Notoamide R: A Prominent Diketopiperazine Fermentation Metabolite amongst Others of Aspergillus ochraceus in the Absence of Ochratoxins.

Ochratoxin A is historically the most notable secondary metabolite of Aspergillus ochraceus on account of its toxicity to animals and fish. Currently, over 150 compounds of diverse structure and biosynthesis is a challenge to predict the array for any particular isolate. A brief focus 30 years ago on the failure to produce ochratoxins in foods in Europe and the USA revealed consistent failures to produce ochratoxin A by isolates from some USA beans. Analysis for familiar or novel metabolites particularly focused on a compound for which mass and NMR analyses were inconclusive. Resort to 14C-labelled biosynthetic precursors, particularly phenylalanine, to search for any close alternative to ochratoxins, was combined with conventional shredded-wheat/shaken-flask fermentation. This yielded, for an extract, an autoradiograph of a preparative silica gel chromatogram, which was subsequently analysed for an excised fraction using spectroscopic methodologies. Circumstances then delayed progress for many years until the present collaboration revealed notoamide R. Meanwhile, pharmaceutical discovery around the turn of the millennium revealed stephacidins and notoamides, biosynthetically combining indole, isoprenyl and diketopiperazine components. Later, in Japan, notoamide R was added as a metabolite of an Aspergillus sp. isolated from a marine mussel, and the compound was recovered from 1800 Petri dish fermentations. Renewed attention to our former studies in England has since shown for the first time that notoamide R can be a prominent metabolite of A. ochraceus, sourced from a single shredded wheat flask culture with its structure confirmed by spectroscopic data, and in the absence of ochratoxins. Renewed attention to the archived autoradiographed chromatogram allowed further exploration, but in particular has stimulated a fundamental biosynthetic approach to considering influences redirecting intermediary metabolism to secondary metabolite accumulation.

Antimicrobial and Cytotoxic Activity of Endophytic Fungi from Lagopsis supina.

In this study, five endophytic fungi belonging to the Aspergillus and Alternaria genera were isolated from Lagopsis supina. The antimicrobial activity of all fungal fermented extracts against Staphylococcus and Fusarium graminearum was tested using the cup-plate method. Among them, Aspergillus ochraceus XZC-1 showed the best activity and was subsequently selected for large-scale fermentation and bioactivity-directed separation of the secondary metabolites. Four compounds, including 2-methoxy-6-methyl-1,4-benzoquinone (1), 3,5-dihydroxytoluene (2), oleic acid (3), and penicillic acid (4) were discovered. Here, compounds 1 and 4 displayed anti-fungal activity against F. graminearum, F. oxysporum, F. moniliforme, F. stratum, Botrytis cinerea, Magnaporthe oryzae, and Verticillium dahliae with diverse MIC values (128-512 µg/ml), which were close to that of the positive control antifungal, actidione (64-128 µg/ml). Additionally, compounds 1 and 4 also exhibited moderate antibacterial activity against S. aureus, Listeria monocytogenes, Escherichia coli, and Salmonella enterica, with low MIC values (8-64 µg/ml). Moreover, compounds 1 and 4 displayed selective cytotoxicity against cancer cell lines as compared with the normal fibroblast cells. Therefore, this study proposes that the endophytic fungi from L. supina can potentially produce bioactive molecules to be used as lead compounds in drugs or agricultural antibiotics.

Growing of fungi on the stored low denatured defatted soybean meals and the hydrolysis of proteins and isoflavone glycosides by fungal enzymes.

Recently, more and more attention has been paid to the effects of fungal contamination and fungal enzymes secreted in raw grain on product quality. As the starting material of protein and active components, the quality of low denatured defatted soybean meals (LDSM) directly determines the qualities of subsequent products. In previous studies, we have revealed that infection with Aspergillus ochraceus protease causes significant hydrolysis of proteins. In this study, growing of fungi on the stored low denatured defatted soybean meals (LDSM) was analyzed by high-throughput sequencing and real-time PCR, which revealed that the abundance of Aspergillus increased significantly after storage. Twenty fungal proteases and 9 fungal glucosidases were found in stored LDSM and zymography showed that the proteases were of serine-type with some cysteine and aspartic activities. Proteolysis of the soybean storage proteins mainly occurred after the hydration of LDSM and the average molecular weight of soy proteins decreased from 57.9 kDa to 30.7 kDa after 60 min's of hydrolysis. Two-dimensional electrophoresis (2-DE) analysis found the polypeptide fragments from soybean 7S and 11S proteins with molecular weight around 10-25 kDa in the hydrated LDSM. Glycosylated isoflavones were hydrolyzed in both dry and hydrated stored LDSM which resulted in significant (p < 0.05) increase in the contents of isoflavone aglycones. This study suggested that fungi contamination be a new factor affecting the properties of LDSM derived soy protein products.

Antifungal activity of puroindoline protein from soft wheat against grain molds and its potential as a biocontrol agent.

Mold growth reduces the quality of stored grains, besides producing toxins that pose a potential threat to human health. Therefore, prevention of grain mold growth during storage is important to ensure a safe and high-quality product, preferably using an eco-friendly antifungal agent. The puroindoline (PIN) protein was extracted by Triton X-114 and identified by QE mass spectrometry. Aspergillus flavus has attracted much attention because of its toxic secondary metabolites, and PIN protein showed a significant inhibition on A. flavus growth. Scanning electron microscopy revealed altered spore morphology of A. flavus following PIN protein treatment, and propidium iodide staining showed incomplete spore cell membranes. The disruption and deformation of A. flavus spores suggest that the cell walls and cell membranes were compromised. Decreased mitochondrial membrane potential and increased levels of intracellular reactive oxygen specieswere detected using JC-1 and 2,7-dichlorodihydrofluorescein diacetate staining, respectively. PIN protein could effectively inhibit the growth and aflatoxins B1 production of A. flavus in stored grains, such as wheat and rice. PIN proteins can inhibit the growth of many common grain storage molds, including Penicillium, Aspergillus spp. (A. flavus, A. glaucus, A. kawachii, A. ochraceus and A. niger), Alternaria and Fusarium graminearum, in a dose-dependent manner. PIN protein has a significant inhibitory effect on the growth of grain molds, with a stronger inhibitory effect noted in wheat and rice. Our study provides a novel and simple theoretical basis for the selection and storage of mold resistance in grains and food during storage.

Prevalence of toxigenic fungi and mycotoxins in Arabic coffee (Coffea arabica): Protective role of traditional coffee roasting, brewing and bacterial volatiles.

Fungal infection and synthesis of mycotoxins in coffee leads to significant economic losses. This study aimed to investigate the prevalence of toxigenic fungi, their metabolites, and the effect of traditional roasting and brewing on ochratoxin A (OTA) and aflatoxins (AFs) contents of naturally contaminated coffee samples. In addition, in vivo biocontrol assays were performed to explore the antagonistic activities of Bacillus simplex 350-3 (BS350-3) on the growth and mycotoxins synthesis of Aspergillus ochraceus and A. flavus. The relative density of A. niger, A. flavus, Penicillium verrucosum and A. carbonarius on green coffee bean was 60.82%, 7.21%, 3.09% and 1.03%, respectively. OTA contents were lowest in green coffee beans (2.15 µg/kg), followed by roasted (2.76 µg/kg) and soluble coffee (8.95 µg/kg). Likewise, AFs levels were highest in soluble coffee (90.58 µg/kg) followed by roasted (33.61 µg/kg) and green coffee (9.07 µg/kg). Roasting naturally contaminated coffee beans at three traditional methods; low, medium and high, followed by brewing resulted in reduction of 58.74% (3.50 µg/kg), 60.88% (3.72 µg/kg) and 64.70% (4.11 µg/kg) in OTA and 40.18% (34.65 µg/kg), 47.86% (41.17 µg/kg) and 62.38% (53.73 µg/kg) AFs contents, respectively. Significant inhibitions of AFs and OTA synthesis by A. flavus and A. carbonarius, respectively, on infected coffee beans were observed in presence of Bacillus simplex BS350-3 volatiles. Gas chromatography mass spectrochemistry (GC-MS/MS) analysis of head-space BS350-3 volatiles showed quinoline, benzenemethanamine and 1-Octadecene as bioactive antifungal molecules. These findings suggest that marketed coffee samples are generally contaminated with OTA and AFs, with a significant level of roasted and soluble coffee contaminated above EU permissible limits for OTA. Further, along with coffee roasting and brewing; microbial volatiles can be optimized to minimize the dietary exposure to mycotoxins.

Effects of Light on the Ochratoxigenic Fungi Aspergillus ochraceus and A. carbonarius.

Ochratoxin A (OTA) usually contaminates agricultural products such as grapes, oatmeal, coffee and spices. Light was reported as an effective strategy to control spoilage fungi and mycotoxins. This research investigated the effects of light with different wavelengths on the growth and the production of OTA in Aspergillus ochraceus and Aspergillus carbonarius. The results showed that the growth of both fungi were extremely inhibited by UV-B. Short-wavelength (blue, violet) significantly inhibited the production of OTA in both fungi, while the inhibitory effect of white was only demonstrated on A. ochraceus. These results were supported by the expression profiles of OTA biosynthetic genes of A. ochraceus and A. carbonarius. To clarify, the decrease in OTA production is induced by inhibition or degradation; therefore, the degradation of OTA under different wavelengths of light was tested. Under UV-B, the degradation rate of 10 µg/mL OTA standard pure-solution samples could reach 96.50% in 15 days, and the degradation effect of blue light was relatively weak. Furthermore, infection experiments of pears showed that the pathogenicity of both fungi was significantly decreased under UV-B radiation. Thus, these results suggested that light could be used as a potential target for strategies in the prevention and control of ochratoxigenic fungi.

Identification and in vitro antifungal susceptibility of causative agents of onychomycosis due to Aspergillus species in Mashhad, Iran.

Aspergillus species are emerging causative agents of non-dermatophyte mold onychomycosis. In this study, 48 Aspergillus isolates were obtained from patients with onychomycosis in Mashhad, Iran, during 2015-2018. The aim is to identify the Aspergillus isolates to the species level by using partial calmodulin and beta-tubulin gene sequencing and MALDI-TOF MS, and to evaluate their in vitro susceptibility to ten antifungal drugs: terbinafine, itraconazole, voriconazole, posaconazole, ravuconazole, isavuconazole, caspofungin, micafungin, anidulafungin and amphotericin B according to CLSI M38-A3. Our results indicate that A.flavus (n = 38, 79%) is the most common Aspergillus species causing onychomycosis in Mashhad, Iran. Other detected species were A. terreus (n = 3), A. tubingensis (n = 2), A. niger (n = 1), A. welwitschiae (n = 1), A. minisclerotigenes (n = 1), A. citrinoterreus (n = 1) and A. ochraceus (n = 1). Aspergillus flavus, A. terreus and A. niger isolates were correctly identified at the species level by MALDI-TOF MS, while all cryptic species were misidentified. In conclusion, A. flavus is the predominant Aspergillus species causing onychomycosis due to Aspergillus spp. in Mashhad, Iran. MALDI-TOF MS holds promise as a fast and accurate identification tool, particularly for common Aspergillus species. It is important that the current database of reference spectra, representing different Aspergillus species is expanded to increase the precision of the species-level identification. Terbinafine, posaconazole and echinocandins were in vitro most active against the studies Aspergillus isolates and terbinafine could be the first choice for treatment of onychomycosis due to Aspergillus.

The Influence of NaCl and Glucose Content on Growth and Ochratoxin A Production by Aspergillus ochraceus, Aspergillus carbonarius and Penicillium nordicum.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin, which deserves particular attention for its widespread contamination of a variety of food and feed. Aspergillus ochraceus, Aspergillus carbonarius, and Penicillium nordicum are an important source of OTA in three different kinds of food commodities, including cereals, grape and dried fruit products, and dry-cured meat products. Deeper knowledge of OTA production and mycelium growth related to the high-sugar or NaCl-rich environments was gained in this manuscript. A. ochraceus and P. nordicum were likely to have greater growth rates in medium supplied with certain concentrations of NaCl (0-80 g/L), and the colony diameter was the largest at the salt content of 40 g/L. P. nordicum was more suitable to grow in NaCl-riched medium, the OTA production was increased to 316 ppb from 77 ppb when 20 g/L NaCl was added. The capability of OTA production was inhibited when salt content was 40 g/L and 60 g/L in A. ochraceus and P. nordicum, respectively. As the glucose content increased to 250 g/L, the capacity of mycelium growth and sporulation was increased significantly in A. ochraceus and A. carbonarius. A. carbonarius was more suitable to grow in high-sugar grape products. OTA production was significantly promoted with an added 100 g/L glucose in A. carbonarius. OTA production was inhibited when glucose content was 150 g/L and in 200 g/L in A. ochraceus and A. carbonarius, respectively. NaCl and glucose have an effect on fungal growth and OTA production, and the activation of biosynthetic genes of OtaA. These results would allow designing new strategies to prevent OTA accumulation on sugar or NaCl-riched foodstuffs and achieve the objective to manufacture cereals, dried vine fruits and dry-cured ham, free of OTA.

Ochratoxin A production by Aspergillus westerdijkiae in Italian-type salami.

The occurrence of ochratoxin A (OTA) in matured meat products can be attributed to mycotoxin coming from raw materials and/or molds that develop on the product surface during ripening. This work aimed to evaluate OTA production by Aspergillus westerdijkiae inoculated on the surface of sausages and its diffusion into the product throughout ripening, study the effect of relative humidity (RH) on the production of this mycotoxin, and investigate the presence of OTA in dry fermented sausages that naturally present intense contamination by A. westerdijkiae. Italian-type sausages were surface inoculated with A. westerdijkiae and two distinct experiments were performed. In the first, the sausages were matured in a chamber with decreasing RH (from 95 to 75%) for 35 days. In the other, the sausages were incubated under different RH (79, 85 or 95%) for 21 days. Samples were taken at the beginning of the experiments and every 7 days, subdividing into casing, outer border, and core for analyses. Sausage samples naturally spoiled by ochratoxigenic fungi were collected during sanitary inspection. Even in the presence of A. westerdijkiae mycelia, no OTA was detected for up to 7 days of sausage maturation. On the other hand, this study demonstrated that the growth of A. westerdijkiae on salami surface produces high amounts of OTA on the casing and allows its diffusion through the casing with contamination to the outer border of sausages. In the same way, it shows that under similar water activity values of substrate, RH influences the amount of OTA produced. Conversely, OTA was restricted to the casing in the naturally contaminated sausages.

Aspergillus species from Brazilian dry beans and their toxigenic potential.

Aspergilli are common contaminants of food and feed and a major source of mycotoxins. In this study, 87 Aspergillus strains were isolated from beans from 14 different cities in Brazil and identified to the species level based on partial calmodulin and ß-tubulin sequence data. All green spored isolates belonged to section Flavi and were identified as A. flavus (n = 39) or A. pseudocaelatus (n = 1). All black spored isolates belonged to section Nigri and were identified as A. niger (n = 24) or A. luchuensis (n = 10), while the yellow spored strains were identified as A. westerdijkiae (n = 7), A. ostianus (n = 3), A. ochraceus (n = 1) or A. wentii (n = 2). The toxigenic potential of these Aspergillus strains from beans was studied by the prospection of genes in three of the major mycotoxin clusters: aflatoxin (seven genes checked), ochratoxin A (four genes) and fumonisin (ten genes and two intergenic regions). Genes involved in the biosynthesis of aflatoxin were only detected in A. flavus isolates: 17/39 A. flavus isolates proved to contain all the aflatoxin genes tested, the others missed one or more genes. The full complement of fumonisin biosynthesis genes was identified in all A. niger isolates. Finally, no genes for ochratoxin A were detected in any of the isolates. Our work suggests that aflatoxin production by some A. flavus strains and fumonisin production by A. niger isolates form the largest mycotoxin risks in Brazilian beans.

Differentiation and quantification of the ochratoxin A producers Aspergillus ochraceus and Aspergillus westerdijkiae using PCR-DGGE.

Ochratoxin A (OTA) is a nephrotoxic, teratogenic, immunotoxic, and carcinogenic mycotoxin which is produced in tropical zones mainly by Aspergillus carbonarius, A. niger, A. ochraceus, and A. westerdijkiae. A. ochraceus and A. westerdijkiae species are phenotypically and genomically very close but A. westerdijkiae produce OTA at a very higher level than A. ochraceus. These species have been differentiated recently. The DNA primer pairs which were drawn so far are not specific and a genomic region of the same size is amplified for both species or they are too specific, and in this case, the DNA of a single species is amplified. To help preventing OTA contamination of foodstuffs, the PCR-DGGE (Denaturing Gradient Gel Electrophoresis) method was used to discriminate between A. ochraceus and A. westerdijkiae DNA fragments of the same size but with different sequences and thus faster access to a diagnosis of the toxigenic potential of the fungal microflora. The proposed methodology was able to differentiate A. westerdijkiae from A. ochraceus with only one primer pairs in a single run. A calibration based on initial DNA content was obtained from image analysis of the DGGE gels and a method of quantification of the two strains was proposed.

Effect of yeast volatile organic compounds on ochratoxin A-producing Aspergillus carbonarius and A. ochraceus.

Many foods and beverages in temperate and tropical regions are prone to contamination by ochratoxin A (OTA), one of the most harmful mycotoxins for human and animal health. Aspergillus ochraceus and Aspergillus carbonarius are considered among the main responsible for OTA contamination. We have previously demonstrated that four low or non- fermenting yeasts are able to control the growth and sporulation of OTA-producing Aspergilli both in vitro and on detached grape berries: the biocontrol effect was partly due to the release of volatile organic compounds (VOCs). Aiming to further characterise the effect of VOCs produced by biocontrol yeast strains, we observed that, beside vegetative growth and sporulation, the volatile compounds significantly reduced the production of OTA by two A. carbonarius and A. ochraceus isolates. Exposure to yeast VOCs also affected gene expression in both species, as confirmed by downregulation of polyketide synthase, non-ribosomal peptide synthase, monooxygenase, and the regulatory genes laeA and veA. The main compound of yeast VOCs was 2-phenylethanol, as detected by Headspace-Solid Phase Microextraction-Gas Chromatography-Tandem Mass Spectrometry (HS-SPME-GC-MS) analysis. Yeast VOCs represent a promising tool for the containment of growth and development of mycotoxigenic fungi, and a valuable aid to guarantee food safety and quality.

Isolation and Evaluation of New Antagonist Bacillus Strains for the Control of Pathogenic and Mycotoxigenic Fungi of Fig Orchards.

Bacillus is an antagonistic bacteria that shows high effectiveness against different phytopathogenic fungi and produces various lytic enzymes, such as chitosanase, chitinase, protease, and gluconase. The aim of this study is to determine Bacillus spp. for lytic enzyme production and to evaluate the antifungal effects of the selected strains for biocontrol of mycotoxigenic and phytopathogenic fungi. A total of 92 endospore-forming bacterial isolates from the 24 fig orchard soil samples were screened for chitosanase production, and six best chitosanolytic isolates were selected to determine chitinase, protease, and N-acetyl-ß-hexosaminidase activity and molecularly identified. The antagonistic activities of six Bacillus strains against Aspergillus niger EGE-K-213, Aspergillus foetidus EGE-K-211, Aspergillus ochraceus EGE-K-217, and Fusarium solani KCTC 6328 were evaluated. Fungal spore germination inhibition and biomass inhibition activities were also measured against A. niger EGE-K-213. The results demonstrated that Bacillus mojavensis EGE-B-5.2i and Bacillus thuringiensis EGE-B-14.1i were more efficient antifungal agents against A. niger EGE-K-213. B. mojavensis EGE-B-5.2i has shown maximum inhibition of the biomass (30.4%), and B. thuringiensis EGE-B-14.1i has shown maximum inhibition of spore germination (33.1%) at 12 h. This is the first study reporting the potential of antagonist Bacillus strains as biocontrol agents against mycotoxigenic fungi of fig orchads.

Mycotoxigenic potentials of the genera: Aspergillus, Fusarium and Penicillium isolated from houseflies (Musca domestica L.).

A study on the potential of houseflies (Musca domestica L.) to spread fungal spores in Gauteng Province, South Africa proved that houseflies are vectors for fungal spores. Therefore, there is a need to determine the toxigenic potentials and to identify the mycotoxins produced by fungal isolates derived from this study. In total 377 potentially toxigenic isolates of Aspergillus (186), Fusarium (85) and Penicillium (106) species (spp.) were isolated. These isolates were further tested for their ability to produce aflatoxins (AFs) [aflatoxin B1, B2, G1 and G2], deoxynivalenol (DON), fumonisin B1 (FB1) ochratoxin A (OTA), and zearalenone (ZEA) by high-performance liquid chromatography (HPLC) respectively. Strains of A. flavus and A. parasiticus belonging to the genera of Aspergillus were found to be the main producers of AFB1, AFB2, AFG1, and AFG2, while A. carbonarius, A. niger and A. ochraceus produced OTA. Fumonisin B1 was produced by F. verticillioides and F. proliferatum with concentrations ranging from 20 to 1834µg/kg and 79 to 262µg/kg respectively. Deoxynivalenol produced mainly by F. culmorum (2-6µg/kg), F. graminearum (1-4µg/kg), F. poae (1-3µg/kg), and F. sporotrichioides (2-3µg/kg) species was the least detected toxin in this study. The high mycotoxins levels produced in isolates from houseflies in this study are regarded as unsafe, especially when international legislated tolerance levels for mycotoxins are considered. Thus, possible human exposure to mycotoxins may pose concerns with respect to human health and demands constant and consistent investigation.

Mycobiota of ground red pepper and their aflatoxigenic potential.

To investigate contamination of ground red pepper with fungi and mycotoxin, we obtained 30 ground red pepper samples from 15 manufacturers in the main chili-pepper-producing areas in Korea. Fungal contamination was evaluated by spreading diluted samples on potato dextrose agar plates. The total fungi counts ranged from 0 to 7.3 × 103 CFU/g. In the samples, the genus Aspergillus had the highest incidence, while Paecilomyces was isolated most frequently. The next most frequent genera were Rhizopus, Penicillium, Cladosporium, and Alternaria. Within Aspergillus, A. ruber was predominant, followed by A. niger, A. amstelodami, A. ochraceus, A. terreus, A. versicolor, A. flavus, and A. fumigatus. The samples were analyzed for aflatoxins, ochratoxin A, and citrinin by ultra-perfomance liquid chromatography (UPLC) with a fluorescence detector. Ochratoxin A was detected from three samples at 1.03‒2.08 µg/kg, whereas no aflatoxins or citrinin were detected. To test the potential of fungal isolates to produce aflatoxin, we performed a PCR assay that screened for the norB-cypA gene for 64 Aspergillus isolates. As a result, a single 800-bp band was amplified from 10 A. flavus isolates, and one Aspergillus sp. isolate. UPLC analyses confirmed aflatoxin production by nine A. flavus isolates and one Aspergillus sp. isolate, which produced total aflatoxins at 146.88‒909.53 µg/kg. This indicates that continuous monitoring of ground red pepper for toxigenic fungi is necessary to minimize mycotoxin contamination.

High-performance liquid chromatography-tandem mass spectrometry method for simultaneous detection of ochratoxin A and relative metabolites in Aspergillus species and dried vine fruits.

A simple, sensitive and reliable quantification and identification method was developed for simultaneous analysis of ochratoxin A (OTA) and its related metabolites ochratoxin alpha (OTα), ochratoxin B (OTB) and mellein. The method was assessed by spiking analytes into blank culture media and dried vine fruits. Performance was tested in terms of accuracy, selectivity and repeatability. The method involves an ultrasonic extraction step for culture samples using methanol aqueous solution (7:3, v/v); the mycotoxin is quantified by high-performance liquid chromatography coupled with electrospray ionisation and triple quadrupole mass spectrometry (LC-ESI-MS/MS). The recoveries were 74.5-108.8%, with relative standard deviations (RSDs) of 0.4-8.4% for fungal culture. The limits of detection (LODs) were in the range of 0.03-0.87 µg l(-)(1), and the limits of quantification (LOQs) ranged from 0.07 to 2.90 µg l(-)(1). In addition, the extraction solutions and clean-up columns were optimised specifically for dried vine fruit samples to improve the performance of the method. Methanol-1% sodium bicarbonate extraction solution (6:4, v/v) was determined to be the most efficient. Solid-phase extraction (SPE) was performed as a clean-up step prior to HPLC-MS/MS analysis to reduce matrix effects. Recoveries ranged from 80.1% to 110.8%. RSDs ranged from 0.1% to 3.6%. LODs and LOQs ranged from 0.06 to 0.40 µg kg(-)(1) and from 0.19 to 1.20 µg kg(-)(1), respectively. The analytical method was established and used to identify and quantify OTA and related compounds from Aspergillus carbonarius and Aspergillus ochraceus in cultures and dried vine fruits. The results showed that A. carbonarius produced OTα, OTB and OTA, whereas A. ochraceus produced OTB, OTA and mellein after 7 days of cultivation. Of 30 commercial samples analysed, 10 were contaminated with ochratoxins; OTB, OTα and mellein were also detected in different samples.

Efficacy of different caffeine concentrations on growth and ochratoxin A production by Aspergillus species.

UNLABELLED: The objective of this study was to evaluate the effect of different caffeine concentrations (0-4%) on (i) lag phase prior to growth, (ii) growth rates and (iii) ochratoxin A (OTA) production by strains from the Aspergillus section Circumdati and Aspergillus section Nigri groups, isolated from coffee, when grown on a conducive medium at 0·98 water activity and 30°C. The lag phases prior to growth increased with caffeine concentration. A strain of Aspergillus niger and Aspergillus carbonarius were the most sensitive to caffeine with growth being inhibited by <1% caffeine. For strains of Aspergillus westerdijkiae, Aspergillus ochraceus and Aspergillus steynii, although growth was inhibited significantly, some growth (10-15% of controls) occurred in 4% caffeine. OTA production was significantly inhibited by only 0·5% caffeine for strains of A. westerdijkiae, A. niger and A. carbonarius. For A. steynii at least 1·5% caffeine was required to inhibit OTA production. In contrast, for the strain of A. ochraceus there was a stimulation of OTA at 3% with a reduction at 4% caffeine. These results are discussed in the context of the different concentrations of caffeine found in Arabica and Robusta coffee and the development of minimization strategies. SIGNIFICANCE AND IMPACT OF THE STUDY: Arabic (0·6%) and Robusta coffee (4%) have significantly different amounts of endogenous caffeine. The growth of six ochratoxigenic fungi which contaminate coffee with ochratoxin A (OTA) had differential tolerance/sensitivity to concentrations of caffeine in vitro in this range. However, low concentrations of caffeine (<0·5%) was inhibitory to OTA production. These results are discussed in the context of the potential for using such information for the design of minimization strategies to control mycotoxin production in such products.

Green synthesis of nanosilver particles by Aspergillus terreus HA1N and Penicillium expansum HA2N and its antifungal activity against mycotoxigenic fungi.

AIMS: The aim of this study was to synthesize silver nanoparticles (AgNPs) by an eco-friendly and low-cost method using the fungi Aspergillus terreus HA1N and Penicillium expansum HA2N as an alternative to chemical procedures mostly requiring drastic experimental conditions emitting toxic chemical byproducts. Also, this study has been extended to evaluate the effect of AgNPs on the growth of some mycotoxigenic fungi and ochratoxin A (OTA) produced by Aspergillus ochraceus. METHODS AND RESULTS: The AgNPs have been characterized by UV-Visible Spectrophotometer, Dynamic Light Scattering (DLS), Fourier Transform Infrared Spectroscopy (FTIR) and Transmission Electron Microscope (TEM). The TEM analysis has revealed that the size of AgNPs ranged between 14 and 25 nm in the case of P. expansum and 10-18 nm in the case of A. terreus. The antifungal activity of AgNP colloids has indicated that the highest inhibition zone was detected with AgNPs synthesized by A. terreus HA1N against all tested fungi. The highest inhibition zone was detected with Aspergillus niger at concentrations 3 and 6 µg of AgNP solution (7·56 ± 0·38 and 11·3 ± 1·8 mm, respectively) while, A. ochraceus showed the maximum inhibition zone (16·33 ± 0·96 mm) at the concentration 9 µg of AgNPs synthesized by A. terreus. The results have also indicated that the AgNPs synthesized by A. terreus and P. expansum at the concentration 220 µg/100 ml media gave the highest reduction of OTA, where the percentages of reduction were 58·87 and 52·18% respectively. CONCLUSIONS: The smallest size AgNPs synthesized by A. terreus HA1N are better in their antifungal activity against all tested mycotoxigenic fungi than the largest one synthesized by P. expansum HA2N. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study focused on using AgNPs in control of OTA production.

Survey of Philippine coffee beans for the presence of ochratoxigenic fungi.

In 2012 to 2014, Philippine green coffee beans from Coffea arabica in Benguet and Ifugao; Coffea canephora var. Robusta in Abra, Cavite, and Ifugao; and Coffea liberica and Coffea excelsea from Cavite were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The presence of fungal species was evaluated both before and after surface sterilization. There were remarkable ecological and varietal differences in the population of OTA-producing species from the five provinces. Aspergillus ochraceus, A. westerdijkiae, and Penicillium verruculosum were detected from Arabica in Benguet and Ifugao while Aspergillus carbonarius, Aspergillus niger, and Aspergillus japonicus were isolated in Excelsa, Liberica, and Robusta varieties from Abra, Cavite, and Davao. Contamination by Aspergillus and Penicillium species was found on 59 and 19 %, respectively, of the 57 samples from five provinces. After disinfection with 1% sodium hypochlorite, the levels of infection by Aspergillus and Penicillium fell to 40 and 17%, respectively. A total of 1184 fungal isolates were identified to species level comprising Aspergillus sections Circumdati (four species), Clavati (one), Flavi (one), Fumigati (one), Nigri (three), and Terrie (one). Within section Circumdati, 70% of A. ochraceus produced OTA as high as 16238 ng g(-1) while 40% of A. westerdijkiae produced maximum OTA of 36561 ng g(-1) in solid agar. Within section Nigri, 16.76% of A. niger produced OTA at the highest 18439 ng g(-1), 10% of A. japonicus at maximum level of 174 ng g(-1), and 21.21% of A. carbonarius yielded maximum OTA of 1900 ng g(-1). Of the 12 species of Penicillium isolated, P. verruculosum was ochratoxigenic, with a maximum OTA production of 12 ng g(-1).

Efficiency of Artemisia nilagirica (Clarke) Pamp. essential oil as a mycotoxicant against postharvest mycobiota of table grapes.

BACKGROUND: In order to get a potent botanical fungicide for the management of fungal decay of table grapes, an experiment was conducted in which 20 essential oils of higher plants were screened at 0.33 µL mL(-1) against dominant fungi causing decay of table grapes, including Aspergillus flavus, A. niger and A. ochraceus. Furthermore, the minimum inhibitory/fungicidal concentration, fungitoxic spectrum and mycotoxin inhibition activity of the most potent oil were determined. The efficacy of the most potent oil in preservation of table grapes, along with organoleptic evaluation, was also carried out by storing 1 kg of grapes in the oil vapour. RESULTS: Artemisia nilagirica oil was found to be most toxic, exhibiting 100% mycelia inhibition of all test fungi. Moreover, 0.29 µL mL(-1) A. nilagirica oil was fungistatic and 0.58 µL mL(-1) was fungicidal for all tested species of Aspergillus. The oil exhibited a broad range of fungitoxicity against other grape berry-rotting fungi. Artemisia nilagirica oil completely suppressed the growth and mycotoxin (AFB1 and OTA) secretion of aflatoxigenic and ochratoxigenic strains of Aspergillus at 1.6 µL mL(-1) . During the in vivo experiment, fumigation of 1 kg of table grapes with 200 and 300 µL dosage of A. nilagirica oil enhanced the shelf life for up to 9 days. The oil did not show any phytotoxic effect. Besides, oil application did not substantively change the sensory properties of the fruits. CONCLUSION: Artemisia nilagirica oil can be used as an alternative botanical fungicide for the control of fruit-rotting fungi of stored grapes.