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Mycotoxin research is facing unprecedented challenges, starting from the urgent need to cope with the consequences of climate change, the global shortage of grain due to unstable political scenarios, and the major transformation of the supply chains after the COVID-19 pandemic. In this scenario, the mycotoxin contamination of human and animal foods is still unavoidable, thus representing a major challenge to global food security. Next to this, the shift to sustainable and circular food production might be accompanied by an increase in food safety issues involving mycotoxins, e.g., when new technologies are applied to reuse side streams from the food industry, it is not known if and how mycotoxins accumulate in these by-products. MycoTWIN is an EU-funded Horizon 2020 project which fosters knowledge transfer and scientific cooperation within the Mediterranean area, involving worldwide experts, decision makers, and stakeholders in the field of mycotoxigenic fungi and mycotoxins. The MycoTWIN project hosted working group meetings, whose aim was to propose operational plans and/or scientific strategic plans to shape the future research directions to better cope with these challenges. In the working group cycle “Future proof approaches for the management of toxigenic fungi and associated mycotoxins along the food chain”, a multi-actor group was guided in co-creation exercises to elaborate on future research directions and propose relevant actions to be implemented for the present to long-term time periods. The discussion focused on three main topics relevant to the assessment and management of risks associated with mycotoxins and toxigenic fungi: (i) needs for the harmonization of molecular and chemical methods and data analysis, (ii) from lab research to marketable solutions: how to fill the gap, and (iii) gaps in data quality for risk assessment.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceous and various Penicillium species, which are known for contaminating agricultural products and posing significant health risks, which include immunotoxicity. This study aims to evaluate the potential of nanostructured lipid carriers (NLCs) loaded with a carotenoid-enriched extract from pumpkin peel (Cucurbita maxima L.) in mitigating the toxic effects of OTA. To address the poor bioavailability and instability of carotenoids, nanoencapsulation techniques were employed to enhance their delivery and efficacy. NLCs were formulated using hydrogenated sunflower oil, pumpkin oil, and soy lecithin using hot high-pressure homogenization. The in vitro study involved co-digesting OTA-contaminated bread with an NLC formulation and assessing the impact of the encapsulated carotenoid on OTA bioaccessibility, bioavailability, and cellular toxicity using Caco-2 and Jurkat T cells. Even though no significant influence was observed on the bioaccessibility and bioavailability of OTA, carotenoid-loaded NLCs exhibited cytoprotective effects by improving cell viability and mitigating OTA-induced toxicity in both Caco-2 and Jurkat T cells. Particularly, the flow cytometry analysis highlighted the ability of carotenoids to mitigate OTA-induced cellular damage by decreasing ROS production and limiting mitochondrial mass changes. The study suggests that the encapsulation of carotenoids in NLCs represents a promising strategy to enhance their protective effects against OTA toxicity, potentially offering a novel approach to food safety and public health protection. The study underscores the potential of nanotechnology in improving the bioavailability and efficacy of natural antioxidants to mitigate mycotoxin-induced damage.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are highly toxic mycotoxins present in food and feed, posing serious health risks to humans and animals. This study aimed to validate an efficient and cost-effective analytical method for quantifying AFB1 and OTA in rat urine using immunoaffinity column extraction and liquid chromatography with fluorescence detection (IAC-LC-FD). Additionally, the study evaluated the effect of incorporating fermented whey and pumpkin into the feed on the urinary excretion of these mycotoxins. The limits of detection and quantification were determined to be 0.1 µg/kg and 0.3 µg/kg, respectively, for both mycotoxins in feed, and 0.2 ng/mL and 0.6 ng/mL, respectively, in urine. The method demonstrated robust recovery rates ranging from 74% to 119% for both AFB1 and OTA in both matrices. In feed samples, the levels of AFB1 and OTA ranged from 4.3 to 5.2 µg/g and from 5.4 to 8.8 µg/g, respectively. This validated method was successfully applied to analyze 116 urine samples from rats collected during the fourth week of an in vivo trial. The results indicated that the addition of fermented whey and pumpkin to the feed influenced mycotoxin excretion in urine, with variations observed based on the sex of the rats, type of mycotoxin, and exposure dosage.

Mycotoxins, secondary metabolites produced by molds, pose significant health risk through contamination of globally consumed cereals. Ochratoxin A (OTA), a prevalent mycotoxin in cereals, is associated with various health hazards, including immunotoxicity. This study explores the bioaccessibility of OTA in bread and its impact on the gastrointestinal barrier. A focus is placed on grape pomace (GP), a by-product of the wine industry, as a potential mitigator of OTA toxicity. Results demonstrate that GP reduces OTA bioaccessibility in the human gastrointestinal system from 94% to 81% at intestinal level, showing promise in limiting the absorption of the harmful toxin. Additionally, GP exhibits cytoprotective effects, enhancing cell viability and mitigating OTA-induced toxicity in both Caco-2 and Jurkat T cells. In view of the above, to understand the mechanisms by which OTA exhibits its toxic effects, flow cytometry was chosen as the main technique for the analysis of cell cycle, reactive oxygen species levels and mitochondrial parameters. Cytofluorimetric evaluation indicates GP's potential in limiting OTA-induced damage at cellular level. The study suggests that GP could serve as functional ingredient to reduce mycotoxin bioaccessibility and toxicity in cereal-based foods, offering a novel and promising approach to enhance food safety and protect public health. The finding highlights the potential of utilizing grape pomace in food formulations to mitigate mycotoxin contamination, providing a valuable contribution to the ongoing efforts to ensure the safety of globally consumed cereal products.

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.