Diseño, caracterización y evaluación de la seguridad de envases activos desarrollados con aceites esenciales.

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Analysis of the Use of Cylindrospermopsin and/or Microcystin-Contaminated Water in the Growth, Mineral Content, and Contamination of Spinacia oleracea and Lactuca sativa

Cyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 μg/L and 50 μg/L, or CYN/MC mixture at 5 + 5 μg/L and 25 + 25 μg/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50µg/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 µg/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxinsEspaña, MINECO/FEDER Project AGL2015-64558-

In vitro study of the viability of Caco-2 cells in presence of two compound of Allium spp essential oil

El aceite esencial de los componentes del género Allium, principalmente ajo y cebolla, presenta propiedades antioxidantes y antibacterianas debidas a la presencia de compuestos azufrados en su composición. La industria alimentaria ha comenzado a desarrollar nuevos sistemas de envasado activo a partir de polímeros seleccionados, a los que se incorporan aceites esenciales que, por sus propiedades, contribuyen a aumentar la vida útil de los alimentos perecederos. En este sentido, se hace necesario evaluar la seguridad asociada al uso de estas sustancias en envases alimentarios que van a estar en contacto con el consumidor a través del alimento. El objetivo del presente estudio fue determinar la citotoxicidad producida por dipropil sulfuro y dipropil disulfuro, dos de los componentes del aceite esencial de ajo y cebolla, en la línea celular Caco-2, células humanas procedentes de carcinoma de colon. Los biomarcadores ensayados fueron el contenido total de proteínas, la captación de rojo neutro y la reducción de la sal de tetrazolio (3-(4,5-dimetiltiazol-2- il)-5-(3-carboximetoxifenil)-2-(4sulfofenil)-2H-tetrazolio). Las células fueron expuestas durante 2, 4 y 8 h a concentraciones comprendidas entre 0 y 200 μM. Los resultados no mostraron diferencias significativas frente al control para ninguno de los tres marcadores, lo que demuestra que bajo las condiciones de los ensayos ambos compuestos azufrados no son citotóxicos para esta línea celular gastrointestinal y podrían ser útiles en la industria alimentaria para desarrollar envases activos.Allium spp. essential oil, mainly from garlic and onion, possesses different beneficial properties, for example antioxidant and antimicrobial effects, due to the presence of sulfur compounds. Food industry is developing new active packaging systems that include the essential oil of garlic in their structure, in order to improve the shelf-life of perishable products. Therefore it is necessary to evaluate the safety associated with the use of these substances in food packaging that will be in contact with the consumer through food. The aim of our study was to evaluate in vitro the cytotoxicity of dipropyl sulfide and dipropyl disulfide. For this purpose, we used the human Caco-2 cell line, from human small intestinal mucosa carcinoma. The assayed cytotoxicity biomarkers were the total protein content, neutral red uptake and reduction of the 3-( 4, 5 - dimethylthiazol - 2 - y l ) - 5 - ( 3 - carboximethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt. Cells were exposed to dipropyl sulfide and dipropyl disulfide in concentrations between 0-200 μM for 2, 4 and 8 h. After periods of exposure, no alterations were observed in any of the biomarkers assayed. These results suggest that both organosulfur compounds are safety options for food industry and could be a choice in the development of active packaging.Ministerio de Ciencia e Innovación (España) AGL2012-38357-C02-01Junta de Andalucía AGR-7252Servicio de Biología del Centro de Investigación, Tecnología e Innovación de la Universidad de Sevilla (CITIUS

New Method for Simultaneous Determination of Microcystins and Cylindrospermopsin in Vegetable Matrices by SPE-UPLC-MS/MS

Cyanotoxins are a large group of noxious metabolites with different chemical structure and mechanisms of action, with a worldwide distribution, producing effects in animals, humans, and crop plants. When cyanotoxin-contaminated waters are used for the irrigation of edible vegetables, humans can be in contact with these toxins through the food chain. In this work, a method for the simultaneous detection of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR), and Cylindrospermopsin (CYN) in lettuce has been optimized and validated, using a dual solid phase extraction (SPE) system for toxin extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Results showed linear ranges (5–50 ng g−1 f.w.), low values for limit of detection (LOD) (0.06–0.42 ng g−1 f.w.), and limit of quantification (LOQ) (0.16–0.91 ng g−1 f.w.), acceptable recoveries (41–93%), and %RSDIP values for the four toxins. The method proved to be robust for the three variables tested. Finally, it was successfully applied to detect these cyanotoxins in edible vegetables exposed to cyanobacterial extracts under laboratory conditions, and it could be useful for monitoring these toxins in edible vegetables for better exposure estimation in terms of risk assessment.España MINECO AGL2015-64558-

A subchronic 90-day oral toxicity study of Origanum vulgare essential oil in rats

Oregano essential oil (Origanum vulgare L. virens) (OEO) is being used in the food industry due to its useful properties to develop new active packaging systems. In this concern, the safety assessment of this natural extract is of great interest before being commercialized. The European Food Safety Authority requests different in vivo assays to ensure the safety of food contact materials. One of these studies is a 90 days repeated-dose oral assay in rodents. In the present work, 40 male and 40 female Wistar rats were orally exposed to 50, 100 and 200 mg/kg body weight (b.w.) OEO during 90 days following the OECD guideline 408. Data revealed no mortality and no treatment-related adverse effects of the OEO in food/water consumption, body weight, haematology, biochemistry, necropsy, organ weight and histopathology. These findings suggest that the oral no-observed-adverse-effect level (NOAEL) of this OEO is 200 mg/kg b.w. in Wistar rats, the highest dose tested. In conclusion, the use of this OEO in food packaging appears to be safe based on the lack of toxicity during the subchronic study at doses 330-fold higher than those expected to be in contact consumers in the worst scenario of exposure.Ministerio de Ciencia e Innovación (AGL2012-38357-C02-01) confinanciado con fondos FEDERJunta de Andalucía (AGR-7252

In vivo genotoxicity evaluation of cylindrospermopsin in rats using a combined micronucleus and comet assay

Cylindrospermopsin (CYN) is a potent cyanotoxin recognized as an emerging human threat due to its cytotoxicity and potential carcinogenicity. Although the genotoxicity of CYN has been extensively studied in vitro, limited data are available on its in vivo genotoxicity. The aim of this study was to evaluate the in vivo genotoxicity of pure CYN (7.5–75 μg/kg body weight) after oral exposure of rats through a combined assay of the micronucleus test (MN) in bone marrow, and the standard and modified comet assay in stomach, liver and blood. Also, histopathological changes in stomach and liver were evaluated. Positive results in the MN test were observed in bone marrow in the exposed rats at all the tested concentrations. However, the comet assay revealed that CYN did not induce DNA strand breaks nor oxidative DNA damage in any of the tissues investigated. Finally, histopathological changes were observed in stomach and liver (7.5–75 μg/kg) in intoxicated rats. These results could indicate that CYN is able to induce irritation in stomach before its biotransformation in rats orally exposed, and genotoxicity in bone marrow.Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE

Estudio de la citotoxicidad basal de compuestos antioxidantes presentes en el aceite esencial de orégano: CARVACROL y TIMOL

Ministerio de Ciencia e Innovación AGL2012-38357-C02-01Junta de Andalucía AGR-725

Characterisation and antimicrobial activity of active polypropylene films containing oregano essential oil and Allium extract to be used in packaging for meat products

Cooked ham is more prone to spoilage than other meat products, making preservation a key step in its commercialisation. One of the most promising preservation strategies is the use of active packaging. Oregano essential oil (OEO) and Proallium® (an Allium extract) have previously been shown to be useful in polylactic acid (PLA)-active films for ready-to-eat salads. The present work aims to study the suitability of polypropylene (PP) films containing OEO and Proallium® in the preservation of cooked ham. Concerning the technological features of the studied material, no significant changes in the mechanical or optical properties of PP films containing the active substances were recorded in comparison to the PP film without extracts. However, films containing both active substances were more flexible than the control film and less strong, highlighting the plasticisation effect of the natural extracts. Moreover, physical properties changed when active substances were added to the film. Incorporation of 4% Proallium® affected the transparency of the film to a higher extent compared to 8% OEO, undergoing decreases in transparency of 40% and 45%, respectively. Moreover, only the film containing the highest amount of OEO (8%) significantly decreased the thickness. Both active substances showed antibacterial properties; however, Proallium®-active films seemed to be more effective against Brochothrix thermosphacta than PP films containing OEO, with all percentages of Proallium® killing the bacterial population present in the ham after 60 days. In addition, materials containing the lowest Proallium® content exhibited higher acceptability by consumers in the sensory analyses with 63–100% willing to purchase, better even than the control package (56–89%). In fact, 2% of Proallium® obtained the best results in the odour study performed by the panellists.Junta de Andalucía (AGR7252)Ministerio de Ciencia e Innovación y Fondos FEDER (AGL2012-38357-C02-01

Estrés oxidativo producido por carvacrol y su combinación con timol en la línea celular CACO-2

Ministerio de Ciencia e Innovación AGL2012-38357-C02-01Junta de Andalucía AGR-725

Cylindrospermopsin-microcystin-LR combinations may induce genotoxic and histopathological damage in rats

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.Ministerio de Economía y Competitividad AGL2015-64558-