Associated factors with mycotoxin exposure in Spanish population.

Human exposure to mycotoxins is a global concern since filamentous fungi can contaminate food and feed from crops to ready-to-eat meals. Human urine biomonitoring is a widely used technique to evaluate mycotoxins exposure, as an alternative to food correlation studies. The aim of this study is to describe human exposure to mycotoxins and to investigate the associated sociodemographic, lifestyle and dietary variables. Participants were 540 women from the Valencia (Spain) cohort of the Spanish Childhood and Environment Project (INMA). A validated multi-mycotoxin method using HPLC-Q-TOF-MS was applied to determine the concentration of ten selected mycotoxins: Enniatin A, Enniatin B, Enniatin A1, Enniatin B1, Beauvericine, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2 and Ochratoxin A. A simultaneous untargeted screening of mycotoxins and their metabolites has been performed. Mycotoxins associations were assessed by bivariate and multivariate regression models using participants' sociodemographic, lifestyle and dietary data collected through questionnaires. Mycotoxins were detected in 81% of urine samples. The method quantified mycotoxins concentrations in up to 151 samples. Most quantified mycotoxins were: Enniatin B [% of detection (concentration range)] = 26% (1.0-39.7 ng/mg) and Enniatin B1 = 7% (0.5-14.4 ng/mg). Besides the ten-targeted mycotoxins, other mycotoxins and metabolites were studied, and higher incidence was observed for Deepoxy-deoxynivalenol (45%), Ochratoxin B (18%) and Ochratoxin α (17%). Higher mycotoxins concentrations were associated with rural areas as well as with participants belonged to lower social class, beer, light sodas and fruit juice consumers. On the contrary, higher processed meat intake was related to lower mycotoxins' levels. Studies are required to better evaluate the exposure to mycotoxins from food and their environmental relationships.

Allium sativum L. var. Voghiera Reduces Aflatoxin B1 Bioaccessibility and Cytotoxicity In Vitro.

The present work focuses on the evaluation of AFB1's bioaccessibility and cytotoxicity in vitro using bread (naturally contaminated) enriched or not enriched with fresh Voghiera garlic (2%). Two different experiments were carried out: experiment 1 (E1), with low-AFB1-concentration breads (1.6-1.7 mg/kg); and experiment 2 (E2), with high-AFB1-concentration breads (96.4-102.7 mg/kg). Eight breads were prepared, four for E1 (experiment 1) and another four for E2 (experiment 2), with each experiment having a control group (C), a garlic-enriched group (2%) (G), an AFB1 group (A), and an AFB1 + garlic group (A + G). Simulated digestion was performed on each type of bread, and gastric and intestinal digests were obtained. AFB1 content in flours, baked bread, and gastric and intestinal digests was measured by High-Performance Liquid Chromatography coupled to Fluorescence Detection. The results demonstrate dose-dependent AFB1 bioaccessibility and that the presence of garlic contributed to its reduction in both doses (7-8%). Moreover, garlic's presence in AFB1-contaminated bread increased cell viability (9-18%) in differentiated Caco-2 cells and mitigated the arrest of S and G2/M phases provoked by AFB1 on Jurkat T cells and reduced apoptosis/necrosis, cellular reactive oxygen species (ROS), and mitochondrial ROS by 16%, 71%, and 24% respectively. The inclusion of garlic as a functional ingredient helped relieve the presence and effects of AFB1.

Use of Mustard Extracts Fermented by Lactic Acid Bacteria to Mitigate the Production of Fumonisin B1 and B2 by Fusarium verticillioides in Corn Ears.

Corn (Zea mays) is a worldwide crop subjected to infection by toxigenic fungi such as Fusarium verticillioides during the pre-harvest stage. Fusarium contamination can lead to the synthesis of highly toxic mycotoxins, such as Fumonisin B1 (FB1) and Fumonisin B2 (FB2), which compromises human and animal health. The work aimed to study the antifungal properties of fermented yellow and oriental mustard extracts using nine lactic acid bacteria (LAB) in vitro. Moreover, a chemical characterization of the main phenolic compounds and organic acids were carried out in the extracts. The results highlighted that the yellow mustard, fermented by Lactiplantibacillus plantarum strains, avoided the growth of Fusarium spp. in vitro, showing Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values, ranging from 7.8 to 15.6 g/L and 15.6 to 31.3 g/L, respectively. Then, the lyophilized yellow mustard fermented extract by L. plantarum TR71 was applied through spray-on corn ears contaminated with F. verticillioides to study the antimycotoxigenic activity. After 14 days of incubation, the control contained 14.71 mg/kg of FB1, while the treatment reduced the content to 1.09 mg/kg (92.6% reduction). Moreover, no FB2 was observed in the treated samples. The chemical characterization showed that lactic acid, 3-phenyllactic acid, and benzoic acid were the antifungal metabolites quantified in higher concentrations in the yellow mustard fermented extract with L. plantarum TR71. The results obtained confirmed the potential application of fermented mustard extracts as a solution to reduce the incidence of mycotoxins in corn ears.

In vitro and in vivo evaluation of AFB1 and OTA-toxicity through immunofluorescence and flow cytometry techniques: A systematic review.

Due to the globalization, mycotoxins have been considered a major risk to human health being the main contaminants of foodstuffs. Among them, AFB1 and OTA are the most toxic and studied. Therefore, the goal of this review is to deepen the knowledge about the toxicological effects that AFB1 and OTA can induce on human health by using flow cytometry and immunofluorescence techniques in vitro and in vivo models. The examination of the selected reports shows that the majority of them are focused on immunotoxicity while the rest are concerned about nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, neurotoxicity, embryotoxicity, reproductive system, breast, esophageal and lung toxicity. In relation to immunofluorescence analysis, biological processes related to AFB1- and OTA-toxicity were evaluated such as inflammation, neuronal differentiation, DNA damage, oxidative stress and cell death. In flow cytometry analysis, a wide range of assays have been performed across the reviewed studies being apoptosis assay, cell cycle analysis and intracellular ROS measurement the most employed. Although, the toxic effects of AFB1 and OTA have been reported, further research is needed to clarify AFB1 and OTA-mechanism of action on human health.

Development and Validation of LC-Q-TOF-MS Methodology to Determine Mycotoxin Biomarkers in Human Urine.

Mycotoxin contamination of foodstuffs is a health concern worldwide and monitoring human exposure to mycotoxins is a key concern. Most mycotoxins and their metabolites are excreted in urine, but a reliable detection method is required, considering the low levels present in this biological sample. The aim of this work is to validate a sensitive methodology capable of simultaneously determining ten targeted mycotoxins as well as detecting untargeted ones by using Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry (LC-Q-TOF-MS). The targeted mycotoxins were: enniatin A, B, A1, and B1, beauvericine, aflatoxin B1, B2, G1 and G2, and ochratoxin A. Several extraction procedures such as liquid-liquid extraction, dilute and shoot, and QuEChERS were assessed. Finally, a modified simple QuEChERS extraction method was selected. Creatinine adjustment and matrix-matched calibration curves are required. The limit of detection and limit of quantification values ranged from 0.1 to 1.5 and from 0.3 to 5 ng/mL, respectively. Recoveries achieved were higher than 65% for all mycotoxins. Later, the method was applied to 100 samples of women's urine to confirm the applicability and determine their internal exposure. The untargeted mycotoxins most found were trichothecenes, zearalenones, and ochratoxins.

Bioaccessibility Study of Aflatoxin B1 and Ochratoxin A in Bread Enriched with Fermented Milk Whey and/or Pumpkin.

The presence of mycotoxins in cereals and cereal products remains a significant issue. The use of natural ingredients such as pumpkin and whey, which contain bioactive compounds, could be a strategy to reduce the use of conventional chemical preservatives. The aim of the present work was to study the bioaccessibility of aflatoxin B1 (AFB1) and ochratoxin (OTA) in bread, as well as to evaluate the effect of milk whey (with and without lactic acid bacteria fermentation) and pumpkin on reducing mycotoxins bioaccessibility. Different bread typologies were prepared and subjected to an in vitro digestion model. Gastric and intestinal extracts were analyzed by HPLC-MS/qTOF and mycotoxins bioaccessibility was calculated. All the tested ingredients but one significantly reduced mycotoxin intestinal bioaccessibility. Pumpkin powder demonstrated to be the most effective ingredient showing significant reductions of AFB1 and OTA bioaccessibility up to 74% and 34%, respectively. Whey, fermented whey, and the combination of pumpkin-fermented whey showed intestinal bioaccessibility reductions between 57-68% for AFB1, and between 11-20% for OTA. These results pointed to pumpkin and milk whey as potential bioactive ingredients that may have promising applications in the bakery industry.

Antifungal Activity of Biocontrol Agents In Vitro and Potential Application to Reduce Mycotoxins (Aflatoxin B1 and Ochratoxin A).

Food bio-preservatives are requested as substituents of chemical pesticides in food. The aim of this study was to carry out a screening of twenty biocontrol agents (BCAs) for their potential fungicidal activity in vitro. Twenty BCAs were tested against ten pathogenic fungi. Some of the cell-free supernatants (CFS) tested showed in vitro antifungal activity versus pathogenic fungi. The highest fungicidal activity was observed in the fermented CFS of Paenibacillus chibensis CECT 375, Bacillus amyloliquefaciens CECT 493, and Pantoea agglomerans CECT 850, which showed a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of 125 and 250 g/L, respectively. The compounds responsible for the antifungal activity, such as organic and phenolic acids, were determined. Lactic acid, acetic acid, benzoic acid, and phenyllactic acid among others can be related to antifungal activity. HPLC-MS/MS analysis showed a reduction of ochratoxin A (OTA) and aflatoxin B1 (AFB1) up to 26% (Paenibacillus alvei CECT 2) and 55% (Paenibacillus polymyxa CECT 155), respectively. The present study prompts that metabolism products of BCAs are propitious for the bioconservation of food, due to their ability to reduce the proliferation of mycotoxigenic fungi and mycotoxins production.

Development of an Antifungal Device Based on Oriental Mustard Flour to Prevent Fungal Growth and Aflatoxin B1 Production in Almonds.

The present study describes the manufacture of an antifungal device composed of oriental mustard flour and hydroxyethyl-cellulose (H-OMF) and evaluates its efficacity in inhibiting Aspergillus flavus growth and aflatoxin B1 (AFB1) production in almonds. Additionally, it compares the H-OMF with allyl isothiocyanate (AITC) and a freeze-dried extract of yellow mustard flour (YMF-E); such substances were previously described as antifungal. Minimum inhibitory concentration (MIC), Minimum fungicidal concentration (MFC), the H-OMF in vitro antifungal activity, and the residual fungal population, as well as the production of AFB1 in almonds were determined. AITC and YMF-E showed significant antifungal activity in vitro. Additionally, the in vitro activity of H-OMF avoided mycelial growth by applying 30 mg/L. Almonds treated with AITC (5.07, 10.13, and 20.26 mg/L) and H-OMF (2000 and 4000 mg/L) showed a reduction in the population of A. flavus and the production of AFB1 to values below the limit of detection. YMF-E showed effectiveness by in vitro methodologies (MIC and MFC) but did not show efficacy when applied in almonds. Our findings indicated that the hydroxyethyl-cellulose-based device containing oriental mustard flour might be utilised as a fumigant to increase the safety of almonds and could be extended to other cereals or dry fruits.

Tri-octahedral bentonites as potential technological feed additive for Fusarium mycotoxin reduction.

In 2009 the EU Regulation 386/2009 established a new functional group of feed additives called "substances for reduction of the contamination of feed by mycotoxin". Later, di-octahedral bentonite (1 m558) was authorised, as an anti-aflatoxin additive, being the only additive of this group authorised to date. This work aims to demonstrate the effectiveness of other bentonites, such as tri-octahedral bentonites, versus Fusarium-mycotoxins, since very few adsorbents have proved their effectiveness in relation to this group of mycotoxins. For this purpose, 7 bentonites (six of them tri-octahedral) and 7 commercial adsorbents, added at 0.02% (w/v), were assayed in an in vitro adsorption experiment using two simulated gastro-intestinal (GI) juices (pH 1.3 and 6.8) versus zearalenone (ZEN: 0.1-5 mg/L), fumonisin B1 (FB1: 1-10 mg/L) and deoxynivalenol (DON: 2-10 mg/L). Mycotoxin adsorption data were fitted to Langmuir and Freundlich isotherms. In vitro adsorption experiments showed that ZEN and FB1 (in the latter case only in acid medium) were partially adsorbed, while the adsorption of DON was negligible. Moreover, the increase of adsorbent dose (up to 0.20%, w/v) significantly improved the in vitro adsorption of ZEN and FB1, reaching >90% of adsorption. The present work proposes the use of some tri-octahedral bentonites as feed additives for Fusarium-mycotoxin reduction.

Evaluation of the hypocholesterolemic effect and prebiotic activity of a lentil (Lens culinaris Medik) extract.

SCOPE: The aim of our work was to produce a hydroalcoholic extract of lentils and to examine (a) the hypocholesterolemic action in an animal model, by studying the plasma cholesterol level and the concentration of bile acids in the feces; (b) the potential prebiotic effect, by conducting an in vitro culture fermentation experiment and assessing the level of SCFAs in the feces of rats. METHODS AND RESULTS: Lentil extract (LE) was obtained by extracting lentils with a solution of H2 0/EtOH (70/30 v/v) for 3 h, and the content of main nutrients was determined. After 71 days of diet-induced hypercholesterolemia in rats, LE reduced the cholesterol level of rats of 16.8% (p < 0.05) and increased the level of bile acids in the feces of rats (p < 0.01). LE revealed the same prebiotic activity of inulin and good bifidogenic activity, inasmuch as it enhanced the growth of Bifidobacterium spp. by 3 log (p < 0.05). The concentration of SCFAs in the feces of rats fed with LE increased during the time of the study. CONCLUSION: This new hydroalcoholic extract obtained from lentils was shown to possess hypocholesterolemic and prebiotic properties, and could have interesting applications in the field of nutraceuticals.

Lipid nutritional value of legumes: Evaluation of different extraction methods and determination of fatty acid composition.

This study sought to contribute to the assessment of the nutritional properties of legumes by determining the fatty acid (FA) composition of 29 legume samples after the evaluation of nine extraction methods. The Folch method and liquid-solid extraction with hexane/isopropanol or with hexane/acetone were investigated, as was the effect of previous hydration of samples. Soxhlet extractions were also evaluated with different solvent mixtures. Results on FA composition using the hexane/isopropanol extraction method were the same in terms of FA composition of the Folch method, but the extraction yield was only around 20-40% of that of the Folch method preceded by hydration. Some types of legumes showed particularly interesting values for the ratio of polyunsaturated fatty acids (PUFAs) n-6/n-3, such as lentils, with the value of 4.0, and Azuki beans, at 3.2. In lentils, the PUFAs% ranged from 42.0% to 57.4%, while in Azuki beans it was 57.5%.

Effective clean-up and ultra high-performance liquid chromatography-tandem mass spectrometry for isoflavone determination in legumes.

Legumes are an excellent source of macronutrients and phytochemicals as isoflavones. The aim of this work was to develop a new analytical method for determining five isoflavone compounds, three of which are aglycons, namely daidzein, genistein, biochanin A, and two of which, daidzin and genistin, are glycosilated, in lentils and other pulses, using an effective clean-up system and UHPLC-MS/MS (triple quadrupole) method. The recoveries obtained by spiking the lentil samples with a standard mixture of isoflavones at three levels of fortification (5, 25 and 100 µg kg(-1)) were in the range of 54.4-111.1%, 68.6-91.1%, and 84.4-114%, respectively. The method was applied to analyse 48 lentil samples from central Italy and pulses for determining the isoflavone content, which was found to range from 1.1 to 95.6 µg kg(-1).

Effects of soyasaponin I and soyasaponins-rich extract on the alternariol-induced cytotoxicity on Caco-2 cells.

Alternariol (AOH) is a mycotoxin produced by Alternaria spp. Soyasaponin I (Ss-I) is present naturally in legumes, and it has antioxidant properties. Cytotoxic and genotoxic effects of AOH have been demonstrated previously in vitro. In the present study, the cytotoxicity of AOH, Ss-I, and soyasaponins-rich extract from lentils was investigated; as well as, the cytoprotective effects of Ss-I and lentil extracts against AOH induced-cytotoxicity on Caco-2 cells. Cytotoxicity was carried out using MTT and PC assays (AOH: 3.125-100 µM, Ss-I: 3.125-50 µM, and lentil extracts: 1:0-1:32) during 24 h of exposure. Only AOH showed cytotoxic effect. The reduction in cell proliferation ranged from 25% to 47%. Simultaneous combination of Ss-I with AOH (1:1) increased cell proliferation (35%) compared to AOH tested alone. The Ss-I and extracts showed synergistic cytoprotective effects against cytotoxicity induced by AOH on Caco-2 cells. Food commodities containing Ss-I could contribute to diminish the toxicological risk that natural contaminant as AOH in diet can produce to humans.