Mycotoxigenic fungi in Portuguese wine regions : fumonisin B2 and ochratoxin A

Studies were conducted between 2001 and 2003 to evaluate the incidence of ochratoxigenic strains in Portuguese wine-grapes. At that time, the main responsible for ochratoxin A production in grapes were found to be Aspergillus carbonarius with a production mean of 1.13 mg/kg and some strains from the Aspergillus niger aggregate with a production mean of 0.14 mg/kg. At that time also, all the black aspergilli isolates were preserved in glycerol and stored at -80 ºC. Since recently A. niger was reported as a producer of the mycotoxin fumonisin B2, its levels produced by black aspergilli isolates from the Portuguese grapes were investigated. Approximately 29% of A. niger aggregate strains were found to produce it between 0.003–6.0 mg/kg with a mean of 0.66 mg/kg and only 6% produced more than 1 mg/kg. The incidence and distribution of fumonisinogenic strains among five Portuguese wine-regions is also presented and correlated with data of the ochratoxigenic ones.A incidência em Portugal de fungos filamentosos produtores de ocratoxina A em uvas para a produção de vinho foi avaliada entre os anos de 2001 a 2003. Nessa altura, o produtor mais frequente nas uvas foi a espécie Aspergillus carbonarius, com uma produção média em laboratório de 1,13 mg/kg, e algumas estirpes do agregado Aspergillus niger, com uma produção média em laboratório de 0,14 mg/kg. Todas as estirpes de Aspergillus negros então isoladas foram conservadas a -80 ºC. Recentemente, estirpes de Aspergillus niger foram descritas como produtores de fumonisina B2, pelo que a produção desta outra micotoxina foi verificada nas cerca de 700 estirpes conservadas. Aproximadamente 29% das estirpes do agregado A. niger produziram fumonisina B2, com produção em laboratório entre 0,003 a 6,0 mg/kg, sendo que apenas 6% produziram mais de 1 mg/kg. A incidência e a distribuição destas estirpes micotoxigénicas será apresentada e discutida.Fundação para a Ciência e a Tecnologia (FCT) - bolsa SFRH/BPD/43922/200

Utilização da extração em fase sólida seguida de análise por GC-MS na caracterização aromática de uvas da casta Alvarinho

Uma das características mais significativas dos vinhos é o aroma[1]. De acordo com a sequência biotecnológica empregue na elaboração do vinho, o aroma está geralmente dividido em três grupos: aroma primário, que engloba o varietal e o préfermentativo, aroma secundário ou fermentativo e aroma terciário ou pós-fermentativo[1]. Embora seja elevado o número de constituintes voláteis de um vinho passíveis de contribuir para o seu aroma, a tipicidade aromática é principalmente devida aos compostos que procedem das uvas, isto é os compostos de origem varietal. O aroma varietal é constituído por substâncias voláteis presentes numa forma livre, odorífera, e na forma de precursores glicoconjugados, inodoros; estes precursores são suscetíveis de ser hidrolisados durante todas as etapas, desde a produção à abertura da garrafa, originando compostos voláteis. Vários estudos têm sido desenvolvidos no sentido de verificar quais os compostos voláteis presentes em diversas variedades de uva e de que modo estes compostos permanecem, desaparecem ou se transformam durante a vinificação, a conservação e o envelhecimento dos vinhos. O presente trabalho, inserido no âmbito do projeto AGROCONTROL, co-financiado pelo “ON.2 – O Novo Norte” e QREN através do Fundo Europeu de Desenvolvimento Regional (FEDER), teve como objetivo a identificação e quantificação dos compostos voláteis de amostras de uvas da casta Alvarinho da colheita de 2011[2-4]. As amostras de uvas deram origem a sumos por trituração num liquidificador, dos quais foram extraídos os compostos voláteis na forma livre e na forma de glicoconjugados, por extração em fase sólida (SPE – Solid Phase Extraction), obtendo-se extratos que foram analisados por GC-MS (GasChromatography-Mass Spectrometry). A extração foi realizada pela passagem de 100 mL de sumo de uva por cartuxos de Lichrolute® EN (Merck), sendo, posteriormente, os compostos aromáticos da fração livre eluídos com 5 mL de mistura azeotrópica pentano-diclorometano (2:1) e os precursores glicoconjugados extraídos com 7 mL de acetato de etilo. Os compostos glicoconjugados foram depois hidrolisados enzimaticamente, com um preparado enzimático comercial (AR 2000), de modo a ser libertados as agliconas aromáticas. A determinação, semi-quantitativa, foi efetuada pelo método do padrão interno, usando 4-nonanol para o efeito. . A identificação dos compostos voláteis foi realizada por comparação dos índices de retenção e dos espectros de massas dos compostos com os de substâncias de referência puras. Os ensaios foram realizados em triplicado. Os resultados obtidos foram comparados com os resultados anteriormente descritos para a mesma casta[5]. Verificou-se que, para os compostos terpénicos analisados, quer na fração livre, quer na fração glicoconjugada, o perfil de compostos terpénicos foi semelhante ao descrito[5]. Os resultados obtidos mostraram uma boa repetibilidade do método utilizado. Este trabalho demonstra a grande aplicabilidade das técnicas analíticas como SPE e GC-MS em áreas como a vinicultura e a enologia

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

Gamma irradiation in ochratoxin A control

ICFC 2017 - International Conference on Food Contaminants (Book of Abstracts)Ochratoxin A (OTA) is one of the main mycotoxins found in food. Gamma radiation is used for preserving foods since it inactivates pathogens. The effect of irradiation on mycotoxins has been studied but results are contradictory. The different matrices and conditions used by several authors may have influenced reported results. The aim of this work was to study the effects of gamma radiation in OTA in order to evaluate its applicability in foods. OTA was irradiated in its dry form, in aqueous and in methanolic solutions to evaluate the water role in OTA irradiation. Then, OTA contaminated food matrices, such as wheat flour, grape juice and wine, were irradiated.In aqueous solutions, high degradation of OTA (≥90%) was obtained with gamma radiation doses as low as 2.5kGy. However, in dried samples, OTA was found extremely resistant to radiation doses of up to 10kGy. In this case, water was a determinant factor for the effectiveness of irradiation process. In the assays with food matrices, radiation doses greater than 10kGy were needed to achieve higher reductions of OTA, being eliminated just 24%. It was also observed that OTA elimination was higher in wheat flour with higher moisture contents but the elimination of OTA in grape juice and wine were not higher than those observed in wheat flour. It is concluded that OTA is very sensitive to irradiation in water solutions but resistant in its dry form and in food matrices. Due to the low elimination percentage observed, it can be considered that gamma radiation is not a suitable technology for the elimination of OTA from foods.Thalita Calado and Luís Abrunhosa received support by grants SFRH/BD/79364/2011 and UMINHO/BPD/51/2015 from FCT, respectively. CEB gratefully acknowledge FCT support through projects UID/BIO/04469/2013, NORTE-01-0145-FEDER-000004, and RECI/BBB-EBI/0179/2012. The C2TN/IST authors gratefully acknowledge their FCT support through projects RECI/AAG-TEC/0400/2012 and UID/Multi/04349/2013.info:eu-repo/semantics/publishedVersio

Aflatoxins detoxification by gamma irradiation

[Excerpt] Mycotoxins are secondary toxic metabolites of filamentous fungi. Aflatoxins (AFs) are produced to Aspergillus species such as A. flavus and A. parasiticus. These fungi are ubiquitous in nature and usually found on agricultural commodities. Therefore, AFs are encountered in many important foodstuff, including wheat, rice, maize, peanuts, sorghum, pearl millet, spices, oilseeds, tree nuts and milk. Due to the high toxicity of AFs, many methods have been studied to reduce or eliminate these mycotoxins from food and feed. Gamma irradiation is one technology that has been investigated with promising results. The aims of this study were (I) to study the effect of gamma radiation on aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 (II) to evaluate the effect of the presence of water on AFs degradation during the irradiation process; and (IV) to evaluate the cytotoxicity of radiolytic products formed. (...

Flow cytometry as a tool for assessing spore cell viability

ICFC 2017 - International Conference on Food Contaminants (Book of Abstracts)Acknowledgment: Thalita Calado, Luís Abrunhosa and Ângela França received support by grants SFRH/BD/79364/2011, UMINHO/BPD/51/2015 and SFRH/BPD/99961/2014 from FCT, respectively. CEB gratefully acknowledge FCT support through projects UID/BIO/04469/2013, NORTE-01-0145-FEDER-000004, and RECI/BBB-EBI/0179/2012.info:eu-repo/semantics/publishedVersio

Gamma irradiation effects on ochratoxin A: degradation, cytotoxicity and application in food

Ochratoxin A (OTA) is one of the main mycotoxins that can be found in food. The use of gamma radiation is a technique for preserving food that may exert some effects on mycotoxins. OTA was irradiated in its dry form, in aqueous and in methanolic solutions, and in wheat flour, grape juice and wine. Additionally, the toxicity of OTA irradiated in water was tested. In aqueous solutions, more than 90% of the OTA was degraded by -radiation doses 2.5 kGy, and a 2-fold reduction in OTA cytotoxicity was observed. In food matrices, the elimination of OTA by -radiation was found more difficult, as radiation doses of 30 kGy eliminate at most 24% of the OTA. Higher moisture content of food matrices did not substantially increase OTA elimination. It is concluded that OTA is very sensitive to irradiation in water solutions but resistant in its dry form and in food matrices.Thalita Calado received support through a grant SFRH/BD/79364/2011 from the Portuguese Foundation for Science and Technology (FCT –Portugal).LuísAbrunhosawassupportedbygrantUMINHO/BPD/51/ 2015 from project UID/BIO/04469/2013 financed by FCT/MEC (OE). This study was supported by the FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-010145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte; and under the scope of the projects RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and RTA2012-00053-00-00 INIA. The C2TN/IST authors gratefully acknowledge their FCT support through projects RECI/AAG-TEC/0400/2012 and UID/Multi/04349/ 2013. The authors are grateful to Luis Alté for his excellent technical assistance.info:eu-repo/semantics/publishedVersio

Mycotoxins in food and mitigation of its effects

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Mycotoxins are toxic compounds mainly produced by fungi of the genera Aspergillus, Fusarium and Penicillium. These toxins are frequently detected in many food commodities including cereals, fruit and vegetables, even after processing [1]. Since the same fungus may produce more than one toxin and the same toxin may be produced by different fungi, mycotoxins often occur as a mixture. Their ubiquitous presence represents a challenge to the health of humans, animals and the environment. Hundreds of compounds are listed as possible mycotoxins; however, only a few occur at levels that may are really challenging. The main focus of AMG is to study and develop integrated and innovative methods, supported in fundamental and applied research, for the mitigation of the occurrence of mycotoxins in food and feed, promoting complex system understanding and knowledge gaps identification. [...]info:eu-repo/semantics/publishedVersio

Effect of Gamma radiation on mycotoxins solutions

Due to the high toxicity of mycotoxins, many methods have been used to reduce or eliminate them from food and feed. Gamma radiation is one technique that has been investigated with some promising results in degradation of some mycotoxins from food. The aim of this study was to clarify the effect of gamma irradiation on aflatoxin B1 (AFB1) aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2), ochatoxin A (OTA) and zearelone (ZEA). The effect of the presence of moisture during the irradiation process was evaluated. Solutions with the same initial mycotoxin concentration were submitted to gamma radiation doses ranging from 0 to 10.0 kGy, at distinct moisture level – dehydrated, in water, and in methanol:water solution. Mycotoxins levels were determined by high-performance liquid chromatography with fluorescence detection (HPLC-FL), and photochemical post-column derivatization (for aflatoxins). The results showed degradation of mycotoxins with doses above 3.0 kGy, but only when irradiated in aqueous environment. With dehydrated samples, no significant reduction was observed. The results showed that gamma radiation was effective in reducing the mycotoxins concentration, but the presence of water (mainly due to the formation of hydroxyl radicals) had a very significant effect.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/79364/201, SFRH/BPD/43922/2008Programa Operacional do Norte (Adl. Portugal) - ChestNutsRad (contract number 13198

Effect of gamma-radiation on zearalenone—degradation, cytotoxicity and estrogenicity

Zearalenone (ZEA) is produced in cereals by different species of Fusarium, being a non-steroidal estrogenic mycotoxin. Despite having a low acute toxicity, ZEA strongly interferes with estrogen receptors. Gamma-radiation has been investigated to eliminate mycotoxins from food and feed, showing promising results. The present study aims to investigate the gamma-radiation effect on ZEA at different moisture conditions and to evaluate the cytotoxicity and estrogenicity of the irradiated ZEA. Different concentrations of dehydrated ZEA and aqueous solutions of ZEA were exposed to gamma-radiation doses ranging from 0.4 to 8.6 kGy and the mycotoxin concentration determined after exposure by high performance liquid chromatography (HPLC) with fluorescence detection. Following this, the cytotoxicity of irradiated samples was assessed in HepG2 cells, by measuring alterations of metabolic activity, plasma membrane integrity and lysosomal function, and their estrogenicity by measuring luciferase activity in HeLa 9903 cells. Gamma-radiation was found to be effective in reducing ZEA, with significant increases in degradation with increased moisture content. Furthermore, a reduction of cytotoxicity with irradiation was observed. ZEA estrogenicity was also increasingly reduced with increasing radiation doses, but mainly in aqueous solutions. These results suggest reduction of ZEA levels and of its toxicity in food and feed commodities may be achieved by irradiation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, C2TN (UIDB/04349/2020) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and by the INIA project RTA2012-00053-00-00.info:eu-repo/semantics/publishedVersio