RÉSUMÉ
An indirect competitive enzyme-linked immunosorbent assay( ic-ELISA) was developed for the rapid detection of ochratoxin A( OTA) in nutmeg( Myristicae Semen),ginger( Zingiberis Rhizoma) and turmeric( Curcumae Longae Rhizoma). The matrix matching standard curve was used instead of the standard curve of sample diluent,and the sample extract and sample diluent were optimized. The sensitivity( IC_(50)) of this method for OTA in nutmeg,ginger and turmeric were determined as 0. 146,0. 157 and 0. 153 ng·m L~(-1),respectively and the limits of detection( LODs) were 0. 040,0. 032 and 0. 031 ng·m L~(-1),respectively. The recovery of samples ranged from 75. 99% to 122. 3%,with RSD<10%. Two positive samples for nutmeg and one positive sample for turmeric occurred in 50 samples,and the highest OTA contamination value was 1 167. 8 μg·kg~(-1). The results were further confirmed by LC-MS/MS. It shows that the developed ic-ELISA method is simple,rapid and sensitive,and can be applied for rapid and high-throughput screening of OTA in nutmeg,ginger and turmeric,as well as some other CHMs.
Sujet(s)
Chromatographie en phase liquide , Contamination de médicament , Médicaments issus de plantes chinoises/analyse , Test ELISA , Tests de criblage à haut débit , Ochratoxines/analyse , Spectrométrie de masse en tandemRÉSUMÉ
A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53‒12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06‒39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.
Sujet(s)
Aliment pour animaux , Calibrage , Chromatographie d'affinité , Chromatographie en phase liquide , Colloïdes , Contamination des aliments/analyse , Sécurité des aliments , Or , Dosage immunologique/méthodes , Concentration inhibitrice 50 , Limite de détection , Nanoparticules métalliques , Ochratoxines/analyse , Analyse de régression , Reproductibilité des résultats , Sensibilité et spécificité , Triticum , Zea mays , Zéaralénone/analyseRÉSUMÉ
Mycotoxins are a group of chemically diverse naturally occurring substances resulting from the secondary metabolism of pathogenic filamentous fungi. They are produced mainly by the genera Fusarium, Alternaria, Aspergillus and Penicillium which can contaminate grains and cereals such as wheat, corn and soy. According to the nature and the concentration levels, mycotoxins can induce toxic effects in food-production animals and humans. An in vitro study was conducted to evaluate the susceptibility of broiler chickens lymphocytes to different concentrations of ochratoxin A, deoxynivalenol and zearalenone. Each toxin was added to the cell medium at different concentrations (0.001, 0.01, 0.1 and 1μg/mL). Cell viability and ecto-adenosine deaminase activity were assessed at 24, 48 and 72 hours by colorimetric assays. Thus, it were used 0.7x10(5) lymphocytes/mL in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 2.5 IU of penicillin/streptomycin per mL, incubated at 37°C in a 5% CO2 atmosphere. All the experiments were carried out in triplicate and the results were expressed as mean ± standard error of the mean. The results showed that OTA and DON induced lymphocyte proliferation and reduced enzymatic activity in vitro (P<0,05), whereas ZEA also promoted proliferation (P<0,05), but neither alteration on enzymatic activity (P>0,05). It was possible to correlate the results about viability cell and ecto-adenosine deaminase activity, suggesting that, at minimal concentrations, the evaluated mycotoxins do not stimulated the enzymatic activity, which has proinflammatory action and contributes for the immunosuppression process, thus, avoiding a decrease on the viability cell. This is the first in vitro study conducted with OTA, DON and ZON in broiler chickens lymphocytes evaluating these parameters.
Micotoxinas representam um vasto grupo de contaminantes químicos naturais originados a partir do metabolismo secundário de fungos filamentosos patogênicos. Elas são produzidas, principalmente, pelos gêneros Fusarium, Alternaria, Aspergillus e Penicillium, os quais podem contaminar grãos e cereais, como trigo, milho e soja. Conforme sua natureza e níveis de concentração, micotoxinas podem induzir efeitos tóxicos em animais de produção e humanos. Um estudo in vitro foi realizado para avaliar a susceptibilidade das células linfocitárias de frangos de corte a diferentes concentrações de ocratoxina A, deoxinivalenol e zearalenona. Cada micotoxina foi adicionada ao meio celular em diferentes concentrações (0,001; 0,01; 0,1 e 1μg/mL). A viabilidade celular e atividade de ecto-adenosina desaminase foram analisadas em 24, 48 e 72 horas através de ensaios colorimétricos. Para isso, foram utilizados 0,7x10(5) linfócitos/mL em meio RPMI 1640, suplementado com 10% de soro fetal bovino e 2,5 UI de penicilina/estreptomicina por mL, incubados em atmosfera de 5% de CO2 a 37 °C. Todos os experimentos foram realizados em triplicata e os resultados foram expressos como média e erro padrão da média. Os resultados obtidos demonstraram que tanto ocratoxina A como deoxinivalenol induziram proliferação linfocitária e baixa atividade enzimática in vitro (P<0,05), enquanto zearalenona também induziu proliferação (P<0,05), mas nenhuma alteração na atividade enzimática (P>0,05). Foi possível correlacionar os dados referentes à viabilidade celular e atividade de ecto-adenosina desaminase, sugerindo que, em concentrações mínimas, as micotoxinas testadas não estimularam a atividade da enzima, que possui ação pró-inflamatória e contribui para o processo de imunossupressão e, portanto, evitando um decréscimo na viabilidade celular. Este é o primeiro estudo feito com OCRA, DON e ZEA sobre linfócitos de frangos de corte em cultivos in vitro na avaliação desses parâmetros.
Sujet(s)
Animaux , Ochratoxines/administration et posologie , Ochratoxines/analyse , Ochratoxines/composition chimique , Techniques in vitro/classification , Techniques in vitro/médecine vétérinaire , Zéaralénone/analyse , Zéaralénone/composition chimiqueRÉSUMÉ
Se realizó un estudio de las condiciones del procesamiento del café de exportación en 15 beneficios, ubicados en Chiriquí, región occidental de Panamá. Además se analizaron 21 muestras de café procesado (grano verde), provenientes de los beneficios. Las muestras fueron analizadas microbiológicamente y se cuantificaron las Aflatoxinas totales (B1, B2, G1 y G2) y Ocratoxina A (OTA), mediante el método de inmunoafinidad ELISA. Se determinó un límite de detección de 0,017 ng/mL, para la Ocratoxina A, lo que equivale a una concentración de 0,829 μg/kg en la muestra, y un límite de detección de 0,027 ng/mL, para las Aflatoxinas totales, lo que equivale a una concentración de 1,350 μg/kg de Aflatoxinas totales. En la muestra, se encontró que cuatro de las 21 (19%) resultaron positivas a la presencia de Ocratoxina A y tres, a la presencia de Aflatoxinas totales (14%). Las muestras presentaron niveles de Ocratoxina A en el rango de 4,90-37,73 μg/kg; sólo tres de ellas superaron el límite máximo permitido por la Unión Europea, para la concentración de Ocratoxina, que es de 5,0 μg/kg. Las Aflatoxinas totales se encontraron en el rango de 1,51- 1,93 μg/kg, por debajo de los 10 μg/kg, que es el límite máximo permitido en el café por la Unión Europea. Los resultados nos indican que el procesamiento de café producido en Panamá cumple satisfactoriamente con los estándares internacionales de manejo poscosecha, lo que conduce a una baja incidencia de hongos productores de micotoxinas y niveles muy bajos de micotoxinas.
Levels of Ochratoxin A and total Aflatoxins in Panamanian exportation coffee by an ELISA Method. A study about processing conditions of exportation coffee in 15 benefits located in Chiriquí, western region of Panama, was conducted. In addition, 21 samples of processed coffee (green beans), from the benefits, were analyzed. The samples were microbiologically tested in order to quantify total aflatoxins (B1, B2, G1 and G2) and Ochratoxin A (OTA), using the immunoaffinity ELISA method. A detection limit of 0.017 ng/mL, was determined for Ochratoxin A, which is equivalent to a concentration of 0.829 μg/kg, and a detection limit of 0.027 ng/mL, for total aflatoxins, which is equivalent to a concentration of 1.350 μg/kg. It was found that four (19%) out of the 21 samples were positive to the presence of Ochratoxin A and three (14%) to the presence of total aflatoxins. Samples showed levels of Ochratoxin A in the range 4.90 - 37.73 μg/kg; only three of them exceeded the maximum limit allowed by the European Union, for the concentration of Ochratoxin, which is of 5.0 μg/kg. Total aflatoxins were found in the range 1.51 - 1.93 μg/kg, below 10μg/kg which is the maximum limit allowed for coffee by the European Union. The results indicate that the processing of coffee produced in Panama successfully meets international standards for postharvest handling, which leads to a low incidence of mycotoxins and very low levels of mycotoxin- producing fungi.
Sujet(s)
Aflatoxines/analyse , Café/composition chimique , Ochratoxines/analyse , Chromatographie en phase liquide à haute performance , Commerce , Test ELISA , Contamination des aliments/analyse , Panama , Normes de référenceRÉSUMÉ
El Análisis de Peligros y Puntos de Control Crítico (HACCP) es una herramienta para la Gestión de Inocuidad de los alimentos que permite identificar los peligros físicos, químicos y biológicos asociados al proceso a través de toda la cadena productiva. Este trabajo tiene por finalidad diseñar el Programa de HACCP para el proceso de producción de cacao en polvo en una industria de alimentos venezolana. Previamente se evaluó el cumplimiento de las Buenas Prácticas de Manufactura (BPM) y los Procedimientos Operativos Estándar de Saneamiento (POES), elementos básicos para el establecimiento del HACCP. Se visitaron las instalaciones de varios proveedores a objeto de observar el cumplimiento de las Buenas Prácticas Agrícolas (BPA). Para el desarrollo del programa HACCP se aplicaron los siete principios básicos del mismo y las cinco tareas preliminares, conforme a la metodología descrita por el Codex Alimentarius.Conducido el análisis de peligros, se identificaron tres puntos de control críticos en la línea de proceso: descascarillado (control de ocratoxina A), fase de tostado (control de Salmonella) y detección de partículas metálicas. Se establecieron los Límites Críticos, los Procedimientos de Vigilancia, las Acciones Correctivas, los Procedimientos de Verificación y de Documentación, recomendándose implementar el Programa HACCP en la industria procesadora de cacao en polvo con la realización de los ajustes correspondientes en los casos donde sea necesario. Recientemente la ocratoxina A (OTA),ha sido relacionada con el cacao en grano. Aunque se ha señalado que el descascarillado es una medida de control efectiva para este peligro químico, se recomienda estudiar la prevalencia de OTA en el cacao producido en el país y validar la etapa del descascarillado como control de micotoxinas.
Design of an HACCP program for a cocoa processing facility. The HACCP plan is a food safety management tool used to control physical, chemical and biological hazards associated to food processing through all the processing chain. The aim of this work is to design a HACCP Plan for a Venezuelan cocoa processing facility.The production of safe food products requires that the HACCP system be built upon a solid foundation of prerequisite programs such as Good Manufacturing Practices (GMP) and Sanitation Standard Operating Procedures (SSOP). The existence and effectiveness of these prerequisite programs were previously assessed.Good Agriculture Practices (GAP) audit to cocoa nibs suppliers were performed. To develop the HACCP plan, the five preliminary tasks and the seven HACCP principles were accomplished according to Codex Alimentarius procedures. Three Critical Control Points (CCP) were identified using a decision tree: winnowing (control of ochratoxin A), roasting (Salmonella control) and metallic particles detection. For each CCP, Critical limits were established,the Monitoring procedures, Corrective actions, Procedures for Verification and Documentation concerning all procedures and records appropriate to these principles and their application was established. To implement and maintain a HACCP plan for this processing plant is suggested. Recently OchratoxinA (OTA) has been related to cocoa beans. Although the shell separation from the nib has been reported as an effective measure to control this chemical hazard, ochratoxin prevalence study in cocoa beans produced in the country is recommended, and validate the winnowing step as well.
Sujet(s)
Cacaoyer/normes , Contrôle des aliments/méthodes , Analyse des risques et maitrise des points critiques/méthodes , Ochratoxines/analyse , Prise décision institutionnelle , Contamination des aliments/prévention et contrôle , Industrie de la transformation des aliments/normes , Mise au point de programmes , Contrôle de qualité , Gestion de la sécurité , Salmonella/croissance et développement , VenezuelaRÉSUMÉ
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.
Sujet(s)
Contamination des aliments/analyse , Chromatographie en phase liquide à haute performance/méthodes , Ochratoxines/analyse , Triticum/composition chimique , Fluorescence , Microbiologie alimentaireRÉSUMÉ
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.
Sujet(s)
Contamination des aliments , Microbiologie industrielle/méthodes , Ochratoxines/analyse , Vin/analyse , Argentine , Éthanol/analyse , Fermentation , Concentration en ions d'hydrogène , Microbiologie industrielle/normes , Projets pilotes , Spécificité d'espèce , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/métabolisme , Vitis/composition chimique , Vitis/classification , Vin/normesRÉSUMÉ
The effect of propionic acid, ammonia and methionine on aflatoxins and ochratoxin A production by Aspergillus parasiticus and Penicillium verrucosum in autoclaved and non-autoclaved corn was investigated. The tested mycotoxins were determined by HPLC using fluorescence detector. The results indicated that the inhibition of toxins production vary between the three tested preservatives and the concentration of each as well. The higher inhibition of toxins production was obtained using a mixture which consists of equal volumes of propionic acid [65%], ammonia [2%] and methionine [0.2%]. In non-autoclaved corn, the inhibition of aflatoxin B1 and aflatoxin G1 production was 99.6% and 99.7% respectively, while aflatoxin B2, aflatoxin G2 and ochratoxin A production were completely inhibited. Meanwhile, in autoclaved corn, the inhibition of aflatoxins B1, B2, G1, G2 and ochratoxin A reached 83.0%, 83.6%, 85.7%, 83.1% and 86.4%, respectively
Sujet(s)
Conservateurs alimentaires/effets indésirables , Ochratoxines/analyse , Zea mays , Ammoniac , Méthionine , Propionates , Chromatographie en phase liquide à haute performanceRÉSUMÉ
Costa Rica no es la excepción en cuanto a la prevalencia de ocratoxina A en plasma, ya que en este estudio se obtuvo la presencia de la micotoxina en el 95% de las 149 muestras estudiadas. También se estudió la presencia de la ocratoxina A en 110 muestras de diferentes marcas de café tostado y molido de las 12 torrefactoras más importantes del país y de 7 supermercados. A excepción de una muestra de café que dio resultados negativos, el resto de muestras analizadas presentaron la micotoxina en cantidades menores a 4000 ng/L o kg. Se trató de encontrar una asociación entre el consumo de café y la presencia de la ocratoxina A en el plasma así como del consumo de cerveza, sin embargo no hubo diferencia estadísticamente significativa en el valor promedio de la micotoxina entre los tomadores y no tomadores de café y tampoco entre los bebedores y no bebedores de cerveza.
Ocratoxin A in human plasma and coffee from Costa Rica by ELISA. Costa Rica is not an exception in the prevalence of ochratoxin A in human plasma, in this research the presence of the micotoxin was found in 95% of the 149 samples studied. The presence of ocratoxina A also was studied in 110 samples of toasted and grounded coffee from the most important 12 coffee factories of the country and from 7 supermarkets. With the exception of one negative sample the rest of them have concentrations of micotoxin below 4000 ng/kg. An association between the coffee consumption and the presence of ochratoxin A in plasma was attempted to be found as well as in the consumption of beer, but there were any statistically significant difference in the average level of mycotoxin between the coffee consumers and non coffee consumers neither between beer consumers and no beer consumers.
Sujet(s)
Humains , Adulte , Café/composition chimique , Analyse d'aliment/méthodes , Contamination des aliments/analyse , Ochratoxines/analyse , Études cas-témoins , Costa Rica , Test ELISA , Ochratoxines/sangRÉSUMÉ
O Laboratório da Seçäo de Química Biológica do Instituto Adolfo Lutz tem sido requisitado desde outubro de 1989 a emitir laudos de análise de ocratoxina A (OA) em café cru em gräo destinado à exportaçäo para Grécia e Líbano, pois estes países impuseram o limite máximo aceitável de 20 mg/kg de OA neste produto. Foram comparados dois métodos de análise de ocratoxina A por cromatografia em camada delgada para café cru em gräo. Um deles descrito no AOAC é específico para a determinaçäo de OA neste produto e o outro foi desenvolvido por Soares e Rodriguez-Amaya (1985) e Sabino (1989). Este último já é utilizado rotineiramente no laboratório nas análises de produtos como arroz, amendoim, mandioca, feijäo, milho e seus derivados. Neste estudo foram levados em consideraçäo os seguintes parâmetros: recuperaçäo e reprodutibilidade do método, facilidade deoperaçäo, disponibilidade de materiais, exposiçäo do analista a reagentes tóxicos, rapidez e custo para implantaçäo na rotina de análise. Os resultados experimentais demonstraram que perante um método específico e consagrado, da AOAC, o método 2 mostrou ser bom e promissor. Escolheu-se a técnica de Soares e Rodriguez-Amaya modificada por Milanez e Sabino pois revelou possuir maior simplicidade de operaçäo e menor custo