Study on the inhibitory effects of native medicinal plants on growth and aflatoxin production by toxigenic Aspergilli isolated from rainbow trout feed

In the present study, natural occurrence of fungi and aflatoxin B1 (AFB1) in pellet feed and feed ingredients used for rainbow trout was investigated with emphasis to Aspergillus section Flavi members and medicinal plants inhibitory to Aspergillus growth and/or AF production. The feed samples were cultured on the standard isolation media including dichloran rosebengal chloramphenicol agar (DRCA) and Aspergillus flavus/parasiticus agar (AFPA) for 2 weeks at 28 °C. Identification of fungal isolates was implemented based on the macro- and microscopic morphological criteria. AFs were detected using high performance liquid chromatography (HPLC). Based on the results obtained, a total of 109 fungal isolates were identified of which Aspergillus was the prominent genus (57.0%), followed by Penicillium (12.84%), Absidia (11.01%) and Pseudallscheria (10.10%). The most frequent Aspergillus species was A. flavus (60.66%) isolated from all the feed ingredients as well as pellet feed. Among 37 A. flavus isolates, 19 (51.35%) were able to produce AFB1 on yeast extract-sucrose (YES) broth in the range of 10.2 to 612.8 [tg/g fungal dry weight. HPLC analyses of trout feed showed that pellet feed and all feed ingredients tested except gluten were contaminated with different levels of AFB1 in the range of 1.83 to 67.35 lig/kg. In order to finding natural inhibitors of fungal growth and/or AF production, essential oils (EOs) and extracts of 49 medicinal plants were studied against an aflatoxin-producing A. parasiticus using a microbioassay technique. The EOs was analyzed by gas chromatography/mass spectrometry (GC/MS). Based on the results obtained, Achillea millefolium sub sp. elborsensis, Ferula gummosa, Mentha spicata, Azadirachta indica, Conium maculatum and Artemisia dracunculus remarkably inhibited A. parasiticus growth without affecting AF production by the fungus. Besides of Thymus vulgaris and Citrus aurantifolia, the EO of Foeniculum vulgare significantly inhibited both fungal growth (-70.0%) and AFs B1 and G1 (-99.0%) production. The EO of Carum carvi and ethyl acetate extract of Platycladus orientalis suppressed AFs B1 and G1 by more than 90.0%, without any obvious effect on fungal growth. The IC50 values of bioactive plants for AFs B1 and G1 were determined in the ranges of 90.6 to 576.2 and 2.8 to 61.9 µg/ml, respectively. Overall, results of the present study indicate the importance of AF contamination of trout feed as a risk factor for fish farming and thus, an urgent necessity for constant monitoring of trout feed for any unacceptable levels of AF contamination. Likewise, antifungal activities of bioactive plants introduced here would be an important contribution to explain the use of these plants as effective antimicrobial candidates to protect feeds from toxigenic fungus growth and subsequent AF contamination.

Biological activities (antibacterial, antifungal and cytotoxic) of secondary metabolites of Ircinia spp.

Sponges are the most primitive of the multicellular, These organisms don’t have any mechanical defense system, so their early appearance in evolution has given them a lot of time for the development of advanced secondary metabolites as chemical defense system. Sponges have the potential to provide drugs from chemical components against diseases. In this investigation the sponge samples, which it is Ircina spp., were collected at depth of 15- 24 meter, from locations on the coastline of Island Kish in Persian Gulf of Iran. For identifying natural components, methanolic and diethyletter were used as extraction solvents, after removal of the solvents, the GC/MS spectra of the fraction were obtained. Then in vitro cytotoxic, antimicrobial and antifungal were identified. In vitro cytotoxity screening, by XTT assay, against KB/ C359 and HUT-56/ C365 cell line, was conducted in this study in 1 - 544 μg/ml. IC54 for winter diethyletter extract was 325 μg/ml, winter methanolic extract was 364 μg/ml, IC54 for summer diethyletter extract was 544 μg/ml, and summer methanolic extract was 454 μg/ml in HUT-56. IC54 for winter diethyletter extract was 454 μg/ml, winter methanolic extract was 444 μg/ml, IC54 for summer diethyletter extract was 344 μg/ml, and summer methanolic extract was 424 μg/ml in KB. In vitro antimicrobial activity by Broth Dilution Methods against clinical gram-positives and gram negatives (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis). The results conducted that the MIC values of winter diethyletter extract for Escherichia coli 24mg/ml, the MIC values of winter diethyletter extract for Escherichia coli 24mg/ml, the MIC and MBC values of winter diethyletter extract for Staphylococcus aureus was 2mg/ml and 24mg/ml. The MIC and MBC values of winter diethyletter extract for Bacillus subtilis was 1.5 mg/ml and 2mg/ml. In vitro antifungal activity by Broth Dilution Methods against clinical pathogens; Candida albicans and Aspergillus fumigatus. The results conducted that the aqueous extracts didn’t have any antifungal activities on pathogens, the MFC of the summer and winter diethyletter extract was 30 mg/ml and 2 mg/ml A. fumigates, the summer and winter methanolic extract was 0722 mg/ml and 2 mg/ml A. fumigates, the summer and winter methanolic was 4/75mg/ml, MFC 5 mg/ml on C. albicans.

Optimização das condições de cultura da Saccharomyces Cerevisiae e de outros fungos produtores de oligossacáridos

Este estudo envolve o controlo e a optimização das condições de culturas dos microrganismos: Saccharomyces cerevisiae CCMI 396, S. cerevisiae v. lab., Aspergillus oryzae CCMI 125, Aspergillus japonicus CCMI 443, Fusarium oxysporum CCMI 866, Aspergillus niger CCMI 296 com vista à produção de oligossacáridos. Determinaram-se os parâmetros característicos das culturas de duas diferentes estirpes de Saccharomyces com diferentes fontes de carbono e em diferentes condições ambientais. O perfil de crescimento da S. cerevisiae CCMI 396 foi semelhante nos diferentes meios de cultura estudados, sendo a velocidade específica de crescimento mais elevada no meio com glucose a pH 5 e a 30°C (0,36h-1). A S. cerevisiae v. lab. Teve velocidade específica de crescimento idêntica nas mesmas condições da outra estirpe, no entanto, o perfil de crescimento foi diferente nos outros meios de cultura. Estudou-se o efeito da adição de sumo de laranja ou de tomate ao meio de cultura com sacarose e avaliou-se a evolução glucídica no meio de cultura durante o ensaio por HPLC com detector RI. Determinou-se a frutosiltransferase no sobrenadante e na fracção intracelular e determinou-se a evolução dos oligossacáridos. Numa segunda parte deste trabalho efectuaram-se culturas dos quatro fungos filamentosos com vista a avaliar a capacidade de produção, nomeadamente, de fruto­oligassacáridos. Os resultados mostraram que a espécie Aspergillus japonicus CCMI 443 originou, nas mesmas condições de cultura, valores superiores, sendo a percentagem de produção FOStotais/GluCtotais de 61% para as enzimas intracelulares e 40% para as enzimas no sobrenadante. ABSTRACT; This study involves control and optimization of the cultures of microorganisms: Saccharomyces cerevisiae CCMI 396, S. cerevisiae v. lab., Aspergillus oryzae CCMI 125, Aspergillus japonicus CCMI 443, Fusarium oxysporum CCMI 866, Aspergillus níger CCMI 296 for oligosaccharides production. Were determined the parameters characteristic of the cultures of two different strains of Saccharomyces with different sources of carbon and in different environmental conditions. The growth profile of S. cerevisiae CCMI 396 was similar in different cultures media, but the highest specific growth was obtained in a medium with glucose, pH 5, at 30°C (0.36h-1). S. cerevisiae v. lab. had similar growth profile in a medium with glucose but with others culture media was different. We studied the effect of adding orange juice or tomato to the culture medium with sucrose and evaluated the evolution glucidic in the culture medium during the test by HPLC with RI detector. Fructosyltransferase was determined in the extracellular and the intracellular fractions and determined the evolution of oligosaccharides. ln the second part of this work were carried out cultures of four filamentous fungi in order to assess production capacity, in particular, fructoligosaccharides. The results showed that the specie Aspergillus japonicus CCMI 443 originated in the same culture conditions, higher values and the percentage of production FOStotal/Guctotal of 61% for intracellular enzymes and 40% for extracellular enzymes.

Comparative study of bioactive potential of Fueniculum vulgare Mill. and Matricaria recutita L. phenolic rich extracts to functionalize cottage chesse

Synthetic additives used in a wide variety of food products have been associated to some toxic effects. This conducted to an increasing interest of consumers for natural additives, including food preservers [1]. Many aromatic herbs have been used to prepare bioactive extracts with benefits to the consumer's health. Foeniculum vulgare Mill. (fennel) and Matricaria recutita L. (chamomile) are examples of popular herbs rich in phenolic compounds with documented antioxidant and antimicrobial properties [2,3]. The present work confirms the antioxidant (DPPH scavenging activity, reducing power and lipid peroxidation inhibition) and antimicrobial (against bacteria such as Bacillus cereus and Salmonella Typhimurium and fungi such as Aspergillus niger, A. versicolor and PenicilliumfimicuJosum) activities of fennel and chamomile extracts, obtained by decoction. The chemical characterization of the extracts, performed by HPLC-DAD-ESIIMS, revealed the presence of five flavonoids (mainly qercetin-3-0- glucoside) and twelve phenolic acids (mainly 5-0-caffeolyquinic acid) for fennel extract and the presence of nine flavonoids (mainly luteolin-0-glucuronide) and ten phenolic acids (mainly di-caffeoyl-2,7- anhydro-3-deoxy-2-octulopyranosonic acid) for chamomile extract. Due to their high antioxidant and antimicrobial activities, both extracts were then incorporated (at DPPH scavenging activity EC25 value: 0.35 mg/mL and 0.165 mg/mL for fennel and chamomile, respectively) in cottage cheeses (prepared by Queijos Casa Matias Lda) as natural additives with two objectives: to increase the shelf-life of the cottage cheeses and to provide bioactive properties to the final products. The results showed that the use of these natural extracts did not alter significantly the nutritional characteristics of the cottage cheese in comparison with control samples (cottage cheese without extracts), but improved its antioxidant potential (more evident in the samples with chamomile extract). After 14 days of storage, only the control samples showed signs of degradation. Overall, the present study highlights the preservation potential of fennel and chamomile extracts in cottage cheeses, improving also their bioactivity.

Análise cromatográfica de um extrato fenólico de funcho utilizado no desenvolvimento de requeijões com propriedades funcionais

Atualmente, existe uma grande procura de alimentos com ingredientes naturais em substituição de aditivos sintéticos que têm sido associados, em determinadas circunstâncias, a alguns efeitos tóxicos [1]. Neste trabalho, preparou-se um extrato aquoso por decocção de Foeniculum vulgare Mill. (funcho) que, após caracterização por HPLC-DAD-ESI/MS, revelou a presença de cinco flavonoides (sendo o maioritário o quercetin-3-O-glucósido) e doze ácidos fenólicos (sendo o maioritário o ácido 5-Ocafeoilquínico). O mesmo extrato revelou um enorme potencial antioxidante (efeito captador de radicais livres DPPH, poder redutor e inibição da peroxidação lipídica) e antimicrobiano (contra bactérias como Salmonella typhimurium e Bacillus cereus, e fungos como Aspergillus niger, A. versicolor e Penicillium funiculosum), o que suscitou o seu potencial de utilização como ingrediente bioativo na funcionalização de alimentos. Assim, procedeu-se à sua incorporação (atendendo ao EC25 =0,35 mg/mL obtido no ensaio de DPPH) em requeijões (preparados na empresa Queijos Casa Matias Lda.). Os resultados mostraram que a presença do extrato não alterou significativamente as características nutricionais (incluindo macronutrientes, valor energético e perfil em ácidos gordos) das amostras controlo (requeijão sem esse ingrediente), no entanto parece aumentar o amarelecimento (parâmetro da cor, b*) após 7 dias de armazenamento. Verificou-se ainda que, após duas semanas de armazenamento apenas as amostras controlo apresentaram sinais de degradação. Além disso, conseguiu-se provar que a incorporação do extrato de funcho conferiu propriedades antioxidantes ao requeijão. Os resultados obtidos provam assim que o extrato fenólico obtido através da decocção de funcho pode ser utilizado como conservante e agente bioativo natural em requeijões.

Incidência de fungos em pós-colheita de banana 'Prata anã'(Musa AAB)

Este trabalho teve por objetivo determinar a ocorrência e a freqüência de fungos em banana 'Prata anã' e elucidar o agente causal das podridões em pós-colheita de frutos provenientes do norte de Minas Gerais. Dois métodos de isolamento foram adotados: diluição em placas, a partir da lavagem de frutos verdes, e direto de frutos maduros. Os fungos Colletotrichum musae, Trichoderma harzianum, Fusarium equisetii, Penicillium sp. Aspergillus parasiticus, Trichothecium roseum, Colletotrichum acutatum, Alternaria sp., Cladosporium musae e Curvularia lunata foram os mais freqüentemente associados aos frutos. A patogenicidade desses fungos foi testada pela substituição de discos da casca de frutos verdes por discos de micélio. Colletotrichum musae apresentou área média lesionada em torno do ponto de inoculação igual a 5,8 cm², enquanto para os demais fungos testados não passou de 1,50 cm². Os resultados mostraram que C. musae é o agente primário das podridões dos frutos examinados com 100 % de incidência e os demais fungos limitaram-se a necrosar os ferimentos em torno do ponto de inoculação. O modo de infecção latente, causada por C. musae, parece favorecer, primeiramente, a colonização interna dos tecidos e, posteriormente, a ação dos fungos oportunistas, que aceleram as podridões nos frutos e na coroa.

GC-MS Analysis, Antimicrobial and Antioxidant Activities of Extracts of the Aerial Parts of Conyza sumatrensis

Phytochemical analyses as well as antimicrobial and antioxidant activities of the extracts of C. sumatrensis aerial parts were investigated in this study. METHODS: The aerial parts of C. sumatrensis were air dried, weighed and exhaustively extracted with hexane, ethyl acetate and methanol successively. The crude extracts were screened for metabolites. These extracts of the plant were evaluated for antimicrobial and antioxidant activities using agar diffusion and DPPH method respectively. The extracts were also analysed using Gas chromatography – Mass spectrometry, and the chromatogram coupled with mass spectra of the compounds were matched with a standard library. RESULTS: Preliminary phytochemical investigation of crude n-hexane, ethyl acetate and methanol extracts of the aerial parts of Conyza sumatrensis revealed the presence of anthraquinones, flavonoids, terpenoids, phenolics, tannin, glycosides and carbohydrate. All the crude extracts gave a clear zone of inhibition against the growth of the test bacteria ( Staphylococcus aureus , Escherichia coli , Bacillus subtilis , Pseudomona aeruginosa, Salmonella typhi , Klebsiellae pneumonae ) at moderate to high concentrations, as well as test fungi ( Candida albicans , Aspergillus niger , penicillium notatum and Rhizopus stolonifer ) at high concentration. Methanolic extract exhibited significant radical scavenging property with IC50 of 17.08 μg/mL while n-hexane and ethyl acetate extracts showed no significant antioxidant activity. GC-MS of N-hexane extract showed a total number of eleven chemical constituents with α-Farnesene and spathulenol being the most abundance compounds constituting 20.27 and 22.28% of the extract respectively. Ethyl acetate extract revealed thirteen compounds with two most abundant compounds, cis-β-farnesene (16.64 %) and cis-pinane (21.09 %). While methanolic extract affords seventeen compounds with Ephytol being the most abundant compound (19.36 %).

Bioactivity, proximate, mineral and volatile profiles along the flowering stages of Opuntia microdasys (Lehm.): defining potential applications

Opuntia spp. flowers have been traditionally used for medical purposes, mostly because of their diversity in bioactive molecules with health promoting properties. The proximate, mineral and volatile compound profiles, together with the cytotoxic and antimicrobial properties were characterized in O. microdasys flowers at different maturity stages, revealing several statistically significant differences. O. microdasys stood out mainly for its high contents of dietary fiber, potassium and camphor, and its high activities against HCT15 cells, Staphylococcus aureus, Aspergillus versicolor and Penicillium funiculosum. The vegetative stage showed the highest cytotoxic and antifungal activities, whilst the full flowering stage was particularly active against bacterial species. The complete dataset has been classified by principal component analysis, achieving clearly identifiable groups for each flowering stage, elucidating also the most distinctive features, and comprehensively profiling each of the assayed stages. The results might be useful to define the best flowering stage considering practical application purposes.

Intoxicaciones por plantas y micotoxinas en rumiantes diagnosticadas en Uruguay

Resumen Las intoxicaciones por plantas y micotoxinas ocasionan importantes pérdidas económicas en países pecuarios, principalmente por muertes de animales y mermas en la producción. Este trabajo fue realizado con el propósito de brindar al veterinario que actúa en el diagnóstico de enfermedades de animales productivos, información breve y accesible sobre las intoxicaciones por plantas y micotoxinas diagnosticadas en rumiantes de Uruguay. Para llevar a cabo el mismo se utilizaron como fuentes bibliográficas: revistas científicas arbitradas, tesis de grado, libros y comunicaciones en congresos, jornadas nacionales e internacionales. Se encontraron 37 géneros y 45 especies de plantas tóxicas, distribuidos en 20 familias botánicas. En cuanto a los hongos toxicogénicos, se hallaron 13 especies pertenecientes a 9 géneros de 6 familias diferentes. Entre las plantas diagnosticadas, la familia Asteraceae (Compositae) cuenta con la mayor cantidad de especies tóxicas, seguida por Gramineae, Solanaceae y Fabaceae (Leguminoseae). Los principales géneros de hongos toxicogénicos Aspergillus, Penicillium, Fusarium y Claviceps han sido diagnosticados en el país. Desde el punto de vista patológico, las plantas y micotoxinas hepatotóxicas resultaron ser las más numerosas. La información presentada en las tablas, permitirá a los veterinarios un fácil abordaje en esta temática

Unravelling the nanostructure of strawberry fruit pectins by atomic force microscopy

Atomic force microscopy (AFM) allows the analysis of individual polymers at nanostructural level with a minimal sample preparation. This technique has been used to analyse the pectin disassembly process during the ripening and postharvest storage of several fleshy fruits. In general, pectins analysed by AFM are usually visualized as isolated chains, unbranched or with a low number of branchs and, occasionally, as large aggregates. However, the exact nature of these structures is unknown. It has been suggested that pectin aggregates represent a mixture of rhamnonogalacturonan I and homogalacturonan, while isolated chains and their branches are mainly composed by polygalacturonic acid. In order to gain insight into the nature of these structures, sodium carbonate soluble pectins from ripe strawberry (Fragaria x ananassa, Duch.) fruits were subjected to enzymatic digestion with endo-Polygalacturonase M2 from Aspergillus aculeatus, and the samples visualized by AFM at different time intervals. Pectins isolated from control, non-transformed plants, and two transgenic genotypes with low level of expression of ripening-induced pectinase genes encoding a polygalacturonase (APG) or a pectate lyase (APEL) were also included in this study. Before digestion, isolated pectin chains from control were shorter than those from transgenic fruits, showing number-average (LN) contour length values of 73.2 nm vs. 95.9 nm and 91.4 nm in APG and APEL, respectively. The percentage of branched polymers was significantly higher in APG polyuronides than in the remaining genotypes, 33% in APG vs. 6% in control and APEL. As a result of the endo-PG treatment, a gradual decrease in the main backbone length of isolated chains was observed in the three samples. The minimum LN value was reached after 8 h of digestion, being similar in the three genotypes, 22 nm. By contrast, the branches were not visible after 1.5-2 h of digestion. LN values were plotted against digestion time and the data fitted to a first-order exponential decay curve, obtaining R2 values higher than 0.9. The half digestion time calculated with these equations were similar for control and APG pectins, 1.7 h, but significantly higher in APEL, 2.5 h, indicating that these polymer chains were more resistant to endo-PG digestion. Regarding the pectin aggregates, their volumes were estimated and used to calculate LN molecular weights. Before digestion, control and APEL samples showed complexes of similar molecular weights, 1722 kDa, and slightly higher than those observed in APG samples. After endo-PG digestion, size of complexes diminished significantly, reaching similar values in the three pectin samples, around 650 kDa. These results suggest that isolated polymer chains visualized by AFM are formed by a HG domain linked to a shorter polymer resistant to endo-PG digestion, maybe xylogalacturonan or RG-I. The silencing of the pectate lyase gene slightly modified the structure and/or chemical composition of polymer chains making these polyuronides more resistant to enzymatic degradation. Similarly, polygalacturonic acid is one of the main component of the aggregates.

UNDERSTANDING LATE SEASON FRUIT ROT PATHOSYSTEMS AND INSECT INTERACTIONS IN MID-ATLANTIC VINEYARDS

Fungal fruit rots and insect pests are among the most important problems negatively affecting the yield and quality of mid-Atlantic wine. In pathogenicity trials of fungi recovered from diseased Chardonnay and Vidal blanc grapes, Alternaria alternata, Pestalotiopsis telopeae, and Aspergillus japonicus were found to be unreported fruit rot pathogens in the region. Additionally, P. telopeae and A. japonicus had comparable virulence to the region’s common fruit rot pathogens. Furthermore, a timed-exclusion field study was implemented to evaluate vineyard insect-fruit rot relationships. It was found that clusters exposed to early-season insect communities that included Paralobesia viteana had a significantly greater incidence of sour rot than clusters protected from insects all season. These results were contrary to the current assumption that fall insects are the primary drivers of sour rot in the region. This research provides diagnostic tools and information to develop management-strategies against fungal and insect pests for mid-Atlantic grape growers.

Aspergiloma pulmonar : A propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Avaliação da atividade fungicida de óleos essenciais e suas substâncias ativas no controlo de fungos de armazenamento

Mestrado em Engenharia Agronómica - Proteção da Plantas - Instituto Superior de Agronomia - UL

Controle biológico de fungos aflatoxigênicos.

2009

Enzyme Optimization for Lignocellulose Hydrolysis using Mechanistic Modeling

A novel mechanistic model for the saccharification of cellulose and hemicellulose is utilized to predict the products of hydrolysis over a range of enzyme loadings and times. The mechanistic model considers the morphology of the substrate and the kinetics of enzymes to optimize enzyme concentrations for the enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Substrates are modeled based on their fraction of accessible sites, glucan content, xylan content, and degree of polymerizations. This enzyme optimization model takes into account the kinetics of six core enzymes for lignocellulose hydrolysis: endoglucanase I (EG1), cellobiohydrolase I (CBH1), cellobiohydrolase II (CBH2), and endo-xylanase (EX) from Trichoderma reesei; β-glucosidase (BG), and β-xylosidase (BX) from Aspergillus niger. The model employs the synergistic action of these enzymes to predict optimum enzyme concentrations for hydrolysis of Avicel and ammonia fiber explosion (AFEX) pretreated corn stover. Glucan, glucan + xylan, glucose and glucose + xylose conversion predictions are given over a range of mass fractions of enzymes, and a range of enzyme loadings. Simulation results are compared with optimizations using statistically designed experiments. BG and BX are modeled in solution at later time points to predict the effect on glucose conversion and xylose conversion.