G. Meca

Comparative cytotoxicity study of enniatins A, A1, A2, B, B1, B4 and J3 on Caco-2 cells, Hep-G2 and HT-29

Enniatins (ENs) are ionophoric, phytotoxic, antihelminthic, and antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The cytotoxicity effect of the ENs A, A(1), A(2), B, B(1), B(4) and J(3) was compared on three tumor cell lines, the human epithelial colorectal adenocarcinoma (Caco-2), the human colon carcinoma (HT-29), and the human liver carcinoma (Hep-G2). The endpoint evaluated was the mitochondrial integrity by using the MTT assays, after 24 and 48 h of incubation. The IC(50) value for EN A(2) on Caco-2 cells, after 24h exposure, was 18.7 ± 4.5 μM and decrease to 2.6 ± 0.7 μM at 48 h of incubation. However, ENs A, A(1), B(1) and B(4) exert pronounced cytotoxic effects in all the cell lines tested by the MTT assay after 24 and 48 h of incubation. The EN A(1) demonstrated to be the most cytotoxic ENs tested. Moreover, no statistical differences were found between the IC(50) values obtained for EN A(1) on Caco-2, HT-29 and Hep-G2, with IC(50) values ranging from 9.1 ± 2.2 μM to 12.3 ± 4.3 μM at 24h and decreasing in a range variable from 1.4 ± 0.7 μM to 2.7 ± 0.8 μM at 48 h. On the other hand, EN A, B(1) and B(4) showed lower cytotoxicity, but in a similar range as the IC(50) values reported on HT-29 (IC(50) values (24h): 16.8 ± 4.3-26.2 ± 6.7 μM), Caco-2 (IC(50) values (24h): 19.5 ± 4.1 μM) and Hep-G2 (IC(50) values (24h): 23.4 ± 5.6-26.2 ± 7.6 μM) cells. Cytotoxic effect with a 48 h of incubation revealed also a significant toxicity of ENs A (IC(50) values ranged from 8.2 ± 1.8 to 11.4 ± 4.6 μM), B(1) (IC(50) values variables from 3.7 ± 0.7 to 11.5 ± 5.3 μM) and B(4) (IC(50) of 4.5 ± 2.9-15.0 ± 4.0 μM). In summary, this study demonstrated that ENs can exert toxic activity at low micromolar concentrations in mammalian cells.

Influence of different soluble dietary fibers on the bioaccessibility of the minor Fusarium mycotoxin beauvericin.

Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains and is known to have various biological activities. This study investigated the bioaccessibility of the BEA tested in concentrations of 5 and 25mg/L, in a model solution and in wheat crispy breads elaborated with different natural binding compounds as the soluble alimentary dietary fibers β-1,3 glucan, chitosan low molecular weight (L.M.W.), chitosan medium molecular weight (M.M.W.), fructooligosaccharides (FOS), galattomannan, inulin and pectin, added at concentrations of 1% and 5%. The bioaccessibility was determinated by employing a simulated gastrointestinal digestion that simulates the physiologic conditions of the digestive tract until the colonic compartment. The determination of BEA in the intestinal fluids was carried out by liquid chromatography-mass spectrometry detection (LC-MS). The mean BEA bioaccessibility data in the model solutions ranged from 31.8% of the samples treated with only the duodenal digestion until 54.0% of the samples processed including the colonic fermentation, whereas in the alimentary system composed by the wheat crispy breads produced with different fiber concentration the duodenal and the duodenal+colonic BEA bioaccessibility resulted in 1.9% and 27.0% respectively.

Chemical reduction of the mycotoxin beauvericin using allyl isothiocyanate.

Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains and known to have various biological activities. This study investigated the reduction of BEA present in the concentration of 25mg/kg on a solution model (phosphate buffer saline at pH 4 and 7) and in wheat flour using allyl isothiocyanate (AITC) as a reactant. The concentration of the mycotoxin studied was evaluated using liquid chromatography coupled to the diode array detector (LC-DAD), whereas adducts formed between the BEA and AITC were examined by liquid chromatography coupled to mass spectrometry-linear ion trap (LC-MS-LIT). In solution, BEA reduction ranged from 20% to 100% on a time-dependent fashion and no significant differences were evidenced between the two pH levels employed. In the food system composed by wheat flour treated with gaseous AITC (50, 100 and 500 μL/L), the BEA reduction varied from 10% to 65% and was dose-dependent. Two reaction products between the bioactive compounds employed in this study were identified, corresponding to BEA conjugates containing one or two AITC molecules.

In vitro antifungal activity of allyl isothiocyanate (AITC) against Aspergillus parasiticus and Penicillium expansum and evaluation of the AITC estimated daily intake

Isothiocyanates (ITCs) are natural compounds derived from cruciferous vegetables produced by enzymatic conversion of metabolites called glucosinolates. They are potentially useful antimicrobial compounds for food applications have been shown to be promising agents against cancer in human cell culture, animal models, and in epidemiological studies. In this study, the antifungal activity of the allyl isothiocyanate (AITC) was evaluated on two mycotoxigenic fungi as Aspergillus parasiticus and Penicillium expnsum, aflatoxins (AFs) and patulin (PAT) producers, employing an assay on solid medium. Also an approximation of the risk evaluation associated to the intake of food treated with the AITC to reduce the risk of fungi spoilage has been evaluated. On solid medium and after 20 days incubation the strain of Penicillium expansum was inhibited with AITC quantities highest than 50 mg, whereas the strain of A. parasiticus was sensible to AITC doses highest than 5 mg. The analysis of the risk assessment associated to the intake of several food classes treated with the bioactive compound AITC to prevent fungi spoilage evidenced that this product can be considered as safe due that the estimated daily intakes (EDIs) are always lower than the AITC Admissible Daily intake (ADI).

Degradation study of enniatins by liquid chromatography-triple quadrupole linear ion trap mass spectrometry.

Enniatins A, A1, B and B1 (ENs) are mycotoxins produced by Fusarium spp. and are normal contaminants of cereals and derivate products. In this study, the stability of ENs was evaluated during food processing by simulation of pasta cooking. Thermal treatments at different incubation times (5, 10 and 15 min) and different pH (4, 7 and 10) were applied in an aqueous system and pasta resembling system (PRS). The concentrations of the targeted mycotoxins were determined using liquid chromatography coupled to tandem mass spectrometry. High percentages of ENs reduction (81-100%) were evidenced in the PRS after the treatments at 5, 10 and 15 min of incubation. In contrast to the PRS, an important reduction of the ENs was obtained in the aqueous system after 15 min of incubation (82-100%). In general, no significant differences were observed between acid, neutral and basic solutions. Finally, several ENs degradation products were identified using the technique of liquid chromatography-triple quadrupole linear ion trap mass spectrometry.

Occurrence, toxicity, bioaccessibility and mitigation strategies of beauvericin, a minor Fusarium mycotoxin

Emerging Fusarium mycotoxins include the toxic secondary metabolites fusaproliferin, enniatins, beauvericin (BEA), and moniliform. BEA is produced by some entomo- and phytopathogenic Fusarium species and occurs naturally on corn and corn-based foods and feeds infected by Fusarium spp. BEA has shown various biological activities (antibacterial, antifungal, and insecticidal) and possesses toxic activity, including the induction of apoptosis, increase cytoplasmic calcium concentration and lead to DNA fragmentation in mammalian cell lines. Cereals food processing has an important effect on mycotoxin stability, leading to less-contaminated food compared to the raw materials. Different industrial processes have shown to be effective practices to reduce BEA contents due to thermal food processing applied, such as cooking, boiling, baking, frying, roasting and pasteurization. Some studies demonstrated the capacity of lactic acid bacteria to reduce the presence of the BEA in model solution and in food chain through fermentation processes, modifying this mycotoxin in a less toxic derivate. Prebiotic and probiotic ingredient can modulate the bioaccessibility of BEA reducing the risk of intake of this minor Fusarium mycotoxin. This review summarizes the existing data on occurrence, toxicity and especially on BEA reduction strategies in food and feed such as chemical reduction, biocontrol and food processing.

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.

A chemical approach for the reduction of beauvericin in a solution model and in food systems.

Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains with a strong antibacterial, antifungal, and insecticidal activities. This study evaluated the reduction of BEA added at 25 mg/kg in phosphate buffer saline (PBS) solutions at pH of 4, 7 and 10, or to different cereal products (kernels and flours) by the bioactive compounds phenyl isothiocyanate (PITC) and benzyl isothiocyanate (BITC). The concentration of the mycotoxin was evaluated using liquid chromatography coupled to the diode array detector (LC-DAD). In solution, BEA reduction ranged from 9% to 94% on a time-dependent fashion and lower pH levels resulted in higher BEA reduction. Cereal kernels and flours treated with gaseous PITC and BITC (50, 100 and 500 μL/L) presented BEA reduction from 9% to 97% and was dose-dependent. Among the crops, corn was the vehicle where BEA was mostly affected by the action of the ITCs, followed by wheat and rice, and lastly barley. Overall, PITC caused higher reduction of BEA and should be chosen over BITC as a fumigant to decrease the levels of this mycotoxin in grains and flours.

Antibacterial activity of the emerging Fusarium mycotoxins enniatins A, A1, A2, B, B1, and B4 on probiotic microorganisms

Enniatins (ENs) are secondary metabolites produced by several Fusarium strains, chemically characterized as N-methylated cyclohexadepsipeptides. These compounds are known to act as antifungal and antibacterial agents, but they also possess anti-insect and phytotoxic properties. In this study, the antimicrobial effect of pure fractions of the bioactive compounds ENs A, A₁, A₂, B, B₁, and B₄ was tested towards nine probiotic microrganisms, twenty-two Saccharomyces cerevisiae strains and nine Bacillus subtilis strains. Antimicrobial analyses were carried out the disc-diffusion method using ENs concentrations ranging from 0.2 to 20,000 ng. Plates were incubated for 24 h at 37 °C before reading the diameter of the inhibition spots. ENs A, A₁, A₂, B, B₁ and B₄, were active against several microorganisms with inhibition halos ranging from 3 to 12 mm in diameter. The most active mycotoxin was the EN A₁, which reduced the microbial growth of 8 strains at the dose of 20,000 ng, with inhibition spots sized between 8 and 12 mm. ENs B and B₄ showed no antimicrobial activity towards the microorganisms tested at doses up to 20,000 ng per disc.

Ciclohexadespipeptide beauvericin degradation by different strains of Saccharomyces cerevisiae

The interaction between the mycotoxin beauvericin (BEA) and 9 yeast strains of Saccharomyces cerevisiae named LO9, YE-2, YE5, YE-6, YE-4, A34, A17, A42 and A08 was studied. The biological degradations were carried out under aerobic conditions in the liquid medium of Potato Dextrose Broth (PDB) at 25°C for 48 h and in a food/feed system composed of corn flour at 37°C for 3 days, respectively. BEA present in fermented medium and corn flour was determined using liquid chromatography coupled to the mass spectrometry detector in tandem (LC-MS/MS) and the BEA degradation products produced during the fermentations were determined using the technique of the liquid chromatography coupled to a linear ion trap (LIT). Results showed that the S. cerevisiae strains reduced meanly the concentration of the BEA present in PDB by 86.2% and in a food system by 71.1%. All the S. cerevisiae strains used in this study showed a significant BEA reduction during the fermentation process employed.