Meat Starter Culture Reduces Aspergillus parasiticus Production of Aflatoxins on Meat-Based and Salami Model Media.

There is great concern about the risk posed by the consumption of food contaminated with aflatoxins (AF), produced mostly by Aspergillus strains, that can also be found in dry-fermented meat products (DFMPs). The aim of this study was to investigate the inhibitory effect of meat starter culture (SC), frequently used for fermentation in the meat industry, on A. parasiticus growth and the production of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and sterigmatocystin (STE) on different meat-based (CMA) and salami model (SM-G) media. Incubation was carried out under optimal conditions for fungal growth and under typical conditions for ripening of DFMPs for 21 days. Reversed-phase UPLC-MS/MS analysis was performed to determine mycotoxin production. SC reduced A. parasiticus growth more on CMA than on SM-G media. AFB1 formation was inhibited on both types of SC-containing media, although SC generally had a stronger inhibitory effect on AFB1 production on CMA than on SM-G. AFB1 and AFB2 were produced on CMA, while AFB1 dominated in SM-G, AFG1, and AFG2 were not detected in any media. The results show that SC inhibited AFB1 formation of A. parasiticus on SM-G media after 21 days of incubation under typical conditions for the production of DFMPs. These results indicate the necessity to investigate AF on natural matrices in an environment that is as similar as possible to real conditions in the production of DFMPs.

A sensitive and rapid bioanalytical method for the quantitative determination of luliconazole in rabbit eye tissues using UPLC-MS/MS assay.

Luliconazole (LCZ) is a novel antifungal imidazole with broad-spectrum and high susceptibility of Aspergillus and Fusarium are the dominant species of fungal keratitis, may potentially be a new medical treatment option for ocular fungal infection. To evaluate LCZ distribution in ocular tissues after topical application for the development of ophthalmic delivery system, it is important to have a bioanalytical method for measuring the drug concentrations in different ocular tissues and aqueous humor (AH). A selective and sensitive ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantification of LCZ in rabbit ocular tissues, including conjunctiva, cornea, AH, iris, lens, vitreous humor (VH), retinal choroid and sclera, using lanoconazole as internal standard (IS). Chromatographic separation was achieved on a Xterra MS, C18 column (2.1 × 50 mm, 3.5 µm) using mobile phase with formic acid solution (0.2%, v/v): acetonitrile (50:50, v/v) at a flow rate of 0.2 ml/min, and the run time was 2.5 min. Detection was performed using the transitions 354.1 → 150.3 m/z for LCZ and 320.1 → 150.3 m/z for IS by positive ion electrospray ionization in multiple reaction monitoring (MRM) mode. Method validation was conducted in accordance with U.S. Food and Drug Administration's regulatory guidelines for bioanalytical method validation. The calibration curves were linear over the concentration range from 2.80 ng/ml to 2038 ng/ml for conjunctiva, cornea and sclera, 2.09 ng/ml to 1019 ng/ml for AH, 2.09 ng/ml to 509.5 ng/ml for iris, 2.09 ng/ml to 203.8 ng/ml for retinal choroid and VH, 2.04 ng/ml to 101.9 ng/ml for lens, with all the squared correlation coefficients (r2) more than 0.99. The accuracy of the method was within the acceptable limit of 89.34%∼112.78% at the lower limit of quantification and other concentrations, Inter-day and intra-day precision values, expressed in terms of RSD (%), in all tissues were within 15% at all concentrations. The mean recoveries of LCZ in rabbit ocular tissues was 84.85%∼100.52%. No interference was found due to matrix components. Luliconazole was stable during the stability studies, including autosampler stability, benchtop stability, freeze/thaw stability and long-term stability. The method was successfully applied to the ocular pharmacokinetic and tissues distribution studies of LCZ in rabbit after topical administration of LCZ ophthalmic drug delivery system.

The Biosynthesis and Transport of Ophiobolins in Aspergillus ustus 094102.

Ophiobolins are a group of sesterterpenoids with a 5-8-5 tricyclic skeleton. They exhibit a significant cytotoxicity and present potential medicinal prospects. However, the biosynthesis and transport mechanisms of these valuable compounds have not been fully resolved. Herein, based on a transcriptome analysis, gene inactivation, heterologous expression and feeding experiments, we fully explain the biosynthesis pathway of ophiobolin K in Aspergillus ustus 094102, especially proved to be an unclustered oxidase OblCAu that catalyzes dehydrogenation at the site of C16 and C17 of both ophiobolin F and ophiobolin C. We also find that the intermediate ophiobolin C and final product ophiobolin K could be transported into a space between the cell wall and membrane by OblDAu to avoid the inhibiting of cell growth, which is proved by a fluorescence observation of the subcellular localization and cytotoxicity tests. This study completely resolves the biosynthesis mechanism of ophiobolins in strain A. ustus 094102. At the same time, it is revealed that the burden of strain growth caused by the excessive accumulation and toxicity of secondary metabolites is closely related to compartmentalized biosynthesis.

Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Antifeedant and Antifungal Activities of Metabolites Isolated from the Coculture of Endophytic Fungus Aspergillus tubingensis S1120 with Red Ginseng.

A new globoscinic acid derivative, aspertubin A (1) along with four known compounds, were obtained from the co-culture of Aspergillus tubingensis S1120 with red ginseng. The chemical structures of compounds were characterized by using spectroscopic methods, the calculated and experimental electronic circular dichroism. Panaxytriol (2) from red ginseng, and asperic acid (4) showed significant antifeedant effect with the antifeedant rates of 75 % and 80 % at the concentrations of 50 µg/cm2 . Monomeric carviolin (3) and asperazine (5) displayed weak attractant activity on silkworm. All compounds were assayed for antifungal activities against phytopathogens A. tubingensis, Nigrospora oryzae and Phoma herbarum and the results indicated that autotoxic aspertubin A (1) and panaxytriol (2) possessed selective inhibition against A. tubingensis with MIC values at 8 µg/mL. The co-culture extract showed higher antifeedant and antifungal activities against P. herbarum than those of monoculture of A. tubingensis in ordinary medium. So the medicinal plant and endophyte showed synergistic effect on the plant disease resistance by active compounds from the coculture of A. tubingensis S1120 and red ginseng.

Fungal Necrotizing Scleritis After Intravitreal Injection Therapy.

PURPOSE: To report a case of infectious necrotizing scleritis secondary to Aspergillus terreus after intravitreal injection therapy. METHODS: This is a case report with literature review. RESULTS: A 98-year-old woman receiving intravitreal aflibercept injections for neovascular age-related macular degeneration in the left eye presented with severe pain, redness, and purulent discharge at the injection site. She was initially treated with topical fortified antibiotics, and clinical improvement was achieved, although microbial cultures showed negative results. Two months later, she presented with severe ocular pain and was diagnosed with anterior necrotizing scleritis. Scleral scrapings were collected for cultures, and intensive topical antibiotic therapy was reintroduced. Evaluation for autoimmune etiology and microbiological testing showed negative results. Because of the progression of the scleral necrotic area, empirical therapy with topical voriconazole was initiated, and surgical debridement was performed. Finally, the culture was positive for A. terreus. The modified therapy consisted of topical voriconazole and oral voriconazole for 3 months with an excellent clinical outcome. CONCLUSIONS: To our knowledge, this is the first case of fungal necrotizing scleritis secondary to intravitreal injection. Diagnosis was delayed due to its chronic clinical course and the slow fungal growth in culture media, but the combined medical and surgical approach resulted in a satisfactory outcome.

Antimicrobial, antioxidant and cytotoxic properties of Chenopodium glaucum L.

We evaluated phytochemical composition, antibacterial, antifungal, anti-oxidant and cytotoxic properties of aqueous (water) and organic extracts (methanol, ethyl acetate and n-hexane) of Chenopodium glaucum. Highest phenolic content 45 mg gallic acid equivalents (GAE)/g d.w was found in aqueous extract followed by ethyl acetate (41mg GAE/g d.w) and methanol extract (34.46 mg GAE/g d.w). Antibacterial potential of aqueous and organic extracts of C. glaucum was examined against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Staphylococcus epidermidis. The aqueous, methanolic, ethyl acetate, and n-hexane extract showed antibacterial activity against A. baumannii, K. pneumoniae, E. coli and S. epidermidis. However, against A. baumannii significantly higher inhibition zone (19 mm and 18.96 mm respectively) was shown by ethyl acetate and methanol extracts. Aqueous extract possessed highest growth inhibition (11 mm) against E. coli. Aqueous, ethyl acetate and methanol extracts showed 9 mm, 10 mm, and 10.33 mm zone of inhibition against the K. pneumoniae. For antifungal activity, the extracts were less effective against Aspergillus niger but showed strong antifungal activity against Aspergillus flavus (A. flavus). The antioxidant activity was measured as DPPH (2, 2-diphenyl-1-picrylhydrazyl), H2O2 and ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity of free radicals. All the organic extracts of C. glaucum possessed ABTS, DPPH and H2O2 scavenging properties. The highest cytotoxic activity measured as half maximal inhibitory concentration (IC50) against human lungs carcinoma cells was recorded for methanolic (IC50 = 16 µg/mL) and n-hexane (IC50 = 25 µg/mL) extracts, respectively. The Gas chromatography-mass spectrometry (GC-MS) analysis showed 4 major and 26 minor compounds in n-hexane extract and 4 major and 7 minor compounds in methanol extract of the C. glaucum. It is concluded that aqueous and organic extracts of C. glaucum would be potential therapeutic agents and could be exploited on a pilot scale to treat human pathogenic diseases.

Characterizing the role of the zinc finger transcription factor AcrpnR in governing development in Aspergillus cristatus.

Filamentous fungi reproduce sexually or asexually, and the developmental processes are strictly regulated by a variety of transcription factors. In this study, we characterized a zinc finger transcription factor, called AcrpnR, in Aspergillus cristatus (GME2916). The ∆AcrpnR strain exhibited decreased asexual reproduction and increased cleistothecium production. The complementation strain showed restoration of these phenotypic differences. Overexpression of AcrpnR resulted in enhanced asexual development and delayed and inhibited sexual reproduction, suggesting that AcrpnR is required for proper asexual and sexual development in A. cristatus. In addition, AcrpnR positively regulated the expression of genes of the central regulatory pathway of conidiation and negatively regulated the expression of sex-related genes. Overall, these results demonstrate that AcrpnR is essential for maintaining a balance between asexual and sexual development.

Anti-Inflammatory, Antiallergic, and COVID-19 Main Protease (Mpro) Inhibitory Activities of Butenolides from a Marine-Derived Fungus Aspergillus terreus.

In December 2020, the U.K. authorities reported to the World Health Organization (WHO) that a new COVID-19 variant, considered to be a variant under investigation from December 2020 (VUI-202012/01), was identified through viral genomic sequencing. Although several other mutants were previously reported, VUI-202012/01 proved to be about 70% more transmissible. Hence, the usefulness and effectiveness of the newly U.S. Food and Drug Administration (FDA)-approved COVID-19 vaccines against these new variants are doubtfully questioned. As a result of these unexpected mutants from COVID-19 and due to lack of time, much research interest is directed toward assessing secondary metabolites as potential candidates for developing lead pharmaceuticals. In this study, a marine-derived fungus Aspergillus terreus was investigated, affording two butenolide derivatives, butyrolactones I (1) and III (2), a meroterpenoid, terretonin (3), and 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde (4). Chemical structures were unambiguously determined based on mass spectrometry and extensive 1D/2D NMR analyses experiments. Compounds (1-4) were assessed for their in vitro anti-inflammatory, antiallergic, and in silico COVID-19 main protease (Mpro) and elastase inhibitory activities. Among the tested compounds, only 1 revealed significant activities comparable to or even more potent than respective standard drugs, which makes butyrolactone I (1) a potential lead entity for developing a new remedy to treat and/or control the currently devastating and deadly effects of COVID-19 pandemic and elastase-related inflammatory complications.

Genome sequence analysis of deep sea Aspergillus sydowii BOBA1 and effect of high pressure on biodegradation of spent engine oil.

A deep-sea fungus Aspergillus sydowii BOBA1 isolated from marine sediment at a depth of 3000 m was capable of degrading spent engine (SE) oil. The response of immobilized fungi towards degradation at elevated pressure was studied in customized high pressure reactors without any deviation in simulating in situ deep-sea conditions. The growth rate of A. sydowii BOBA1 in 0.1 MPa was significantly different from the growth at 10 MPa pressure. The degradation percentage reached 71.2 and 82.5% at atmospheric and high pressure conditions, respectively, within a retention period of 21 days. The complete genome sequence of BOBA1 consists of 38,795,664 bp in size, comprises 2582 scaffolds with predicted total coding genes of 18,932. A total of 16,247 genes were assigned with known functions and many families found to have a potential role in PAHs and xenobiotic compound metabolism. Functional genes controlling the pathways of hydrocarbon and xenobiotics compound degrading enzymes such as dioxygenase, decarboxylase, hydrolase, reductase and peroxidase were identified. The spectroscopic and genomic analysis revealed the presence of combined catechol, gentisate and phthalic acid degradation pathway. These results of degradation and genomic studies evidenced that this deep-sea fungus could be employed to develop an eco-friendly mycoremediation technology to combat the oil polluted marine environment. This study expands our knowledge on piezophilic fungi and offer insight into possibilities about the fate of SE oil in deep-sea.

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.

Deep convolutional neural network: a novel approach for the detection of Aspergillus fungi via stereomicroscopy.

Fungi of the genus Aspergillus are ubiquitously distributed in nature, and some cause invasive aspergillosis (IA) infections in immunosuppressed individuals and contamination in agricultural products. Because microscopic observation and molecular detection of Aspergillus species represent the most operator-dependent and time-intensive activities, automated and cost-effective approaches are needed. To address this challenge, a deep convolutional neural network (CNN) was used to investigate the ability to classify various Aspergillus species. Using a dissecting microscopy (DM)/stereomicroscopy platform, colonies on plates were scanned with a 35× objective, generating images of sufficient resolution for classification. A total of 8,995 original colony images from seven Aspergillus species cultured in enrichment medium were gathered and autocut to generate 17,142 image crops as training and test datasets containing the typical representative morphology of conidiophores or colonies of each strain. Encouragingly, the Xception model exhibited a classification accuracy of 99.8% on the training image set. After training, our CNN model achieved a classification accuracy of 99.7% on the test image set. Based on the Xception performance during training and testing, this classification algorithm was further applied to recognize and validate a new set of raw images of these strains, showing a detection accuracy of 98.2%. Thus, our study demonstrated a novel concept for an artificial-intelligence-based and cost-effective detection methodology for Aspergillus organisms, which also has the potential to improve the public's understanding of the fungal kingdom.

Antifungal activity of Saccharomyces cerevisiae and assessment of ochratoxigenic load on currants by means of Real Time PCR.

Currants are prone to contamination by ochratoxin during cultivation, processing and storage conditions. Saccharomyces cerevisiae is considered to be among the main species of grape yeast flora able to control antagonistic fungi. In this study, the potential of S. cerevisiae Y33 was investigated to inhibit the growth of several fungal species indigenous to the microbiota of grapes. Moreover, the efficacy of this yeast species was investigated to inhibit OTA by toxin producing fungi both in vitro and in situ. For this purpose thirty-five different fungal species, belonging to the genera Aspergillus, Penicillium, Cladosporium, Fusarium and Alternaria interacted in vitro with S. cerevisiae on Malt Extract agar plates, stored at 25 °C for 14 days. Results showed that the highest OTA producer A. carbonarius F71 was inhibited more than 99% from day 7, in contrast to A. niger strains that presented enhanced OTA production at day 14 due to interaction with S. cerevisiae Y33. Additionally, the antifungal potential of the selected yeast was also studied in situ on currants subjected to different treatments and stored at 25 °C for 28 days. Microbiological analysis was undertaken for the enumeration of the bacterial and fungal flora, together with OTA determination at 7 and 21 days. To quantify A. carbonarius on all treated currant samples, molecular analysis with Real Time PCR was employed. A standard curve was prepared with A. carbonarius DNA. The efficiency of the curve was estimated to 10.416, the slope to -3.312 and the range of haploid genome that could be estimated was from 1.05 to 105∙105. The amount of A. carbonarius DNA in all treated currants samples, where the fungus was positively detected, ranged from as low as 0.08 to 562 ng DNA/g currants. The antifungal activity of S. cerevisiae Y33 was observed in all studied cases, causing inhibition of fungal growth and OTA production.

Trade-off between Plasticity and Velocity in Mycelial Growth.

Tip-growing fungal cells maintain cell polarity at the apical regions and elongate by de novo synthesis of the cell wall. Cell polarity and tip growth rate affect mycelial morphology. However, it remains unclear how both features act cooperatively to determine cell shape. Here, we investigated this relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 µm-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, any hypha growing through the channel must adapt its morphology. Live-cell imaging analyses revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels, or had lost apical polarity after emerging from the other end of the channel. Fluorescence live-cell imaging analyses of the Spitzenkörper, a collection of secretory vesicles and polarity-related proteins at the hyphal tip, in Neurospora crassa indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, including phytopathogenic fungi. This comparative approach revealed that the growth defects induced by the channels were not correlated with their taxonomic classification or with the width of hyphae, but, rather, correlated with the hyphal elongation rate. This report indicates a trade-off between morphological plasticity and velocity in mycelial growth and serves to help understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology, and pathogenicity.IMPORTANCE Cell morphology, which is controlled by polarity and growth, is fundamental for all cellular functions. However how polarity and growth act cooperatively to control cell shape remains unclear. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 µm-width channels of microfluidic devices. We found that most fast growing hyphae often lost the cell polarity after emerging from the channels, whereas slow growing hyphae retained polarity and continued growing, indicating a trade-off between plasticity and velocity in mycelial growth. These results serve to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.

Influence of type, concentration, exposure time, temperature, and presence of organic load on the antifungal efficacy of industrial sanitizers against Aspergillus brasiliensis (ATCC 16404).

Parameters such as type and concentration of the active compound, exposure time, application temperature, and organic load presence influence the antimicrobial action of sanitizers, although there is little data in the literature. Thus, this study aimed to evaluate the antifungal efficacy of different chemical sanitizers under different conditions according to the European Committee for Standardization (CEN). Aspergillus brasiliensis (ATCC 16404) was exposed to four compounds (benzalkonium chloride, iodine, peracetic acid, and sodium hypochlorite) at two different concentrations (minimum and maximum described on the product label), different exposure times (5, 10, and 15 min), temperatures (10, 20, 30, and 40 °C), and the presence or absence of an organic load. All parameters, including the type of sanitizer, influenced the antifungal efficacy of the tested compounds. Peracetic acid and benzalkonium chloride were the best antifungal sanitizers. The efficacy of peracetic acid increased as temperatures rose, although the opposite effect was observed for benzalkonium chloride. Sodium hypochlorite was ineffective under all tested conditions. In general, 5 min of sanitizer exposure is not enough and >10 min are necessary for effective fungal inactivation. The presence of organic load reduced sanitizer efficacy in most of the tested situations, and when comparing the efficacy of each compound in the presence and absence of an organic load, a difference of up to 1.5 log CFU was observed. The lowest concentration recommended on the sanitizer label is ineffective for 99.9% fungal inactivation, even at the highest exposure time (15 min) or under the best conditions of temperature and organic load absence. Knowledge of the influence exerted by these parameters contributes to successful hygiene since the person responsible for the sanitization process in the food facility can select and apply a certain compound in the most favorable conditions for maximum antifungal efficacy.

Interacting climate change factors (CO2 and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix.

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2-5 °C and CO2 from 400 to 800/1200 ppm. This study examined the effect of (i) current and increased temperature in the alternating 11.5 h dark/12.5 h light cycle (15-28 °C vs 18-34 °C), representative of the North Apulia area, South Italy and (ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), on growth, expression of biosynthetic genes (AcOTApks, AcOTAnrps, AcOTAhal, AcOTAp450, AcOTAbZIP) and regulatory genes of Velvet complex (laeA/veA/velB, "velvet complex") involved in OTA biosynthesis and OTA phenotypic production by three strains of A. carbonarius. The experiments made on a grape-based matrix showed that elevated CO2 resulted in a general stimulation of growth and OTA production. These results were also supported by the up-regulation of both structural and regulatory genes involved in the OTA biosynthesis. Our work has shown for the first time that elevated CO2 concentration in the Mediterranean region may result in an increased risk of OTA contamination in the wine production chain.

Inducing secondary metabolite production of Aspergillus sydowii through microbial co-culture with Bacillus subtilis.

BACKGROUND: The co-culture strategy which mimics natural ecology by constructing an artificial microbial community is a useful tool to activate the biosynthetic gene clusters to generate new metabolites. However, the conventional method to study the co-culture is to isolate and purify compounds separated by HPLC, which is inefficient and time-consuming. Furthermore, the overall changes in the metabolite profile cannot be well characterized. RESULTS: A new approach which integrates computational programs, MS-DIAL, MS-FINDER and web-based tools including GNPS and MetaboAnalyst, was developed to analyze and identify the metabolites of the co-culture of Aspergillus sydowii and Bacillus subtilis. A total of 25 newly biosynthesized metabolites were detected only in co-culture. The structures of the newly synthesized metabolites were elucidated, four of which were identified as novel compounds by the new approach. The accuracy of the new approach was confirmed by purification and NMR data analysis of 7 newly biosynthesized metabolites. The bioassay of newly synthesized metabolites showed that four of the compounds exhibited different degrees of PTP1b inhibitory activity, and compound N2 had the strongest inhibition activity with an IC50 value of 7.967 µM. CONCLUSIONS: Co-culture led to global changes of the metabolite profile and is an effective way to induce the biosynthesis of novel natural products. The new approach in this study is one of the effective and relatively accurate methods to characterize the changes of metabolite profiles and to identify novel compounds in co-culture systems.

Functional Role of Aspergillus carbonariusAcOTAbZIP Gene, a bZIP Transcription Factor within the OTA Gene Cluster.

Aspergillus carbonarius is the principal fungal species responsible for ochratoxin A (OTA) contamination of grapes and derived products in the main viticultural regions worldwide. In recent years, co-expressed genes representing a putative-OTA gene cluster were identified, and the deletion of a few of them allowed the partial elucidation of the biosynthetic pathway in the fungus. In the putative OTA-gene cluster is additionally present a bZIP transcription factor (AcOTAbZIP), and with this work, A. carbonarius ΔAcOTAbZIP strains were generated to study its functional role. According to phylogenetic analysis, the gene is conserved in the OTA-producing fungi. A Saccharomyces cerevisiae transcription factor binding motif (TFBM) homolog, associated with bZIP transcription factors was present in the A. carbonarius OTA-gene cluster no-coding regions. AcOTAbZIP deletion results in the loss of OTA and the intermediates OTB and OTß. Additionally, in ΔAcOTAbZIP strains, a down-regulation of AcOTApks, AcOTAnrps, AcOTAp450, and AcOTAhal genes was observed compared to wild type (WT). These results provide evidence of the direct involvement of the AcOTAbZIP gene in the OTA biosynthetic pathway by regulating the involved genes. The loss of OTA biosynthesis ability does not affect fungal development as demonstrated by the comparison of ΔAcOTAbZIP strains and WT strains in terms of vegetative growth and asexual sporulation on three different media. Finally, no statistically significant differences in virulence were observed among ΔAcOTAbZIP strains and WT strains on artificially inoculated grape berries, demonstrating that OTA is not required by A. carbonarius for the pathogenicity process.

New alkaloids from the diversity-enhanced extracts of an endophytic fungus Aspergillus flavus GZWMJZ-288.

Three new alkaloids (1-3) together with four previously reported compounds (4-7) were identified from the extracts and the diversity-enhanced extracts of the fermentation broth of the endophytic fungus, Aspergillus flavus GZWMJZ-288 associated with Garcinia multiflora. The structures of new compounds were respectively determined as 19-amino-19-dehydroxy 5-epi-α-cyclopiazonic acid (1), 2-hydroxymethyl-5-(3-oxobutan-2-yl)aminopyran-4(4H)-one (2) and 4-amino-2-hydroxymethylpyridin-5-ol (3) by spectroscopic analysis, ECD calculation and X-ray single crystal diffraction. Compounds 1 and 4 with 19-enamine were dynamic equilibrium of Z- and E- isomers in the solution but favored in Z- isomers in the solid state, while compound 7 with 19-enol was favored in Z- isomer in the solution but a mixture of Z- and E- isomers in solid state. This phenomenon could be explained by the quantum-mechanical energies calculations. Among the isolated compounds 1-7, compounds 1, 4 and 7 with a rare 1,3,4,5-tetrahydro-1-azaacenaphtho[3,4-c]pyrrolizidine skeleton showed α-glucosidase inhibitory activity with the IC50 values of 41.97 ± 0.97, 232.57 ± 11.45 and 243.95 ± 3.36 µM, respectively, and the binding modes were performed by silico docking studies.

Key parameters optimization of chitosan production from Aspergillus terreus using apple waste extract as sole carbon source.

Chitosan is commonly obtained from shrimp and crab shell chitin by deacetylation; however, such supplies appear limitation. An alternative source of chitosan is cell wall in certain fungi. In this study, chitosan production through submerged fermentation of Aspergillus terreus on apple waste extract as sole carbon source was investigated. Monod equation with a maximum specific growth rate of 0.083 h-1 and half-saturation constant of 6.67 w/v% was best described the kinetic of growth. Results of response surface methodology showed the highest chitosan to substrate yield of 49.32 mg gsubstrate-1, chitosan to fungal biomass yield of 140.9 mg gcell-1, and fungal biomass to substrate yield of 0.387 gcell gsubstrate-1 were simultaneously obtained at temperature 30.0 °C, initial pH 5.98, and ammonium nitrate concentration 5.0 g L-1. The chitosan produced at the optimum condition was characterized by FTIR, TGA, and DSC analysis, and degree of deacetylation was 88.2%.