Antimicrobial packaging based on ɛ-polylysine bioactive film for the control of mycotoxigenic fungi in vitro and in bread.

ɛ-Poly-l-lysine (ɛ-PL) is a cationic peptide with a broad-spectrum antimicrobial activity. This study investigates the use of ɛ-PL as natural antimicrobial to inhibit fungal growth and to reduce aflatoxins (AFs) production. Antifungal activity of starch biofilms with different concentrations of ɛ-Poly-l-lysine (ɛ-PL) was determined in solid medium against Aspergillus parasiticus (AFs producer) and Penicillium expansum. Then, biofilms were tested as antimicrobial devices for the preservation of bread loaf inoculated with A. parasiticus CECT 2681 and P. expansum CECT 2278. Shelf life and AFs content were examined. Biofilms with concentrations of ɛ-PL less than 1.6 mg/cm2 showed no fungal growth inhibition in solid medium, while the antifungal activity of the films with greater than 1.6 mg/cm2 of ɛ-PL was dose dependent. The shelf life of bread inoculated with A. parasiticus was increased by 1 day with the use of films containing 1.6-6.5 mg ɛ-PL/cm2, while shelf life of bread tainted with P. expansum was increased by 3 day with 6.5 mg ɛ-PL/cm2. AFs production was greatly inhibited by ɛ-PL biofilms (93-100%). Thus, ɛ-PL biofilms could be potentially used as antimicrobial device during bread storage as a natural alternative to the synthetic preservatives. Practical applications: Ɛ-Polylysin is a natural substance from microbial metabolism. Polylysine has a function to prevent a microbe from proliferating by ionic adsorption in the microbe. ɛ-polylysine has a wide antibacterial spectrum and has an obvious lethal effect on Gram-positive and Gram-negative bacteria, yeast, mold, viruses, etc. It has a good antibacterial effect on the Gram-negative bacteria E. coli and Salmonellae, which are difficult to control with other natural preservatives. ɛ-Polylysine has already been used generally as a food additive in Japan, Korea and other part of world. In the United States, FDA has recognized the polylysine as a GRAS material. Considered the positive results obtained in the study, this compound could be used for the production of antimicrobial biofilms, applied as separator slices in the loaf bread production, to prevent the growth of the mycotoxigenic fungi A. parasiticus and P. expansum, contributing to reduce the use of the synthetically preservatives in bakery industry and also of the negative impact that these compounds could generate on the health of the end users.

Reaction of zearalenone and α-zearalenol with allyl isothiocyanate, characterization of reaction products, their bioaccessibility and bioavailability in vitro.

This study investigates the reduction of zearalenone (ZEA) and α-zearalenol (α-ZOL) on a solution model using allyl isothiocyanate (AITC) and also determines the bioaccessibility and bioavailability of the reaction products isolated and identified by MS-LIT. Mycotoxin reductions were dose-dependent, and ZEA levels decreased more than α-ZOL, ranging from 0.2 to 96.9% and 0 to 89.5% respectively, with no difference (p⩽0.05) between pH 4 and 7. Overall, simulated gastric bioaccessibility was higher than duodenal bioaccessibility for both mycotoxins and mycotoxin-AITC conjugates, with duodenal fractions representing ⩾63.5% of the original concentration. Simulated bioavailability of reaction products (α-ZOL/ZEA-AITC) were lower than 42.13%, but significantly higher than the original mycotoxins. The cytotoxicity of α-ZOL and ZEA in Caco-2/TC7 cells was also evaluated, with toxic effects observed at higher levels than 75µM. Further studies should be performed to evaluate the toxicity and estrogenic effect of α-ZOL/ZEA-AITC.

Detección de siete micotoxinas tricotecenos en cereales infantiles mediante extracción QuEChERS y cromatografía líquida espectrometría de masas en tándem / Detection of seven trichothecene mycotoxins in infant cereal foods by QuEChERS extraction and liquid chromatography coupled to tandem mass spectrometry

Las micotoxinas tricotecenos se encuentran comúnmente en los cereales, como el trigo, la cebada, el maíz, la avena, el centeno y los productos derivados. Los bebés y los niños pequeños se consideran un grupode alto riesgo debido al gran consumo de alimentos a base de cereales en relación con su peso corporal. En este estudio se ha desarrollado y validado un método rápido, selectivo y sensible para la cuantificación simultánea de 7 micotoxinas tricotecenos (HT-2 toxina, T-2, diacetoxiscirpenol (DAS), deoxinivalenol (DON), 3-acetil-DON, 15-acetil DON, y fusarenon-X (FUS-X) en cereales infantiles. Las micotoxinas se han extraído de las muestras mediante QuEChERS modificado (acrónimo de rápido, fácil, barato, eficaz, robusto y seguro). El método se basa en una única extracción con una mezcla de metanol y acetonitrilo, seguido de una etapa de extracción/partición después de la adición de la sal, y un paso de limpieza utilizando extracción en fase sólida de dispersión (EPS-D). El análisis se llevó a cabo usando cromatografía líquida combinada con un espectrómetro de masas en tándem de triple cuadrupolo (EM/EM) con ionización por electrospray en modo positivos (ESI+) con monitorización de reacción múltiple (MRM). El método validado demostró tener una buena exactitud (>72% de recuperación), reproducibilidad (<10% de desviación estándar relativa interdia) y sensibilidad para las micotoxinas seleccionadas (los límites de detección del método oscilaron entre 0,02 mg kg-1 a 0,15 mg kg-1). Se analizaron 57 muestras de cereales infantiles comercializadas en España revelando la presencia de FUS-X, 15-acetil DON, 3-acetil-DON y T-2 en 9 (15,5%) muestras (AU)

Trichothecene mycotoxins are commonly found in cereals, including wheat, barley, maize, oats, rye and derived products. Infants and young children are considered the highest risk group because their large dietary intake of cereal-based foods in relation to their body weight. In this study, a rapid, selective and sensitive method was developed and validated for the simultaneous quantification of 7 trichothecenes (HT-2 toxin, T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), 3-acetyl-DON, 15-acetyl-DON, and fusarenon-X (FUS-X) in infant cereal food. Mycotoxins have been extracted from the samples using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure. The method was based on a single extraction with a mixture methanol and acetonitrile, followed by an extraction/partitioning step after the addition of salt, and a cleanup step utilizing dispersive solid-phase extraction (D-SPE). The analysis was carried out using a liquid chromatography combined with a triple–quadrupole tandem mass spectrometer (MS/MS) by electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). The validated method showed to be accurate (>72% recovery), reproducible (<10% relative standard deviation interday) and sensitive for the selected mycotoxins (method detection limits ranged from 0.02 mg kg-1 to 0.15 mg kg-1). The screening of 57 samples of infant cereal food commercialized in Spain revealed the presence of FUS-X, 15-Acetyl-DON, 3-Acetyl- DON and T-2 in 9 (15.5%) samples (AU)

Study of the chemical reduction of the fumonisins toxicity using allyl, benzyl and phenyl isothiocyanate in model solution and in food products.

Fumonisins (FBs) are bioactive compounds produced by several strains of Fusarium spp. which contain a polyketide structure similar to sphinganine. These mycotoxins contain a free amino group that could work as an electron donor and react with the electrophile carbon present within the isothiocyanate (ITC) group. The objective of this study was to determine the effect of ITCs (allyl, benzyl and phenyl) on the stability of FB(1), FB(2) and FB(3). Firstly, PBS solutions at three pH levels (4, 7 and 9) were prepared and added with pairs of one FB (1 mg/L) plus one ITC (1 mg/L). Then, gaseous ITC was used to fumigate corn kernels and corn flour contaminated with FBs produced by Gibberella moniliformis CECT 2987 in situ. Mycotoxin levels were evaluated using liquid chromatography coupled to mass spectrometry in tandem (LC-MS/MS), while products formed from the reaction of FBs and ITCs were examined by liquid chromatography coupled to mass spectrometry-linear ion trap (LC-MS-LIT). The reduction of FB(1) and FB(2) in solution ranged from 42 to 100% on a time-dependent manner. This variance was greatly influenced by pH. In general, lower pH levels eased the reaction between ITCs and FBs. ITC fumigation treatment (50, 100 and 500 µL/L) was able to reduce 53-96% of FB(1) levels, 29-91% of FB(2) and 29-96% of FB(3). Four reaction products between the bioactive compounds employed in this study were identified, corresponding to FB + ITC conjugates.

Toxicity evaluation of individual and mixed enniatins using an in vitro method with CHO-K1 cells.

Enniatins (ENs) A, A1, B and B1 are produced by Fusarium species. They are known as emerging fusariotoxins, and can cause outbreaks in both humans and animals. ENs elicits a wide range of different biological properties and toxicological effects, and their co-occurrence may enhance the extent of these hazards. As the potential toxins reach in vitro cells in the same way as they would in vivo, cytotoxicity was studied with CHO-K1, which is considered one of the most sensitive cell lines for preliminary screening of cytotoxicity studies. In this study, individual cytotoxic effects of ENs were evaluated by MTT assay after exposing ENs to CHO-K1 cells for 24, 48, and 72h. The IC50 values obtained for ENs A, A1, B and B1 ranged from >7.5 to 2.83±0.49, from 8.8±2.29 to 1.65±0.06, from 11.0±2.65 to 2.80±0.16 and from 4.53±1.23 to 2.47±0.29µM, respectively. The ENs cytotoxic interaction was evaluated by the isobologram method. The IC50 values for ENs combination ranged from 0.44±0.15 (ENs A1+B1) to 0.97±0.48 (ENs A1+B+B1). The binary combinations ENs A+B1, ENs A1+B and ENs B+B1 showed additive effect thought all concentrations tested. Sinergistic effect of combined ENs A+A1, A+B, A1+B1, A+A1+B, A+A1+B1, A+B+B1 and A1+B+B1 at higher concentrations occurred. Synergism effect (CI from 0.37±0.08 to 0.74±0.22) was observed at higher concentrations with binary and tertiary combinations of EN A, while antagonism effect was obtained at lower concentrations for ENs A+A1+B1 (CI=2.67±1.32) and ENs A1+B+B1 (CI=3.14±1.91). These results provide quantitative evidence that ENs cytotoxicity depend on their concentrations, and also on their combination with other mycotoxins.

Simultaneous determination of eight underivatised biogenic amines in fish by solid phase extraction and liquid chromatography-tandem mass spectrometry.

Biogenic amines on fish tissue are formed as a result of bacterial contamination and spoilage during storage. A new method based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using a triple quadrupole (QqQ) analyser was developed for the analysis of eight biogenic amines (cadaverine, histamine, phenylethylamine, putrescine, spermine, spermidine, tyramine and tryptamine) in fish tissues. Sample preparation was performed by extraction with trichloroacetic acid 5% and solid phase extraction clean up with STRATA X cartridge. The MS/MS method was validated and compared with a method based on the analysis of dansyl derivatives by LC and fluorescence detector (FD). MS/MS achieved higher sensitivity (from 0.02mgkg(-1) for spermidine and phenylethylamine to 0.2mgkg(-1) for spermine) when compared to FD (from 1mgkg(-1) for putrescine and tyramine to 4mgkg(-1) for histamine); MS/MS method showed higher precision too, with intraday relative standard deviations (RSDs) from 1% to 4% with respect to those obtained with FD method (from 3% to 17%). Recovery study was conducted at two different fortification levels and the average ranged from 71% to 93% for all of the studied compounds with RSDs lower than 18%. Matrix-matched standards were used to counteract matrix effect observed in MS/MS determination. The applicability of the method was demonstrated by the analysis of biogenic amines in fish obtained from commercials of Valencia.

Effects of aldicarb and propoxur on cytotoxicity and lipid peroxidation in CHO-K1 cells.

Cytotoxic effects of aldicarb, its sulfone and sulfoxide, and propoxur, lipid peroxidation and antioxidant parameters in Chinese Hamster Ovary (CHO-K1) cells were determined. D,L-buthionine-(S,R)-sulfoximine (BSO) was assayed to determine the role of GSH in the protection against carbamate cytotoxicity. Pre-treatment with 60 microM BSO, induced a significant decrease in the glutathione reductase (GR; 64-141%), the glutathione peroxidase (GPx; 10-30%) and the glutathione S-transferase (GST; 59-93%) activities, and its GSH levels (79-85%), while the oxidized glutathione (GSSG) levels significantly increased (64-78%) respect to experiment non-BSO-pretreated. Carbamates BSO pre-treated vs. non-BSO pre-treated showed a significant increase in malondialdehyde (MDA) production (from 13% to 52% vs. 25% to 93%). These data suggest that carbamates could injure CHO-K1 cells via oxidative stress by the increase of MDA production; moreover, BSO enhance the oxidative damage caused by carbamates. However, the glutathione system protects cells from carbamates damage.

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)

Pesticide residue determination in surface waters by stir bar sorptive extraction and liquid chromatography/tandem mass spectrometry.

In this stir bar sorptive extraction (SBSE) method, 16 pesticides were extracted from surface water samples by sorption onto 1 mm polydimethylsiloxane layer coated on a 10-mm-length stir bar magnet. After liquid desorption of the analytes with 1 ml of methanol, the detection was performed on a liquid chromatography-tandem mass spectrometry with a triple quadrupole (QqQ) analyzer using selected reaction monitoring mode via electrospray ionization. Parameters affecting SBSE operation, including sample volume, salt addition, extraction time, stirring rate, and desorption conditions, have been evaluated. The optimized SBSE method required two 50 ml aliquots of surface water samples, one aliquot was added of 30% NaCl and stirred at 900 rpm during 1 h for testing five pesticides with log K(o/w) < 3, and the other aliquot was directly extracted following the same procedure for the rest of the pesticides with log K(o/w) > 3. The method was validated in spiked surface water samples at limits of quantifications (LOQs) and ten times the LOQs showing recoveries <62%, and the LOQs reached were from 0.03 microg l(-1) for diazinon to 3 microg l(-1) for simazine. The proposed methodology was applied to the determination of these compounds in samples from Albufera Lake and surrounding channels, showing that SBSE is a powerful tool for routine control analysis of pesticide residues in surface water.

Survey of fumonisins B1, B2 and B3 in conventional and organic retail corn products in Spain and Italy and estimated dietary exposure.

A survey was conducted to determine the occurrence of fumonisin B1, B2 and B3 during 2007 in 186 samples of organic and conventional locally available corn products. Samples included baby food (n = 62), corn flour (11), cornflakes (23), pasta (14), cookies (17) and other corn products (59) were obtained from popular markets of Valencia (Spain) and Perugia (Italy). The analytical method used pressurized liquid extraction and liquid chromatography/electrospray ionization tandem mass spectrometry with a triple quadrupole (QqQ) analyser. Of the 104 Spanish samples, 22% contained levels in the range of 2-449 µg kg(-1), 2-229 µg kg(-1) and 6-105 µg kg(-1) for FB1, FB2 and FB3, respectively, while 19 (23%) of the 82 Italian samples were positive with quantifiable levels between 2-235 µg kg(-1), 3-187 µg kg(-1), and 4-40 µg kg(-1) for fumonisins B1, B2 and B3, respectively. Overall, none of the Italian samples and only one organic baby food sample from a Spanish market was above the maximum permitted levels established by European legislation. Fumonisins were found mostly in corn flour followed by cookies and cornflakes. Eleven samples from Spain and nine samples from Italy were organic products, being contaminated the 72% and 77% of the samples, respectively. Analysis of the results showed that levels of fumonisins in corn products were similar in Italy and Spain. The safety of fumonisin intake through corn products was demonstrated by the calculation of the estimated daily intake of both populations considering organic and conventional products separately, which ranged from 1.7 × 10(-3) to 0.72 µg kg(-1) bw day(-1) and comparing them with the provisional maximum total daily intake (PMTDI) of 2 µg kg(-1) bw day(-1) established by the European Union.