Aflatoxigenic Fungi and Aflatoxins in Portuguese Almonds

Aflatoxin contamination of nuts is an increasing concern to the consumer's 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

Identification of aflatoxigenic and non‐aflatoxigenic strains of Aspergillus section Flavi isolated from Portuguese almonds

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.Fundação para a Ciência e a Tecnologia (FCT) - bolsa SFRH/BD/28332/200

Effect of γ-radiation in the Survival of Aspergillus parasiticus in Chestnuts

Castanea sativa produces the European chestnuts, also known as sweet chestnuts. C. sativa is adapted to regions with humid and temperate to cold climate, and does not withstand long hot and dry periods. Chestnuts are a seasonal product, being Portugal the fourth country in terms of worldwide chestnut production. After harvest and during storage, problems with deterioration may arise, mainly by plagues or microorganisms, such as the development of fungi. Fewer studies have been devoted to determining fungal contamination of chestnuts from the main producing countries (Turkey, Bolivia, Italy and Portugal). In fact, most of those studies are relative to marketed chestnuts with unknown origin. Reports on chestnuts marketed (origin not reported) in cold and humid countries, like Canada and Switzerland refer to contaminations strongly dominated by Penicillium spp., with Aspergillus spp. being of no significance, while studies from drier and warmer regions, like Georgia, USA and Ar’Ar, Saudi Arabia, report important incidences of Aspergillus (sections Wentii, Flavi and, to a lesser extent, Nigri) [1]. Currently, the safety of the chestnuts chain is based on good manufacturing practices. One of the few processing steps in this industry is the washing of chestnuts in hot water. After washing and before commercialization, a chemical treatment by fumigation with methyl bromide is required. However, recently, this treatment was banned from European Union. The hot water treatment may not be enough to guarantee the safety of chestnuts and, as a side effect, a detrimental effect on some quality traits may be observed. So, an alternative for safe processing of chestnuts is needed. The irradiation is one promising alternative for chestnuts treatment. This method is more environmental friendly and could be more effective in microorganisms destruction; however, studies that confirm the efficacy of irradiation (both in terms of safety and of sensorial traits) are needed. In this study, the use of irradiation with γ-rays as an inactivation agent against one of the most ubiquitous and mycotoxigenic fungi – Aspergillus parasiticus – was studied. Inactivation of filamentous fungi was evaluated by exposing chestnuts to known levels of γ-rays (0.25 kGγ; 0.5 kGγ; 3.0 kGγ and 10.0 kGγ). Chestnuts were previously inoculated with a spore suspension of one strain of A. parasiticus. After irradiation, chestnuts were washed with peptone (0.1%), being the washing solution spread in MEA10 and DG18 plates for yeasts, filamentous fungi and A. parasiticus counting. The growth of colonies was observed after 4 days. In general, the higher the level of irradiation the lower the survival rate. Regarding yeast, at the two lower irradiation levels, no effect on the yeast load was observed, while a slight decrease was observed at 3.0 kGγ and no growth observed at 10.0 kGγ. For filamentous fungi (and particularly for A. parasiticus) a similar trend was observed, although a not significant higher resistance of filamentous fungi (including A. parasiticus) to irradiation was observed.ON.2 -O Novo Norte (Programa Operacional do Norte) and the Portuguese Government, financed by European Regional Development Fund (ERDF) under the project CHESTNUTSRAD/1319

Anti-N-Methyl-D-Aspartate Receptor Encephalitis with Positive Serum Antithyroid Antibodies, IgM Antibodies Against Mycoplasma Pneumoniae and Human Herpesvirus 7 PCR in the CSF

We report the case of a boy with an encephalopathy associated with extrapyramidal and psychiatric symptoms and anti-N-methyl-D-aspartate receptor antibodies. He had positive serum antithyroid antibodies, IgM antibodies against Mycoplasma pneumoniae and human herpesvirus 7 polymerase chain reaction in the cerebrospinal fluid. He was successfully treated with rituximab, after steroids, intravenous immunoglobulin and plasma exchange. The pathophysiology of this disorder may be post-infectious and autoimmune

MYCOTOX-PALOP - Multi-actor partnership for the risk assessment of mycotoxins along the food chain in African Portuguese speaking countries (PALOP)

The health impact of mycotoxin exposure is grossly underreported in Angola (AN) and Mozambique (MZ) due to the lack of coordinated monitoring and medical surveillance, and its control is inadequately addressed. Due to inherent climatic, agricultural and political conditions, these countries are subjected to major food security issues, which could be partially mitigated by increasing the awareness to crop losses due to fungal and mycotoxin contamination. The aims of the project are to: gather knowledge on food and feed fungal losses and mycotoxin contamination in MZ and AN; set mycotoxin risk assessment programmes; and establish intervention strategies to reduce human exposure to mycotoxins and their negative impacts, by means of safe and efficient intervention strategies. Access, training and extension actions at the scientific, technical and community levels will be strongly promoted to build human and technical capacity, bridging the gap between research and the various stakeholders, including farmers, retailers, trading companies, and regulatory agencies. Integrated actions and policies will expectedly be adopted, in a way of contributing to the improvement of the governance policies and programmes. This project finally aims at contributing to long-term benefits for citizens, economy and society, as it meets important Development Goals set by the UN 2030 Agenda for Sustainable Development, in particular Goal 2 End hunger, achieve food security and improved nutrition, and promote sustainable agriculture. MYCOTOX-PALOP builds on the scientific, technical and field expertise of a joint task force including CIMO-IPB and CEB-UMinho (Portugal), ISPKS (AN) and UEM (MZ).Fundação para a Ciência e Tecnologia (FCT)Aga-Khan Development Networkinfo:eu-repo/semantics/publishedVersio

Polyphasic approach including MALDI-TOF mass spectrometry to characterise aflatoxigenic species of Aspergillus section flavi

Aflatoxins are toxic compounds which are produced as secondary metabolites by the fungi Aspergillus flavus, A. parasiticus and A. nomius growing on a variety of food products and are known to be carcinogenic, mutagenic, teratogenic and immunosuppressive1,2. Aspergillus is a large genus, with a complex taxonomy. The genus is easily identified by its characteristic conidiophore, but species identification and differentiation is complex, mainly because it is traditionally based on a range of morphological features. One includes the aflatoxigenic species referred above 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. Species from A. flavus group are morphologically and genetically very similar, and are therefore difficult to differentiate by both cultural and molecular methods. Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry has already shown high potentialities in discriminating very closely related taxa. In this work is presented a polyphasic approach including MALDI-TOF MS to discriminate A. flavus group strains.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28332/200

Worldwide interlaboratory study on the determination of ochratoxin A in different wine type samples

Interlaboratory studies are decisive tools to help the validation of a specific analytical methodology or to assess the reproducibility of the use of different methods to analyze a given compound or compounds in certain sample matrices. In this work, homogeneous samples of two white wines (“White Wine” and “White Liqueur Wine”) and one red wine (“Red Fortified Wine”) from Portugal with different production techniques and characteristics, namely in alcohol strength (10.5%, 16.0% and 19.0% ethanolic content, respectively), were analyzed for their contents in ochratoxin A (OTA), a mycotoxin generated from fungal contamination. White Liqueur Wine was naturally contaminated, whereas the other two wine type were spiked with ethanolic OTA solutions. The participation of 24 laboratories from 17 countries of five continents was ensured for this study. Although with no restrictions in terms of analytical methodology to employ, 75% of the laboratories resorted to immunoaffinity columns clean-up followed by high performance liquid chromatography with fluorescence detection (HPLC-FD), most of them in accordance with the European Standard EN 14133. For White Wine samples, the general mean OTA concentration was 1.96 μg/l (two outliers) with interlaboratorial standard deviation (sL) of 0.53 μg/l; for White Liqueur Wine, mean of 1.59 μg/l (one outlier), with sL = 0.59 μg/l; and for Red Fortified Wine, mean of 2.73 μg/l (no outliers), with sL = 0.96 μg/l. Outliers were determined by Cochran and Grubbs tests. The Horrat index, recommended by the Association of Official Analytical Chemists (AOAC) for the quality assurance of the collaborative study was, on average, 1.7. This study proved that OTA determination in wines is reproducible, regardless of the methodology employed.INIAP (Portugal), through the Program AGRO