Toxigenic fungi in coffee samples: a menace to public health

Introduction - Mycotoxin contamination was reported to occur in some food and commodities, such as coffee, particularly due to the presence of toxigenic fungi such as Aspergillus, Penicillium and Fusarium spp. Aspergilli are known to produce high levels of mycotoxins, such as ochratoxin and aflatoxin. Aspergillus ochraceus has been proposed as the major cause of ochratoxin A contamination in coffee beans. Aim of the study - The aim of this work was to evaluate the prevalence of Aspergillus sections Circumdati, Flavi and Fumigati in 28 green coffee samples to be used by Portuguese coffee industry, from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) species from different origins.

Importance of size-selective particle measuring for assessing occupational exposures: a case study “from field to fork”

Sampling the total air concentration of particulate matter (PM) only provides a basic estimate of exposure that normally not allows correlating with the observed health effects. Therefore is of great importance to recognize the particles size distribution and, particularly, the exposure to fine particles (≤ 2.5 μm). This particles dimension corresponds to the respirable fraction, the one that can implicate local and systemic effects due to particle deposition and clearance from the lungs and transport within the organism. This study intended to describe occupational exposure to PM2.5 in three units related with swine production and consumption, namely: feed production, swine production and swine slaughterhouse. A size-selective particle measuring in five to six workplaces of each unit was performed. Measurements of PM were done using a portable direct-reading hand-held equipment (Lighthouse, model 3016 IAQ). Data showed slaughterhouse unit with higher values, with values ranging from 0.030 to 0.142 mg/m3 (0.073 + 0.043), being the cutting room the workplace with higher values. In feed production unit, values were between 0.026 and 0.033 mg/m3 (0.028 + 0.003) with the warehouse of pharmacy products as the workplace with higher values. Finally, in swine unit values ranged from 0.006 to 0.048 mg/m3 (0.023 + 0.017) with the batteries area presenting the higher values. PM can be rich in fungi and bacteria and their metabolites, such as endotoxins and mycotoxins. Previous publications already showed high contamination in these occupational settings and particles can have an important role in exposure since can easily act as carrier of these agents. Data acquired allow not only a better prediction of particle penetration into respiratory regions of the respiratory tract, but also a better estimation of PM health effects. Moreover, data permit to identify the workplaces where investment should be made to prevent and reduce exposure.

Fungal contamination in coffee samples: a public health concern

Introduction - Fungi are natural coffee contaminants and under certain environmental conditions have the potential to produce toxins. Many studies revealed that the important toxigenic fungal genera (Aspergillus and Penicillium) are natural coffee contaminants, and are present from the field to storage. Aspergilli from the Circumdati and Nigri sections are known to produce high levels of ochratoxin A, a mycotoxin known as nephrotoxic for animals and humans. This work aimed to evaluate fungal distribution and also the prevalence of Aspergillus sections Fumigati, Flavi, Nigri and Circumdati from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) green samples.

Assessment of genotoxicity of aflatoxin M1 and B1 contaminated milks after in vitro human digestion

Introduction - Milk is considered a complete food from the nutritional point of view. Milk can be exposed to various types of contamination, such as mycotoxins. These metabolites are naturally occurring toxic compounds produced by fungi. Several studies on milk samples have reported the presence of aflatoxin B1 (AFB1) and M1 (AFM1), due to the high incidence in samples intended for human consumption, carcinogenicity proven AFB1 and resistance of the contaminants to the process of digestion, making those available for intestinal absorption. Considering these aspects, the objective of this study was to evaluate the genotoxicity of milk samples contaminated by AFB1 and AFM1 before and after the action of lactic acid bacteria using Caco-2 intestinal human cells.

Bibliotecas na saúde… e a saúde nas bibliotecas?

Em arquivos e bibliotecas a presença de fungos é considerada nefasta pelas suas implicações na conservação e leitura de documentos históricos e pela sua associação a problemas de saúde sentidos pelos funcionários e utentes que frequentam estes locais. De acordo com alguns autores, os problemas de saúde mais reportados por funcionários em Bibliotecas e Arquivos são dermatite, rinite, alergias e asma. Embora revestida de inegável importância, existem poucos estudos internacionais sobre a temática e, em Portugal, a contaminação fúngica em ambiente arquivístico e em bibliotecas é ainda muito pouco conhecida. O estudo realizado em quatro Arquivos Portugueses teve como objectivo conhecer a contaminação fúngica, contribuindo para a análise da qualidade do ar interior desses espaços e sua comparação com estudos internacionais. Para isso foram recolhidas amostras de ar e de superfícies e estas foram analisadas por métodos clássicos de cultura e, quando necessário, por métodos de biologia molecular. A avaliação foi feita quantitativa e qualitativamente, considerando os requisitos legais em vigor. No que respeita à análise do ar, o número de unidades formadoras de colónias (UFC)/m3 nunca excedeu as 500 (limite legislado), tendo sido verificada contaminação interior em todos os locais estudados. Comparativamente aos estudos realizados anteriormente em contextos semelhantes foram encontrados níveis elevados de contaminação por leveduras nas amostras de ar analisadas em Arquivos Portugueses. Não foi identificado nenhum fungo patogénico neste estudo, mas em quase todas as amostras estavam presentes fungos potencialmente toxinogénicos. Dentro do grupo dos Aspergillus, o A.versicolor mostrou predominância, tendo este fungo reconhecidas capacidades de emissão de micotoxinas em ambiente de interior. A inclusão de amostras de superfície revelou-se vital para conhecer todo o espectro fúngico existente em cada um dos locais estudados, incluindo a detecção de Stachybotrys chartarum e a do fungo potencialmente queratinofílico, Chrysosporium carmichaelli. Tanto para a saúde como para a conservação, o recente estudo realizado em quatro arquivos permitiu retirar importantes conclusões e reforçar a necessidade de vigilância, sendo também útil para a definição de padrões de qualidade no campo do património cultural.

Avaliação da interação entre aflatoxina M1 e B1 com a fração proteica do leite

Dissertação composta por 02 artigos.

Novas tecnologias para o controle da giberela do trigo na safra 2014 no sudoeste do Paraná

The work consists in wheat production system and the evolution of damage caused by wheat scab. The study considers the lack of resistant cultivars to the disease, the low efficiency of chemical control and the presence of mycotoxins in grains. The study aimed to confirm the effectiveness of fungicides prothioconazole and metconazole; prove the bar deposition efficiency with nozzles directed jets the side of the ear resulting in full coverage of trapped anthers; prove the viability of using a warning system (application after the start of flowering before the onset of rains provided for future 24-48 hours and try to improve the efficiency in controlling scab. The experiment was conducted in the experimental field of Technological University Federal do Paraná in the city of Pato Branco, Paraná, conducted in two seasons, the first established on May 6, 2014 and the second on 21 May 2014. It was used to cultivate Amethyst field of OR Improvement seeds, . Passo Fundo, Rio Grande do Sul Treatments consisted of a control without fungicide application and one and two applications of prothioconazole + 17.5% trifloxystrobin (Fox) 500 ml / ha; metconazole 8.0% + pyraclostrobin 13% ( Opera Ultra) 750 mL / ha In the evaluation, second season, metconazole + pyraclostrobin with two application statistically was the treatment that showed a lower incidence in spikes reducing from 100 to 63.3%;. metconazole + pyraclostrobin performed better also in reducing the incidence of spikelets, 80.3% to 34.7%; the control ears had better results in the second season with two applications of metconazole + pyraclostrobin; in spikelets, in his first season, metconazole + pyraclostrobin with two application resulted in better control with 67%; with two applications of metconazole + pyraclostrobin gave the highest yield even under climatic conditions favorable to disease; metconazole + pyraclostrobin in both seasons showed superior efficiency in controlling FHB giving greater weight gain; with two application of both fungicides, the hectoliter weight was 81 and 79, respectively, each fungicide; the application of fungicides was made in advance of expected rain, occurring seven days of rain and a total volume of 85,2mm the first time, and 16 days of rain and an accumulation of 400 mm in the second period; the test efficiency can be confirmed when the direction of the spray jets launched wings coupled in double nozzle bodies perpendicular to the target upright reaches the sides of the spike. The metconazole + pyraclostrobin proved the most effective fungicide against the prothioconazole + trifloxystrobin in wheat scab control in the 2014 harvest.

Professional exposure to fungal pathogens: an update to exposure conditions and exposure measurement

In many occupational settings an exposure to fungi occurs. Fungal exposure may occur for instance in the form of dermatocytes, yeasts or mold. Associated to the fungi themselves an exposure to cell wall components like ß(1 ? 3)-D-glucans, to mycotoxins or to microbial volatile compounds can occur. Health hazards may differ across species because fungi may produce different allergens and mycotoxins, and some species can infect humans. Occupational settings are often characterized by special exposure conditions with respect to duration, frequency and especially to the level of exposure resulting at least sometimes to high or very high fungal exposure. Because of these special conditions occupational settings are suitable for epidemiologic studies. However, the knowledge about occupational exposure to fungi and associated compounds like mycotoxins is still fragmentary and not well disseminated. An indication for a high fungal exposure is for instance the handling of dry natural products like grain, hay or herbal plants with a high specific surface and the tendency to release dust during handling. The fungal components often form the determinative part of such dusts and might be a vehicle to respiratory airways. The authors will present results of exposure measurements of occupational settings and exposure conditions which are only rarely investigated.

Biodegradação de deoxinivalenol por aspergillus oryzae e rhizopus sp.: um estudo bioquímico de degradação e toxicidade

A contaminação aleatória de alimentos por micotoxinas afeta as condições de sanidade das dietas de humanos e animais. Dentre as toxinas fúngicas, deoxinivalenol (DON) se destaca pela freqüente contaminação de produtos agrícolas e alimentos e pela sua resistência a degradação pelo emprego de métodos tradicionais de processamento, o que motiva políticas de controle e a busca por técnicas de descontaminação. A descontaminação biológica empregando processsos fermentativos tem sido apontada como uma alternativa promissora, pois permite degradar micotoxinas através do sistema enzimático microbiano e melhorar características funcionais e sensoriais de matérias-primas e insumos alimentícios. Este trabalho teve por objetivo estudar condições e mecanismos de biodegradação de deoxinivalenol empregando Aspergillus oryzae e Rhizopus sp. em sistemas fermentativos submersos. Para tanto, foi necessário adequar metodologia para reação de derivação na determinação cromatográfica de DON; estudar o potencial e condições de degradação via fermentação submersa por Aspergillus oryzae e Rhizopus sp.; e avaliar a atividade de oxidoredutases e a citotoxicidade dos extratos fementados. A otimização da metodologia estabeleceu a melhor condição para a reação de derivação com 200 µL de anidrido trifluoroacético e 18 mg de bicarbonato de sódio, durante 6 minutos a 74 °C na faixa entre 7 e 21 µg de DON. A quantificação de DON residual no meio fermentado mostrou que as espécies fúngicas Rhizopus sp. e Aspergillus oryzae possuem a capacidade de degradar DON demonstrando índices médios de 87,4 e 62,4% respectivamente, principalmente quando o meio submerso foi água estéril e fermentação realizada durante 48 horas. A velocidade máxima de degradação neste intervalo foi de 10,8 e 12,4 ppb/h, observando também um aumento na atividade específica da enzima peroxidase. Os extratos dos fermentados com A. oryzae e Rhizopus sp. apresentaram efeito de inibição de proliferação celular (IC50) quando concentrados 10 vezes em 48 e 72 horas respectivamente. Os meios fermentados com Rhizopus sp. apresentaram menor efeito (1,5 vezes) quando comparado com Aspergillus oryzae.

Exposure and risk assessment in occupational exposure to fungi: aspects to consider in highly contaminated settings

Although a clear correlation between levels of fungi in the air and health impacts has not been shown in epidemiological studies, fungi must be regarded as potential occupational health hazards. Fungi can have an impact on human health in four different ways: (1) they can infect humans, (2) they may act as allergens, (3) they can be toxigenic, or (4) they may cause inflammatory reactions. Fungi of concern in occupational hygiene are mostly non-pathogenic or facultative pathogenic (opportunistic) species, but are relevant as allergens and mycotoxins producers. It is known that the exclusive use of conventional methods for fungal quantification (fungal culture) may underestimate the results due to different reasons. The incubation temperature chosen will not be the most suitable for every fungal species, resulting in the inhibition of some species and the favouring of others. Differences in fungi growth rates may also result in data underestimation, since the fungal species with higher growth rates may inhibit others species’ growth. Finally, underestimated data can result from non-viable fungal particles that may have been collected or fungal species that do not grow in the culture media used, although these species may have clinical relevance in the context. Due to these constraints occupational exposure assessment, in setings with high fungal contamination levels, should follow these steps: Apply conventional methods to obtain fungal load information (air and surfaces) regarding the most critical scenario previously selected; Guideline comparation aplying or legal requirements or suggested limits by scientific and/or technical organizations. We should also compare our results with others from the same setting (if there is any); Select the most suitable indicators for each setting and apply conventional-culture methods and also molecular tools. These methodology will ensure a more real characterization of fungal burden in each setting and, consequently, permits to identify further measures regarding assessment of fungal metabolites, and also a more adequate workers health surveillance. The methodology applied to characterize fungal burden in several occupational environments, focused in Aspergillus spp. prevalence, will be present and discussed.

Fumonisina B1 em arroz: validação de método e efeito de tratamento térmico nos níveis da micotoxina

O arroz (Oryza sativa, L.), como todos os cereais, pode ser contaminado por fungos responsáveis por danos tecnológicos, nutricionais e toxicológicos, dentre eles a produção de micotoxinas. Diversas toxinas fúngicas produzidas pelo gênero Fusarium tem sido relatadas em arroz, no entanto a fumonisina B1 (FB1) é pouco estudada neste grão. As principais características da FB1 é a alta solubilidade em solventes polares, estabilidade a altas temperaturas além de efeitos neurotóxicos e carcinogênicos. Assim o objetivo deste trabalho foi avaliar o efeito do tratamento térmico e hidrotérmico nos teores de fumonisina B1 e nas características químicas de arroz comercial. Na primeira etapa do trabalho foi adaptado um método para detecção e quantificação de FB1 em arroz cru e após cocção, por HPLC-FL. O método foi avaliado quanto aos indicativos de eficiência destacando-se o LOD (30 µg.kg-1) e a recuperação ( 90% para arroz cru e 86% pra arroz cozido). Na segunda etapa realizou-se o levantamento de ocorrência de FB1 em 05 diferentes amostras comerciais de arroz integral, branco e parboilizado da cidade de Rio Grande, RS, totalizando 9 amostras. Foi detectada a presença de FB1 em 7 das 9 amostras, sendo que os maiores índices foram encontrados em amostras de arroz parboilizado e integral apresentando níveis de contaminação entre 30 e 170 µg.kg-1. A terceira etapa do trabalho consistiu no estudo do efeito de tratamentos térmicos sobre os níveis de FB1 em amostras após aplicação de calor. Foram testados tratamento hidrotérmico com evaporação, tratamento hidrotérmico com autoclavagem e tratamento térmico seco. O maior nível de redução dos teores iniciais de FB1 foi 82,8% quando se empregou tratamento térmico seco a 125 °C/3 min. Ainda foram avaliados os efeitos do t ratamento hidrotérmico com evaporação de água na composição química e na digestibilidade protéica. Esta característica proporcionou aumento de até 100% na digestibilidade in vitro das proteínas e reduziu em média 73% do teor de contaminação com FB1.

Caraterização in vitro dos efeitos tóxicos da patulina na integridade do epitélio intestinal e potenciais efeitos protetores da cisteína

A mucosa intestinal é a primeira barreira biológica encontrada pelas micotoxinas presentes nos alimentos, sendo a patulina, uma micotoxina produzida por fungos do género Penicillium spp., uma preocupação particular atendendo a que a exposição humana a esta micotoxina pode conduzir a efeitos imunológicos, neurológicos e gastrointestinais. Considerando estes efeitos para a saúde, o presente estudo tem como objetivos a avaliação do efeito tóxico da exposição intestinal a patulina, bem como a determinação do potencial efeito protetor da coadministração de patulina e cisteína na membrana intestinal, utilizando para o efeito células Caco-2. A integridade da membrana intestinal foi determinada pela medição da resistência elétrica transepitelial (TEER). Os resultados evidenciaram um decréscimo acentuado nos valores de TEER após 24 horas de exposição celular a 95 μM de patulina. Para as concentrações mais reduzidas verificou-se uma redução máxima inferior a 25% após 24 horas de exposição. A coadministração de patulina (95 μM) e cisteína (40 μM) revelou um decréscimo nos valores de TEER. O tratamento com cisteína em concentrações superiores ( 400 μM) revelou efeito protetor da membrana intestinal, tendo em conta os valores de TEER. Estes resultados contribuem para uma avaliação do risco mais precisa associada à exposição a contaminantes alimentares.

Potencial antifúngico, antioxidante e inibidor da produção de aflatoxina por extratos fenólicos de Chlorella sp. e Spirulina platensis

A contaminação fúngica acarreta alterações na qualidade nutricional e no valor econômico de produtos alimentícios podendo causar danos patológicos em plantas, animais e humanos. A identificação da atividade antioxidante, antifúngica e antimicotoxinas, em extratos de microalgas com propriedade de inibir a multiplicação de fungos e subseqüente produção de micotoxinas abre a perspectiva de empregar substâncias mais eficientes e com maior ação específica contra estes microorganismos. Entre os compostos com propriedades inibidoras de radicais livres, de crescimento fúngico e produção de micotoxinas, destacam-se os compostos fenólicos, que podem inibir a atividade metabólica microbiana, dificultando a atividade de enzimas. Neste estudo foram avaliados o poder de inibição de multiplicação fúngica de Rhizopus oryzae e Aspergillus flavus pelos extratos fenólicos de Chlorella sp. e Spirulina platensis, bem como sua atividade antioxidante, e a atividade antimicotoxinas da última microalga contra Aspergillus flavus. O conteúdo de fenóis totais foi em média 1000 µgfenóis/g Spirulina platensis e 600 µgfenóis/g Chlorella sp., sendo que o acido gálico e o cafeíco foram identificados como compostos majoritários na Spirulina platensis. As determinações de glicosamina (parede celular) e ergosterol (membrana celular) mostraram-se bons indicativos do desenvolvimento microbiano permitindo uma boa estimativa da inibição dele. O extrato fenólico de Spirulina platensis apresentou capacidade de inibir cerca de 50% a formação da parede e da membrana celular para ambos os fungos estudados e de 100% a produção de aflatoxina B1 até o 10º dia de cultivo do Aspergillus flavus. Além disso, o extrato metanólico de Spirulina platensis inativou 53,5% o DPPH reativo, limitou o escurecimento enzimático ocasionado pela peroxidase em 55% e inibiu a peroxidação lipídica em 46% após 14 dias de armazenamento sob luz. Estes resultados mostram que a ação antifúngica, antimicotoxinas e antioxidante está naturalmente presente em alguns tecidos microbianos e que encontrar a forma de extraí-los e aplicá-los como conservantes alimentícios é muito promissor para substituição aos antifúngicos e outros conservantes químicos.

Contribute of the MYCOMIX project and future perspectives for Human Biomonitoring and health risk assessment in Portugal

There is a growing concern within public health about mycotoxin involvement in human diseases, namely those related to children. The MycoMix project (2012-2015), funded by the Portuguese Foundation for Science and Technology, gathered a multidisciplinary team aiming at answering several questions: 1) Are Portuguese children exposed daily to one or several mycotoxins through food? 2) Can this co-exposure affect children´s health? and 3) Are there interaction effect between mycotoxins? Mycomix results revealed that Portuguese children (< 3 years old, n=103) are exposed to multiple mycotoxins through food consumption. Cumulative risk assessment results revealed a potential health concern for the high percentiles of intake, specially for aflatoxins which are carcinogenic compounds. This fact assumes particular importance considering the interactive effects found in in vitro bioassays. These results highlight the need for a more accurate approach to assess the human exposure to mycotoxins6. Within the Mycomix project the assessment of mycotoxin exposure was based on calculations combining mycotoxin data in food with population data on food consumption. This approach does not consider some aspects as the inter-individual metabolism variation, the exposure through sources other than food and the heterogeneous distribution of mycotoxins in food. Exposure assessment of mycotoxins in Portuguese population through biomarkers is still missing and further studies are urgent to be developed. The European Human Biomonitoring Initiative (EHBMI), a proposal within the European Joint Programme, aims to advance the understanding of the extent of exposure to environmental chemicals across Europe and the impact on human health, by gathering national expertise in human biomonitoring domain. At national level Mycomix project uncovered the potential health risk of exposure of Portuguese children to multiple mycotoxins. The risk assessment expertise acquired within Mycomix, namely in analysis and toxicology of chemical mixtures, will be brought together as a contribute to EHBMI objectives.

Is there a health risk concerning the children´s consumption of cereal-based products? A cumulative mycotoxin risk assessment approach

Historically, the health risk of mycotoxins had been evaluated on the basis of single-chemical and single-exposure pathway scenarios. However, the co-contamination of foodstuffs with these compounds is being reported at an increasing rate and a multiple-exposure scenario for humans and vulnerable population groups as children is urgently needed. Cereals are among the first solid foods eaten by child and thus constitute an important food group of their diet. Few data are available relatively to early stages child´s exposure to mycotoxins through consumption of cereal-based foods. The present study aims to perform the cumulative risk assessment of mycotoxins present in a set of cereal-based foods including breakfast cereals (BC), processed cereal-based foods (PCBF) and biscuits (BT), consumed by children (1 to 3 years old, n=75) from Lisbon region, Portugal. Children food consumption and occurrence of 12 mycotoxins (aflatoxins, ochratoxin A, fumonisins and trichothecenes) in cereal-based foods were combined to estimate the mycotoxin daily intake, using deterministic and probabilistic approaches. Different strategies were used to treat the left censored data. For aflatoxins, as carcinogenic compounds, the margin of exposure (MoE) was calculated as a ratio of BMDL (benchmark dose lower confidence limit) and aflatoxin daily exposure. For the remaining mycotoxins, the output of exposure was compared to the dose reference values (TDI) in order to calculate the hazard quotients (HQ, ratio between exposure and a reference dose). The concentration addition (CA) concept was used for the cumulative risk assessment of multiple mycotoxins. The combined margin of exposure (MoET) and the hazard index (HI) were calculated for aflatoxins and the remaining mycotoxins, respectively. Main results revealed a significant health concern related to aflatoxins and especially aflatoxin M1 exposure according to the MoET and MoE values (below 10000), respectively. HQ and HI values for the remaining mycotoxins were below 1, revealing a low concern from a public health point of view. These are the first results on cumulative risk assessment of multiple mycotoxins present in cereal-based foods consumed by children. Considering the present results, more research studies are needed to provide the governmental regulatory bodies with data to develop an approach that contemplate the human exposure and, particularly, children, to multiple mycotoxins in food. The last issue is particularly important considering the potential synergistic effects that could occur between mycotoxins and its potential impact on human and, mainly, children health.