Giuseppe Meca

Further data on the presence of Fusarium emerging mycotoxins enniatins, fusaproliferin and beauvericin in cereals available on the Spanish markets

In this work, 64 samples of cereals purchased from local markets in the Valencian community (Spain) were investigated for the presence of six emerging mycotoxins: enniatins ENs (ENA, ENA1, ENB and ENB1), beauvericin (BEA) and fusaproliferin (FUS). Samples were extracted with a mixture of water/acetonitrile (85/15, v/v) by using an Ultra-turrax homogenizer. Mycotoxins were then identified and quantified with a liquid chromatography (LC) with diode array detector (DAD). Positive samples were confirmed with an LC-MS/MS. Analytical Results showed that the frequencies of contamination of samples with ENs, BEA and FUS were 73.4%, 32.8% and 7.8%, respectively. ENA1 was the most mycotoxin found and levels ranged from 33.38 to 814.42 mg/kg. ENB levels ranged between 2.23 and 21.37 mg/kg. ENB1 levels varied from 4.34 to 45.94 mg/kg. All samples were free of ENA. BEA levels ranged from 0.51 to 11.78 mg/kg and FUS levels varied between 1.01 and 6.63 mg/kg. It could be concluded from this study that the high contamination levels found especially for ENs could be of a negative impact on the population. This is the first paper on the presence of emerging mycotoxins in cereals available in Spain.

Bioaccessibility of Deoxynivalenol and its natural co-occurrence with Ochratoxin A and Aflatoxin B1 in Italian commercial pasta

Cereals products for direct human consumption are rarely contaminated by moulds, unlike raw materials, which are often infected, either in the field or during storage. In this study, 27 samples of dried pasta characterised by size, packaging and marketing intended for young children consumption were collected and analysed by liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for Deoxynivalenol (DON), Ochratoxin A (OTA) and Aflatoxin B(1) (AFB(1)) determination. The samples that showed the highest amounts of one of the mycotoxins were cooked for 10min, digested with an in vitro gastrointestinal protocol and bioaccessibility values were calculated. Seven of the 27 samples exceeded from 120% to 225% the legal limit of 200μg/kg for DON fixed for processed cereal-based baby foods by an European Regulation; all the collected samples were under the OTA legal limit (0.05μg/kg) fixed by the European Regulation and no sample was contaminated by AFB(1) over the instrumental limit of detection of 0.10μg/kg. The mean value of gastric bioaccessibility verified for the DON resulted of 23.1%, whereas mean duodenal bioaccessibility was 12.1%.

Interactive effects of zearalenone and its metabolites on cytotoxicity and metabolization in ovarian CHO-K1 cells.

Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin with high binding affinity to estrogen receptors. ZEA is rapidly absorbed and metabolized in vivo to α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). So, mixtures of them may be present in biological systems and suppose a hazard to animals and human health. The aims of this study were to determine the cytotoxic effects of ZEA and its metabolites, alone and in combination in ovarian (CHO-K1) cells during 24, 48 and 72h by the MTT assay; and to investigate the metabolism of the CHO-K1 cells on ZEA, and its conversion into α-ZOL and β-ZOL by CHO-K1 cell after 24 and 48h of exposure. The IC50 value obtained for individual mycotoxins range from 60.3 to >100.0μM, from 30.0 to 33.0μM and from 55.0 to >75.0μM for ZEA, α-ZOL and β-ZOL, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the degree of the three mycotoxin interaction. The CI values for binary combinations ranged from 0.56±0.15 (synergism at low concentrations) to 5.25±5.10 (addition at high concentrations) and tertiary combinations from 2.95±0.75 (antagonism at low concentrations) to 0.41±0.23 (synergism at high concentrations). The concentration of ZEA and its metabolites was determined with liquid chromatography coupled to the mass spectrometer detector-linear ion trap (LC-MS-LIT). The percentage of ZEA degradation ranged from 4% (24h) to 81% (48h). In the same conditions, α-ZOL and β-ZOL concentration decreased from 8% to 85%. No conversion of ZEA in α-ZOL and β-ZOL was detected. However, at 24h of exposure other degradation products of ZEA and its derived were detected.

Risk analysis of main mycotoxins occurring in food for children: An overview.

Mycotoxins are secondary metabolites produced by fungi contaminating the food chain that are toxic to animals and humans. Children up to 12 years old are recognized as a potentially vulnerable subgroup with respect to consumption of these contaminants. Apart from having a higher exposure per kg body weight, they have a different physiology from that of adults. Therefore they may be more sensitive to neurotoxic, endocrine and immunological effects. For these reasons, a specific and up-to-date risk analysis for this category is of great interest. In this review, an accurate analysis of the main mycotoxins occurring in food intended for children (deoxynivalenol, aflatoxins, ochratoxins, patulin and fumonisins) is presented. In particular, known mechanisms of toxicity and levels of exposure and bioaccessibility in children are shown. In addition, recent discoveries about the strategies of mycotoxins managing are discussed.

Antifungal effects of the bioactive compounds enniatins A, A1, B, B1.

To produce enniatin (ENs), Fusarium tricinctum CECT 20150 was grown in a liquid medium of potato (PDB), being mycotoxin purified by high performance liquid chromatography (HPLC) with a reverse phase semipreparative column using a mobile phase of acetonitrile/water using gradient condition. The purity of the ENs fractions was verified by analytical HPLC and LC/MS-MS. The pure fractions of ENs were utilized to study the biological activity on several mycotoxigenic moulds as Fusarium verticilloides, Fusarium sporotrichioides, Fusarium tricinctum, Fusarium poae, Fusarium oxysporum, Fusarium proliferatum, Beauveria bassiana, Trichoderma harzianum, Aspergillus flavus, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus ochraceus and Penicillium expansum. The results obtained demonstrated that in several antibiograms, ENs induced the inhibition of the grown microorganisms tested.

Isolation and purification of enniatins A, A1, B, B1, produced by Fusarium tricinctum in solid culture, and cytotoxicity effects on Caco-2 cells

Enniatins (ENs) are antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The ENs A, A(1), B, B(1) were purified from extracts of Fusarium tricinctum grown on a solid medium of corn, by a low pressure liquid chromatography (LPLC) on reverse phase of Amberlite XAD-7 followed by semipreparative LC. The purity and the structure of the isolated compounds were confirmed by LC-MS/MS. The technique of the purification of the fungal extract enabled complete separation of the ENs A, A(1), B, B(1) with a mean purity of 97% for all the compounds. The cytoxicity of the ENs was tested in the cell lines of human origin (epithelial colorectal adenocarcinoma cells, Caco-2) by MTT assays. Only EN A(1) and B(1) evoked toxicity at the tested concentrations. The inhibitory concentration (IC(50)) for EN A(1) on Caco-2 cells was 12.3 microM, whereas the IC(50) produced by the EN B(1) was 19.5 microM. This study indicates that ENs, fungal metabolites that are commonly found in corn and in general in product composed by corn, may have a toxic potential for human health.

Antibacterial effect of the bioactive compound beauvericin produced by Fusarium proliferatum on solid medium of wheat.

To obtain the bioactive compound beauvericin (BEA), Fusarium proliferatum CECT 20569 was grown on a solid medium of wheat, utilizing the technique of the solid state fermentation (SSF), being this mycotoxin purified by high performance liquid chromatography (HPLC) with a reverse phase semi-preparative column using as the mobile phase acetonitrile/water in gradient condition. The purity of the BEA was verified by analytical HPLC and liquid chromatography tandem mass spectrometry (LC/MS-MS). The pure fractions of BEA were utilized to determinate the antibiotic effects on several bacterial strains that are considered normally pathogens of the intestinal tract as: Escherichia coli, Enterococcus faecium, Salmonella enterica, Shigella dysenteriae, Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfringens, Pseudomonas aeruginosa and Staphylococcus aureus.

Study of the cytotoxic activity of beauvericin and fusaproliferin and bioavailability in vitro on Caco-2 cells.

Beauvericin (BEA) is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and produces cytotoxic effects in mammalian cells. Fusaproliferin (FUS) is a mycotoxin that has toxic activity against brine shrimp, insect cells, and teratogenic effects on chicken embryos. The aim of this study was to determine the cytotoxicity of BEA and FUS in human epithelial colorectal adenocarcinoma HT-29 and Caco-2 cells, the transepithelial transport and the bioavailability using Caco-2 cells as a simulated in vitro gastrointestinal model of the human intestinal epithelium. The inhibitory concentration (IC(50)) evidenced by BEA in the Caco-2 cells was 24.6 and 12.7 μM at 24 and 48 h exposure, respectively, whereas the IC(50) values evidenced in HT-29 cells were 15.0 and 9.7 μM, respectively. FUS was cytotoxic, but no IC(50) data were observed in the range of concentration tested. BEA bioavailability was variable from 50.1% to 54.3%, whereas FUS presented a bioavailability variable from 80.2% to 83.2%. Results obtained demonstrated a potential risk for human health.

Study of the potential toxicity of commercial crispy breads by evaluation of bioaccessibility and bioavailability of minor Fusarium mycotoxins.

Enniatins (ENs) are bioactive compounds produced by the secondary metabolism of several Fusarium strains and known to have several biological activities, such as acting as enzyme inhibitors, antifungal and antibacterial agents, and immunomodulatory substances. This study has investigated the ENs bioaccessibility, spiked in commercial wheat crispy bread at 1.5 and 3.0μmol/g concentrations, their transepithelial transport and bioavailability using Caco-2 cells as a model of the human intestinal epithelium. The content (%) of the four ENs contained in the gastric fluid has resulted variable from 69% to 91%, considering the two concentrations assayed. The mean bioaccessibility data for the compounds studied, resulted of 80%. The compounds that evidenced the highest absorption, using the in vitro model which simulated the transepithelial transport, were the EN A (70.8±1.3% of absorption) and A(1) (73.8±0.9%) at 1.5 and 3.0μmol/g concentrations, respectively. The compound with the lowest transport value (50.7±1.3%) was the EN A at 3.0μmol/g concentration. The bioavailability data evidenced by the other ENs employed ranged from 55.2±1.1% to 66.1±1.0%.

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.