Armando Venâncio

A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almonds

A polyphasic approach consisting of morphological, chemical and molecular characterization was applied to 31 isolates of Aspergillus Section Flavi originating from Portuguese almonds, with the aim of characterizing and identifying aflatoxigenic and non-aflatoxigenic strains. On the basis of morphological characters (mainly colony color on Czapek-Dox agar and conidia morphology), we found two distinct groups among the population under study: 18 isolates (58%) had dark-green colonies and rough conidia, and were classified as Aspergillus parasiticus; the remaining 13 isolates (42%) had yellow-green colonies and smooth to finely rough globose conidia, and were classified as Aspergillus flavus. Chemical characterization involved the screening of the isolates for aflatoxins B (AFB) and G (AFG), and also for cyclopiazonic acid (CPA), by HPLC with fluorescence and UV detection, respectively. All A. parasiticus isolates were strong AFB and AFG producers, but no CPA production was detected, showing a consistent mycotoxigenic pattern. The A. flavus isolates showed to be more diversified, with 77% being atoxigenic, whereas 15% produced CPA and low levels of AFB and 8% produced the 3 groups of mycotoxins. Aflatoxin production was also screened on Coconut Agar Medium (CAM), and the results were consistent with the HPLC analysis. Sclerotia production showed no correlation to aflatoxigenicity. Molecularly, two genes of the aflatoxin biosynthetic pathway, aflD (=nor1) and aflQ (=ord1=ordA) were tested for presence and expression (by PCR and RT-PCR, respectively). The presence of both genes did not correlate with aflatoxigenicity. aflD expression was not considered a good marker for differentiating aflatoxigenic from non-aflatoxigenic isolates, but aflQ showed a good correlation between expression and aflatoxin-production ability.

A practical approach for identifications based on mycotoxin characters of Penicillium

The taxonomy of the penicillia is unstable particularly in the important antibiotic and mycotoxin-producing subgenus Penicillium. There are difficulties relating identifications to mycotoxin production. Also, the validity of dual nomenclature for pleomorphic fungi is under discussion increasingly. Patulin is an important mycotoxin produced by various fungi and has strict limits in the European Union. The mycotoxin and/or the isoepoxydon dehydrogenase (IDH) gene of the metabolic pathway have been assessed in 318 strains predominately of subgenus Penicillium. These data were used to classify the isolates. Subgenus Penicillium contained most of the IDH and patulin positives. The species and varieties in subgenus Penicillium which were associated with patulin detection can be reduced to one name, viz. Penicillium Pen p+ (p = patulin). This has been extended to other mycotoxin producing penicillia to indicate the scope of the scheme. The classification will lead to the number of taxa being reduced, while avoiding species names and hence dual nomenclature. Culture independent analysis of environmental samples is mentioned. The scheme could be used with advantage for other fungi.

Interaction with Penicillium expansum enhances Botrytis cinerea growth in grape juice medium and prevents patulin accumulation in vitro.

Interactions between fungi occur when they grow on the same host plant. This is the case of Botrytis cinerea and Penicillium expansum on grape. P. expansum is also responsible for production of the mycotoxin patulin. In this study, the influence of the interaction between both fungi on fungal growth parameters was studied as well as the effect on the accumulation of patulin by P. expansum. For that purpose, spores of B. cinerea and P. expansum were inoculated together (mixed inoculum), and the parameters growth rate, time for growth and patulin accumulation were assessed. The presence of P. expansum conidia shortened the time for growth of mixed inoculum colonies which, at the end of incubation, were B. cinerea‐like. Although some P. expansum growth was observed in mixed inoculum colonies, very low levels of patulin were observed. In assays carried out in patulin‐spiked medium, B. cinerea was capable to metabolize the mycotoxin. The capabilities of B. cinerea to shorten time for growth and prevent patulin accumulation are competing abilities that facilitate grape colonization.

Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes

To evaluate the incidence of fungi producing ochratoxin A (OA) in Portuguese wine grapes, a survey was conducted in 11 vineyards, from four winemaking regions each with distinct climatic conditions. From setting to the harvesting period, a total of 1,650 berries were sampled by plating methods. Out of 370 aspergilli and 301 Penicillium strains isolated, 14% of the aspergilli were OA-producing strains. None of the penicillia were OA-producing strains. The black aspergilli were predominant (90%). All Aspergillus strains were tested in vitro for OA production and all were preserved in the Micoteca da Universidade do Minho (MUM) culture collection. Most of the Aspergillus carbonarius (97%) and 4% of the Aspergillus niger aggregate strains were OA producers. Almost all ochratoxigenic strains were isolated at harvest time, mainly in the regions with a Mediterranean climate. In the vineyards sampled, the percentage of colonized berries with ochratoxigenic strains was up to 38%. The vineyards from the region with Atlantic influences, with high rainfall, exhibited the lowest occurrence of Aspergillus and ochratoxigenic strains, 0% to 10% and 0% to 2% colonized berries, respectively. Data obtained here supports the hypothesis that A. carbonarius and occasionally A. niger, are the main producers of OA in grapes. In this study, the highest incidence of these fungi occurred in vineyards with a Mediterranean climate.

Filamentous fungal characterizations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight intact cell mass spectrometry (MALDI‐TOF ICMS) is coming of age for the identification and characterization of fungi. The procedure has been used extensively with bacteria. UV‐absorbing matrices function as energy mediators that transfer the absorbed photoenergy from an irradiation source to the surrounding sample molecules, resulting in minimum fragmentation. A surprisingly high number of fungal groups have been studied: (i) the terverticillate penicillia, (ii) aflatoxigenic, black and other aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting fungi (e.g. Serpula lacrymans) and (v) dermatophytes. The technique has been suggested for optimizing quality control of fungal Chinese medicines (e.g. Cordyceps). MALDI‐TOF ICMS offers advantages over PCR. The method is now used in taxonomic assessments (e.g. Trichoderma) as distinct from only strain characterization. Low and high molecular mass natural products (e.g. peptaibols) can be analysed. The procedure is rapid and requires minimal pretreatment. However, issues of reproducibility need to be addressed further in terms of strains of species tested and between run variability. More studies into the capabilities of MALDI‐TOF ICMS to identify fungi are required.

Mycotoxin production from fungi isolated from grapes

Aims: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes.

Biodegradation of ochratoxin a for food and feed decontamination.

Ochratoxin A (OTA) is one of the most important mycotoxins that is found in food and feed products. It has proven toxic properties, being primarily known for its nephrotoxicity and carcinogenicity to certain animal species. OTA is produced by several species of Aspergillus and Penicillium that can be found in a wide variety of agricultural products, which makes the presence of OTA in these products common. Many countries have statutory limits for OTA, and concentrations need to be reduced to as low as technologically possible in food and feed. The most important measures to be taken to control OTA are preventive in order to avoid fungal growth and OTA production. However, these measures are difficult to implement in all cases with the consequence of OTA remaining in agricultural commodities. Remediation processes are often used to eliminate, reduce or avoid the toxic effects of OTA. Biological methods have been considered increasingly as an alternative to physical and chemical treatments. However, examples of practical applications are infrequent. This review will focus on the (i) known microorganisms and enzymes that are able to biodegrade OTA; (ii) mode of action of biodegradation and (iii) current applications. A critical discussion about the technical applicability of these strategies is presented.

Aflatoxigenic fungi and aflatoxins in Portuguese almonds

Aflatoxin contamination of nuts is an increasing concern to the consumers health. Portugal is a big producer of almonds, but there is no scientific knowledge on the safety of those nuts, in terms of mycotoxins. The aim of this paper was to study the incidence of aflatoxigenic fungi and aflatoxin contamination of 21 samples of Portuguese almonds, and its evolution throughout the various stages of production. All fungi belonging to Aspergillus section Flavi were identified and tested for their aflatoxigenic ability. Almond samples were tested for aflatoxin contamination by HPLC-fluorescence. In total, 352 fungi belonging to Aspergillus section Flavi were isolated from Portuguese almonds: 127 were identified as A. flavus (of which 28% produced aflatoxins B), 196 as typical or atypical A. parasiticus (all producing aflatoxins B and G), and 29 as A. tamarii (all nonaflatoxigenic). Aflatoxins were detected in only one sample at 4.97 μg/kg.

Reactivity of human salivary proteins families toward food polyphenols.

Tannins are well-known food polyphenols that interact with proteins, namely, salivary proteins. This interaction is an important factor in relation to their bioavailability and is considered the basis of several important properties of tannins, namely, the development of astringency. It has been generally accepted that astringency is due to the tannin-induced complexation and/or precipitation of salivary proline-rich proteins (PRPs) in the oral cavity. However, this complexation is thought to provide protection against dietary tannins. Neverthless, there is no concrete evidence and agreement about which PRP families (acidic, basic, and glycosylated) are responsible for the interaction with condensed tannins. In the present work, human saliva was isolated, and the proteins existing in saliva were characterized by chromatographic and proteomic approaches (HPLC-DAD, ESI-MS, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and MALDI-TOF). These approaches were also adapted to study the affinity of the different families of salivary proteins to condensed tannins by the interaction of saliva with grape seed procyanidins. The results obtained when all the main families of salivary proteins are present in a competitive assay, like in the oral cavity, demonstrate that condensed tannins interact first with acidic PRPs and statherin and thereafter with histatins, glycosylated PRPs, and bPRPs.

The Condensation of Salicylaldehydes and Malononitrile Revisited: Synthesis of New Dimeric Chromene Derivatives

The reaction of salicylic aldehydes with malononitrile was reinvestigated, and the reaction pathway was followed by 1H NMR spectroscopy. A delicate control of the experimental conditions allowed the synthesis of 2-imino-2H-chromene-3-carbonitriles 1, (2-amino-3-cyano-4H-chromen-4-yl)malononitriles 2, 4-amino-5-imino-2,7-dimethoxy-5H-chromeno[3,4-c]pyridine-1-carbonitrile 12, and (4,5-diamino-1-cyano-1,10b-dihydro-2H-chromeno[3,4-c]pyridin-2-ylidene)malononitrile 13. Two novel 2-iminochromene dimers, with structures 8 and 9, were isolated and fully characterized. The activity of compound 8a on Aspergillus spp. growth and on ochratoxin A production was evaluated. The results of the bioassays indicate that compound 8a, applied at concentrations of 2 mM, totally inhibited the growth of the fungi tested. Ochratoxin A production by Aspergillus alliaceus was reduced by about 93% with a 200 microM solution of this compound. A moderate inhibitory effect was observed for the analogous structure 8b, and no inhibition was registered for compounds 2 and 1, used as synthetic precursors of the dimeric species 8.