Mónica Fernández-Franzón

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.

Analysis of fumonisins B1, B2 and B3 in corn-based baby food by pressurized liquid extraction and liquid chromatography/tandem mass spectrometry

A sensitive and reliable method using pressurized liquid extraction (PLE) and liquid chromatography (LC)/electrospray ionization (ESI) tandem mass spectrometry with a triple quadrupole (QqQ) analyzer has been developed for the analysis of fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) in corn-based baby foods. Influence of several extraction parameters that affect PLE efficiency such as temperature, pressure, solvent extraction, number of cycles and dispersant/clean-up agents were studied. The selected PLE operating method was: 3g of sample was packed into 11 ml stainless-steel cell and fumonisins were extracted with methanol at 40 degrees C, 34 atm in one cycle of 5 min at 60% flush. The analytes were ionized in ESI operating with positive ion mode and identified by selecting two monitoring transitions, permitting quantification and confirmation in a single injection. Recoveries ranged from 68% to 83% at fortification levels of 200 microg kg(-1) with relative standard deviation (RSD) from 4% to 12%. The limits of quantification were from 2 microg kg(-1) for FB(1) and FB(2), and 5 microg kg(-1) for FB(3), which are below the maximum residue level established by the European Union legislation in infant formulas. The proposed method was successfully applied to the analysis of twenty seven samples of baby food products collected from different markets, and one positive sample with a content of 15.9 microg kg(-1) for FB(1), 9.2 microg kg(-1)for FB(2) and 5.8 microg kg(-1) for FB(3) was obtained. Given the simplicity and potential of the proposed procedure, its application for safety control is recommended.

Presence of Ochratoxin A (OTA) Mycotoxin in Alcoholic Drinks from Southern European Countries: Wine and Beer

The main filamentous fungi producers of mycotoxins are Aspergillus spp., Penicillium spp., and Fusarium spp. Their effect can provoke a broad range of toxic properties including carcinogenicity and neurotoxicity, as well as reproductive and developmental toxicities. Ochratoxin A (OTA) is produced by Aspergillus and Penicillium spp. The purpose of this review was to evaluate the risk assessment of OTA in alcoholic drinks (beer and wine) by compiling the results obtained from studies and reviews related to the presence of OTA in these two drinks from southern European countries in the period 2005-2013 and comparing those results with the legislation available in the European Union.

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.

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.

Formation of Fumonisin B1−Glucose Reaction Product, in Vitro Cytotoxicity, and Lipid Peroxidation on Kidney Cells

Fumonisin B(1) (FB(1)) content in corn products decreases during the heating process in foods containing reducing sugars, mainly because of the formation of N-(carboxymethyl)fumonisin B(1). In this study, a rapid method has been developed for the determination of both compounds in corn products using a high-speed blender, Ultra-Turrax, for solvent extraction and liquid chromatography-tandem mass spectrometry. The kinetics of FB(1) degradation and the formation of the Maillard adduct were studied in a model system constituted by corn bread spiked with FB(1) and heated at 160, 180, and 200 degrees C for 3, 6, 10, 15, and 20 min. FB(1) decreased from 0.96 to 0.3 mg/kg and N-(carboxymethyl)fumonisin B(1) increased to 0.1 mg/kg. Cytotoxicity and lipid peroxidation were studied in monkey kidney cells (Vero cells). After 24 h exposure, FB(1) revealed an IC(50) (median inhibitory concentration) of 55 +/- 7 microM with neutral red uptake, but no IC(50) was obtained after N-(carboxymethyl)fumonisin B(1) exposure at the studied concentrations. Lipid peroxidation was assessed using the thiobarbituric acid reactive substance (TBARS) method for 90 min and 24 and 48 h. FB(1) significantly increased the production of malondialdehyde in Vero cells exposed to 1 microM FB(1) after 24 h, while malondialdehyde increased after 5 microM N-(carboxymethyl)fumonisin B(1) exposure. These findings showed that the transformation products exhibit lower cytotoxicity than fumonisin B(1) and lipid peroxidation may be involved in the cytotoxicity induced by both toxins.

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.

Multi-mycotoxin contamination of couscous semolina commercialized in Morocco.

The multi-mycotoxin contamination of ninety-eight (98) couscous semolina samples collected from various areas in Morocco was investigated in this study. Samples were surveyed for the presence of 22 mycotoxins (four aflatoxins, ochratoxin A, diacetoxiscyrpenol (DAS), three fumonisins, beauvericin (BEA), deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15-ADON), 3-acetyl-deoxynivalenol (3-ADON), nivalenol (NIV), sterigmatocystin (STG), zearalenone (ZEA), four enniatins, T-2 and HT-2 toxins). Results showed that 96 out of 98 total couscous samples (98%) were contaminated by at least one mycotoxin. Enniatin B (ENB), Enniatin B1 (ENB1), Enniatin A1 (ENA1) and zearalenone (ZEA) have shown the highest incidences in contaminated samples. The dietary exposure was estimated to be 1.02, 0.57, 0.06, 0.57 and 0.3μg/kgbw/day for the sum of (DON+3-ADON+15-ADON), fumonisins (FB1+FB2+FB3), the sum of (T2+HT-2), NIV and ZEA, respectively.

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.

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

Abstract 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.