Mycotoxins in raw materials, beverages and supplements of botanicals: A review of occurrence, risk assessment and analytical methodologies.

Over recent years, consumer interest in natural products, such as botanicals has increased considerably. One of the factors affecting their quality is the presence of mycotoxins. This review focuses on exploring the mycotoxin occurrence in botanicals (raw material and ready-to-eat forms such as infusions or tablets) and the risk assessment due to their ingestion. Aflatoxins, Ochratoxin A, and Fumonisins are the most commonly studied mycotoxins and data in the literature report levels ranging from traces to 1000 µg/kg in raw materials. In general, the highest contents observed in raw materials decreased to unconcerning levels after the preparation of the infusions, reaching values that generally do not exceed 100 µg/L. Regarding botanical dietary supplements, the levels observed were lower than those reported for other matrices, although higher levels (of up to 1000 µg/kg) have been reported in some cases. Risk assessment studies in botanicals revealed a higher risk when they are consumed as tablets compared to infusions. Analytical methodologies implied in mycotoxin determination have also been contemplated. In this sense, liquid chromatography coupled to fluorescence detection has been the most frequently employed analytical technique, although in recent years tandem mass spectrometry has been widely used.

Presence of mycotoxins in ready-to-eat food and subsequent risk assessment.

A study on a set of ready-to-eat meals (n = 328) based on cereals, legumes, vegetables, fish and meat was carried out to determine the natural presence of twenty-seven mycotoxins by both liquid chromatography and gas chromatography coupled mass spectrometry in tandem (MS/MS) after QuEChERS extraction. The occurrence of mycotoxins was headed by cereal samples with 35% of samples contaminated by at least one mycotoxin followed by vegetables (32%), legumes (15%) and lastly, 9% of fish and meat samples were contaminated. DON was the most detected mycotoxin in vegetables, meat, fish and cereals with an incidence of 13% 18% 19% and 60%, respectively, and the highest mean levels were found in fish (1.19 µg/kg) and vegetable (1.53 µg/kg), respectively. The highest levels means were for HT-2 toxin ranging from 4.03 to 7.79 µg/kg, in cereal and legume samples respectively. In this last, HT-2 toxin was also the most prevalent (54%). In meat samples, OTA resulted with highest value with 8.09 µg/kg. Likewise, PCA analysis revealed a high correlation between the mycotoxins and the food groups analyzed. The findings indicate that there is no toxicological concern associated with exposure to mycotoxins for consumers as all levels were in accordance with the legislation.

Multi-mycotoxin determination in barley and derived products from Tunisia and estimation of their dietary intake.

A study on raw barley and derived products (barley soup and beers) was carried out to determine the natural presence of twenty-four mycotoxins by both liquid chromatography and gas chromatography coupled to tandem mass spectrometry (MS/MS). The developed multi-mycotoxin procedure was based on both SLE and QuEChERS extraction steps. 66% of analyzed samples presented mycotoxin contamination and only one sample, which was soup of barley (6 ng/g), exceeded the maximum level (ML) established by EU for OTA (5 ng/g). Raw barley was the most contaminated matrix (62%), which concentrations ranged from 1.70 to 287.13 ng/g) and type of detected mycotoxins (DON, 15AcDON, NEO, NIV, HT2, FB1, OTA, ENA, ENA1, ENB and ENB1). DON was the most detected mycotoxin with an incidence of 56%, 29% and 23% in beer, soup of barley and barley, respectively. However, the highest levels detected were for ENA, in raw barley with 287 ng/g. In beer and soup of barley samples, the mycotoxins with highest level were 15AcDON (15.6 ng/g) and ENB1 (55.1 ng/g), respectively. Furthermore, 80% of positive soup of barley samples showed co-occurrence. No toxicological concern was associated to mycotoxins exposure for consumers.

Quantification of Listeria monocytogenes in salads by real time quantitative PCR.

A real time quantitative PCR (RTQ-PCR) was carried out purifying DNA extracts of Listeria monocytogenes using a High Pure Listeria Sample Preparation Kit and quantifying in a LightCycler system with hybridisation probes. A standard curve was constructed with serial dilutions. A range linear relationship, from 10 to 10(5)L. monocytogenes colony forming units (CFU), was observed between threshold cycle (Ct) and logarithmic concentration of the serial dilutions. The assay was linear in a range from 10 to 10(5)L. monocytogenes CFU and the coefficient of determination (r2) was >0.98. RTQ-PCR presented an efficiency of >85%. The accuracy of the PCR-based assay, expressed as % bias, ranged from 9% to 26% and the precision, expressed as % CV, ranged 9-22%. Intraday and interday variabilities were studied at 10(2) CFU/g and resulted in 12% and 14%, respectively. The proposed RTQ-PCR method and classical cultural methods were applied to analyse 77 salads from restaurants in Valencia (Spain). All culture positive samples were also RTQ-PCR positive.

Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals.

Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.

Indirect analysis of urea herbicides from environmental water using solid-phase microextraction.

We described here a solid-phase microextraction procedure used to extract six urea pesticides-- chlorsulfuron, fluometuron, isoproturon, linuron, metobromuron and monuron--from environmental samples. Two polydimethylsiloxanes and a polyacrylate fiber (PA) are compared. The extraction time, pH control, addition of NaCl to the water and the influence of organic matter such as humic acid on extraction efficiency were examined to achieve a sensitive method. Determination was carried out by gas chromatography with nitrogen-phosphorus detection. The proposed method requires the extraction of 2 ml of sample (pH 4, 14.3%, w/v, NaCl) for 60 min with the PA fiber. The limits of detection range from 0.04 for linuron to 0.1 microg/l for fluometuron and monuron and the relative standard deviations at the 1 microg/l level are between 15% and 9%. The apparent fiber-water distribution constants (Kfw) calculated in the proposed conditions were in the order of 10(3). Phenylurea herbicides were indirectly determined in the form of their derived anilines and chlorsulfuron in the form of an aminotriazine as confirmed by gas chromatography-mass spectrometry. Natural waters were utilized to validate the final procedure. However, a unequivocal identification in unknown environmental samples should be done by LC-MS. The presence of dissolved organic matter such as humic acid produces losses during the extraction step. Adding sodium chloride to the sample compensates for this effect.

Application of solid-phase microextraction for determining phenylurea herbicides and their homologous anilines from vegetables.

Residues of metobromuron, monolinuron and linuron herbicides and their aniline homologous were analyzed in carrots, onions and potatoes by solid-phase microextraction (SPME) performed with a polyacrylate fiber. A juice was obtained from food samples that were further diluted, and an aliquot was extracted after sodium chloride (14%) addition and pH control. At pH 4 only the phenylureas were extracted. A new extraction at pH 11 allowed the extraction of phenylureas plus homologous aniline metabolites. Determination was carried out by gas chromatography with nitrogen-phosporus detection (NPD) the identity of the determined compounds was studied by gas chromatography-mass spectrometry. Limits of quantification (LOQs) obtained with NPD and MS (selected-ion monitoring) were in the microg/kg order allowing determination of maximum residue levels (MRLs) established in the Spanish regulations. MRLs ranged from 0.02 to 0.1 mg/kg depending on the kind of food and herbicide. Under the proposed conditions matrix effects were low enough to permit calibration with samples proceeding from ecological (non-pesticide treated) crops. Twelve commercial samples of each carrots, onions and potatoes were analyzed and only three samples of potatoes contained residues of linuron at levels below MRLs.

Rapid determination of ochratoxin A in cereals and cereal products by liquid chromatography.

A new method based on extraction with octylsilica (C8) followed by liquid chromatography coupled with fluorescence detection (LC-FLD) was studied to determine ochratoxin A (OTA) from cereals and cereal products. Optimization of different parameters, such as type and amount of solid phase, type and volume of eluent and amount of sample were carried out. Recovery of OTA from rice samples spiked at 10 ng/g level was of 86% with relative standard deviation of 5%. The limits of detection and quantification of the proposed method were 0.25 and 0.75 ng/g, respectively. Furthermore, LC-FLD after of OTA methylation and liquid chromatography coupled to mass spectrometry with an electrospray interface were used for confirmation of OTA in all studied samples. The proposed method was applied to 62 samples of cereals and cereal products and the presence of OTA was found in seven samples.

[Prosthetic hip joint infection caused by Listeria monocytogenes]. / Infection d'une prothèse totale de hanche par Listeria monocytogenes.

The authors report an unusual case of prosthetic hip joint infection caused by Listeria monocytogenes. The patient, an 87-year-old lady who had undergone a right total hip replacement 10 years previously, presented with pain and restriction of hip motion three weeks after an episode of abdominal pain. Aspiration of the joint yielded a dark fluid, from which Listeria Monocytogenes type 4-b was isolated. Blood cultures remained negative. After prolonged antibiotic therapy, symptoms gradually resolved. A few months later, pain recurred with radiological signs of loosening of the femoral component. One-stage revision arthroplasty was performed combined with antibiotic treatment. The patient remains asymptomatic at one year follow-up. Laboratory data and x-ray control are normal. Prosthetic hip joint infection with Listeria monocytogenes is uncommon; few cases have been reported. The literature review shows that prolonged antibiotic therapy alone may be used in patients for whom removal of the prosthesis is not desirable, although revision arthroplasty or prosthesis removal remains necessary in the other cases.

Defensa Alimentaria:revisión de herramientas y estrategias / Food Defense: tools review and strategies

La defensa alimentaria se refiere principalmente a la contaminación intencional del suministro de alimentos, amenazando de esta manera la seguridad de la cadena agroalimentaria lo que puede significar grandes riesgos e inmensurables impactos. La prevención de la contaminación de los alimentos y la reducción de la probabilidad de la contaminación deliberada y el conocimiento de los peligros son los primeros pasos para construir unas estrategias efectivas de food defense y requieren un esfuerzo recurrente en toda la cadena de suministro de alimentos. La naturaleza intencional de estos riesgos ofensivos que se pueden originar en el entorno interno o externo de la industria alimentaria, significa que se deben considerar diferentes factores y se requieren diferentes tácticas para avanzar más allá de los enfoques de seguridad alimentaria comunes. Sin embargo, el conocimiento sobre técnicas básicas de defensa alimentaria sigue siendo limitado. En el presente estudio, se exploran conceptos básicos de food defense para delimitar la vulnerabilidad alimentaria a nivel del fabricante, los proveedores y los clientes de la industria alimentaria así como las bases de un sistema de vigilancia sensible y programas de capacitación en comunicación

Food defense refers mainly to the intentional contamination of the food supply, thus threatening the safety of the agri-food chain, which can mean great risks and huge impacts. The prevention of food contamination and the reduction of the likelihood of deliberate contamination and knowledge of hazards are the first steps to build effective food defense strategies and require a recurring effort throughout the food supply chain. The intentional nature of these offensive risks that may originate in the internal or external environment of the food industry means that different factors must be considered and different tactics are required to move beyond common food security approaches. However, knowledge about basic food defense techniques remains limited. In the present study, basic food defense concepts are explored to delimit food vulnerability at the level of the manufacturer, suppliers and customers of the food industry, as well as the foundations of a sensitive surveillance system and communication training programs

Dietary exposure to mycotoxins through fruits juice consumption / Exposición a micotoxinas a través del consumo de zumo de frutas

A study on fruit juice products (apple, pineapple, apricot, orange and pear) was carried out to determine the natural occurrence of fifteen mycotoxins by gas chromatography coupled to tandem mass spectrometry (MS/MS). A developed multi-mycotoxin procedure was carried out by dispersive liquid-liquid microextraction (DLLME). 36% of the analyzed samples presented mycotoxin contamination. PAT was detected in orange, apple, mixed fruits and pineapple juices with prevalence of 86%, 60%, 29%, 14% at mean concentrations of 34.57 µg/L, 33.41 µg/L, 8.59 µg/L, 8.02 µg/L, respectively. One orange juice sample, exceeded the maximum level (ML) established by EU for PAT (50 µg/L). HT-2 toxin was found in mixed juice (43%) at mean level of 22.38 µg/L. Overall no toxicological concern was associated to mycotoxins exposure for children and adult population and the results obtained highlight the necessity for rigorous monitoring studies on HT-2 in fruit juice

Se presenta un estudio sobre zumos de frutas a base de manzana, piña, albaricoque, naranja y pera para determinar la presencia natural de quince micotoxinas mediante cromatografía de gases acoplada a espectrometría de masas en tándem (EM/EM). El procedimiento desarrollado de multi-micotoxinas se llevó a cabo mediante micro-extracción líquida-líquida dispersiva (DLLME). El 36% de las muestras analizadas presentaron contaminación con micotoxinas y una muestra de jugo de naranja, superó el nivel máximo (ML) establecido por la UE para PAT (50 μg/L). Se detectó PAT en naranja, manzana, frutas mezcladas y jugos de piña con una prevalencia de 86%, 60%, 29%, 14% a concentraciones promedio de 34.57 μg/L, 33.41 μg/L, 8.59 μg/L, 8.02 μg/L, respectivamente. La toxina HT-2 estaba presente en el jugo mixto (43%) a un nivel medio de 22.38 μg/L. En general, ninguna preocupación toxicológica se asoció con la exposición a micotoxinas en la población de niños y adultos, los resultados ponen de relieve la necesidad de estudios rigurosos de monitoreo de HT-2 en el zumo de fruta

Estudio de los parámetros que afectan al contenido de deoxinivalenol en cereales o alimentos a base de cereales obtenidos en comercios de Valencia (España) / Study of several parameters affecting the deoxynivalenol content in cereal and cereal-based products from Valencia (Spain)

Las micotoxinas son metabolitos fúngicos que aparecen comúnmente como contaminantes de muchos cereales y pueden causar una amplia variedad de efectos tóxicos. En el presente estudio, se analizó un total de 182 muestras de diferentes cereales (arroz, trigo, maíz, avena, espelta, soja y tapioca) obtenidos en establecimientos de la Comunidad Valenciana (España). Se examina la presencia de deoxinivalenol (DON) y se realiza un estudio estadístico de las muestras según el tipo de cultivo (orgánico o convencional), su composición, el tipo de producto y la marca (comercial o blanca) para estimar si estos parámetros influyen en la concentración de DON. Del total de muestras, 111 estaban contaminadas con DON, pero en concentraciones inferiores a los límites permitidos por la legislación europea. Se observó mayor incidencia de DON en muestras de trigo (80%) seguidas de maíz (35%) y arroz (13%), pero sin diferencia significativa (p>0,05) entre el tipo de cereal; ni con la práctica agrícola. Sin embargo, los resultados demostraron que el tipo de producto en el cereal procesado (p<0,001; los productos a base de lluvia y sémola presentaran valores mayores de DON que los fideos), los componentes del alimento procesado (p<0,01; los niveles de DON eran superiores en las rosquilletas de pan simples respecto a las rosquilletas con queso y con chocolate ) y la marca (p<0,05; las marcas blancas contienen más DON que las comerciales) si influyen en los niveles de DON hallados en las muestras de trigo (AU)

Mycotoxins are fungal metabolites that commonly appear as contaminants in many cereals and can cause a variety of toxic effects. In this study, a total of 182 samples of different cereals (rice, wheat, corn, oats, spelt, soy and tapioca) obtained in establishments of Valencia (Spain) were analyzed. The presence of deoxynivalenol (DON) was determined and a statistical study of the samples was performed according to the type of crop (organic or conventional), its composition, the type of product and brand (commercial or white) to estimate whether these parameters influence DON concentration. Of the total samples, 111 were contaminated with DON, but their concentration were below the limits allowed by European legislation. Higher DON concentration was observed in wheat samples (80%) followed by corn (35%) and rice (13%), but no significant difference (p> 0.05) between the type of cereal; or harvest conditions. However, the results showed that the type of product in the processing cereal (p <0.001; the rain-based products and grits presented higher values of DON than noodles), processed food components (p <0.01; DON levels were higher in simple snacks than snacks bread with cheese and chocolate) and brand (p <0.05; white brands contain more DON than commercial ones) have related to DON concentration found in wheat samples (AU)

Exposure assessment of fruits contaminated with pesticide residues from Valencia, 2001- 03.

A total of 634 samples of oranges, tangerines, peaches, nectarines, khakis and watermelons were collected from an Agricultural Valencia Community Cooperative during the May 2001 to April 2003 campaigns and they were analysed for 15 pesticides among those recommended for pest treatment. A conventional multiresidue analytical procedure based on ethyl acetate extraction was used followed by gas chromatography coupled to a nitrogen phosphorus detector for routine analysis; and mass spectrometry was performed for confirmation. Recovery studies with spiked samples at 0.5 mg kg-1 for each pesticide ranged from 52% for acephate to 87% for fenthion with a standard deviation <20%. Limits of quantification ranged from 0.1 to 100 microg kg-1. A total of 43% of samples contained pesticide residues; and 5% exceeded the maximum residue levels (MRLs). Nine of the pesticides studied were found. Malathion, which was the most frequently detected, was found in 121 samples (19%) at 0.002-4.25 mg kg-1; followed by fenthion in 104 samples (16%) at 0.005-2.3 mg kg-1; and methidation in 68 samples (10%) at 0.008-1.3 mg kg-1. Khaki showed the highest contamination rates with 60% of contaminated samples that exceeded more often the MRLs; and fenthion was the pesticide more frequently detected in all the commodities studied at levels above the European MRLs. The estimated daily intakes of each pesticide calculated from the results obtained were much lower than the acceptable daily intakes.

Determinación de deoxinivalenol en rosquilletas mediante cromatografía de gases acoplada a espectrometría de masas en tándem / Determination of deoxynivalenol in wheat-based snacks by gas chromatography-triple quadrupole tandem mass spectrometry

El deoxinivalenol (DON) es la micotoxina producida por hongos del género Fusarium que con más frecuencia se detecta en cereales y productos a base de cereales. Por ello, es recomendable realizar una continua monitorización de su incidencia en los alimentos. Este trabajo propone un procedimiento analítico basado en una extracción tipo QuEChERS seguido de una cromatografía de gases acoplada a un detector de triple cuadrupolo para la determinación de DON en rosquilletas. Se analizaron un total de 40 muestras las cuales se dividieron según su composición en dos grupos. El DON fue identificado en el 67,5% de las muestras analizadas con un contenido máximo de 61 μg/kg. A pesar de su incidencia elevada, los niveles de DON hallados fueron muy inferiores a los límites máximos legislados en la actual legislación europea (500 μg/kg). Los resultados obtenidos muestran una baja exposición a DON a través del consumo de esta matriz alimentaria (AU)

Deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal and cereal-based products, and a continuous monitoring of this toxin in foodstuffs is highly desirable. In this sense, a QuEChERS based extraction and gas chromatography-tandem mass spectrometry detection is proposed to determine DON in an appetizer largely consumed, the wheat-based snacks. In this study, a total of 40 samples were analyzed. The samples were divided into two groups based on the composition. Extraction was carried out with acetonitrile followed by a dispersive solid phase extraction and analyzed for DON content by gas chromatography-tandem mass spectrometry (GC-QqQ-MS/MS) method. The overall occurrence of samples with DON was 67.5%, with maximum content of 61μg/kg. In spite of its high incidence, DON concentrations found in samples were much lower than the maximum limit established in the current European legislation (500 μg/kg) for the foodstuff evaluated. Data obtained indicated a low exposure to DON through the consumption of this food commodity (AU)

La docencia de la Toxicología en la Universitat de Valencia ante la convergencia europea / Teaching toxicology in the University of V alencia through the European convergence

La incorporación del Espacio de Europeo de Educación Superior (EEES) al sistema de enseñanza universitario ha supuesto un cambio en las metodologías enseñanza-aprendizaje de las distintas titulaciones que comprende la Universitat de València (UV). En este artículo se realiza un repaso a la transformación que supuesto el EEES en la enseñanza de la toxicología en la UV tanto en el pregrado como en el grado. En los grados de Farmacia, Ciencias Ambientales, Ciencias y Tecnología de los Alimentos y Nutrición Humana y Dietética se han mantenido las asignaturas obligatorias que se impartían, pero además la Toxicología participa en otras asignaturas obligatorias y nuevas asignaturas optativas. Además, con los nuevos Grados se han incluido enseñanzas en Toxicología en el Grado de Medicina y Criminología. Actualmente, el área de toxicología participa en el Master oficial de Calidad y Seguridad Alimentaria y en el Doctorado en Ciencias de la Alimentación. Este aumento de la participación de la Toxicología en los nuevos Grados y postgrados responde a la demanda por parte de la sociedad de profesionales con mayores conocimientos toxicológicos (AU)

The incorporation of the European Higher Education Area (EHEA) to the university education system has meant a change in the teaching-learning methodologies of the different qualifications that are included in the Universitat de València (UV). This work takes a look at the transformation that the EHEA has brought to the teaching of toxicology in the UV, in both graduate and postgraduate degrees. In grades of Pharmacy, Environmental Sciences, Sciences and Food Technology and Human Nutrition and Dietetics have remained the core subjects that were taught, but also Toxicology participates in other core subjects and new electives ones. In addition, the Toxicology studies have been included in the Grade of Medicine and Criminology. Currently, the area of toxicology participates in the of ficial Master of Quality and Food Safety and PhD in Food Science. This increased participation of Toxicology in the new graduate degrees responds to the demand from the society of professional with more knowledge in toxicology (AU)