Fungal infection and mycotoxin contamination in agricultural products are a serious food safety issue. The detection of fungal infection and mycotoxin contamination in food products should be in a rapid way. A Near-infrared (NIR) hyperspectral imaging system was used to detect fungal infection in 2013 crop year canola, wheat, and barley at different periods after inoculation and different concentration levels of ochratoxin A in wheat and barley. Artificially fungal infected (Fungi: Aspergillus glaucus, Penicillium spp.) kernels of canola, wheat and barley, were subjected to single kernel imaging after 2, 4, 6, 8, and 10 weeks post inoculation in the NIR region from 1000 to 1600 nm at 61 evenly distributed wavelengths at 10 nm intervals. The acquired image data were in the three-dimensional hypercube forms, and these were transformed into two-dimensional data. The two-dimensional data were subjected to principal component analysis to identify significant wavelengths based on the highest principal component factor loadings. Wavelengths 1100, 1130, 1250, and 1300 nm were identified as significant for detection of fungal infection in canola kernels, wavelengths 1280, 1300, and 1350 nm were identified as significant for detection of fungal infection in wheat kernels, and wavelengths 1260, 1310, and 1360 nm were identified as significant for detection of fungal infection in barley kernels. The linear, quadratic and Mahalanobis statistical discriminant classifiers differentiated healthy canola kernels with > 95% and fungal infected canola kernels with > 90% classification accuracy. All the three classifiers discriminated healthy wheat and barley kernels with > 90% and fungal infected wheat and barley kernels with > 80% classification accuracy. The wavelengths 1300, 1350, and 1480 nm were identified as significant for detection of ochratoxin A contaminated wheat kernels, and wavelengths 1310, 1360, 1480 nm were identified as significant for detection of ochratoxin A contaminated barley kernels. All the three statistical classifiers differentiated healthy wheat and barley kernels and ochratoxin A contaminated wheat and barley kernels with a classification accuracy of 100%. The classifiers were able to discriminate between different durations of fungal infections in canola, wheat, and barley kernels with classification accuracy of more than 80% at initial periods (2 weeks) of fungal infection and 100% at the later periods of fungal infection. Different concentration levels of ochratoxin A contamination in wheat and barley kernels were discriminated with a classification accuracy of > 98% at ochratoxin A concentration level of ≤ 72 ppb in wheat kernels and ≤ 140 ppb in barley kernels and with 100% classification accuracy at higher concentration levels. / May 2016

Šio darbo tiksas - nustatyti ochratoksino A kiekius žaliose ir skrudintose kavos pupelėse. Mėginiai tyrimams buvo paimti iš dviejų Lietuvos kavos skrudinimo įmonių. Kavos pupelių mėginiai buvo iš Amerikos, Afrikos ir Azijos. Žalių kavos pupelių užterštumas ochratoksinu A buvo 1,0 – 3,5 µg/kg, o po skrudinimo jis padidėjo 14,12 – 63,64%. Taip pat buvo identifikuojami ochratoksiną A gaminantys grybai ir skaičiuojami jų kolonijas sudarantys vienetai. Nustatyta, kad kavos pupelės buvo užterštos A. niger, A. ochraceus, A. parasiticus, A. sydowii ir A. flavus. / The experiments was done in LUHS VA in the Animal Welfare Reaserch Laboratory. The aim of this study were to determinate ochratoxin A amounts in green and roasted coffee beans. Samples were taken from two coffee roasters companies in Lithuania. Coffee samples were from America, Asia and Africa. The determination of OTA in green coffee samples was 1,0 – 3,5 µg/kg, but after roasting it increase to 14,12 – 63,64%. Also were identified OTA-producing species of fungi and their colony formed quantities. It was found that determination of fungi were 50% of A. niger colonies, other 50% were of A. ochraceus, A. parasiticus, A. sydowii and A. flavus.

Univ., Diss., 2006--Giessen

Zugl.: Berlin, Techn. Univ., Diss., 2008

A vitivinicultura é uma atividade extremamente importante no estado do Rio Grande do Sul, representando cerca de 50% das uvas produzidas e 90% dos vinhos em todo o país. Diferentes gêneros fúngicos são comumente encontrados infectando as bagas, os principais são Alternaria, Botrytis, Cladosporium e Aspergillus. Esta contaminação é influenciada por diferentes fatores, entre eles, características climáticas da região, variedades da uva, tipo de cultivo, entre outros. O gênero Aspergillus destaca-se dos demais, pois as espécies frequentemente encontradas nas uvas, os chamados Aspergillus seção Nigri, são potenciais produtores de Ocratoxina A, micotoxina nefrotóxica e possivelmente carcinogênica para humanos. Os objetivos deste trabalho foram a caracterização micotoxicológica de uvas das variedades Merlot e Cabernet Sauvignon, produzidas em três regiões vitivinícolas do Rio Grande do Sul, coletadas em dois estágios de maturação das bagas. As amostragens foram realizadas no inicio da mudança de cor e na colheita nas regiões da Campanha, Serra do Sudeste e Serra do Nordeste. Foram isolados oito gêneros fúngicos, com destaque para Alternaria, que foi predominante em todas as regiões, estágios de cultivo e nas duas variedades. Os Aspergillus seção Nigri foram predominantes no gênero Aspergillus, representando 88% dos isolados, que se destacaram na região da Campanha e na variedade Cabernet Sauvignon. O período da colheita demonstrou ser critico para a contaminação por Aspergillus negros na variedade Cabernet Sauvignon, enquanto na variedade Merlot, maior frequência desses fungos foi observada durante o início da mudança de cor. Uma cepa de Aspergillus japonicus foi capaz de produzir ocratoxina A na concentração de 148 ng/mL/106 conídios. Não foram encontrados níveis detectáveis de OTA nas amostras de uvas. A caracterização da contaminação fungica e por OTA em uvas cultivadas no estado é relevante para garantir a qualidade destes produtos e a segurança da população consumidora. / The viticulture is an important activity in Brazil. Rio Grande do Sul, the southern state of Brazil, responsible for almost 90% of wines production. Different fungal genera are commonly found infecting the berries, mainly Alternaria, Botrytis, Cladosporium and Aspergillus. Grape infection and fungal growth in influenced by different factors, including climatic characteristics of the region, grape varieties, crop type, among others. Among the genus Aspergillus, Aspergillus section Nigri stands out from the others, because its ability to produce ochratoxin A, mycotoxin with nephrotoxic characteristics and classified as a possible carcinogenic to humans. The aim of this study was the mycological characterization of wine grapes of Merlot and Cabernet Sauvignon varieties, cultivated at three wine producer regions of Rio Grande do Sul, collected during two stages of berries maturation. The sampling was conducted at the early ripeness and harvesting from the Campanha, Serra do Sudeste and Serra do Nordeste regions. Eight fungal genera were isolated, highlighting Alternaria, which was predominant in all regions, growing stages and varieties. Aspergillus section Nigri were prevalent in the genus Aspergillus, representing 88% of the isolates mainly at Campanha region as well as Cabernet Sauvignon veriety. The harvest period proved to be critical for contamination by Aspergillus section Nigri in C. Sauvignon variety; nevertheless, in Merlot, the greater frequency of these fungi was observed at early ripeness. One Aspergillus japonicus isolate was able to produce OTA at a concentration of 148 ng mL-1/106 conidia. No detectable levels of OTA were found in any grape samples. The characterization of fungal and OTA contamination in grapes cultivated in Rio Grande do Sul is extremely important to ensure the quality of these products and consumers safety.

A vitivinicultura é uma atividade extremamente importante no estado do Rio Grande do Sul, representando cerca de 50% das uvas produzidas e 90% dos vinhos em todo o país. Diferentes gêneros fúngicos são comumente encontrados infectando as bagas, os principais são Alternaria, Botrytis, Cladosporium e Aspergillus. Esta contaminação é influenciada por diferentes fatores, entre eles, características climáticas da região, variedades da uva, tipo de cultivo, entre outros. O gênero Aspergillus destaca-se dos demais, pois as espécies frequentemente encontradas nas uvas, os chamados Aspergillus seção Nigri, são potenciais produtores de Ocratoxina A, micotoxina nefrotóxica e possivelmente carcinogênica para humanos. Os objetivos deste trabalho foram a caracterização micotoxicológica de uvas das variedades Merlot e Cabernet Sauvignon, produzidas em três regiões vitivinícolas do Rio Grande do Sul, coletadas em dois estágios de maturação das bagas. As amostragens foram realizadas no inicio da mudança de cor e na colheita nas regiões da Campanha, Serra do Sudeste e Serra do Nordeste. Foram isolados oito gêneros fúngicos, com destaque para Alternaria, que foi predominante em todas as regiões, estágios de cultivo e nas duas variedades. Os Aspergillus seção Nigri foram predominantes no gênero Aspergillus, representando 88% dos isolados, que se destacaram na região da Campanha e na variedade Cabernet Sauvignon. O período da colheita demonstrou ser critico para a contaminação por Aspergillus negros na variedade Cabernet Sauvignon, enquanto na variedade Merlot, maior frequência desses fungos foi observada durante o início da mudança de cor. Uma cepa de Aspergillus japonicus foi capaz de produzir ocratoxina A na concentração de 148 ng/mL/106 conídios. Não foram encontrados níveis detectáveis de OTA nas amostras de uvas. A caracterização da contaminação fungica e por OTA em uvas cultivadas no estado é relevante para garantir a qualidade destes produtos e a segurança da população consumidora. / The viticulture is an important activity in Brazil. Rio Grande do Sul, the southern state of Brazil, responsible for almost 90% of wines production. Different fungal genera are commonly found infecting the berries, mainly Alternaria, Botrytis, Cladosporium and Aspergillus. Grape infection and fungal growth in influenced by different factors, including climatic characteristics of the region, grape varieties, crop type, among others. Among the genus Aspergillus, Aspergillus section Nigri stands out from the others, because its ability to produce ochratoxin A, mycotoxin with nephrotoxic characteristics and classified as a possible carcinogenic to humans. The aim of this study was the mycological characterization of wine grapes of Merlot and Cabernet Sauvignon varieties, cultivated at three wine producer regions of Rio Grande do Sul, collected during two stages of berries maturation. The sampling was conducted at the early ripeness and harvesting from the Campanha, Serra do Sudeste and Serra do Nordeste regions. Eight fungal genera were isolated, highlighting Alternaria, which was predominant in all regions, growing stages and varieties. Aspergillus section Nigri were prevalent in the genus Aspergillus, representing 88% of the isolates mainly at Campanha region as well as Cabernet Sauvignon veriety. The harvest period proved to be critical for contamination by Aspergillus section Nigri in C. Sauvignon variety; nevertheless, in Merlot, the greater frequency of these fungi was observed at early ripeness. One Aspergillus japonicus isolate was able to produce OTA at a concentration of 148 ng mL-1/106 conidia. No detectable levels of OTA were found in any grape samples. The characterization of fungal and OTA contamination in grapes cultivated in Rio Grande do Sul is extremely important to ensure the quality of these products and consumers safety.

A vitivinicultura é uma atividade extremamente importante no estado do Rio Grande do Sul, representando cerca de 50% das uvas produzidas e 90% dos vinhos em todo o país. Diferentes gêneros fúngicos são comumente encontrados infectando as bagas, os principais são Alternaria, Botrytis, Cladosporium e Aspergillus. Esta contaminação é influenciada por diferentes fatores, entre eles, características climáticas da região, variedades da uva, tipo de cultivo, entre outros. O gênero Aspergillus destaca-se dos demais, pois as espécies frequentemente encontradas nas uvas, os chamados Aspergillus seção Nigri, são potenciais produtores de Ocratoxina A, micotoxina nefrotóxica e possivelmente carcinogênica para humanos. Os objetivos deste trabalho foram a caracterização micotoxicológica de uvas das variedades Merlot e Cabernet Sauvignon, produzidas em três regiões vitivinícolas do Rio Grande do Sul, coletadas em dois estágios de maturação das bagas. As amostragens foram realizadas no inicio da mudança de cor e na colheita nas regiões da Campanha, Serra do Sudeste e Serra do Nordeste. Foram isolados oito gêneros fúngicos, com destaque para Alternaria, que foi predominante em todas as regiões, estágios de cultivo e nas duas variedades. Os Aspergillus seção Nigri foram predominantes no gênero Aspergillus, representando 88% dos isolados, que se destacaram na região da Campanha e na variedade Cabernet Sauvignon. O período da colheita demonstrou ser critico para a contaminação por Aspergillus negros na variedade Cabernet Sauvignon, enquanto na variedade Merlot, maior frequência desses fungos foi observada durante o início da mudança de cor. Uma cepa de Aspergillus japonicus foi capaz de produzir ocratoxina A na concentração de 148 ng/mL/106 conídios. Não foram encontrados níveis detectáveis de OTA nas amostras de uvas. A caracterização da contaminação fungica e por OTA em uvas cultivadas no estado é relevante para garantir a qualidade destes produtos e a segurança da população consumidora. / The viticulture is an important activity in Brazil. Rio Grande do Sul, the southern state of Brazil, responsible for almost 90% of wines production. Different fungal genera are commonly found infecting the berries, mainly Alternaria, Botrytis, Cladosporium and Aspergillus. Grape infection and fungal growth in influenced by different factors, including climatic characteristics of the region, grape varieties, crop type, among others. Among the genus Aspergillus, Aspergillus section Nigri stands out from the others, because its ability to produce ochratoxin A, mycotoxin with nephrotoxic characteristics and classified as a possible carcinogenic to humans. The aim of this study was the mycological characterization of wine grapes of Merlot and Cabernet Sauvignon varieties, cultivated at three wine producer regions of Rio Grande do Sul, collected during two stages of berries maturation. The sampling was conducted at the early ripeness and harvesting from the Campanha, Serra do Sudeste and Serra do Nordeste regions. Eight fungal genera were isolated, highlighting Alternaria, which was predominant in all regions, growing stages and varieties. Aspergillus section Nigri were prevalent in the genus Aspergillus, representing 88% of the isolates mainly at Campanha region as well as Cabernet Sauvignon veriety. The harvest period proved to be critical for contamination by Aspergillus section Nigri in C. Sauvignon variety; nevertheless, in Merlot, the greater frequency of these fungi was observed at early ripeness. One Aspergillus japonicus isolate was able to produce OTA at a concentration of 148 ng mL-1/106 conidia. No detectable levels of OTA were found in any grape samples. The characterization of fungal and OTA contamination in grapes cultivated in Rio Grande do Sul is extremely important to ensure the quality of these products and consumers safety.

Orientador: Jose Luiz Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-13T15:09:21Z (GMT). No. of bitstreams: 1 Copetti_MarinaVenturini_D.pdf: 6048122 bytes, checksum: 93c1ab6938ae661500c36bb19ce26312 (MD5) Previous issue date: 2009 / Resumo: O Brasil se encontra entre os maiores produtores mundiais de cacau. Parte da sua produção é destinada à exportação, principalmente após passar por algum processamento industrial, e o restante se destina ao consumo interno. A qualidade obtida ao final do processamento do cacau depende de uma ampla variedade de fatores, dentre os quais os microrganismos desempenham um papel fundamental. A presença de fungos é observada em várias fases do processamento, o que além do aspecto deteriorativo representa um perigo toxicológico devido a possibilidade de síntese de micotoxinas. Na presente tese são apresentados os resultados sobre a presença de fungos, ocratoxina A e aflatoxinas em um total de 494 amostras de cacau analisadas em diferentes fases do processamento. A maior quantidade e diversidade de fungos foi encontrada em amostras coletadas durante o processamento na fazenda, especialmente durante a secagem e estocagem das amêndoas. Foram isolados 1132 espécies potencialmente toxigênicas do gênero Aspergillus: A. flavus, A. parasiticus, A. niger, A. carbonarius e grupo dos A. ochraceus. Com relação à presença de micotoxinas, as amostras da secagem e estocagem foram as que apresentaram maiores níveis de ocratoxina A e aflatoxinas na fazenda, enquanto que nas amostras coletadas em indústrias processadoras de cacau os maiores níveis de ocratoxina A foram detectados na casca, torta e cacau em pó e os de aflatoxina, nos nibs e liquor. Nas amostras de chocolate observou-se uma relação direta entre a quantidade de cacau no produto e os níveis de aflatoxinas e ocratoxina A, sendo estes maiores em chocolate em pó e chocolate meio amargo. Também foram conduzidos experimentos para avaliar as condições que afetam a produção de ocratoxina A no cacau por A. carbonarius. Foi observado que, diante da presença de inóculo, a produção de ocratoxina A já ocorre durante a fermentação. Os níveis de ocratoxina A foram menores quando sementes de cacau com polpa integral foram fermentadas durante 6 dias em caixas, se comparados aos obtidos com sementes parcialmente despolpadas e conduzidas à secagem sem prévia fermentação. Os resultados em geral mostraram a existência de uma grande diversidade de fungos, muitos dos quais toxigênicos, durante o processamento do cacau e a inter-relação de fatores intrínsecos, ambientais e de processamento pareceram determinar a ocorrência ou não de micotoxinas / Abstract: Brazil is one of the biggest cocoa producing countries. Part of its production is exported, mainly after undergoing some industrial processing, and the other supplies the internal market. The quality of the final cocoa product depends on several factors, and microorganisms play an important role on this. Investigations have demonstrated the presence of filamentous fungi in many processing stages. Besides the spoilage potential, those are a cause for concern from the toxicological aspect, since mycotoxins may be produced. This thesis presents the results concerning the presence of fungi, ochratoxin A and aflatoxins in a total of 494 cocoa samples collected at different processing stages, from farm to market. The greatest occurrence and the major diversity of fungi were found in samples collected during the drying and storage of the beans on the farm. A total of 1132 potentially toxigenic fungi were isolated, belonging to the species A. flavus, A. parasiticus A. niger, A. ochraceus group and A. carbonarius. Regarding the presence of mycotoxins, samples collected during drying and storage presented the highest ochratoxin A and aflatoxin levels amongst samples collected at farm. During industrialization, the most contaminated products were the cocoa shell, cocoa cake and cocoa cake with respect to ochratoxin A. The aflatoxin levels were higher in the nibs and liquor. A direct correlation between the amount of cocoa in the product and the ochratoxin A level in the chocolate was observed. Dark chocolate and powdered commercial cocoa were the most contaminated samples. An additional experiment was carried out to evaluate the conditions affecting ochratoxin A production by A. carbonarius in cocoa on the farm. It was observed that ochratoxin A production could start during fermentation if toxigenic fungi were present, and lower ochratoxin levels were found when the integral beans were fermented during 6 days in box if compared to those with pulp partially removed and submitted to drying fermentation. In general, during cocoa processing, a wide variety of fungi was found, some of these toxigenic. Intrinsic, environmental and processing factors acting together seemed to determine the production of mycotoxins in cocoa / Doutorado / Doutor em Ciência de Alimentos

Le travail réalisé au cours de cette thèse a porté sur le développement de biocapteurs électrochimiques d’affinité, sensibles et sélectifs, pour la détection de l’ochratoxine A (OTA) et l’aflatoxine M1 (AFM1). Les biocapteurs développés reposent sur l’association de différents nanomatériaux pour une meilleure performance analytique. Pour construire notre transducteur, nous avons associé le polypyrrole à des dendrimères poly(amido-amine) PAMAM, ce qui a permis d’avoir de très bon rendements grâce au propriétés électriques du polypyrrole et à l’augmentation de la surface active due à la structure tridimensionnelle des dendrimères. L’utilisation d’aptamères spécifiques pour la détection des différentes mycotoxines a permis leur détection et quantification à des concentrations de l’ordre des nM, ainsi que l’élargissement des gammes dynamiques. Nous avons pu démontrer grâce à l’utilisation de dendrimères de différentes tailles que la sensibilité des biocapteurs ne provient pas uniquement de l’affinité qui existe entre les biorécepteurs et leurs molécules cibles, mais aussi des propriétés physico-chimiques du biocapteur. / This aim of this work is to develop ultrasensitive electrochemical biosensors with high affinity toward ochratoxine (OTA) and aflatoxine M1 (AFM1). In order to obtain the best analytical performances, we associated nano-materials in the transducer construction: conducting polypyrrole polymer and poly(amido-amine) dendrimères. Thanks to this association, we benefited from the conducting material’s electrical properties, and the large active detection surface dendrimers. For the bimolecular sensing part, we used specific DNA aptamers which allowed us to quantify mycotoxines at nM concentrations. In addition, the different aptamer based biosensors present a very large dynamic ranges. We also demonstrated through the use of different sizes of dendrimers, that the sensitivity depend not only in the affinity between bioreceptors and their target molecules, but also in the physico-chemical properties of the biosensor.

This thesis summarizes knowledge about mycotoxins, with focus to ochratoxin A. It also summarizes its tolerable levels of food intake, detoxification and analytical methods for mycotoxins. The work also includes a chapter describing liposomes that were used for the analysis of ochratoxin A by liposomal electrokinetic capillary electrophoresis (LECK). The practical part includes the analysis of ochratoxin A from Aspergillus ochraceus Wilhelm and Aspergillus melleus Yukawa fungi cultivated on a rye and optimization of the method for analysis of ochratoxin A based on liposomes of different compositions. By capillary zone electrophoresis, ochratoxin A is not sufficiently separated and detected in the extracted mixture; conversely, when liposome solutions are used, different migration behavior can be achieved while stabilizing ochratoxin A in solution due to amphiphilic interactions between mycotoxins and liposomes. Therefore, the LEKC method was used for this work. The most suitable liposome composition has been shown to be molar ratios of 25% cholesterol (membrane stabilization) / 50% 2-oleoyl-1-palmitoyl-sn-glycerol 3-phosphocholine (main zwitterionic lipid) / (25% 1,2-diacyl-sn-glycerol)-3-phospho-L-serine (introduction of negative charge).