosyl euphorbioside A (5) and euphorbioside A (6) showed a discoloration effect that was possibly related to mitochondrial functionality in yeast, and also cytotoxicity only on the cancer cell line that was tested. Interestingly, treatment of MCF7 cells with 7-p-metoxyphenylacetate-3,8,12-triacetate ingol (3) and deglucosyl euphorbioside A (5) not only led to a specific cytotoxic effect but also to the increase in the level of intracellular ROS.

Transcriptomic analysis of inhibition by eugenol of ochratoxin A biosynthesis and growth of Aspergillus carbonarius

Aspergillus carbonarius is a saprophytic filamentous fungus that produces the mycotoxin ochratoxin A (OTA), a powerful hepatotoxin, nephrotoxin and teratogen, which causes global problems with food contamination during storage. Various methods were applied to assess the impact of eugenol on the OTA production and mycelial growth of A. carbonarius. The minimum inhibitory concentration (MIC) for eugenol treatment was 0.8 μL/mL. Eugenol treatment at 0.2 μL/mL (1/4 MIC) resulted in 87.7% decrease in OTA production in spite of only a 26.9% inhibition of mycelial growth, and actually caused marked morphological changes of spores and mycelia by Scanning electron microscopy (SEM). A total of 400 differentially expressed genes (DEGs) were detected by comparatively transcriptomic analysis of A. carbonarius in the presence or absence of eugenol, including 324 down-regulated and 76 up-regulated DEGs. The transcription of the clustered genes for OTA biosynthesis was significantly reduced under eugenol stress and further confirmed by RT-qPCR. LaeA, a global regulator for secondary metabolites biosynthesis, was markedly downregulated by eugenol. Eugenol disrupted cell integrity by the disturbance of the DEGs expression in relation with cellular structure, which was also proved by monitoring chitinase activity, malondialdehyde (MDA) and ergosterol content. Changes in superoxide dismutase (SOD) and catalase (CAT) activity, and glutathione (GSH) content showed that eugenol induced oxidative stress, which was also consistent with the changes in gene expression. These findings provide useful insights into the antifungal and antimycotoxigenic mechanisms of eugenol and improve understanding of the effects of eugenol on A. carbonarius.

A novel strain Lactobacillus brevis 8-2B inhibiting Aspergillus carbonarius growth and ochratoxin a production

Aspergillus carbonarius is a typical OTA production strain in genus Aspergillus. Inhibiting the growth of A. carbonarius during planting, storage and processing is the most effective way to reduce OTA contamination in grapes. The objective of this study was to screen the microorganisms that can inhibit the growth of A. carbonarius, study the inhibition mechanism and evaluate the inhibitory effect on A. carbonarius in grapes. The results showed that the new isolated strain Lactobacillus brevis 8-2B could significantly inhibit the growth and OTA production of A. carbonarius at a certain concentration, and could significantly down regulate the expression of OTA synthesis genes. Through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), it was found that L. brevis 8-2B had obvious destructive effect on the hyphae, cell walls, cell membranes and organelles of A. carbonarius. It was found that L. brevis 8-2B could significantly inhibit grape decay and prolong the shelf life of grapes in the vitro test. In conclusion, the elucidation of the inhibition mechanism of L. brevis 8-2B provides theoretical support for the development of biocontrol agents.

In-vitro Application of a Qatari Burkholderia cepacia strain (QBC03) in the Biocontrol of Mycotoxigenic Fungi and in the Reduction of Ochratoxin A biosynthesis by Aspergillus carbonarius.

Mycotoxins are secondary metabolites produced by certain filamentous fungi, causing human and animal health issues upon the ingestion of contaminated food and feed. Among the safest approaches to the control of mycotoxigenic fungi and mycotoxin detoxification is the application of microbial biocontrol agents. Burkholderia cepacia is known for producing metabolites active against a broad number of pathogenic fungi. In this study, the antifungal potential of a Qatari strain of Burkholderia cepacia (QBC03) was explored. QBC03 exhibited antifungal activity against a wide range of mycotoxigenic, as well as phytopathogenic, fungal genera and species. The QBC03 culture supernatant significantly inhibited the growth of Aspergillus carbonarius, Fusarium culmorum and Penicillium verrucosum in PDA medium, as well as A. carbonarius and P. verrucosum biomass in PDB medium. The QBC03 culture supernatant was found to dramatically reduce the synthesis of ochratoxin A (OTA) by A. carbonarius, in addition to inducing mycelia malformation. The antifungal activity of QBC03’s culture extract was retained following thermal treatment at 100 °C for 30 min. The findings of the present study advocate that QBC03 is a suitable biocontrol agent against toxigenic fungi, due to the inhibitory activity of its thermostable metabolites.

Competitive yeast action against Aspergillus carbonarius growth and ochratoxin A production

The aim of the present study was to investigate the interaction between 67 different yeast isolates and 3 wild isolates of Aspergillus carbonarius originated from Greek vineyards and characterized by high ochratoxigenic potential. The selected fungi were used either as single cultures or combined in a mixed culture. Yeasts and fungi were grown as mono-cultures and co-cultures in solid (MEA, CYA) and liquid (CY broth) media, grape berries and sterilized grape juice. Fungal growth was monitored by means of colony area measurements. The model of Baranyi and Roberts was further fitted to growth data to provide estimates of the colony area growth rate (cm2/day). Moreover, OTA analysis was undertaken for CY broth and agar as well as for grape berries and juice, on the 8th and 15th days of incubation at 25 °C. A significant reduction in fungal growth rate, final colony size and toxin production was observed in both liquid and solid media by the different yeast species assayed. The most competitive strains belonged to Saccharomyces, Pichia, Metschnikowia, Dekkera and Rhodotorula genera. Similar results were obtained from inoculated grape berries and grape juice. Specifically, Saccharomyces cerevisiae Y33 resulted in a decrease in fungal colony area of >90% and 93% after 3 and 4 days of co-culture, respectively. Similar results were obtained for OTA, where toxin concentration of the highest producer (A. carbonarius F3) was reduced from 14,983 and 31,565 ng/mL at 8 and 15 days, respectively, to 5 ng/mL and below detection limit (1 ng/g) when co-cultured with S. cerevisiae Y33. The results of this study could provide a pool of yeast species that must be further investigated for potential application as biological control agents at pre- and post-harvest level in wine and grape juice processing.

Use of lactic acid bacteria for the inhibition of Aspergillus flavus and Aspergillus carbonarius growth and mycotoxin production

Almonds and peanuts are a rich source of proteins, vitamins and unsaturated fatty acids. However, they can be also contaminated by mycotoxigenic fungi; a reason that has enhanced to investigate efficient strategies of management of these fungal contaminations. Some Lactic acid bacteria have been proven capable of inhibiting growth and mycotoxin production in livestock and transform it into nontoxic derivatives. In this work, four lactic acid bacteria (LAB) were tested for their abilities to inhibit the growth and mycotoxin production of Aspergillus flavus and Aspergillus carbonarius. Antifungal activity was evaluated in agar medium as well as in almonds and peanuts. Results showed that LAB significantly inhibited Aspergillus flavus and Aspergillus carbonarius in agar medium but none of the strains were able to completely inhibit fungal growth. The highest fungal growth inhibition was obtained using L. kefiri FR7 (51.67% and 45.56% growth inhibition of A. flavus and A. carbonarius, respectively). The cell-free supernatants (CFS) from LAB reduced fungal growth with average growth inhibitions ranging from 13.33% to 40.56% and 12.78% to 37.78% for A. flavus and A. carbonarius, respectively. We noted also that cell-free supernatants at pH7 (CFS-pH7) from the entire tested LAB did not inhibit fungal growth. L. kefiri FR7 was the most effective strain in mycotoxin suppression with a reduction percentage reaching 97.22%, 95.27% and 75.26% for AFB1, AFB2 and OTA respectively. Moreover, the inoculation of L. kefiri FR7 in almonds artificially contaminated with A. flavus decrease 85.27% of AFB1 and 83.94% of AFB2 content after 7 days of incubation. On the other hand, application of L. kefiri FR7 in peanuts artificially contaminated with A. carbonarius reduced OTA content to 25%. Our study revealed the potential use and application of L. kefiri FR7 in the control of fungi growth and mycotoxins production in almonds and peanuts.

Sodium alginate–cinnamon essential oil coated apples and pears: Variability of Aspergillus carbonarius growth and ochratoxin A production

The scope of the present study was to use selected fruits as model foods (wounded skin or slices of apples and pears), for the in situ assessment of the potential of natural antimicrobials to control fungal growth and OTA production and the investigation of alternative ways of their application, e.g., via edible coatings. Fresh fruits were cut: i) in halves or ii) across in round slices of ca. 1 cm thickness. Wounds were introduced into the skin and the center of the slice (5 mm deep; 4 mm diameter) and inoculated with a range of 2.0–7.5 × 103spores per wound of Aspergillus carbonarius. Following inoculation, samples were coated with Na – alginate supplemented with 0.3 (0.3% ECC) and 0.9% v/v (0.9% ECC) cinnamon ΕΟ. Inoculated samples without edible coating and EO (C) or with edible coating and without EO (EC) were used as negative or positive controls, respectively. All samples were stored under aerobic conditions at 15, 20, and 25 °C. Fungal growth was estimated by colony diameter measurements (n = 30), while OTA production was determined by HPLC (n = 4). Antimicrobial treatment with 0.9% EO was more effective on fungal growth when the inoculation took place on slices than in wounded skin (p < .05), regardless of storage temperature and fruit. The variability of μmax increased with EO concentration, except for the coated slices of apples with 0.9% v/v EO (at all temperatures), or pears with 0.3% and 0.9% v/v EO (at 15 and 20 °C), where no growth was observed. OTA was below the detection limit (1 ppb) on the majority of 0.9% ECC apples slices and in 0.3% ECC and 0.9% ECC pears slices, stored at 20 and 25 °C. However, the sample to sample variation in the produced amounts of OTA was remarkable. Thus, considering that inhibition of growth and toxin production do not always concur, the present study provided quantitative information on the variability in A. carbonarius growth and OTA production in real model foods in response to antimicrobial coating with natural active compounds. Such data could be of relevance to risk assessment and assist in designing effective control strategies for limiting OTA levels in foods and thus, protecting consumer health.

Effect of potassium sorbate (E-202) and the antifungal PgAFP protein on Aspergillus carbonarius growth and ochratoxin A production in raisin simulating media.

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi. The presence of OTA in raisins is mainly related to black Aspergillus spp. contamination. This toxin poses risks to human and animal health due to its high toxicity and carcinogenicity. New strategies to avoid the risk associated with OTA are therefore necessary. A comparison was made between the effects of the antifungal protein PgAFP and potassium sorbate (E-202) on the growth of Aspergillus carbonarius, biosynthetic- and stress-related gene expression and its OTA production at two water activity (aw ) levels, 0.95 and 0.93 aw . The results showed that PgAFP successfully controlled OTA production, whereas E-202, despite being able to reduce Aspergillus carbonarius growth, caused a significant increase in OTA production by the fungus. PgAFP protein, a biological compound with an antifungal activity, is safer to use than E-202 and may be proposed as a food preservative and a useful strategy to control ochratoxigenic A. carbonarius in raisins. © 2018 Society of Chemical Industry.

Effect of Fungicides on Growth and Ochratoxin A Production by Aspergillus carbonarius from Brazilian Wine Grapes

Prevention in the field of mycotoxin-producing fungi is the most effective strategy for controlling the presence of mycotoxins in foods. Chemical fungicides are widely used to protect crops, so their implications on mycotoxin production need to be considered. Therefore, the aim of this study was to evaluate the effect in vitro and on grapes of five fungicides commonly used on grape cultures in Brazil on Aspergillus carbonarius growth and ochratoxin A (OTA) production. At the doses recommended by manufacturers, most fungicides significantly reduced A. carbonarius growth and OTA production in vitro, whereas this effect was influenced by the type of fungicide, dose, and temperature. Temperature was the main factor that influenced the effectiveness of fungicides. In general, at 15°C, fungicides showed the greatest reduction on fungal growth and OTA production. Fungicide effect on grapes was different to that on a semisynthetic grape medium. All fungicide doses were not effective at controlling A. carbonarius in grapes. Thus, the direct effect of fungicides on grapes must be studied to obtain a better approximation of field conditions. The results indicate that the use of fungicides at the doses recommended by manufacturers is better than the application at low doses. This study showed that at the lowest doses, where fungal growth is not inhibited, fungicides positively stimulate OTA production.

Antifungal Activity of the Onion (Allium cepaL.) Essential Oil AgainstAspergillus, FusariumandPenicilliumSpecies Isolated from Food

The aim of this study was to investigate the antifungal potential of onion (Allium cepa L. cultivar Kupusinski jabucar) essential oil against fungal species isolated from food. Twenty one compounds were identified in the onion essential oil and the major components were dimethyl-trisulfide (16.64%) and methyl-propyl-trisulfide (14.21%). Applied concentrations of onion essential oil (3.5, 7.0, 14.0, and 28.0 μL/100 mL) caused the delay or absence of fungal growth with different inhibitory effects on the deceleration in the growth rate. Onion essential oil at a concentration of 28.0 μL/100 mL demonstrated a fungicidal effect on the growth of Aspergillus carbonarius, Aspergillus wentii, Aspergillus versicolor, Penicillium brevicompactum, Penicillium glabrum, Penicillium chrysogenum, and Fusarium spp. and an inhibitory effect on Aspergillus niger and Penicillium aurantiogriseum. Macroscopic and microscopic investigations showed that the application of onion essential oil caused changes in macro-morphology and micro-morphology of the investigated fungi. Practical Applications The obtained results show that onion essential oil, in addition to the basic sensory role in a food product, can also be used as antifungal agent in food protection from mycological and mycotoxicological contamination.

Effect of water activity and temperature on growth ofAspergillus carbonarius andAspergillus tubingensis and their interactions on ochratoxin A production

Aspergillus sectionNigri are described as the main source of ochratoxin A (OTA) contamination in grapes and wine worldwide. The grape-growing area in Argentina has a wide latitudinal extension with ecological variations that allow the classification of well-demarcated regions. The aims of this study were: to determine the effect of eco-physiological parameters on growth ofAspergillus tubingensis andAspergillus carbonarius and to evaluate the interaction between these species on OTA production in synthetic grape juice medium under different water activity (aw) and temperature conditions. The results showed that optimal growth conditions forA. tubingensis andA. carbonarius were 0.980 aw and 28 °C, howeverA. tubingensis grew faster thanA. carbonarius at all temperatures and aw tested. OTA production byA. carbonarius was favoured at 20 °C and 0.950-0.965 aw, during 14 days of incubation. The effect ofA. tubingensis on OTA production byA. carbonarius was mainly dependent on temperature. At 35 °C,A. tubingensis reduced the OTA production while this effect was not observed at 20 °C. More OTA could be produced as a defence reaction against fungal competitors to maintain niche colonisation, but in this study no effects by a related species were observed.

A Saccharomyces cerevisiae wine strain inhibits growth and decreases Ochratoxin A biosynthesis by Aspergillus carbonarius and Aspergillus ochraceus.

The aim of this study was to select wine yeast strains as biocontrol agents against fungal contaminants responsible for the accumulation of ochratoxin A (OTA) in grape and wine and to dissect the mechanism of OTA detoxification by a Saccharomyces cerevisiae strain (DISAABA1182), which had previously been reported to reduce OTA in a synthetic must. All of the yeast strains tested displayed an ability to inhibit the growth of Aspergillus carbonarius both in vivo and in vitro and addition of culture filtrates from the tested isolates led to complete inhibition of OTA production. S. cerevisiae DISAABA1182 was selected and further tested for its capacity to inhibit OTA production and pks (polyketide synthase) transcription in A. carbonarius and Aspergillus ochraceus in vitro. In order to dissect the mechanism of OTA detoxification, each of these two fungi was co-cultured with living yeast cells exposed to yeast crude or to autoclaved supernatant: S. cerevisiae DISAABA1182 was found to inhibit mycelial growth and OTA production in both Aspergilli when co-cultured in the OTA-inducing YES medium. Moreover, a decrease in pks transcription was observed in the presence of living cells of S. cerevisiae DISAABA1182 or its supernatant, while no effects were observed on transcription of either of the constitutively expressed calmodulin and β-tubulin genes. This suggests that transcriptional regulation of OTA biosynthetic genes takes place during the interaction between DISAABA1182 and OTA-producing Aspergilli.

Grape variety related trans-resveratrol induction affects Aspergillus carbonarius growth and ochratoxin A biosynthesis

The paper reports the results of a study performed to investigate the influence of the grape variety on the growth of Aspergillus carbonarius on grape berries and the correlation between the amount of ochratoxin A (OTA) and the content of trans-resveratrol produced after fungal contamination. Variations in the amount of OTA produced by the fungus are observed depending on both grape variety and on the induction of trans-resveratrol determined during the infection. The obtained data suggest that if an increase in trans-resveratrol production in grape berries occurs early after the fungal infection, the berry exploits this compound to control OTA synthesis. If the increase in trans-resveratrol concentration is delayed after fungal infection (40h), a control of OTA accumulation can not be achieved. The possibility of exerting significant control of OTA biosynthesis by this phytoalexin seems to rely in the promptness of its production, as occurs also in other fungus plant interactions and, in turn, seems to be dependent also on grape cultivar. In this fungus-plant system, trans-resveratrol appears to represent a defence-related compound toward A. carbonarius and OTA contamination.

Inhibition of A. carbonarius growth and reduction of ochratoxin A by bacteria and yeast composites of technological importance in culture media and beverages

Five composites of yeast and six of bacterial isolates from fermented products were studied, in order to assess their ability to inhibit Aspergillus carbonarius growth and reduce OTA concentration in culture media and beverages. The antagonistic effect of the above composites against A. carbonarius growth was studied in synthetic grape medium of pH 3.5 and a(w) 0.98, 0.95, 0.92 after incubation at 25°C. Different combinations of initial inocula of bacteria or yeast composites and fungi were used (10(2)cfu/mL vs 10(5)spores/mL; 10(5)cfu/mL vs 10(2)spores/mL; and 10(5)cfu/mL vs 10(5)spores/mL). Regarding the OTA reduction experiment, 10(3) and 10(7)cfu/mL of the bacteria and yeast composites were inoculated in liquid media of different pH (3.0, 4.0, 5.0, and 6.1 or 6.5) and initial OTA concentration (50 and 100μg/L) and incubated at 30°C. Moreover, grape juice, red wine, and beer were supplemented with 100μg/L of OTA and inoculated with composites of 16 yeasts (16YM) and 29 bacterial (29BM) strains (10(7)cfu/mL) to estimate the kinetics of OTA reduction at 25°C for 5days. Fungal inhibition and OTA reduction were calculated in comparison to control samples. None of the bacterial composites inhibited A. carbonarius growth. The high inoculum of yeast composites (10(5) cfu/mL) showed more efficient fungal inhibition compared to cell density of 10(2) cfu/mL. All yeast composites showed higher OTA reduction (up to 65%) compared to bacteria (2-25%), at all studied assays. The maximum OTA reduction was obtained at pH 3.0 by almost all yeast composites. For all studied beverages the decrease in OTA concentration was higher by yeasts (16YM) compared to bacteria (29BM). The highest OTA reduction was observed in grape juice (ca 32%) followed by wine (ca 22%), and beer (ca 12%). The present findings may assist in the control of A. carbonarius growth and OTA production in fermented foodstuffs by the use of proper strains of technological importance.

Aspergillus carbonarius growth and ochratoxin A production on irradiated dried grapes under different water activity and temperature conditions

Grapes have different destinations. The most important in the national and international market is wine production, but another is dehydration to produce raisins. Dried vine fruits are at greater risk of ochratoxin A (OTA) contamination than wine grapes because the ratio of Aspergillus carbonarius to Aspergillus niger aggregate increases during drying. The growth of these species, and consequently OTA production, can be influenced by different environmental factors, the two most important being water activity (a W) and temperature. The objective of the present work was to evaluate the lag phase, growth rate and OTA production by two A. carbonarius isolates on irradiated dried grapes at different a W (0.910, 0.928, 0.955, 0.973 and 0.995), temperatures (15, 25 and 30 °C) and incubation times (7, 14 and 21 days). Growth was observed at all a W and temperature ranges assayed. No significant differences between the growth rates reached at 25 °C and 30 °C by both isolates were observed. At the assayed conditions, OTA production occurred over the whole range of temperatures (15-30 °C), with the maximum at 25 and 30 °C depending on the a W. In general, OTA concentration increased as a W increased with no statistically significant differences at the tested incubation times. This work provides information that can be used by companies for the purpose of preventing A. carbonarius and OTA contamination during storage on this and other substrates (e.g. dried prunes, figs and apricots) destined for human consumption.