Application of Syzygium aromaticum, Ocimum sanctum, and Cananga odorata essential oils for management of Ochratoxin A content by Aspergillus ochraceus and Penicillium verrucosum: An in vitro assessment in maize grains

The study is directed to establish the minimizing effects of Syzygium aromaticum, Ocimum sanctum, and Cananga odorata essential oils on the growth and ochratoxin A (OTA) level of Aspergillus ochraceus and Penicillium verrucosum in maize grains. S. aromaticum essential oil (SAEO), O. sanctum essential oil (OSEO), and C. odorata essential oil (COEO) were extracted by hydro-distillation technique, and a total of 50, 44, and 48 chemical constituents were identified by gas chromatography-mass spectrometry (GC-MS), respectively.The SAEO and OSEO belong to the chemotype of eugenol, whereas, COEO was found to be the chemotype of thymol, limonene, and ?-ylangene. The antifungal activity of essential oils (EOs) was determined by the micro-well dilution technique. The SAEO showed superior antifungal activity compared to OSEO, COEO, and synthetic antifungal agent nystatin, and its minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values against A. ochraceous and P. verrucosum were noticed as 1251 ± 42.32 and 1878 ± 28.47 µg/mL, and 0815 ± 22.69 and 1146 ± 51.19 µg/mL, respectively.The antifungal mechanism of EOs was unveiled by assessing the intracellular reactive oxygen species (ROS), ergosterol content, and membrane integrity. The antifungal investigations found that EOs caused fungal mortality by increasing the intracellular ROS, depleting ergosterol synthesis, and distracting membrane integrity. Finally, antifungal and antimycotoxin activity of EOs was demonstrated in maize grains. The SAEO, OSEO, and COEO have reduced the complete fungal growth and OTA level of A. ochraceous and P. verrucosum correspondingly at 2500 and 2500, 3500 and 2500, and 3500 and 3500 µg/g in maize. The EOs could act as natural antifungal agents; protect foodstuffs from fungal infection and mycotoxins during storage.

Determination of ochratoxin A in coffee by ELISA method and its relationship with the physical, physicochemical and microbiological properties / Determinación de ocratoxina A en café mediante método ELISA y su relación con las propiedades físicas, fisicoquímicas y microbiológicas

Background: Coffee is one of the most consumed beverages in the world; however, it may contain toxic compounds such as ochratoxin A (OTA). Objectives: Determine the OTA's presence in different types of coffee, intended for beverage preparation and marketed in Colombia through the application of the enzyme-linked immunosorbent assay (ELISA) and analyze its relationship with the physical, physicochemical and microbiological properties. Methods: 8 samples of coffee commercialized in the Colombian market were selected, in which the OTA content was determined by applying the ELISA method. Likewise, a microbiological analysis was performed, and physicochemical properties were determined, such as moisture content, aw, percentage total dissolved solids (%TDS), and extraction yield (%EY). Physical properties such as free-flow densities, compacted bulk densities (CBD), porosity, average particle size (ASP), and color. The data were treated with multivariate analysis using Principal Component Analysis (PCA) and Cluster Analysis (CA) to quantitatively investigate the relationships between the coffee samples concerning their physical, physicochemical properties, and OTA content. LSD test was applied with a significance level of 95 % and Pearson correlation test. Results:All the samples had OTA content, but only 2 exceeded the limits allowed by the regulations, with a maximum value of 15.449 µg/Kg, which represents 31.449 % of the tolerable daily intake according to the parameters defined by Joint FAO/WHO Expert Committee on Food Additives (JECFA). According to the PCA and CA, the samples were grouped harmonically according to the type of coffee associated with its commercial presentation and industrial process, OTA content, and ASP. OTA content was significantly and positively correlated (p< 0.05) with %EY, %TDS, ASP, porosity, CBD and moisture. Conclusions: The coffees marketed in Colombia showed a variable range of OTA, where soluble coffees had higher OTA contents than roasted coffees, and 25 % of the coffees analyzed do not meet the levels defined by Colombian regulations. The OTA content in coffee is related to properties that define the ability to extract solutes from coffee

Antecedentes: El café es una de las bebidas más consumidas en el mundo, sin embargo, puede contener compuestos tóxicos como la ocratoxina A (OTA). Objetivos: Determinar la presencia de OTA en diferentes tipos de café destinados a la preparación de bebida y comercializados en Colombia mediante la aplicación del ensayo inmunoabsorbente ligado a enzimas (ELISA) y analizar su relación con las propiedades físicas, fisicoquímicas y microbiológicas. Métodos: Se seleccionaron 8 muestras de café comercializado en el mercado colombiano, en las cuales se determinó el contenido de OTA mediante la aplicación del método ELISA. Así mismo se realizó análisis microbiológico y se determinaron propiedades fisicoquímicas como contenido de humedad, aw, porcentaje de sólidos disueltos totales (%TDS) y rendimiento de extracción (%EY); y propiedades físicas como densidad por caída libre, densidad compactada (CBD), porosidad, tamaño promedio de partícula (ASP) y color. Los datos fueron tratados con análisis multivariado empleando análisis de componentes principales (PCA) y análisis de conglomerados (CA) para investigar cuantitativamente las relaciones entre las muestras de café con respecto a sus propiedades físicas, fisicoquímicas y contenido de OTA. Se aplicó prueba LSD con un nivel de significación del 95 % y prueba de correlación de Pearson. Resultados: Todas las muestras presentaron contenido de OTA, pero solo 2 sobrepasaron los límites permitidos por la normatividad, con un valor máximo de 15.449 µg/Kg, el cual representa un 31.449 % de la ingesta diaria tolerable según los parámetros definidos por el Comité Mixto FAO/OMS de Expertos en Aditivos Alimentarios (JECFA). De acuerdo al PCA y CA, las muestras se agruparon armónicamente de acuerdo al tipo de café asociado a su presentación comercial y proceso industrial, contenido de OTA y ASP; el contenido de OTA se correlacionó significativa y positivamente (p < 0.05) con el %EY, %TDS, ASP, porosidad, CBD y humedad. Conclusión: Los cafés comercializados en Colombia presentan un rango variable de OTA, en donde los cafés solubles presentan contenidos de OTA mayores que los cafés tostados y el 25 % de los cafés analizados no cumplen con niveles definidos por la normatividad colombiana. El contenido de OTA en el café está relacionado con propiedades que definen la capacidad de extracción de solutos del café

Survey of Aflatoxins and Ochratoxin A in Spices from Brazilian Market

Abstract The incidence of aflatoxins and ochratoxin A in spices purchased from São Paulo State, Brazil was investigated. A total of 180 black pepper (Piper nigrum L.), colorífico (mixture of cornmeal or cassava flour with powdered annatto, Bixa orellana) ginger (Zingiber officinale Roscoe), nutmeg (Myristica fragrans), paprika (Capsicum annuum L.), and turmeric (Curcuma longa) were analyzed with a modified methods by using immunoaffinity column for clean-up and liquid chromatography/fluorescence detector for separation and quantification. Analytical methods were optimized for each spice, focusing mainly on the extraction step. OTA recoveries ranged from 65-102%. AFs recoveries were >70% except for AFG2. The average levels of AFs and OTA in black pepper, colorífico and turmeric samples were less than 2 ng/g. Twenty-five ginger samples (100%) contained OTA 0.10 - 7.10 ng/g and 21 samples (84%) contained AFs 0.10 - 9.55 ng/g. Twenty-nine nutmeg samples (100%) contained OTA 0.92 - 65.49 ng/g and AFs 2.71 - 48.67 ng/g. Thirty paprika samples (100%) contained OTA, 0.75 - 147.18 ng/g and twenty-two samples (73%) contained AFs, 0.11 - 14.92 ng/g. AFs and OTA in nutmeg and paprika could represent a food safety issue in Brazil.

Efecto antifúngico y antimicotoxigénico de Lactobacillus plantarum CRL 778 a diferentes valores de actividad de agua / Antifungal and antimycotoxigenic effect of Lactobacillus plantarum CRL 778 at different water activity values

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi with high impact Lactic acid bacteria; in food safety due to its toxicity. In the last decade, the presence of OTA was widely reported in different foods. In this study, the ability of Lactobacillus (L.) plantarum CRL 778 to control growth and OTA production by Aspergillus (A.) niger 13D strain, at different water activity (a w) values (0.955, 0.964, 0.971, 0.982, and 0.995) was determined in vitro. Both parame ters were significantly (p<0.05) reduced by the lactobacilli and the effect depended on a w. Greatest growth rate inhibition (46.9%) was obtained at a w = 0.995, which is the most suitable value for growth and production of antifungal metabolites (lactic acid, acetic acid, phenyllac-tic and hydroxyl-phenyllactic acids) by L. plantarum CRL 778. Besides, morphological changes and inhibition of melanin synthesis were observed in colonies of A. niger 13D in presence of L. plantarum CRL 778 at a w ranged between 0.971 and 0.995. In addition, maximum reduction (90%) of OTA production took place at a w = 0.971, while inhibition of fungi growth was more evident at a w =0.995. These findings suggest that L. plantarum CRL 778 could be used for control of ochratoxigenic fungal growth and OTA contamination in different fermented foods with a w values between 0.971 and 0.995.

Ocratoxina A (OTA) es una micotoxina producida por hongos filamentosos con un alto impacto en la seguridad alimentaria debido a su toxicidad. En la última década se ha reportado ampliamente a nivel mundial, la presencia de OTA en diversos alimentos. En este estudio se evaluó in vitro, la capacidad de Lactobacillus (L.) plantarum CRL 778 de controlar el crecimiento y la producción de OTA por Aspergillus (A.) niger 13D, a diferentes valores de actividad de agua (a w): 0.955, 0.964, 0.971,0.982 y 0.995). La cepa láctica redujo significativamente (p <0.05) ambos parámetros, siendo el efecto dependiente del valor de a w. La mayor inhibición del crecimiento (46.9%) se obtuvo a a w =0.995, valor más adecuado para el crecimiento y producción de metabolitos antifúngicos (ácido láctico, ácido acético, ácidos fenil-láctico e hidroxi-fenil láctico) por la cepa láctica. Además, se observaron cambios morfológicos en las colonias de A. niger 13D, crecidas en presencia de L. plantarum CRL 778 a valores de a w de 0.971 y 0.995. El porcentaje máximo de reducción en la producción de OTA (90%) por la cepa láctica se observó a un valor de a w = 0.971, mientras la inhibición del crecimiento fúngico fue mayor cuando a w = 0.995. Estos hallazgos sugieren que L. plantarum CRL 778 podría emplearse para el control de la contaminación por hongos ocratoxigénicos en alimentos con valores de aw comprendidos entre 0.971-0.995.

Aspergillus niger as the Source of Ochratoxin A of Contaminated Pyrus communis in Taif Market

Fruits are one of the most important agricultural products that supply the body with vitamins and essential minerals elements, but it is contaminated by fungi during the period of growth, harvesting and storage. A. niger is one of the species that grows on the fruit during the period of storage, and secretes mycotoxins especially ochratoxin A. This study was conducted with the purpose of isolating and identifying different strains of A. niger from 20 samples of pear collected from Taif markets and to determine the ability of these strains to produce OTA. It was observed that showed that out of 20 pear samples collected, 19 samples were detected to be contaminated with different strains of A. niger and the strains were able to produce OTA. From 27 isolates of A. niger which was used to test the ability of production OTA, 10 strains only produced OTA. The range of OTA in all strains were 0.18 to 9.5 ppb. Representative 27 strains of ochratoxigenic and non ochratoxigenic black Aspergilli isolated were subjected for detection of ochratoxin biosynthesis genes, by using two sets of primer for two genes involved in ochratoxin biosynthetic pathway. Bands of the fragments of PKS15C-MeT and PKS15KS genes visualized at 998 and 776 bp, respectively. Whereas, the presence of four tested genes is not sufficient marker for differentatin between aflatoxigenic and non aflatoxigenic isolates.

Aspergillus species from groundnuts (Arachis hypogaea) and mycotoxin production by toxigenic species

Aims@#Groundnut is an important food crop and is susceptible to contamination by Aspergillus. The present study was conducted to identify Aspergillus spp. from groundnuts as well as to detect mycotoxin production by toxigenic species. @*Methodology and results@#Molecular identification using ITS region, β-tubulin and calmodulin genes identified six species, A. niger, A. tubingensis, A. flavus, A. aculeatus, A. sydowii and A. fumigatus. Phylogenetic tree of combined sequences showed the isolates from the same species were grouped with reference strains in the same clade, thus the species identity was confirmed. Detection of mycotoxin biosynthesis genes can give an indication of mycotoxin production. Two ochratoxin A genes, PKS15KS and PKS15C-MeT were detected in seven A. niger isolates but none of the isolates produced ochratoxin A when quantification was conducted using Ultra-High Performance Liquid Chromatography. Two aflatoxin B1 biosynthesis genes, Nor-1 (norsolorinic acid) and Ver-1 (Versicolorin) genes were detected in A. flavus but only KDH7 and KL27b isolates produced aflatoxin B1 with concentrations of 1.0 μg/g and 1.1 μg/g, respectively. @*Conclusion, significance and impact of study@#Various species of Aspergillus found on groundnuts may lead to potential mycotoxin contamination as toxigenic species were also recovered. The occurrence of Aspergillus spp. can reduce the quality of the legumes as well as reducing their shelf life.

Toxicological characteristics of Ochratoxin A and its impact on male reproduction / 中华男科学杂志

Ochratoxin A (OTA) is found not only nephrotoxic, teratogenic, neurotoxic, and immunotoxic, but also reprotoxic for human and animals. In the recent decade, more attention has been paid to the impact of OTA on human reproduction and the studies of its underlying mechanisms. Many studies show that OTA affects the function of the reproductive system by acting as an endocrine disrupter and, as a testicular toxin, decreases sperm quality and even induces testis cancer. This review summarizes the toxicological characteristics and toxicokinetic process of OTA as well as recent progress in the studies of various toxic effects of OTA and their underlying mechanisms, hoping to call the attention from more people to the toxicity of OTA to male reproductive health.

Research progress of rapid detection of ochratoxin A and its application prospect in traditional Chinese medicine / 中国中药杂志

Ochratoxin A (OTA) is a toxic secondary metabolite mainly produced by Aspergillus and Penicillium species, with strong renal toxicity, teratogenic, carcinogenic, mutagenic effect. Studies have shown that OTA is not only widely contaminated in food and feed crops, but also has been widely contaminated in Chinese herbal medicines such as spices, licorice and so on. In view of OTA's universality and harmfulness, this paper summarizes the flow visualization test strip, microsphere, electrochemical sensor, surface enhanced Raman spectroscopy technology in OTA rapid detection, which provides reference for the research and application of high throughout detection instrument miniaturization in order to achieve OTA quick detection and simple operation.

Honey mediated silver nanoparticles and their inhibitory effect on aflatoxins and ochratoxin A.

Silver nanoparticles (AgNPs) have potential antimicrobial activity against bacteria and fungi. The synthesis of AgNPs have been reported using several chemical and physical methods which are not friendly environment. Therefore, our technique has focused on the synthesis of AgNPs by natural compounds. The aim of this study has been to synthesis AgNPs by safe nontoxic method using Egyptian honey (EH) as reducing and capping agents and to investigate its ability to reduce the mycelial growth and the production of aflatoxins (AFs) and ochratoxin A (OTA) by Aspergillus flavus and Aspergillus ochraceus, respectively. AgNPs have been characterized by UV-Visible Spectrophotometer, Dynamic Light Scattering (DLS), Fourier Transform Infrared Spectroscopy (FTIR), and Transmission Electron Microscope (TEM).The obtained results indicated that the synthesis of honey AgNPs depends on the concentration of bulk metal (AgNO3) used in the synthesis process. The TEM image has revealed the formation of spherical well dispersed AgNPs, while the main size of AgNPs detected by DSL is 9.9 nm. Our results have indicated that 3 mg -100 ml media of honey derived AgNPs have reduced the aflatoxin (AF) G1, G2, B1 andB2 production by A. parasiticus to 77.55, , 62.91, 58.76 and 66.56%, respectively and ochratoxin A (OTA) by A. ochraceus to 79.85 % with significantly inhibitory effect on mycelial growth. The percentage of reduction depends on the AgNPs concentration.

Development of ochratoxin A detection based on electrochemical sensor using Au-ball labels

Aims: The present study was aimed to develop a highly sensitive and rapid method for ochratoxin A (OTA) detection. Methodology and results: In this study, an electrochemical peptidesensor for OTA detection was developed using numerous number of Au particles coated on the surface of silica particle (Au-ball). Moreover, this assay was performed in 384 well plate, so the multiple detections was done. The synthesized silica particle was spherical in shape and size was 275±17 nm. After coated with Au layer, size of Au-ball was 280±14 nm. Au particles loaded can be taken up resulting in approx. 1×107 Au3+ molecules per silica particle. Moreover, the optimization conditions were studied. The limit of detection of this assay showed as low as 2 ppb. Conclusion, significance and impact study: This platform showed rapid, sensitive and specific to ochratoxin A detection. In spite of these advantages, this Au-ball based peptidesensors is suitable to use in the detection of ochratoxin A contaminated in coffee or seed industry.

Occurrence, harmful effects and analytical determination of Ochratoxin A in coffee.

Coffee is an important stimulant and a good source of bioactive constituents with beneficial health effects. Coffee beans are readily attacked by the growth and proliferation of several fungal species from the genera Aspergillus and Penicillium. Ochratoxin A, an important toxin produced by these fungal entities, is commonly found in coffee products, from farm to cup-thus challenging the popularity of this world-acclaimed beverage. The toxin has been shown to be nephrotoxic and hepatotoxic, among other deleterious effects. This has prompted the European Commission to set limits for ochratoxin A in coffee products. In order to abide by these regulatory codes, various analytical methods have been developed and deployed in the analysis of the toxin in green coffee beans, roasted coffee and instant coffee. This review will discuss the occurrence of ochratoxin A, its harmful effects and various analytical methods that have been applied in its detection and quantification in coffee products.

Development and characterization analysis of a new type of electrochemical immunosensor for the detection of ochratoxin A / 国际检验医学杂志

Objective To develop a new type of electrochemical immunosensor for the detection of ochratoxin A (OTA ) . Methods Double layers of self‐assembly immunosensor for the detection of OTA were constructed based on the composite single‐walled carbon nanotubes(SWNTs)/chitosan(CS) membrane immobilized on glassy carbon electrode(GC) .Scanning electron mi‐croscopy(SEM) ,square wave voltammetry and cyclic voltammetry were used to analyze the characterization of the sensor ,then its specificity for detection was studied .Results SWNTs/CS composit membrane could increase the sensitivity of OTA detection sig‐nificantly ,and effectively distinguish the different types of mycotoxins .Conclusion The electrochemical immunosensor developed in the study is easy to operate and could detect OTA rapidly with good specificity and low detection limit .

Safety issues and new rapid detection methods in traditional Chinese medicinal materials

The safety of traditional Chinese medicine (TCM) is a major strategic issue that involves human health. With the continuous improvement in disease prevention and treatment, the export of TCM and its related products has increased dramatically in China. However, the frequent safety issues of Chinese medicine have become the 'bottleneck' impeding the modernization of TCM. It was proved that mycotoxins seriously affect TCM safety; the pesticide residues of TCM are a key problem in TCM international trade; adulterants have also been detected, which is related to market circulation. These three factors have greatly affected TCM safety. In this study, fast, highly effective, economically-feasible and accurate detection methods concerning TCM safety issues were reviewed, especially on the authenticity, mycotoxins and pesticide residues of medicinal materials.

Rhodotorula glutinis and Its Two Mutants Ameliorate Hepato-Renal Dysfunction Induced by Ochratoxin A on Rats.

Aim: This study was designed to investigate the possible curative effect of Rhodotorula glutinis (R. glutinis) and its two mutants (Col-1R1 and Col-1R3) against hepatorenal toxicity induced by ochratoxin (OA) in rat. Methods: The strains of yeast Col- 1R1 and Col- 1R3 have been genetically improved and isolated from R. glutinis after colchicines treatment. OA was produced and determined from Aspergillus ochracus isolate from Egyptian corn. Experimental design: Five groups of rats were treated as follows: group 1, was the control group orally given 4 ml / Kg 0.1 M NaCOH3; group 2 treated with OA (1.7 mg /Kg).Groups 3, 4 and 5 orally administered the R. glutinis and its two mutants (50 X106 colony forming unit (cfu) / 10 ml saline / kg body weight) prior 1hr of OA -treatment for 15 successive days. Results: The studied autoploidy strains showed significant increase in caratenoids level, protease, β-1, 3-glucanase and chitinase activities when compared with the parental strain. Biochemical results revealed that OA significantly decreased serum total antioxidant capacity (TAC) and it caused elevation inserum transaminases (AST, ALT), creatinine, uric acid, nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and carcinoembryonic antigen (CEA) (P <0.05) as compared with the control group. The three tested yeasts significantly decreased the elevated values toward the normal levels and improved the pathological feature in liver and kidney tissues. Moreover, R. glutinis and the two mutants significantly reduced hepatorenal damaged arias, increased optical density of DNA and alleviated ochratoxin A-induced caspase-3 activation. The resultant effect of the two mutant strains had more powerful effect more than the wild strainto ameliorate hepatorenal dysfunction in ochratoxicosis-rat. Conclusion: Col-1R3 was more effective than Col-1R1 may be due to its higher contents of carotenoids, glucane and chitine, which act as antioxidants.

Fungal Flora and Ochratoxin A Associated with Coffee in Cameroon.

Aims: 104 samples were collected from the west region and the coastal plain of Cameroon during two coffee campaigns, 2009 and 2010. Two coffee processes were evaluated (wet and dry processes) at different stages from harvesting to storage. Study Design: Food contaminants. Place and Duration of Study: Food Microbiology Laboratory, Department of Food Science and Nutrition (ENSAI) University of Ngaoundere; UMR 95 Qualisud, CIRAD of Montpellier, between May 2009 and September 2012. Methodology: Fungi profile was evaluated by direct plating techniques and identified using morphological and molecular tools. OTA levels were analyzed using HPLC technique after extraction and filtration using an immunoaffinity column. Results: Results obtained revealed an overall percentage of fungal contamination between 60-92% in 2009 and 70-90% in 2010. There was no ecological difference in the composition of ochratoxigenic species present in five sites. Coffee beans sampled in 2009 had a colonization incidence of 18-40% A. carbonarius, 12-22% A. niger, 3-15% A. ochraceus while those of 2010 had a colonization incidence of 15-30% A. carbonarius, 35- 40% A. niger, and 2-7% A. ochraceus. Fungal diversity was not correlated with the geographical origin, coffee cultivar and processing method. There was no difference between the processes studied in terms of occurrence of ochratoxigenic fungi. OTA levels were mostly below the recommended standards although some isolated cases of extreme contamination were observed in 2009. A higher level of OTA was detected in the presence of A. niger, A. carbonarius and A. ochraceus than when only A. niger was present. Conclusion: The important fungi with the potential to produce OTA in Cameroonian coffee beans are A. carbonarius and A. niger. These two species were predominant on each type of coffee beans. It was also observed that once a toxigenic strain was isolated from a coffee sample, the sample contained OTA.

Ochratoxigenic fungi associated with green coffee beans (Coffea arabica L.) in conventional and organic cultivation in Brazil

The genera Aspergillus comprises species that produce mycotoxins such as aflatoxins, ochratoxins and patulin. These are cosmopolitan species, natural contaminants of agricultural products. In coffee grains, the most important Aspergillus species in terms of the risk of presenting mycotoxins belong to the genera Aspergillus Section Circumdati and Section Nigri. The purpose of this study was to assess the occurrence of isolated ochratoxigenic fungi of coffee grains from organic and conventional cultivation from the South of Minas Gerais, Brazil, as well as to evaluate which farming system presents higher contamination risk by ochratoxin A (OTA) produced by fungi. Thirty samples of coffee grains (Coffea arabica L.) were analysed, being 20 of them of conventional coffee grains and 10 of them organic. The microbiological analysis was done with the Direct Plating Technique in a Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. The identification was done based on the macro and micro morphological characteristics and on the toxigenic potential with the Plug Agar technique. From the 30 samples analysed, 480 filamentous fungi of the genera Aspergillus of the Circumdati and Nigri Sections were isolated. The ochratoxigenic species identified were: Aspergillus auricoumus, A. ochraceus, A. ostianus, A. niger and A. niger Aggregate. The most frequent species which produces ochratoxin A among the isolated ones was A. ochraceus, corresponding to 89.55%. There was no significant difference regarding the presence of ochratoxigenic A. ochreceus between the conventional and organic cultivation systems, which suggests that the contamination risk is similar for both cultivation systems.

Determinación de ocratoxina-a por HPLC con detección por fluorescencia (HPLC-FL): un nuevo método estandarizado para muestras de trigo / Ochratoxin-a determination by HPLC with fluorescence detection(HPLC-FL): a new standardization method for wheat samples

El objetivo principal de este estudio fue evaluar la presencia de Ocratoxina-A (OTA) en los granos del trigo y harina del trigo realizadas por un nuevo método de determinación que usa la cromatografía líquida de alta resolución (CLAR) acoplada al descubridor delfluorimetrio. El experimento usó seis muestras de grano de trigo del lugar del almacenamiento diferente a la industria local de Chapeco (SC), Brasil Sur, en agosto, 2008. El extracto de OTA era llevado a cabo usando el acetonitrila: agua (120:80 vlv) como solventes. Después el suprenadante fue filtrado, y aplicado en la columna del inmunoafinidad específica a OTA. Además, la columna se lavó con agua y la toxina era el eluido con el metanol. La determinación del OTA se realizó por detección de fluorescencia acoplado al aparato de HPLC. Los volúmenes de OTA en los granos del trigo y harina del trigo eran entonces los determínate y los resultados mostraron una concentración de OTA menor que los límites exigidos por la legislación internacional.

The main objective of this study was to evaluate the presence of Ochratoxin A (OTA) in wheat grains and wheat flour samples using a new high performance liquid chromatography (HPLC) method. The experiment used six wheat grain samples from different industry storage place from Chapeco (SC), South Brazil, on August 2008. The OTA extraction was carried out using acetonitrile: water (120:80 v/v) as solvent. Thereafter, the supernatant was filtered, and applied on OTA-specific immunoafinity column to HPLC Furthermore, the column was washed with water and the toxin was eluted with methanol. The OTA wheat grains and wheat flour concentration were analyzed by a fluorescence detector coupled to the HPLC apparatus. The results showed a smaller OTA concentration than the limits set by international legislation.

Fate of ochratoxin A content in Argentinean red wine during a pilot scale vinification / Evolución del contenido de ocratoxina A en vinos tintos argentinos durante el proceso de vinificación a escala piloto

The aim of this work was to evaluate the fate of ochratoxin A (OTA) content from must to wine during the red wine making process in a pilot scale vinification. The study was done using musts obtained from two red grape varieties (Bonarda and Tempranillo) artificially contaminated with two OTA levels. A duplicate set of tanks of 100 l each was established for each must (Bonarda and Tempranillo). The fermentations were initiated by inoculation of two Saccharomyces spp. strains having different fermentation performance. The must from the Tempranillo variety was spiked with 6 μg/l of OTA while that from the Bonarda variety with 0.3 μg/l of the toxin. Samples were collected at different stages of the process. Performance of the alcoholic and malolactic fermentations was monitored. Titratable and volatile acidity, pH, ethanol, sugar and SO2 concentrations were determined following standard methods proposed by the Office International de la Vigne et du Vin (OIV). OTA analysis was done by HPLC. Detection and quantification limits were 0.01 and 0.1 ng/ml, respectively. The OTA levels during the vinification trials dropped to an average of about 86.5%. The type of Saccharomyces strains used showed no effect on toxin reduction.

El objetivo del presente trabajo fue evaluar la evolución del contenido de ocratoxina A (OTA) en mostos durante un proceso de vinificación a escala piloto. Se utilizaron mostos de dos variedades de uvas tintas (Bonarda y Tempranillo) contaminados artificialmente con dos niveles distintos de OTA. El ensayo fue llevado a cabo por duplicado en tanques de fermentación de 100 l cada uno. La fermentación se inició mediante la inoculación de dos cepas de Saccharomyces spp. con diferentes características fermentativas. El mosto de la variedad Tempranillo fue contaminado con 6 μg/l de OTA y el mosto de la variedad Bonarda con 0,3 μg/l de la toxina. Se colectaron muestras durante los diferentes estadios del proceso de vinificación. Se estableció el avance de dicho proceso sobre la base de la evolución de las fermentaciones alcohólica y maloláctica. Se determinó la acidez total y volátil, el pH y el contenido de etanol, de azúcar y de SO2 siguiendo los protocolos estándares propuestos por la Oficina Internacional de la Vid y el Vino (OIV). El contenido de OTA se evaluó por HPLC. Los límites de detección y cuantificación fueron 0,01 y 0,1 ng/ml, respectivamente. Los niveles de OTA disminuyeron alrededor del 86,5% al final del proceso de vinificación. El tipo de cepa de Saccharomyces spp. utilizada no tuvo efecto sobre la reducción de OTA.

Genetic relationships among strains of the Aspergillus niger aggregate

We analyzed the genetic relationships between 51 fungal isolates previously identified as A. niger aggregate, obtained from dried fruit samples from worldwide origin and 7 A. tubingensis obtained from Brazilian coffee beans samples. Greater fungal diversity was found in black sultanas. Aspergillus niger sensu stricto was the most prevalent species. It was found in all fruit substrates of all geographical origins. Based on Random Amplification of Polymorphic DNA (RAPD) and β-tubulin sequences data two groups of A. niger were found. In spite of the small number of isolates from Group IV an association between extrolite patterns and molecular clustering is speculated. A. tubingensis were the second most frequent species and this species were clearly subdivided into two groups. The finding of two groups for A. tubingensis strains could not yet explain the contradictions found in the literature about the capability this species for ochratoxin production, because both of them were formed by only non-ochratoxin-producing strains.

Neste trabalho foi analisada a relação genética entre 51 isolados obtidos de amostras de frutas secas provenientes de diferentes regiões do previamente identificados como pertencentes ao agregado A. niger e 7 isolados de Aspergillus tubingensis obtidos de amostras de café do Brasil. Maior diversidade fúngica foi encontrada em uvas passas escuras. Aspergillus niger sensu stricto foi a espécie mais frequente. Esta espécie foi encontrada em todos os substratos e origens geográficas analisadas. Baseando-se nos dados de Polimorfismo de DNA Amplificado ao Acaso (RAPD) e sequências de nucleotídeos do gene da β-tubulina, dois grupos de A. niger foram observados. Apesar do pequeno número de isolados do grupo IV uma associação entre padrão de extrólitos e agrupamento molecular foi encontrada. A. tubingensis foi a segunda espécie mais frequente e foi claramente subdivida em dois grupos. Como os grupos de A. tubingensis são formados somente por linhagens não produtoras de ocratoxina A, a identificação destes grupos não explica a controvérsia encontrada na literatura sobre a capacidade desta espécie em produzir a referida toxina.

Aspectos relacionados à presença de fungos toxigênicos em uvas e ocratoxina A em vinhos / Aspects related to the presence of toxigenic fungi in grapes and ochratoxin A in wines

A colonização dos Aspergillus da secção Nigri nas uvas durante o cultivo é a principal fonte de ocratoxina A (OTA) nos vinhos. A. carbonarius e A. niger são os principais produtores desta micotoxina em uvas e são fungos oportunistas que, se desenvolvem, principalmente, nas bagas danificadas durante seu amadurecimento. A produção de OTA em uvas é influenciada pelas condições climáticas e áreas geográficas, bem como pela variedade de uva, pelo sistema de cultivo e pelos danos causados nas uvas por insetos, infecção fúngica ou excesso de irrigação e chuva. As medidas para o controle de fungos toxigênicos devem considerar esses pontos críticos de controle. A OTA presente nas uvas é transferida para o vinho durante o processo de vinificação, sendo que um aumento na concentração de OTA ocorre após a maceração das uvas. Durante o envelhecimento do vinho, observa-se que a toxina permanece estável, pois a mesma concentração de OTA é encontrada no vinho após um ano de armazenamento. Boas práticas de produção, como, por exemplo, a seleção e separação dos cachos de uva com desenvolvimento fúngico visível auxilia, consideravelmente, na redução dos níveis de contaminação por fungos produtores de OTA, bem como dos níveis dessa micotoxina nos vinhos.

The infection of grapes by 'black aspergilli' in the field is the main source of ochratoxin A (OTA) in the wine. Aspergillus carbonarius and A. niger fungi are the main producers of this mycotoxin in grapes. They are opportunistic fungi that develop mainly on damaged berries at ripening. The production of OTA in grapes is influenced by climatic conditions, geographical location, grape varieties, crop system, berries damage caused by insets, fungal infection or excessive irrigation and rainfall. Control measures for toxigenic mycoflora in the vineyards must consider these critical control points. OTA in grapes is transferred to wine during vinification and an increase of OTA concentration in must was observed during maceration. The toxin remains stable during wine aging because the same OTA concentration is found in wine after 1 year. Good Agriculture Practices including balanced soil tillage, irrigation, nitrogen fertilization and pruning associated with Good Manufacturing Practices, such as separation of rot bunches helps considerably to reduce OTA-producing fungi and levels of mycotoxin in wine.