Report of the IVth Workshop of the Spanish National Network on Mycotoxins and Toxigenic Fungi and Their Decontamination Processes (MICOFOOD), Held in Pamplona, Spain, 29–31 May 2019

The present publication collects the communications presented in the IV Workshop of the Spanish National Network on Mycotoxins and Toxigenic Fungi and their Decontamination Processes (MICOFOOD), held in the School of Pharmacy and Nutrition of the Universidad de Navarra (Pamplona, Spain) from the 29 to the 31 May 2019. More than 70 professionals from academia, the industry and public services have participated. The scientific program included: five sessions: sponsors (presentation and services), toxigenic fungi, toxicology, analysis and control, and reduction and prevention strategies. In total, 18 oral communications and 24 posters were presented. It is worth mentioning the high participation and quality of the communications from PhD students. The invited conference, entitled: “Mycotoxins within the framework of exposure assessment: past present and future”, was given by Dr. Barbara de Santis (Istituto Superiore di Sanità, Rome, Italy). The meeting ended with the roundtable: “From feed to fork: safe food without mycotoxins”, where representatives of feed and agrofood companies and public administrations discussed about the current situation and problems related with mycotoxins. Different prizes were awarded for the best oral presentation (Effect of Staphylococcus xylosus on the growth of toxigenic moulds in meat substrates, by E. Cebrian et al., University of Extremadura), and the best posters (Combined toxicity of aflatoxins and ochratoxin A: A systematic review by M. Alonso-Jaúregui et al., Universidad de Navarra; and Application of natamycin in products affected by toxigenic fungi by Torrijos et al., Universitat de València). The participants had the opportunity to learn about the history and gastronomy of Pamplona. Situated in the north of Spain, Pamplona is a city of Roman origin featuring a large gothic cathedral complex and a Vauban citadel of the 16th century.


Introduction
Mycotoxins are secondary metabolites produced by fungi and they are considered by the scientific community as one of the most important public health problems (https://www.who.int/news-room/ MICOFOOD aims to strengthen the networking among Spanish researchers in the field of toxigenic fungi and mycotoxins. Moreover, its objective is to foster the connection with other similar international networks, industry and public administrations. To this aim, one of the main activities carried out by the Network is the organization of an annual workshop to share knowledge on: • Characterization of mycotoxin-producing fungi, methodologies for detection and control, biosynthetic pathways and genetic/environmental regulatory mechanisms. • Development of multi-mycotoxin detection methods in food and biological matrices to be applied for exposure assessment purposes.

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Evaluation of the effects of thermal treatments on the stability and on mycotoxin content during food production, processing and storage.

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Toxicological characterization of individual and combination of mycotoxins.
Four workshops have been organized so far in venues of the different participating research centers: University of Valencia, University of Extremadura, University of Zaragoza and, the most recent one, in the Universidad de Navarra (Figure 1).

Scientific Committee
and metabolites (including isotopically marked standards) and to the possibility of detecting multiple toxins within a single sample. Exposure assessment requires that the limits of detection be as low as possible, while at the same time maintaining satisfactory levels of precision. Quality of data plays a central role in the EFSA forum, where particular attention is given to continuity in data collection of contaminants and to the development of harmonized methodologies. Mycotoxin exposure assessment methodologies are mainly focused on the deterministic approach but probabilistic calculations are becoming a supportive tool for refinement of exposure scenarios. Mycotoxins represent an extremely important issue within the program DG SANTE (Directorate General of Health and Food Safety of the European Commission). In the Commission agenda on agricultural contaminants, both classical and emerging mycotoxins are considered, and during the last 15 years, in addition to the definition of maximum limits the commission has also dealt with applying legislative measures and defining the standards to be applied by the actors of the agro-food chain to allow a correct and effective application of prevention measures. Despite the efforts made, there are still sensitive issues, which are not dealt with by the authorities: the role of the scientific community is to be vigilant and to work to fill the existing gaps. Abstract: Ochratoxin A (OTA) is a mycotoxin that can be found in a variety of common foods and beverages. This mycotoxin is produced by several species of Penicillium and Aspergillus among which Aspergillus carbonarius is recognized as the main OTA source on grapes and derived products [1]. OTA production is a very consistent property of A. carbonarius and nearly 100% of the isolates of this species produce OTA [2]. In the present study, we present the genome resequencing of an OTA producer strain of A. carbonarius and three atypical non-OTA producing strains [3]. These strains were sequenced using Illumina technology and compared with the genome reference Acv3. We performed some new specific bioinformatics analyses in genes involved in OTA biosynthesis. We focused these analyses on nonsense and missense mutation detection, and also in to identify large DNA deletions in the genome of the three non-OTA producing strains. The OTA-producer strain showed variants in AcOTApks, AcOTAnrps, AcOTAbZIP and AcOTAp450. In atoxigenic strains, five common missense variants in AcOTApks gene were found. Although some gaps of more than 1,000 bp were identified in nonochratoxigenic strains, no large deletions in functional genes related with OTA production were found. Moreover, the expression of five genes of the putative OTA biosynthetic cluster was downregulated under OTA-inducing conditions in the nonochratoxigenic strains. Abstract: Enniatins (ENs) are cyclic depsipeptide mycotoxins produced by a group of Fusarium fungi. They have been found as natural contaminants in food and feed and they can severely interfere with human health. In particular, it has been shown they affect mitochondrial processes by modifying oxidative phosphorylation. The aim of the present study was to evaluate toxicological effects of ENs in the electron transport chain in vivo. A total of 14 female, two-month-old Wistar rats were employed divided in three groups: control, medium and high exposure. The four rats of the control group were exposed to the vehicle (PBS) by esophageal intubation, while the five of the treated ones were intoxicated with medium concentrations: single dose of EN A 256, EN A1 353, EN B 540, EN B1 296 g/mL; and other five with the higher ones: single dose of EN A 513, EN A1 706, EN B 1021, EN B1 593 g/mL. They were sacrificed after eight hours of exposure. The rats' liver, stomach and kidneys were analysed. RNA was extracted from tissues and transcriptional analysis was carried out by qPCR using the SYBR TM Green method. Four genes of the electronic transport chain were selected by using KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Database. In particular, ND1, CO1, ATP5, Sdha and S18 as a reference gene. Gene expression was calculated by StepOnePlus software. Statistical analysis was performed by applying the Student's t-test. Results in the liver showed considerable group variability in gene expression among all the animals employed. In the stomach, the main differences in gene expression have been reported between control and medium-dose treated rats, divided into upregulation and downregulation for all the analysed genes. In kidneys, ND1 and CO1 expression was clearly affected by the medium concentration used while Sdha expression varies according to the concentration employed. No changes have been observed for ATP5. To sum up, despite the high concentration of ENs used, no significant changes in gene expression were observed. Keywords: oxidative phosphorylation; gene expression; in vivo; qPCR ionophoric activity and subsequent mitochondriotoxic properties among others, slight adverse effects in vivo, such as loss of weight, have been found. At transcriptomic level in Jurkat lymphoblastoid T-cell line, both revealed a similar downregulation pattern affecting most of the genes involved in the electron transport chain pathway. In order to explore the adverse outcome pathway that leads to loss of homeostasis, it was proposed to investigate the changes in mitochondrial protein expression in Jurkat cells. The chosen combination of beauvericin and enniatin B was 1:1 at three different doses: 0.01-0.1-0.5 µM. Control cells were exposed to 0.5% dimethylsulfoxide (DMSO). Cells were treated during 24 h and then mitochondria were extracted. Three biological replicates and technical duplicates from each treatment were injected in a Nano-LC-Q-TOF ultra-definition MS E and raw data were processed using Swiss-Prot database. After comparing the control and the three doses results from the 1821 proteins identified and quantified, 340 proteins were selected using the parameters: max fold change ≥ 1.5 and ANOVA p-value ≤ 0.05. These selected proteins were analyzed by different bioinformatics tools for proteomics data interpretation. The most overrepresented pathways using Reactome version 66 were the citric acid cycle and respiratory electron transport, mitochondrial protein import and respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins. PANTHER (Protein Analysis Through Evolutionary Relationship) version 14.1 indicated protein transmembrane import into intracellular organelle as the most overrepresented biological process and disulfide oxidoreductase activity as molecular function. Moreover, Jurkat cells exposed to beauvericin and enniatin B low concentration mixture highly overexpress mtRF1a, mitochondrial peptide chain release factor 1-like showing versus control a fold change > 34 in all conditions. Abstract: Metabolomics is an emerging 'omics' discipline that measures the dynamic responses of the metabolome to various stimuli. Mycotoxins are ingested through contaminated food and feed and are able to reach the bloodstream and cross the blood-brain barrier (BBB). Among more than 400 mycotoxins identified, beauvericin and enniatins are cytotoxic compounds, with the ability to alter intracellular ion homeostasis; aflatoxins are classified as human carcinogens, their toxic effects include genotoxicity, teratogenicity and immunosuppressive activity; ochratoxin is a potent nephrotoxic and zearalenone shows endocrine disrupting effects. BBB is a permeable but selective cell wall that defends the brain from toxic compounds by separating brain extracellular fluid from blood. The aim of this study is to investigate mycotoxins BBB passage, individually and mixed, and their neurotoxicity mechanisms from a metabolomics perspective. The BBB in vitro model was prepared using ECV 304 endothelial human cells over an insert and C6 glial rat cells on the bottom of the well, both plated at a density of 50,000 cel/mL. Transendothelial electric resistance was measured every day after day 4 to ensure the integrity of BBB. At around day 9 of coculture, coinciding with the highest integrity of the barrier, the experiment was carried out. ECV 304 differentiated endothelial human cells were exposed to beauvericin, zearalenone, enniatins, aflatoxins and ochratoxin, individually or in combination, at concentration of 100 nM for 2 h. Apical and basal media were collected separately for each insert-well for the extraction of extracellular metabolites. The extraction method distinguishes between lipidic and aqueous phases. Extracts were lyophilized (aqueous phase) or dried under N2 stream (lipidic phase) and maintained at −20 • C before resuspension in mobile phase for injection. Finally, the extracellular metabolites were profiled using an Agilent 6540 Ultra High Definition UHD Accurate Mass Quadrupole Q-TOF LC/MS instrument. Results were analyzed by MassHunter software and metabolites were identified via METLIN (Metabolite and Chemical Entity Database).

Abstract:
The comet assay is included in the EFSA guidance on genotoxicity testing strategies for chemical substances present in food and feed, including mycotoxins. The comet assay on its standard-alkaline version is able to detect strand breaks and alkali-labile sites at the single-cell level. The assay has been combined with DNA repair enzymes to augment its sensitivity towards other lesions. The aim of this work was to optimize the conditions and evaluate the performance of different enzymes in combination with the comet assay to detect altered bases. Different DNA repair enzymes were used in combination with the comet assay in TK-6 cells treated with non-cytotoxic concentrations of methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), to induce alkylation damage, or potassium bromate (KBrO 3 ) and H 2 O 2 , to induce oxidized bases. The enzyme-modified comet assay was also applied in cells treated with cytotoxic concentrations of Triton X-100. The DNA repair enzyme activity was also tested on GlycoSpot biochips by LXRepair (France). Results showed that the DNA repair enzymes used were able to detect different levels of the oxidized and alkylated bases in a specific manner. None of the enzymes showed activity in untreated or Triton X-100-treated cells. Moreover, the expected specificity of the enzymes was detected in the GlycoSpot biochips. The enzyme-modified comet assay using a combination of DNA repair enzymes is a valuable tool, not only to study the potential genotoxic effect of compounds in food, but also to elucidate the mechanism of action of genotoxic compounds detected in food, such as some mycotoxins. Abstract: Aflatoxins (AFs) are the most important mycotoxins due to the risk they pose to food safety, and Aspergillus flavus is the most relevant producing species. Currently, essential oils (EOs) are considered efficient sustainable methods to control fungal growth since their potent fungicide activity is well known. In the present work, the in vitro effect of seven EOs extracted from aromatic plants from two different years (2015 and 2016) was evaluated regarding A. flavus growth and its ability to produce AFs. The experiments were performed in CYA (Czapek Yeast Extract Agar) medium supplemented with EOs at concentrations from 10 to 1000 µg/mL. All EOs analyzed reduced fungal growth and AFL production to some extent, although those from winter savory (Satureja montana) and origanum (Oreganum virens) were by far the most effective ones. Subsequently, the effect of both EOs was evaluated in a wide range of water activities (a w 0.94-0.96-0.98) and concentrations (350, 700 y 1000 µg/mL) using two A. flavus isolates (A7 y A10). Fungal growth was measured by turbidimetry using a Bioscreen C analyzer and AFL concentration was determined by HPLC. Winter savory EO was able to retard fungal growth at all concentrations tested and its effect was more significant than that observed using origanum EO. In both cases, the effect was related to a w and growth was reduced more at a w 0.94. No statistically significant differences were found regarding both isolates. In general, AFL production was reduced at a w 0.94 and 0.98 although the presence of EOs at low levels at a w 0.96 supposed an induction in AFL production. The results showed that beer samples were the most contaminated samples, even at concentrations ranging from 0.24 to 54.76 µg/L. A significant incidence of alternariol was found in wine reaching concentration levels of up to 43.48 µg/L. Patulin and ochratoxin A were the most frequently detected mycotoxins in cava and cider samples with incidences of 26% and 40%, respectively. Ochratoxin A was found in one wine sample exceeding the maximum level established by the EU. A combined assessment of exposure was performed, based on the sum of mycotoxin concentrations detected in the same sample to approach the dietary exposure magnitude to mycotoxins through these beverages. The risk associated with mycotoxin levels obtained in the analysed beverages was not significant.

Keywords: mycotoxins; LC-MS/MS; GC-MS/MS
Abstract: In the last 15 years, Galician dairy cattle has lost 30% of its population, even though, in 2017, Galician dairy cattle accounted for 42% of the Spanish dairy cattle. In the last years, a progressive and worrisome decline in fertility in the breeding of dairy cattle, the data indicate a decrease of approximately 20% in the last 30 years. Factors such as genetics, the absence of well-being, inadequate nutrition, poor reproductive management and the increase of reproductive diseases may be the cause of this decline. A diet with toxic substances of environmental origin can have effects on follicular fluid and uterine content and therefore pose harmful consequences on reproductive efficiency. Mycotoxins are a clear example of toxic substances of environmental origin that can be ingested by the animal through the feeding provided. For all this and given the importance of reproductive efficiency in milk production influencing the profitability of farms, the objective of this research is to conduct a preliminary study to estimate the prevalence of mycotoxins in bile. Fifty samples of bile were taken from animals slaughtered in a Lugo slaughterhouse and the presence of 10 mycotoxins was evaluated with a method developed by the laboratory based on immunoaffinity columns and subsequent detection of mycotoxins by HPLC-MS/MS. None of the bile samples analyzed showed residues of aflatoxin B1, B2, G1 and G2, deoxynivalenol, diacetoxyscirpenol, fumonisin B2, ochratoxin A, T2-toxin and zearalenone. These results agree with data provided by the Galician Association of Compound Food Manufacturers since they also did not detect the presence of the mycotoxins investigated in the raw materials evaluated.
of the extracts in solid medium (PDA) was carried out. The BALs that showed antifungal potential were selected to establish the minimum inhibitory concentration (MIC) and the minimum fungicide concentration (MFC). At the same time, the extracts were characterized, evaluating the antioxidant capacity by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) test and the content of phenolic acids previous extraction by QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methodology and identification by Liquid Chomatography-Electrospray/Mass Spectrometry Quadropole Time of Flight (LC-ESI/MS Q-TOF). Four strains of Lactobacillus plantarum were selected for their antifungal properties. The fermented YMF extracts showed a higher antifungal activity, with MIC values between 7.8 and 31.3 g/L and MFC values between 15.6 and 62.5 g/L. The fermentation increased the antioxidant capacity of the extracts tested, with a higher content of antioxidant compounds in the extracts made from YMF. Regarding the content of phenolic compounds, nine phenolic acids with powerful antimicrobial capacity were identified. Future assays will proceed to study the protein and peptide content, focusing on their respective antimicrobial activity.

Engineered Silver Nanoparticles, a Possible Tool in the Management of Aflatoxigenic and Ochratoxigenic Fungi and Aflatoxins and Ochratoxin A Production in Food
potency to disrupt mycotoxins production. Since the nineteenth century, silver-based compounds have been used in many antimicrobial applications, but there is a need to understand the risk that this material poses to human health. A bibliographic revision of 29 scientific articles has been made to batch and compare the silver nanoparticles genotoxicity results. From 49 comet, 15 FPG, 8 Endo-III and 1 OGG1 modified comet, 51 micronucleus and six mouse lymphoma in vitro assays, 40, 10, 6, 1, 31 and 6 showed significant genotoxic results, respectively. From 10 comet, 2 Endo-III and 2 OGG1 modified comet, 16 micronucleus, 2 Pig-a, 3 chromosome aberration, 2 DNA deletion and 4 HX2A determination in vivo assays, 4, 2, 2, 9, 0, 3, 2 and 3 reported positive genotoxic results, respectively. Smaller nanoparticles produce higher genotoxic effects. The results showed increased genotoxicity of nanoparticles coated with citrate compared to PVP. AgNPs accumulation in liver leads to long-term effects. Pig-a was shown to be inappropriate for the study of AgNPs genotoxicity.
1 Abstract: One of the main hazards in dry-cured meat products is ochratoxin A (OTA), which is mostly produced by Penicillium nordicum and Penicillium verrucosum. Although antifungal compounds are currently applied on the surface of dry-cured fermented sausages to avoid the growth of undesirable moulds, there is an increasing trend to replace these chemical preservatives with others of natural origin. Within the latter, there are essential oils from aromatic plants whose effect against ochratoxigenic moulds of concern in cured meat products has been scarcely studied. The objective of this work was to evaluate the antagonistic activity of the essential oils obtained from three spices (rosemary, thyme and oregano) commonly added to dry-cured fermented sausages against ochratoxigenic moulds. For this, P. nordicum FHSCC Pn15 was inoculated in a dry-cured fermented sausage-based medium with and without the addition of different concentrations of each essential oil. As positive control, a batch with two concentrations of a commercial preparation with potassium sorbate and natamycin was prepared. After incubating at 12 • C for 15 days the mould growth was visually evaluated and OTA was quantified by uHPLC-MS/MS QqQ. Although no differences in the mould growth were detected among the treatments, OTA levels showed a significant increase with the addition of the highest concentration of the antifungal preparation. On the contrary, a significant decrease of OTA levels below the detection limit was observed when the rosemary essential oil was added at the highest concentration. Consequently, the use of rosemary essential oil could be considered an alternative strategy to commercial antifungal compounds to control the hazard posed by OTA in dry-cured fermented sausages.
Keywords: Penicillium nordicum; dry-cured fermented sausages; ochratoxin A; antagonistic activity; essential oils were found in samples of maize, grass, alfalfa, sugar beet pulp and immature corn silage. Among the different types of silages studied, maize silage samples were the most heavily contaminated. Out of 44 analysed samples, 30 were contaminated by at least one mycotoxin: 41% were positive for the presence of FBs, 14% for deoxynivalenol, 23% for 15-acetyldeoxynivalenol and 16% for zearalenone. The levels of mycotoxins detected in the samples did not exceed the guidance values recommended by the EU. The lack of relationship between Fusarium counts in the microbiological study and the mycotoxin analysis pointed that these mycotoxins were probably synthetized before or immediately after ensiling. Mould growth and mycotoxin contamination in silages and crops, which are subjected to ensiling, should be regularly monitored in order to minimize the chronic exposure of dairy cows to mycotoxins through the intake of contaminated feed.
Keywords: silage; fungi; multi-mycotoxin analysis; UHPLC-FLD; HPLC-MS/MS The aim of this work is in one side to collect the studies and effects of various mycotoxin on SH-SY5Y; on the other side, to study the cytotoxic effects of beauvericin (BEA), and ochratoxin A (OTA) in that cell line. All articles selected address on in vitro cellular based assays. Cell viability has been studied by: propidium iodide assay, MTT (3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, neutral red assay, lactate dehydrogenase leakage assay or reduction of the total cellular protein concentration. Here, cytotoxicity was measured by MTT assay, over 24, 48 and 72 h with a single treatment at the concentration range of 0.01 to 2.5 µM for bea and, from 0.2 to 50 µM for OTA at 1:2 dilutions. Based on the conversion of MTT into formazan crystal by cells that are alive, this assay determines mitochondrial activity and concentration that reach 50% inhibition of cellular proliferation (IC50). Individual IC50 values detected was diverse and BEA resulted to present higher toxic potential than OTA on SH-SY5Y cells. Abstract: Aflatoxins pose a serious risk to food safety and cause high economic losses, contaminating crops in the field and during storage and affecting a wide variety of raw materials and processed foods. In addition, the incidence of these mycotoxins in the EU food supply chain has increased in recent years by factors related to the climate change. Foodstuffs contaminated with aflatoxins are a major threat to food safety, potentially affecting the most vulnerable population groups, such as children. Therefore, it is relevant to investigate the presence of aflatoxins in cocoa, which is frequently consumed by this high-risk population group. In addition, there is currently a certain legal gap in EU legislation since the content of aflatoxins B1, B2, G1 and G2, which are carcinogenic to humans (Group 1 of IARC), is not regulated for cocoa and derived products. The objective of this study was to evaluate the contamination by aflatoxins B1, B2, G1 and G2 in 50 commercial samples of branded cocoa powder (11 organic and 39 conventional). The mycotoxins were extracted with a methanol/water mixture (80:20) followed by cleanup using immunoaffinity columns. Finally, the determination was made by high-performance liquid chromatography coupled to photochemical (PHRED) and fluorescence (FLD) detectors, with a limit of detection of 0.02 µg/kg for each of the aflatoxins. The percentage of positivity to total aflatoxins was 44% (22 out of 50), with levels ranging from 0.02 µg/kg to 3.33 µg/kg. The incidence of the different aflatoxins was B1 (13 samples), G1 (11 samples) and B2 (3 samples); no aflatoxin G2 was detected. Aflatoxins B1, B2 and G1 were detected simultaneously in one sample, B1 and G1 in three samples, while B1 and B2 coexisted in two samples. The incidence of total aflatoxins was very similar in organic (45.5%) and conventional (43.6%) cocoa samples. To the best of our knowledge, this is the first survey of Tunisian barley varieties contamination by the emerging mycotoxins for a period of consecutive four years. Even though meteorological conditions may be favorable for the growth of toxigenic fungi-mainly Fusarium avenaceum in our case-the varietal resistance itself and the agronomic pack performed within the field were able to insure a production with no toxicological issues. Such results may be used in national breeding programs in order to continue improving barley active films to reduce/inhibit the growth of A. steynii in maize grains under different environmental conditions was determined, and iii) the effect of these active films to inhibit OTA accumulation in the seeds under the assayed conditions was tested. ANOVA showed that film class, a w , temperature and their interactions significantly affected growth rates and OTA production. The most effective films were those containing CINHO. ED 50 , ED 90 and ED 100 (Minimum lethal concentration, MLC) ranged 165-350, 297-601, 333-666 µg EVOH-CINHO/25 g maize grains, respectively, depending on environmental conditions. The least efficient were EVOH-LIN films, since the ED 50 , ED 90 and ED 100 were 2800->3330, >3330 and >3330 µg EVOH-LIN/25 g maize grains, respectively. The effectiveness of bioactive films increased with increasing doses. Optimal fungal growth and OTA production happened at 32 • C. This species can be very competitive in warm climates and under storage conditions. The EVOH-CINHO films followed by EVOH-IEG and EVOH-CIT films, designed in this study can be potent antifungal agents against A. steynii and strong inhibitors of OTA biosynthesis in maize grains at very low doses. This is the first study on the impact that interacting environmental conditions and bioactive films have on the growth of A. steynii and OTA production.

Keywords
Keywords: Aspergillus steynii; bioactive films; essential oils; effective doses; ochratoxin A Abstract: Toxigenic fungi and mycotoxins cause devastating effects on agricultural crops, economy, food security and human and animal health. Ochratoxin A (OTA) is a potent nephrotoxin also known to be teratogenic, immunosuppresive and carcinogenic. In Spain, Aspergillus steynii and A. carbonarius are the main ochratoxigenic species in cereals and grapes, respectively. Copper oxychloride and sulfur are non-systemic fungicides, widely used against many fungal diseases, especially in ecological agriculture. Mancozeb is a broad range contact non-selective fungicide but it is not authorized in organic farming. The aims of this study were: (i) to assay the effect of these fungicides on OTA biosynthesis by A. steynii and A. carbonarius isolates from Spanish oat grains and grapes, respectively, under different temperatures and (ii) to study the possibility of using artificial neural networks (NNs) encompassing both multilayer perceptrons (MLP-NN) and radial-basis function networks (RBFN) to predict OTA accumulation over time in cultures. Oat-based agar medium and grape-based agar medium were used for A. steynii and A. carbonarius cultures, respectively. Media were supplemented with mancozeb (1-30 mg/L), copper oxychloride (5-500 mg/L) and sulfur (10-8000 mg/L). Incubation contaminated corn with F. graminearum ITEM 126 at doses of 25, 50 and 100 µg/g, determining the content of mycotoxins at 20 and 40 days of incubation by LC-ESI/MS Q-TOF. Natamycin was effective against all strains tested. The strategies evaluated for the reduction of the fungal population of P. commune in mozzarella cheese were effective, increasing the shelf life of the product. Three mycotoxins (Fumonisin B1, Neosolaniol and Fusarenon X) and two metabolites (Deoxynivalenol-3-glucoside, T2-triol) were detected in corn at 40 days of incubation. The dose of 100 µg/g of natamycin reduced the presence of these mycotoxins between 31.7 and 77.1%, while for the rest of mycotoxins a complete inhibition was achieved. Natamycin has shown to be a substance of interest in the application of products commonly affected by toxigenic fungi. This substance could be an alternative to the use of synthetic chemical additives.