CARACTERIZAÇÃO DA MICOFLORA E DO PERFIL DE MICOTOXINAS NA AMÊNDOA E CASTANHA DE TRÁS-OS-MONTES DESDE A PRODUÇÃO À COMERCIALIZAÇÃO

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Publications

MALDI-TOF ICMS as a modern approach to identify potential aflatoxigenic fungi

Publication . Rodrigues, Paula; Santos, Cledir; Venâncio, Armando; Lima, Nelson

The Aspergillus section Flavi is among the best studied fungi, having different commercial applications, but also causing biodeterioration of commodities and food spoilage. Fungi from this Section are also responsible for the production of highly toxic secondary metabolites – the aflatoxins. They are morphologically and genetically very similar, and can be difficult to differentiate by both cultural and molecular biology methods. Besides that, new species are continuously being described in this Section. A reliable identification typically implies the analyses of a variety of morphological, biochemical and molecular traits. Recently, Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) has been used to generate spectra of protein masses in a range of 2,000 to 20,000 Da that are a taxa specific fingerprinting. This technique has already shown high potentialities to discriminate very closely related taxa and, it has been used as a new tool in the polyphasic approach to identify potential aflatoxigenic fungi. Aim: This work aims to validate the MALDI-TOF ICMS technique on Aspergillus Section Flavi identification. As a matter of consequence, obtained results by spectral analysis were compared to those obtained by morphological, biochemical and molecular biology methods. Materials and Methods: 1. Morphological analysis: fungi were cultured on three different media (Malt Extract Agar [MEA], Czapek Yeast Autolysate [CYA] and CYA supplemented with 20% sucrose [CY20S]); 2. Biochemical analysis: Aflatoxins and Cyclopiazonic Acid analyses were performed by HPLC; 3. Molecular biology analysis: Partial calmodulin gene was sequenced; 4. MALDI-TOF ICMS analysis: spectra of protein masses, on 2,5-dihydroxybenzoic acid (DHB) in a range of 2,000 to 20,000 Da, were obtained using Shimadzu Axima-LNR equipment and treated for fungal identification using SARAMISTM Package. Results and Discussion: 1. A good agreement between methods on species level identification was obtained; 2. Molecular biology and spectral data analyses generated similar dendrograms with concomitant strains clustering; 3. Under the experimental conditions used spectral analyses were able to identify potential aflatoxigenic species. Conclusion: MALDI-TOF ICMS has shown a very good resolution on the identification of Aspergillus Section Flavi species. Results obtained with MALDI‐TOF ICMS were similar to those obtained by DNA sequence analysis, with the advantage of being (a) rapid, (b) inexpensive in terms of labour and consumables, and (c) reliable when compared with other biological techniques. Using MALDI‐TOF ICMS the results showed a great potential to the fungal identification and it is another additional step for our polyphasic fungal identification approach. However, even with the polyphasic approach fungal identifications remain in some situations time‐consuming and decisions regarding what represents a species tend to be subjective.

2010Conference object

Open access

Identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almonds

Publication . Rodrigues, Paula; Venâncio, Armando; Kozakiewicz, Zofia; Lima, Nelson

Aspergillus subgenus Circumdati section Flavi, also referred to as the A. flavus group, has attracted worldwide attention for its industrial use and toxigenic potential. Section Flavi is divided in two groups of species. One includes the aflatoxigenic species A. flavus, A. parasiticus and A. nomius, which cause serious problems in agricultural commodities, and the other one includes the non‐aflatoxigenic species A. oryzae, A. sojae and A. tamarii, traditionally used for production of fermented foods. Differentiating aflatoxigenic from non‐aflatoxigenic species and strains in food commodities is of major importance in food quality control. 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, we found two distinct groups among the population under study: 58% were classified as A. parasiticus and the remaining 42% were classified as A. flavus. Chemical characterization involved the screening of the isolates for aflatoxins B (AFB) and G (AFG), and also for cyclopiazonic acid (CPA), by HPLC. All A. parasiticus isolates were strong AFB and AFG producers, but no CPA production was detected. 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. 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.

2009Conference object

Open access

Characterization and identification of aspergillus section flavi isolates from portuguese almonds using a polyphasic approach including MALDI-TOF ICMS

Publication . Rodrigues, Paula; Santos, Cledir; Venâncio, Armando; Lima, Nelson

Aspergillus is a large genus, with a complex and ever evolving taxonomy. Section Flavi is one of the most significant sections in the genus, and is one of the best studied among fungi, for the numerous industrial applications as well as for food safety issues. Section Flavi is composed of a large number of very closely related species. While these species are difficult to differentiate morphologically and even genetically, they differ in a characteristic that is of paramount importance for food safety, as some are responsible for the production of the highly toxigenic aflatoxins. Taxonomy and species identification are therefore subject of great interest for scientists aiming to clarify the species concept and limits within the section. In this sense, the establishment of schemes for species and for aflatoxigenic strains identification that are simultaneously accurate, sensitive, robust and expedite is mandatory. At present, reliable identification schemes typically imply the analysis of a wide variety of morphological, biochemical and molecular traits. Recently, Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) has been used to generate spectra of protein masses in a range of 2,000 to 20,000 Da, which result in a taxa specific fingerprint. This technique has already shown high potentialities to discriminate very closely related taxa, but has rarely been used in fungal species identification, either on its one or as part of a polyphasic scheme of identification. This work aimed to: i) characterize the population of Aspergillus section Flavi collected from Portuguese almonds in relation to their aflatoxigenic potential; ii) identify the isolates by applying a set of morphological, biochemical, molecular and spectral analyses (polyphasic approach); iii) compare the data obtained from the various approaches in terms of sensitivity, reliability and user-friendliness; and iii) determine the validity of MALDI-TOF technique for the identification of closely related field isolates of section Flavi.

2010Conference object

Open access

Characterisation of mycoflora and aflatoxigenic fungi from portuguese almonds: from production to commercialization

Publication . Rodrigues, Paula; Venâncio, Armando; Lima, Nelson

Aflatoxins are among the most carcinogenic natural compounds known, and have been frequently detected in numerous agricultural commodities, nut fruits being among the most contaminated ones. These toxins are a result of the secondary metabolism of fungi belonging to the genus Aspergillus Section Flavi, namelly Aspergillus flavus, A. parasiticus and A. nomius. The early detection of aflatoxigenic fungal contamination and the preventive control of aflatoxin production seems to be the only viable way to respond to the rigorous limits imposed by EU legislation. In Portugal, knowledge on mycoflora and mycotoxigenic profiles of almonds is scarce. Our objective was to characterise portuguese almonds in respect to mycoflora, with strong emphasis on aflatoxigenic fungi, from production to commercialization. Various samples of almonds were collected from the field, as well as during storage and processing. Environmental data have also been recorded. Samples were plated directly on MEA+10% NaCl and all fungi belonging to the genus Aspergillus isolated and identified to the group or species level. All Aspergillus belonging to Section Flavi were further characterised based on their mycotoxigenic profile, with the aim of identifying aflatoxigenic and non-aflatoxigenic strains. Fungal diversity and relative frequencies of Aspergillus spp. were determined for the various samples and correlated with environmental conditions. We have observed a progressive reduction on fungal diversity of almonds from the field to the end of processing, as well as an increase of the frequency of more xerophilic fungi, including Aspergillus Section Flavi. Concerning the latter, we report that Aspergillus parasiticus are predominantly aflatoxigenic and Aspergillus flavus are predominantly non-aflatoxigenic. Other less common species have also been identified.

2009Conference object

Open access

Incidence and diversity of the fungal genera Aspergillus and Penicillium in Portuguese almonds and chestnuts

Publication . Rodrigues, Paula; Venâncio, Armando; Lima, Nelson

Almonds (Prunus dulcis (Miller) D.A. Webb) and European (sweet) chestnuts (Castanea sativa Miller) are of great economic and social impact in Mediterranean countries, and some areas they constitute the main income of rural populations. Despite all efforts to control fungal contamination, toxigenic fungi are ubiquitous in nature and occur regularly in worldwide food supplies, and these nuts are no exception. This work aimed to provide knowledge on the general mycobiota of Portuguese almonds and chestnuts, and its evolution from field to the end of storage. For this matter, 45 field chestnut samples and 36 almond samples (30 field samples and 6 storage samples) were collected in Trás-os-Montes, Portugal. All fungi belonging to genus Aspergillus were isolated and identified to the section level. Fungi representative of other genera were identified to the genus level. In the field, chestnuts were mainly contaminated with the genera Fusarium, Cladosporium, Alternaria and Penicillium, and the genus Aspergillus was only rarely found, whereas almonds were more contaminated with Aspergillus. In almonds, Aspergillus incidence increased significantly from field to the end of storage, but diversity decreased, with potentially toxigenic isolates belonging to sections Flavi and Nigri becoming more significant and widespread throughout storage. These fungi were determined to be moderately associated, which can be indicative of mycotoxin co-contamination problems if adequate storage conditions are not secured.

2013Journal article

Open access