Avances en la evaluación del riesgo alimentario por micotoxinas: bioaccesibilidad, biodisponibilidad, biomarcadores e impacto de los compuestos bioactivos

• Autores: Paula Llorens Castelló
• Directores de la Tesis: Cristina Juan García (dir. tes.), Juan Carlos Moltó Cortés (codir. tes.)
• Lectura: En la Universitat de València ( España ) en 2025
• Idioma: inglés
• Tribunal Calificador de la Tesis: María José Esteve Mas (presid.), Renata Pietrzak-Fiecko (secret.), Souheib Oueslati (voc.)
• Programa de doctorado: Programa de Doctorado en Ciencias de la Alimentación por la Universitat de València (Estudi General)
• Menciones:
  • Internacional
• Calificación: Sobresaliente Cum Laude
• Materias:
  • Química
    • Química analítica
      • Análisis cromatográfico
  • Ciencias de la vida
    • Biología celular
      • Cultivo celular
  • Ciencias médicas
    • Ciencias de la nutrición
      • Digestión
      • Sustancias toxicas naturales
• Enlaces
  • Tesis en acceso abierto en: webges.uv.es (pdf)
• Resumen

  • Bioactive compounds are natural substances found in plant-based foods such as beetroot, tiger nut, and pumpkin, known for their antioxidant, anti-inflammatory, and cell-protective properties. These benefits are attributed to compounds such as polyphenols, betalains, and other bioactives that can contribute to improved health. Nutritional valorization of these products and their by-products involves using them to develop enriched functional foods, such as breads, which can offer protection against contaminants like mycotoxins. Mycotoxins are secondary metabolites produced by fungi such as Aspergillus and Fusarium, which can contaminate foods during cultivation, storage, or processing, posing a significant risk to human health. To evaluate mycotoxin exposure in plant-based foods and their interaction with bioactive compounds, in vitro studies of bioaccessibility and bioavailability were conducted, along with the determination of exposure biomarkers in biological matrices. A review of the incidence of mycotoxins in plant-based meat alternatives (PBMAs) and their ingredients was carried out. Aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), fumonisins (FBs), and enniatins (ENNs) were identified as the most frequent mycotoxins. An analytical method was developed and validated for the extraction and quantification of mycotoxins AFB1, AFB2, AFG1, AFG2, and OTA in the oral, gastric, and intestinal phases of digestion. This method demonstrated high sensitivity, with limits of quantification (LOQ) ranging from 2.01 to 3.51 ng/g, and good precision, with recoveries between 83% and 110%. In wheat bread, OTA exhibited higher bioaccessibility in the gastric phase, while AFB1 showed a significant decrease in the intestinal phase. In pumpkin-enriched bread, the mycotoxins showed lower bioaccessibility. The bioaccessibility of AFB1, OTA, ZEN, and the betalains betanin, betanidin, and vulgaxanthin-III was studied in beetroot-enriched bread (Beta vulgaris) to assess how the matrix influences the release of these compounds during digestion. ZEN bioaccessibility decreased to 5% under combined fortification conditions, while AFB1 and OTA reached 15%. Betalains showed bioaccessibility of up to 27%. Total betalains (TBC) demonstrated high bioaccessibility (up to 99%), while total polyphenols (TPC) achieved 27%. In the bioavailability study using Caco-2 cells, AFB1 and OTA exhibited high bioavailability under triple combination conditions, reaching up to 90%, while ZEN showed limited bioavailability, especially in double combinations. Bioactive compounds such as TBC and TPC also showed high bioavailability, reaching 45% for TBC and between 32% and 66% for TPC, especially in the presence of mycotoxins. Furthermore, the bioaccessibility of polyphenols from tiger nut horchata (TNB) and its solid by-product (TNBP) was evaluated through in vitro digestion, and the bioavailability of individual polyphenols and TPC was assessed alongside AFB1, OTA, and ZEN in Caco-2 cells. Polyphenols showed a mean bioaccessibility of 27% in TNB and 57% in TNBP. At the individual level, certain polyphenols, such as ferulic acid acyl-ß-D-glucoside in TNB and caffeic acid hexoside in TNBP, exhibited high bioaccessibility (over 99%), likely due to enzymatic conversion processes during digestion. In terms of bioavailability, polyphenols displayed high values, exceeding 80% for TNB and ranging from 61% to 86% for TNBP, with trans-cinnamic acid and epicatechin derivative being the most bioavailable, showing an increase in the presence of mycotoxins. TPC also increased in the presence of mycotoxins, exceeding 72% in both matrices. The presence of mycotoxins (AFB1, OTA, ZEN) was affected by polyphenols, with a synergistic effect observed in TNB for OTA (up to 80%) and AFB1 (up to 71%), while ZEN showed a significant reduction in TNBP, likely due to competitive absorption mechanisms. Focusing on ZEN, due to its high incidence in cereals and plant products, a scientific review was conducted on ZEN exposure biomarkers and their metabolites in biological fluids to assess their utility for estimating human and animal exposure to this mycotoxin. This review highlighted that the primary biomarkers of ZEN exposure are its metabolites -zearalenol (-ZEL) and ß-zearalenol (ß-ZEL), which exhibit differences in oestrogenic activity, with -ZEL being more potent than ß-ZEL. Studies reviewed showed that the concentration of these biomarkers in urine and serum depends on factors such as species, sex, diet, and physiological status. In humans, -ZEL is the predominant biomarker, while significant differences were observed between species in animals: -ZEL is the main metabolite in pigs, whereas ß-ZEL predominates in poultry. The review emphasised that the simultaneous detection of multiple metabolites allows for a more accurate assessment of exposure, reflecting both the intake and metabolism of the mycotoxin. To further understand the impact of ZEN, its exposure was investigated through biomarkers in the liver of chicken, pork, and lamb. The results revealed that 69% of the samples contained at least one mycotoxin or metabolite, with ß-zearalenol being the most frequently detected compound. Species differences were evident: lamb liver showed the highest prevalence (93%), followed by chicken (46%) and pork (40%). In chicken, ß-ZOL was the predominant metabolite (42%, max. 152.62 ng/g), while in pork, ZAN stood out (33%, max. 1.36 ng/g), and in lamb, -ZOL was the most abundant (40%). These results highlight the utility of biomarkers in assessing dietary exposure. In conclusion, the results obtained in this doctoral thesis confirm that mycotoxins are still a significant risk to food safety. Although progress has been made in their detection and toxicological evaluation, it is essential to deepen the understanding of their behaviour during digestion and intestinal absorption, as well as their interactions with bioactive compounds present in foods or added to them. These interactions can influence their toxicity and bioavailability, highlighting the need to develop effective mitigation strategies.