Application of Syzygium aromaticum, Ocimum sanctum, and Cananga odorata essential oils for management of Ochratoxin A content by Aspergillus ochraceus and Penicillium verrucosum: An in vitro assessment in maize grains

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

Secondary metabolites of endophytic fungus Aspergillus ochraceus SX-C7 from Selaginella stauntoniana / 中草药

Objective: To study the secondary metabolites of endophytic fungus Aspergillus ochraceus SX-C7 from Setaginella stauntoniana. Methods: The compounds were isolated and purified by silica gel, Sephadex LH-20, C-18 reversed phase column chromatography, and their structures were identified by MS and NMR analyses. Antibacterial activity and inhibitory activity against acetylcholinesterase were studied by doubling dilution and Ellman methods. Results: Ten known compounds were isolated from the secondary metabolites of A. ochrateus SX-C7, and identified as: semivioxanthin (1), 6-hydroxy-p-menth-4 (5)-en-3-one (2), flavacol (3), magnolin (4), preussin B (5), circumdatins D (6), isofucosterol (7), sclerotiamide (8), 3β-hydroxyergosta-8,24(28)-dien-7-one (9), and 3-O-β-D-glucopyranosyl stigmasta-5(6),24(28)-diene (10). Conclusion: Compounds 1, 2, 4, 7, and 10 were isolated from A. ochraceus SX-C7 for the first time. The results of the bioactivity assay showed that compound 10 possessed obvious inhibitory activity against Bacillus subtilis with a MIC value of 2 μg/mL, which was at the same grade with positive control. Compound 1 exhibited potent inhibitory activity against acetylcholinesterase with an inhibitory rate of 62.3% (the final concentration was 50 μmol/L.

Phytochemical profile of methanolic extracts from Chilca (Baccharis glutinosa) roots and its activity against Aspergillus ochraceus and Fusarium moniliforme

Aim: The present study aimed to identify the phytochemicals of methanolic extract from Baccharis glutinosa (chilca) roots (MEBg) and to evaluate its antifungal activity on two major fungal pathogens of agricultural importance. Methodology: The antifungal activity was evaluated by inhibition halo, minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and % sporulation against A. ochraceus and F. moniliforme. As a preliminary test, inhibition halo was tested using 1, 10, 100 and 270 mg ml-1 of MEBg. Different concentrations of MEBg were applied for MIC and MFC tests. Ketoconazole was used as positive control. The treatments were applied in triplicate. The phytochemical compounds of MEBg were determined by GC-MS analysis. Results: The MEBg produced an inhibition zone of 2 to 4 mm in the inhibition halo test, with concentrations of 100 and 270 mg ml-1 for A. ochraceus and F. moniliforme, respectively. Reduction in % sporulation above 50 was shown in concentrations over 8 mg ml-1. MEBg were reported to exhibit antifungal activities against A. ochraceus and F. moniliforme with the MIC values ranging from 2 to 5.6 mg∙ml-1 and the MFC from 12 to 15 mg ml-1. GC-MS analysis of Chilca extracts revealed that the most abundant metabolites were furfural compounds and organic acids. The most abundant furfural compounds were 5-(hydroxymethyl) furan-2-carbaldehyde (38.59%), furan-2-carbaldehyde (4.103%) and 5-methylfuran-2-carbaldehyde (2.1%). Interpretation: The MEBg revealed efficient antifungal activity, likely due to the presence of bioactive compounds, which could be used as an alternative for biological control of pathogenic fungi in maize and coffee crops.

Screening of cytotoxic activities toward WiDr and Vero cell lines of ethyl acetate extracts of fungi-derived from the marine sponge Acanthostrongylophora ingens

Ethyl acetate extracts of fungi-derived from the marine sponge Acanthostrongylophora ingens were tested forcytotoxic activity against WiDr and Vero cell lines. Three of fungi extracts exhibited strong cytotoxicity with percentageof viability (≤50%) occurring at concentrations of 100 µg/ml. One isolate (IB141) showed specific cytotoxicityagainst WiDr cells whreas not against Vero cells. This isolate was identified based on molecular characterizationusing sequence analysis of the partial 18S rRNA gene. The result indicated that IB141 was identical to Aspergillusochraceus. A comparatively high part of positive bio-activity screening results were acquired in this study, displayingthat the fungi-derived from the marine sponge A. ingens have potential as a source of new anti-cancer agents.

Study on secondary metabolites of endophytic fungus Aspergillus ochraceus from Polygonatum cyrtonema / 中草药

Objective: To investigate the secondary metabolites of Aspergillus ochraceus, an endophytic fungus isolated from Polygonatum Cyrtonema. Methods: Compounds were isolated and purified from the EtOAc extract by using chromatography technology and their structures were established on the basis of comprehensive spectroscopic analysis. Results: A total of 15 compounds were obtained and their structures were elucidated as 6,7-dihydroindolizin-8(5H)-one (1), polygonatine A (2), 8-oxo-5,6,7,8-tetrahydro-3-indolizinyl methyl acetate (3), 8-hydroxyketone (4), cyclo-(L-Leu-L-Ile) (5), alternariol (6), seco- patulolide C (7), n-butyl-β-D-fructopyranoside (8), Nb-acetyltryptamine (9), N-trans-cinnamoyltyramine (10), 5-hydroxy- methylfurfural (11), 5,7-dihydroxy-6,8-dimethyl-3-(4’-hydroxybenzyl) chroman-4-one (12), 5,7-dihydroxy-6-methyl-8- methoxy-3- (4’-hydroxybenzyl) chroman-4-one (13), 25R-3-β-hydroxyspirost-5-en-12-one (14), and 25S-3-β-hydroxyspirost-5-en-12-one (15). Conclusion: All chemical constituents are isolated from A. ochraceus for the first time.

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Antimicrobial activity of endophytic fungi from coffee plants / Atividade antimicrobiana de fungos endofíticos de plantas de café

Endophytic fungi are a promising source for discovery of compounds with biotechnological potential. The aim of this study was to select and identify endophytic fungi from Coffea arabica that produce volatile organic compounds (VOCs), evaluate the effect of the VOCs produced by endophytic fungi on the growth of Rhizoctonia solani, Fusarium oxysporum, Phoma sp., Botrytis cinerea, Fusarium solani, Fusarium verticillioides, Cercospora coffeicola and Pestalotia longisetula, and select endophytic fungi with potential for biological control of Aspergillus ochraceus inoculated in coffee beans and F. verticillioides inoculated in corn seeds. An isolate of Muscodor albus was used as selection tool for VOC producing fungi. Among the 400 endophytic fungi isolates, 11 were able to grow in the presence of VOCs produced by M. albus. These fungi were identified as Muscodor spp. (9) and Simplicillium sp. according to searches in UNITE database using DNA sequences of internal transcribed spacer (ITS). The VOC's produced by endophytic fungi inhibited the growth the phytopathogenic fungi with different efficacies, compared to the control. The VOCs produced by Muscodor coffeanum (COAD 1842) showed fungicidal effect against A. ochraceus on coffee beans. Six endophytic fungi completely inhibited growth of F. verticillioides inoculated in corn seeds. This study demonstrates that the volatile-compound producing endophytic fungi, isolated from Coffea arabica, are promising sources of bioactive compounds.

Fungos endofíticos são uma fonte promissora para a descoberta de compostos com potencial biotecnológico. O objetivo deste estudo foi selecionar e identificar fungos endofíticos de Coffea arabica que produzem compostos orgânicos voláteis (COVs), avaliar o efeito dos compostos orgânicos voláteis produzido por fungos endofíticos sobre o crescimento de Rhizoctonia solani, Fusarium oxysporum, Phoma sp., Botrytis cinerea, Fusarium solani, Fusarium verticillioides, Cercospora coffeicola e Pestalotia longisetula e selecionar fungos endofíticos com potencial para controle biológico de Aspergillus ochraceus inoculado em grãos de café e F. verticillioides inoculado em sementes de milho. Um isolado de Muscodor albus foi utilizado como ferramenta de seleção para fungos endofíticos produtores de COVs. Dentre os 400 fungos endofíticos isolados, 11 foram capazes de crescer na presença de COVs produzidos por M. albus. Estes fungos foram identificados como Muscodor spp. (9) e Simplicillium sp. de acordo com pesquisas na base de dados UNITE usando sequências de DNA do espaçador transcrito interno (ITS). Os COVs produzidos por fungos endofíticos inibiram o crescimento dos fungos fitopatogênicos em comparação com o controle com diferentes eficácias. Os COVs produzidos por Muscodor coffeanum (COAD 1842) apresentou efeito fungicida contra A. ochraceus em grãos de café. Seis fungos endofíticos inibiram completamente o crescimento de F. verticillioides inoculado em sementes de milho. Este estudo demonstra que os fungos endofíticos produtores de compostos voláteis isolados de Coffea arabica são fontes promissoras de compostos bioativos.

Capacidad antifúngica de sobrenadantes libres de células obtenidos de la fermentación de un sustrato de "panela" con gránulos de kéfir de agua / Antifungal capacity of cell-free supernatants obtained from fermentation of a substrate of brown sugar with water kefir grains

El kéfir de agua (KA) es una bebida fermentada medianamente ácida elaborada con soluciones azucaradas y fermentada por un consorcio de microorganismos, principalmente bacterias ácido lácticas (BAL) y levaduras (LEV), embebidas en un polisacárido llamado gránulo de KA. La presencia de hongos y sus toxinas es un problema de la producción de alimentos, como Aspergillus ochraceus y sus micotoxinas especialmente en café y vino. Entre algunas alternativas que se han evaluado para su inhibición se incluyen las bacterias ácido lácticas y productos fermentados en general. El objetivo principal de esta investigación fue evaluar la capacidad del KA en inhibir o retrasar el crecimiento de A. ochraceus. Se emplearon 8 sobrenadantes libres de células (SLC) obtenidos de diferentes fermentaciones de panela con gránulos de KA y con diferentes concentraciones de ácidos orgánicos (láctico y acético). Se hicieron fermentaciones con gránulos de KA en solución de panela por periodos de 32,5 h, a 25, 30 y 37 °C. Se determinaron la cinética de acidificación; el incremento de biomasa y se hizo el recuento de los grupos de microorganismos que componen el gránulo. A 25 °C se determinó el mayor aumento de biomasa (92%). La temperatura de fermentación afectó el recuento de los microorganismos que conforman el gránulo, principalmente las BAL, disminuyendo su cantidad a la máxima temperatura de fermentación (37 °C) (1x10ˆ6 UFC ml-1), comparado con la mínima temperatura (25 °C) (6x10ˆ7 UFC ml-1). El fermento que presentó mayor actividad antifúngica fue el SLC5 (pH: 3,2; temperatura de fermentación: 30 °C). El poder inhibitorio se atribuyó a los ácidos orgánicos producidos durante la fermentación, aunque no se puede descartar que hayan actuado otras sustancias no cuantificadas. Se pudo comprobar que el KA puede fermentar y aumentar su biomasa en un sustrato como el agua de panela y que sus SLC tienen la capacidad de reducir el crecimiento de A. ochraceus.

Water kéfir (WK) is a moderately sour fermented beverage elaborated in sugar-containing solutions through fermentation by a microorganism consortium, principally lactic acid bacteria (LAB) and yeasts, embedded in a matrix of polysaccharide so-called WK grains. The presence of fungi and their toxins it is a problem for the food industry, such as Aspergillus ochraceus and their mycotoxin production especially in coffee and wine. Some alternatives that have been evaluated for their inhibition include lactic acid bacteria and generally fermented products. The main objective was to evaluate the capacity of WK to ferment a Colombian beverage made with Panela and to assess capability of this product to retard the germination of a common toxigenic fungi like Aspergillus ochraceus. 8 cell-free supernatants (CFS) were obtained from separate fermentations and with different organic acids (OA) concentrations (e. g., lactic and acetic). Different fermentations were conducted with WK in Panela broth during 32.5 h periods at 25, 30, and 37°C. We determined the kinetics of acidification of WK along with the corresponding increment in biomass and conducted a quantitation of microorganisms groups that composed the grain. The greatest biomass increase occurred at 25 °C (92%); but the fermentation temperature affected the composition of microorganisms conforming the grain, with the quantity of LAB decreasing at the highest fermentation temperatures (37 °C) (1x10ˆ6cfu ml-1), compared with the minimum temperature (25 °C) (6x10ˆ7 cfu ml-1). The CFS5 (pH 3.2; fermentation temperature 30 °C) exhibited the greatest antifungal activity. We attribute the inhibitory power of these ferments to the OA produced during fermentation, although we cannot discard the possibility of the action of other substances not specifically quantified. We concluded that WK can grow and fermentate Panela broth and that its CFS can exert an antifungal effect against A. ochraceus.

Fungal Flora and Ochratoxin A Associated with Coffee in Cameroon.

Aims: 104 samples were collected from the west region and the coastal plain of Cameroon during two coffee campaigns, 2009 and 2010. Two coffee processes were evaluated (wet and dry processes) at different stages from harvesting to storage. Study Design: Food contaminants. Place and Duration of Study: Food Microbiology Laboratory, Department of Food Science and Nutrition (ENSAI) University of Ngaoundere; UMR 95 Qualisud, CIRAD of Montpellier, between May 2009 and September 2012. Methodology: Fungi profile was evaluated by direct plating techniques and identified using morphological and molecular tools. OTA levels were analyzed using HPLC technique after extraction and filtration using an immunoaffinity column. Results: Results obtained revealed an overall percentage of fungal contamination between 60-92% in 2009 and 70-90% in 2010. There was no ecological difference in the composition of ochratoxigenic species present in five sites. Coffee beans sampled in 2009 had a colonization incidence of 18-40% A. carbonarius, 12-22% A. niger, 3-15% A. ochraceus while those of 2010 had a colonization incidence of 15-30% A. carbonarius, 35- 40% A. niger, and 2-7% A. ochraceus. Fungal diversity was not correlated with the geographical origin, coffee cultivar and processing method. There was no difference between the processes studied in terms of occurrence of ochratoxigenic fungi. OTA levels were mostly below the recommended standards although some isolated cases of extreme contamination were observed in 2009. A higher level of OTA was detected in the presence of A. niger, A. carbonarius and A. ochraceus than when only A. niger was present. Conclusion: The important fungi with the potential to produce OTA in Cameroonian coffee beans are A. carbonarius and A. niger. These two species were predominant on each type of coffee beans. It was also observed that once a toxigenic strain was isolated from a coffee sample, the sample contained OTA.

Inhibición del crecimiento de Aspergillus ochraceus mediante "panela" fermentada con gránulos de kefir de agua / Fungal growth inhibition of Aspergillus ochraceus with "panela" fermented with water kefir grains

Antecedentes: El kéfir de agua (KA) es una bebida producida con soluciones azucaradas fermentadas con gránulos constituidos por bacterias ácido lácticas (BAL) y levaduras embebidas en una matriz de polisacárido. Aspergillus ochraceus es un hongo filamentoso micotoxigénico, contaminante frecuente y productor de ocratoxina en café, uvas y vino. Se ha estudiado la actividad antifúngica de productos fermentados por BAL pero hasta ahora no se conocen estudios sobre la capacidad antifúngica del KA. Objetivos: Evaluar la capacidad antifúngica de soluciones de panela fermentadas a diferentes tiempos y temperaturas con KA sobre A. ochraceus. Métodos: Se inocularon 10 µL de una suspensión (3x107 conidios. ml-1) de A. ochraceus en agar malta mezclado con sobrenadantes libres de células (SLC) de los productos fermentados, incubando a 25°C y midiendo diariamente el diámetro del micelio, con lo cual se determinó el efecto antifúngico sobre la tasa de crecimiento y la fase de latencia. Se determinó la concentración de ácidos orgánicos (AO) en los SLC mediante HPLC. Resultados: Los productos fermentados por mayor tiempo y temperatura tuvieron mayor concentración de (AO) y mayor descenso de pH siendo el SLC8 (pH: 2,8) el más inhibitorio. Conclusiones: La solución de panela en agua fue adecuada para fermentación con gránulos de KA, obteniéndose metabolitos con propiedades antifúngicas como los AO.

Ochratoxigenic fungi associated with green coffee beans (Coffea arabica L.) in conventional and organic cultivation in Brazil

The genera Aspergillus comprises species that produce mycotoxins such as aflatoxins, ochratoxins and patulin. These are cosmopolitan species, natural contaminants of agricultural products. In coffee grains, the most important Aspergillus species in terms of the risk of presenting mycotoxins belong to the genera Aspergillus Section Circumdati and Section Nigri. The purpose of this study was to assess the occurrence of isolated ochratoxigenic fungi of coffee grains from organic and conventional cultivation from the South of Minas Gerais, Brazil, as well as to evaluate which farming system presents higher contamination risk by ochratoxin A (OTA) produced by fungi. Thirty samples of coffee grains (Coffea arabica L.) were analysed, being 20 of them of conventional coffee grains and 10 of them organic. The microbiological analysis was done with the Direct Plating Technique in a Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. The identification was done based on the macro and micro morphological characteristics and on the toxigenic potential with the Plug Agar technique. From the 30 samples analysed, 480 filamentous fungi of the genera Aspergillus of the Circumdati and Nigri Sections were isolated. The ochratoxigenic species identified were: Aspergillus auricoumus, A. ochraceus, A. ostianus, A. niger and A. niger Aggregate. The most frequent species which produces ochratoxin A among the isolated ones was A. ochraceus, corresponding to 89.55%. There was no significant difference regarding the presence of ochratoxigenic A. ochreceus between the conventional and organic cultivation systems, which suggests that the contamination risk is similar for both cultivation systems.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2 percent recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40ºC and 5.0, respectively. The enzyme was fully stable from 40ºC to 60ºC during 1 hr. The activity was enhanced by Mn2+ (33-39 percent) and NH4+ (15 percent). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described.

Obtenção de protoplastos do fungo filamentoso Aspergillus ochraceus / Production of protoplasts from the filamentous fungus Aspergillus ochraceus

A obtenção de protoplastos é uma ferramenta importante para a transformação genética de fungos. Neste trabalho foi estudada a influência de fatores como idade micelial, tipo e concentração de enzimas e estabilizadores osmóticos na produção de protoplastos de Aspergillus ochraceus. Os melhores resultados de produção de protoplastos foram obtidos utilizando-se NH4Cl 0,8mol L-1 como estabilizador osmótico, micélio com 24h de crescimento e a combinação de Lysing Enzymes e Meicelase ambas, a 20mg mL-1. Entretanto, bons resultados foram também obtidos com a utilização apenas de Lysing Enzymes.

Production of protoplasts is an important tool for genetic transformation of fungi. A protocol for protoplasts production in Aspergillus ochraceus was developed, evaluating culture aging of mycelium, different commercial enzymes and osmotic stabilizers. The best results were obtained with NH4Cl 0.8mol L-1 as osmotic stabilizer, mycelial age of 24 hours and Lysing Enzymes (20mg mL-1) plus Meicelase (20mg mL-1) as lytic enzymes. Good results were also obtained with Lysing Enzymes alone.