Report of the Vth Workshop of the Spanish National Network on Mycotoxins and Toxigenic Fungi and Their Decontamination Processes (MICOFOOD), 10–11 December 2020

The Spanish National Network on Mycotoxins and Toxigenic Fungi and their Decontamination Processes (MICOFOOD) held its V Workshop on 10-11 December 2020. The venue was the University of Valencia, although, due to the pandemic situation, most of the participants followed the event online. Over 100 scientists, researchers, and representatives of the industry followed the Workshop, with the aim of discussing the different aspects of mycotoxin research and their impact on human and animal health, including: Study of mycotoxin-producing fungi, toxicology, analytical methods for the determination of mycotoxins, occurrence studies, reduction, and prevention, among others[...].

The present study aimed at characterizing the genotoxicity of mycotoxins in silico by means of two predictive tools: DEREK Nexus ® (Lhasa Limited ® ), a knowledge-based expert system for qualitative toxicity prediction, and VEGA (vegahub.eu), a qualitative structure-activity relationship (QSAR) model platform. The mycotoxins chosen were clustered in groups: (i) Group 1 included aflatoxin B1 and sterigmatocystin that have a central scaffold in common (furo [2,3] benzofuran) are produced by the Aspergillus species within the same biosynthetic route; (ii) Group 2 included type A (T-2 toxin and HT-2 toxin) and type B thrichothecenes (nivalenol, deoxinivalenol and its acetylated forms of the OH located at position 3 and 15 and fusarenon-X). All are produced by Fusarium species and share a central scaffold (1,5-dimethylspiro[8-oxatricyclo[7.2.1.02,7]dodec-5-ene-12,2'-oxirane]) and (iii) Group 3 with mycotoxins with no common features (ochratoxin A, zearalenone, and fumonisin B1). The predictions from DEREK were classified as "positive" (certain, probable, plausible, or equivocal) or "negative" (improbable or nothing to report) for genotoxicity; while VEGA classification was "non-mutagenic," "mutagenic," and "suspected to be mutagenic" and was accompanied with a level of prediction reliability.
The predictions for aflatoxins and sterigmatocystin with DEREK indicated plausible chromosome damage in vitro and in vivo, plausible in vitro mutagenic potential, and equivocal in vivo mutagenicity. With VEGA, almost all QSAR models agreed on the mutagenic potential of both mycotoxins with good reliability. For group 2, the common prediction from DEREK showed a plausible chromosome aberration and mutagenic potential in vitro and in vivo. The overall VEGA conclusion for nivalenol, deoxynivalenol, and F-X was no-mutagenicity, with just one QSAR model presenting an appropriate reliable prediction. With respect to type A thrichothecenes, T-2 toxin was no mutagenic, though HT-2 Microbial fermentations with lactic acid bacteria (LAB) produce bioactive compounds, including phenolic acids, that are released during human digestion, may have antioxidant, antimicrobial, and antifungal activity against several mycotoxigenic fungi. The objective of the present study was to evaluate the bioaccessibility and bioavailability of phenolic acids from two food matrices-whey powder and yellow mustard flour-fermented with Lactobacillus plantarum, respectively. Fermented and non-fermented extracts were analyzed by LC-qTOF-MS (initial extracts). For bioaccessibility determination, each extract was subjected to a simulated human gastrodigestion system reproducing physiological steps (oral, gastric, and pancreatic digestion) by incubation at 37.5 • C with (i) artificial saliva containing α-amylase; pepsin (pH = 2; 2 h incubation); and (ii) pancreatin and bile salts (pH = 6.5; 2 h incubation). Intestinal digest (pH = 7.2) was analyzed by LC-qTOF-MS (digested extracts), and frozen (−80 • C), lyophilized, and resuspended in Hanks Balanced Salt Solution (HBSS). Bioavailability evaluation was performed on in vitro intestinal epithelium Caco-2 cells model cultured in Transwell plates (225,000 cells/well) until complete differentiation (day 21; verified by the neutral red assay). Four concentrations of digested extracts (0.2%, 0.4%, 0.6%, and 0.8%) were added in triplicates into the apical area, and aliquots of both apical and basolateral compartments were collected at different times (0, 1, 2, 3, 4 h) and analyzed by LC-qTOF-MS (absorbed extracts). Analysis of initial extracts showed the presence of phenolic acids including DL-3-Phenyllactic acid and Lactic acid in whey and mustard extracts; while Benzoic acid, P-Coumaric acid, and Sinapic acid were only present in mustard extracts. Preliminary results showed bioaccessibility of DL-3-Phenyllactic ranging between 2.8-6.0% and 1.2-3%, in mustard and whey extracts, respectively; being slightly higher in the case of fermented whey compared to the nonfermented extract. The analysis of collected apical and basolateral aliquots will allow the study of the human absorption process, as well as the bioavailability determination of these phenolic compounds.

Victor D Opazo *, Carlos Luz, Juan Manuel Quiles, Raquel Carbonell, Jordi Mañes and Giuseppe Meca
Fermentation with lactic acid bacteria (LAB) is studied as a strategy to decrease fungal contamination and mycotoxins production since it originates bioactive compounds, such as polyphenols, with antimicrobial, antifungal, and antioxidant activity. The aim of the present study was to evaluate total polyphenols and antioxidant activity of mustard flour and whey extracts fermented with LAB after simulated human digestion and in vitro absorption in Caco-2 cells. Fermented and non-fermented extracts were subjected to a digestion process in different steps by enzyme addition (α-amylase, pepsin; pancreatin, bile salts), pH adjustment (pH = 2; 6.5; 7.2), and incubation (37.5 • C; 4 h). Digested extracts were lyophilized and resuspended at different concentrations (0.2, 0.4, 0.6, and 0.8%) in HBSS. Extracts were added into the apical part of the intestinal epithelium model once the intestinal barrier was completely formed (differentiation day 21). Cells were incubated for 4 h, and both apical and basolateral aliquots were collected at 1, 2, 3, and 4 h to be analyzed. Total polyphenols were determined by Folin-Ciocalteu Reagent microprocedure. Briefly, a 25 µL sample (or gallic acid as standard curve) were mixed with 125 µL diluted Folin (1/5) and 25µL NaCO 3 (20%) after vigorous agitation. Absorbance was measured at 750 nm after 1 h incubation in darkness. Antioxidant activity was determined by the reduction of 100 µL 2,2-diphenyl-1-picrylhydrazyl (DPPH) after its reaction with 50 µL sample, 1 h incubation in darkness, and absorbance measurement at 517 nm. Preliminary results showed increased concentration of total polyphenols with dose increment in flour extracts collected at 2, 3, and 4 h, with values ranging between 0.3 and 20.8 mg/L. Whey extracts showed detectable polyphenols concentrations only at the highest doses (0.6, 0.8%) and at 3, 4 h (0.5-1.2 mg/L). Low antioxidant activity was observed (<6%) mainly at 3, 4 h. Results indicate that polyphenols and antioxidant compounds are present in small amounts in mustard flour and whey extracts after simulated human digestion and absorption. Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are mycotoxins produced by filamentous fungi belonging to Aspergillus and Penicillium genera. They are currently considered the most important mycotoxins in terms of food safety in both humans and animals. Over the years, the ability of bioactive compounds to prevent mycotoxins adverse effects have been investigated. For this purpose, the beneficial effect of milk fermented whey and pumpkin extract rich in carotenoids on AFB1 and OTA cytotoxicity will be evaluated in Jurkat T cells through a proteomic approach. Jurkat T cell culture exposed to: (a) AFB1, (b) OTA, (c) AFB1 and OTA, (d) fermented whey, (e) carotenoids, (f) fermented whey and carotenoids, (g) AFB1, OTA, and fermented whey (h) AFB1, OTA and carotenoids (i) AFB1, OTA, fermented whey and carotenoids and to control DMSO 0.5%. Proteins will be extracted from exposed cells by means of a lysis buffer (Urea 8M/Thiourea 2M/Tris-HCl 50 mM) and subsequently quantified by using a NeoDot nano-spectrophotometer. Afterward, a concentration of 1000 ppm protein extract will be reduced with dithiothreitol and alkylated with iodoacetamide at a concentration of 200 mM in order to disrupt polypeptide chains. Lastly, peptides will split in Lysine-Arginine bonds through tryptic digestion overnight. Three biological and two technical replicates of each sample will be analyzed with an LC system coupled with quadrupole time of flight (Q-TOF, Agilent) in a concentration of 100 µg/µL by using a C18 column during a 40 min run time at a flow rate of 0.5 mL/min. The data obtained will be processed with Spectrum Mill software, and the differentially expressed proteins will be statistically evaluated by using Mass Professional Profiler software (Agilent). Results will show significant differentially expressed proteins involved in Jurkat T cell functions and the impact which they may produce on human health.

Keywords: mycotoxin; proteomics; lymphoblastoid cells; Q-TOF; prevention
Acknowledgments: This work was supported by Spanish Ministry of Economy and Competitiveness (PID2019-108070RB-I00-ALI) and grant (GVPROMETEO2018-126). Near-infrared hyperspectral imaging (HSI-NIR) introduces the spatial recognition ability to conventional NIR devices. This facility makes HSI-NIR suitable for single kernel spectral features recognition as the region of interest. Thus, the trouble of heterogeneous contamination of wheat batches by mycotoxins can be managed by sorting of wheat batches in grain industry arrival. Moreover, it would be an alternative as a routine analytical method to analyze samples taken from batches. Unlike wet chemistry analytic techniques, HSI-NIR is faster, environmentally-friendly, and non-destructive. The present work had two main objectives. The first one was to standardize HSI-NIR for individual kernel DON analysis to build a prediction model able to quantify the concentration of this mycotoxin. The second objective was the calibration of HSI-NIR to discriminate kernels above and below the EU legal limit (1250 µg/kg). UHPLC analysis of DON for individual kernels, previously scanned by HSI-NIR, was used as reference method. The kernels were scanned in both a crease-up and crease-down position and for different image captures. The spectra were pretreated by Multiplicative Scatter Correction (MSC) and Standard Normal Variate (SNV), 1st and 2nd derivatives and normalization, and they were also evaluated by removing spectral tails. Firstly, the results showed that the best fitted predictive model was on SNV pretreated data with a performance of R 2 0.88 and RMSECV 4.8 mg/kg, for which 7 characteristic wavelengths were used. For the second objective, Linear Discriminant Analysis (LDA), Naïve Bayes, and K-nearest Neighbors models were classified with 98.9 and 98.4% of correctness 1st derivative and SNV spectra, respectively. The results demonstrated the ability of HSI-NIR as a starting point for DON management in wheat, and they are encouraging for future investigations in wheat single kernel quantification and sorting according to DON contamination in the grain industry. The presence of moulds on the surface of dry-cured ham is common due to the environmental conditions reached during the ripening process. Some fungi are producers of ochratoxin A (OTA). This mycotoxin is highly toxic, thus it is necessary to apply preventive measures to reduce its presence. A promising strategy is the use of microorganisms usually found in this product, such as Cocci Gram +, catalase +. The aim of this work was to evaluate the antifungal effect of S. xylosus and S. equorum isolated from dry-cured ham on the growth of P. nordicum and A. westerdijkiae and OTA production at two different concentrations (10 3 and 10 5 cfu/mL) in dry-cured ham-based agar at 15 and 20 • C. The diameter of the colonies was measured daily for 21 days. OTA was extracted by QuEChERS methodology and quantified by Orbitrap Q Exactive Plus analysis. The two staphylococci isolates significantly decreased the growth of both moulds and OTA production at 15 and 20 • C. The reduction of both parameters by S. xylosus was directly proportional to the inoculated concentration of the bacteria. S. xylosus provoked a drop in OTA production of more than 99% in both P. nordicum and A. westerdijkiae. S. equorum significantly decreased the fungal growth even to the lowest inoculum concentration, although the reduction of OTA production, between 98% and 99%, was very similar to both concentrations. In conclusion, both staphylococci isolates showed a great antifungal activity against ochratoxigenic moulds commonly found on dry-cured ham. Consequently, their use as biocontrol agents could be an appropriate preventive measure to control the hazard associated with the presence of OTA in dry-cured ham. New sensing strategies are demanded by European stakeholders to reduce the cost of analysis, to fulfill the increasingly stringent European regulations, and to satisfy the pressing consumer demand for safer food products. Our current research lines focus on the synthesis of novel selective (bio)mimetic recognition elements for developing optical biosensors and separation methods to satisfy mycotoxin analysis's legal, economic, and societal needs. We have applied the phage display technique to the selection of mycotoxin peptide mimetics, or mimopeptides, that have led to optical biosensors and bioassays. Random 12-mer and cyclic 7-mer peptide libraries have allowed the identification of mycotoxin epitope mimics that might replace the conventional hapten conjugates in competitive immunoassays. We have identified mimopeptides of fumonisin B 1 , zearalenone, and HT-2 toxin that turned out to be excellent mimics for biosensing with microarrays, magnetic bead-based, and homogeneous immunoassays. The peptides have been fused by genetic engineering with fluorescent or bioluminescent proteins providing more simple alternatives for target analysis, as neither secondary antibodies nor further labeling are required for the assay. The novel platforms feature superb sensitivities (detection limits below the LMRs for all the mycotoxins tested) and have been validated using certified reference materials. Molecularly imprinted polymers (MIPs) are synthetic antibody mimics that selectively recognize molecular targets. MIPs are highly robust materials, showing superior operational stability under a wide variety of conditions. We have developed selective MIPs for the analysis of zearalenone, alternariol, alternariol monomethyl ether, citrinin, and tenuazonic acid mycotoxins, exploring new sensing mechanisms for developing optical sensors and as selective solid-phase extraction sorbents for those mycotoxin analyses in foodstuff. All these approaches have demonstrated the applicability of MIPs in the field of food safety and quality assurance and control.

Keywords
Keywords: bio(mimetic); molecularly imprinted polymers; phage display; optical sensors; solid-phase extraction Ergot alkaloids (EAs) are toxins produced mainly by fungi of the Claviceps genus, prevalent in cereals, whose ingestion can cause ergotism. Although the European Commission has established a maximum content of 0.5 g/kg of ergot sclerotia in most unprocessed cereals, the maximum content for EAs in food is still under study. QuEChERS methodology has been widely applied for the multi-class analysis of mycotoxins in cereals, but EAs have been rarely included. In this work, an improved QuEChERS-UHPLC-MS/MS method is proposed to determine the major EAs [ergometrine (Em), ergosine (Es), ergotamine (Et), ergocornine (Eco), ergokryptine (Ekr), ergocristine (Ecr)], and their corresponding epimers [ergometrinine (Emn), ergosinine (Esn), ergotaminine (Etn), ergocorninine (Econ), ergokryptinine (Ekrn) and ergocristinine (Ecrn)] in cereals and cereal-based products. To improve sensitivity and to reduce organic solvent consumption, only 4 mL of acetonitrile and 3.0 mmol/L ammonium carbonate (85:15, v/v) were used as extractant. Moreover, 150 mg of Z-Sep+/C18 were selected as dispersive sorbents, decreasing matrix effect (<20% for most EAs) and improving recoveries (between 85-109%). Extraction under alkaline conditions as well as the rapid sample treatment enabled to minimize the EAs epimerization during the procedure. Procedural calibration curves were established for each matrix; LOQs were below 3.9 µg/kg in all cases, significantly lower in comparison with those obtained from other procedures. Precision (intra and inter-day) was lower than 15% (RSD) in all cases. Finally, 85 cereal samples were analyzed (30 of barley, 30 of wheat, and 25 of oat-based products), with the following positive results: One oat bran sample (total EAs content of 10.7 µg/kg), four barley samples (total EAs content between 18.0-54.0 µg/kg), and eight wheat samples (total EAs contents between 6.5-77.4 µg/kg). Em was the most frequently found, being in 70% of the positive samples. Soil is the main reservoir of mycotoxigenic fungi in vineyards, and the application of biocontrol agents (BCA) might be a good option to reduce mycotoxin in grapes and their derivatives. This fact is especially relevant in organic management because the use of conventional fungicides is not permitted.
In this work, we performed a metagenomic approach to unveil the differences between organic and conventionally managed vineyards regarding (i) the diversity of soil microbiota, (ii) the occurrence of mycotoxigenic species, and (iii) the presence of potential BCA candidates. Ten samples from vineyard soils were collected from 5 locations in Spain. Next-generation sequencing was performed using 16S and ITS2 libraries.
The analysis of Shannon and Inverse Simpson indexes were used to evaluate diversity and species richness. In all cases, their values were related to rich communities, which are uniform, and no species were found to be dominant.
Potential mycotoxigenic species were present in all samples, although no evident relationships were found between management or location and contamination levels. At least two potential mycotoxigenic Aspergillus species were detected in all samples, being Aspergillus niger aggregate and Aspergillus flavus the most frequently occurring. However, the most unexpected result was the presence of high levels of Fusarium incarnatum-equiseti species complex since its relevance has not been previously reported in grapes and derivatives.
Potential fungal and bacterial BCA were found in all soil samples regardless of vineyard management. It is important to highlight the high occurrence of the actinomycetes Pseudoarthrobacter in all samples as well as Hanseniaspora uvarum that represented more than 50% of the fungal diversity in a sample collected in a conventional vineyard from Valencia.
These results revealed the potential of metagenomics to perform integrated approaches to study soil communities and their relation to mycotoxigenic species and potential BCA.
Keywords:next-generation sequencing; biological control; soil microbiota; diversity index 4.14. Antifungal Activity of Peracetic Acid against Aspergillus flavus in Corn

Juan Manuel Quiles *, Raquel Carbonell, Jordi Mañes and Giuseppe Meca
Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain * Correspondence: Juan.quiles@uv.es Peracetic acid (APA) is a compound used in the food industry for the disinfection of food and food-contact surfaces. Its mechanism of action is to oxidize lipid membranes, DNA, metabolites, and proteins with sulfhydryl groups and double bonds. Its main advantages are its wide antimicrobial spectrum and that its decomposition products (CH 3 COOH, O 2 , and H 2 O) are substances with low toxicity.
This study aims to quantify the antifungal activity of APA against the toxigenic fungi Aspergillus flavus (A. flavus), a common corn contaminant. First, the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of APA were established against A. flavus in liquid medium, using the 96-well plate method. Secondly, the use of APA released through a hydroxyethyl cellulose (HEC) gel in 1 L jars was tested to inhibit the growth of different concentrations of A. flavus inoculated into PDA solid media plates. Micellar growth was observed for 10 days. Finally, the same methodology was used to evaluate the antifungal activity and the reduction of aflatoxin B1 (AFB1) production caused by the APA-releasing HEC gel in A. flavus contaminated corn. Fungal growth was studied by colony counting in PDA plates seeded with decimal serial dilutions of contaminated corn; AFB1 production was determined in methanol extracted samples of contaminated corn analyzed by mass spectrometry-associated liquid chromatography (HPLC-MS/MS).
The results of MIC and MFC for APA against A. flavus in liquid medium were 125 mg/L and 187.5 mg/L, respectively. The antifungal doses for APA released through an HEC gel were estimated in the range between 10 mg/L and 25 mg/L for A. flavus inoculated in PDA plates and 300 mg/L for the contaminated corn, where this dose was also able to reduce AFB1 production completely. The aflatoxins AFB1, AFB2, AFG1, and AFG2 are mycotoxins produced primarily by toxigenic strains of the fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxinproducing fungi are found in areas with a hot, humid climate, and aflatoxins in food are a result of both pre-and post-harvest fungal contamination. The aflatoxins are genotoxic, and they are classified by IARC as Group 1 (carcinogenic to humans). Maximum levels are set for AFB1 and the sum of AFB1, AFB2, AFG1, and AFG2 in tree nuts, groundnuts (peanuts), and other oilseeds, dried fruits, cereals, and some spices as well as processed products thereof. For AFB1, maximum levels are also set for baby foods and processed cereal based foods for infants and young children, as well as in dietary foods for special medical purposes intended especially for infants.
This study was conducted to screen the occurrence of the 4 aflatoxins in 114 samples (24 peanuts, 21 almonds, 20 walnuts, 29 dried figs, and 20 dates) purchased in retail shops and local markets in different regions from Algeria during 2019. The contamination levels were assessed, after an immunoaffinity column cleanup, by high-performance liquid chromatography coupled to fluorescence detector using photochemical derivatization (HPLC-PHRED-FLD). The LOD and the LOQ of the used method were 0.02 and 0.06 µg/kg, respectively. The results revealed that 27 out of 114 samples (23.7%) were positive for at least one aflatoxin. About 22 samples (19.3%) were contaminated by AFB1, with levels ranging from 0.02 to 5.25 µg/kg. The most frequently found aflatoxin in contaminated food samples was AFB1, and the three others were generally not reported in the absence of AFB1.  Aspergillus carbonarius consistently produces large amounts of ochratoxin A (OTA), a mycotoxin with nephrotoxic effects on animals and humans. In previous studies, we resequenced the genome of three atypical and unique non-OTA-producing strains of A. carbonarius [1,2]. Although no large deletions in functional genes related to OTA production were found, some private missense variants of non-ochratoxigenic strains in the AcOTApks gene were detected [2]. The rest of the OTA cluster genes (AcOTAnrps, AcOTAp450, AcOTAhal, and AcOTAbZIP) showed no privative mutations in the non-OTAproducing strains. In this study, we applied the RNA Sequencing technology to carry out a global transcriptional analysis on four A. carbonarius strains, one OTA producer, and the three atypical non-OTA-producing strains, and to analyze the differentially expressed genes directly or indirectly related to OTA biosynthetic pathway [3]. A total of 696 differentially expressed genes (DEGs) were identified comparing the OTA-producing strain vs. the three non-OTA-producing strains. Among these DEGs, 280 genes were up-regulated, and 333 genes were down-regulated in the non-OTA-producing strains. Among downregulated DEGs, over-represented biological process categories were oxidation-reduction and metabolic processes. Where molecular function is concerned, oxidoreductase and hydrolase activities were over-represented. Within the most down-regulated genes in the three non-OTA-producing strains, we found the AcOTApks and AcOTAnrps genes. The AcOTAp450 gene the transcription factor AcOTAbZIP were also down-regulated. Besides, the AcOTAhal gene was down-regulated, although no significant differences were observed. We also confirmed the five common missense variants found in the AcOTApks gene of these atoxigenic strains, and we showed that one of these mutations can have a deleterious impact on the biological function of the acyltransferase domain of this gene. Aspergillus carbonarius is one of the main species responsible for toxin contamination of grapes and their derivatives, wine, coffee, and cocoa. Ochratoxin A (OTA), the major mycotoxin produced by A. carbonarius, is a secondary metabolite classified as a possible human carcinogen due to its high nephrotoxic character and the immunosuppressive effects that it triggers. It is known that growth, development, and secondary metabolite production are interconnected processes controlled by global regulatory factors in filamentous fungi. Besides that, these regulatory factors are encoded by genes that are generally located outside the gene clusters involved in the biosynthesis of secondary metabolites. An example of these global regulatory factors is the VELVET Complex (VelB/VeA/LaeA), a complex that couples fungal development with secondary metabolism, in response to light. Studies have demonstrated the relation between the loss of veA and laeA and the drastic reduction of OTA and conidial production. One of the biosynthetic genes within the putative OTA cluster of A. carbonarius is the polyketide synthase pks gene. In the present study, we have independently deleted veA, laeA and pks genes in the ochratoxigenic A. carbonarius ITEM 5010 strain by targeted gene replacement using Agrobacterium tumefaciens-mediated transformation. Finally, we are developing the CRISPR-Cas9 system mediated by A. tumefaciens transformation by using the alb1 gene as a phenotypic marker because the deletion of this gene produces white spores.  Aspergillus carbonarius is the main Ochratoxin A (OTA) producer in grapes destined for wine production. Recently, biological control methods have been used due to their high efficiency, the absence of resistance development, and low environmental impact. In this study, we screened the biocontrol capabilities of three Bacillus isolates, which previously showed antagonistic proprieties towards other mycotoxigenic fungi, towards A. carbonarius growth and OTA production. Bacterial suspensions were mixed with molten Czapek yeast extract (CYA) medium and pour plated into petri dishes. After solidification, an agar plug of A. carbonarius was inserted in the center of the plate. Cultures were incubated at 25 • C for 7 days and the radial mycelium was measured daily to determine mycelial growth rate. Subsequently, the colonies were washed twice with aqueous tween 80, and the number of conidia was determined. To evaluate the biocontrol effect on OTA production, both microorganisms were inoculated in CYA broth, which was incubated at 25 • C for 7 days on a rotary shaker. After incubation, 10 mL aliquots of the culture were removed, and OTA was extracted with chloroform. The extracts were analyzed by UPLC/MS-MS.

Tiago C. Einloft *, Patricia Bolzan de Oliveira and Rafael Gomes Dionello
A. carbonarius mycelial growth rate was drastically reduced by all bacteria, ranging from 76% to 96% of inhibition. Conidial production was also significantly reduced by all three bacteria. Bacillus sp. RP103 and RP242 were most effective, reducing conidial production by 10 times. OTA production by A. carbonarius was reduced in a range from 86 to 93%. The results obtained in this study indicate that these bacteria have promising biocontrol characteristics and could characterize a new strategy for the management of A. carbonarius growth and OTA contamination in wine grapes. The surface of dry-cured fermented sausages is colonized during their ripening by molds, being some of them ochratoxigenic. Nowadays, the meat industry uses synthetic antifungal compounds to avoid the growth of such molds, together with other unwanted ones. Nevertheless, several strategies based on using natural preservatives, such as plant extracts and native microorganisms, are gaining interest. The aim of this study was to evaluate the antagonist activity of the essential oil of rosemary (REO), a spice commonly used in the dry-cured fermented sausages processing, and the acorn extract (AE) in the presence and absence of a Debaryomyces hansenii strain with known ability to be used as a protective culture. Slices of raw dry-cured fermented sausages "chorizo" were thus placed in pre-sterilized receptacles with 86% relative humidity. After inoculating them with the treatments alone and in combination, three ochratoxigenic strains of Penicillium nordicum (PN856, PN92, and PN15) were inoculated. As a negative control, only the molds were separately inoculated. After incubating all the treatments at 12 • C for 15 days, ochratoxin A (OTA) was quantified using an uHPLC coupled to a Q exactive Plus detector. Different patterns in the ability of the biocontrol agents to alter the OTA production were detected at the mold strain level. In PN92 and PN15, a significant positive synergic effect was detected in the presence of AE and the yeast. Similarly, the combination of AE with REO significantly reduced the OTA level in PN15. Nonetheless, REO was the unique treatment provoking a significant reduction of OTA production in PN856. When REO, AE, and the yeast were inoculated together, a significant increase in the OTA levels was observed in PN92 and PN15. Consequently, further studies to evaluate increasing concentrations of the biopreservative agents without modifying the sensorial characteristics of the products and able to reduce the OTA production in all the ochratoxigenic strains should be developed. Keywords: ochratoxin A; plant extracts; dry-cured fermented sausages; antagonist activity presence are nowadays focused on vegetal and microbial agents. Previous in vitro studies have shown efficiency for reducing OTA of rosemary leaves (RL), rosemary essential oil (REO), and Debaryomyces hansenii (Dh). The objective of the study was to test their antifungal effect when applied alone or in combination. Dry-cured fermented sausages were processed in a pilot plant using 18 combinations of these biocontrol agents. Batch 1 was uninoculated with them. Batch 2 included 2 g/kg of RL added to the meat mix before stuffing. In batch 3, the casings were macerated in water with RL for one day before stuffing. In batch 4, REO was applied after stuffing. Batch 5 included a combination of REO and RL. In batch 6, a commercial antifungal (natamycin + potassium sorbate) was applied. Besides, Dh was applied together with the other treatments inside the meat (batches 7-12) and after stuffing (batches 13-18). P. nordicum was finally inoculated on the surface of all treated sausages. After ripening, OTA was quantified by UHPLC-MS/MS. Significant reductions of OTA presence due to most of the treatments with RL and Dh in the meat mix (batches 2, 3, 7, 8, 9, 11f, and 12) were detected, being the majority of the obtained levels below the limit of quantification (<0.25 ng/g). The presence of the yeast on the surface only reduced OTA levels when it was inoculated alone (batch 13), with RL (batches 14 and 17) and with the commercial antifungal (batch 18). Therefore, the incorporation of rosemary and D. hansenii in the meat mix before stuffing can be successfully implemented as synergic antifungal agents during the processing of dry-cured fermented sausages.

Veronica Zingales *, Mónica Fernández-Franzón and María José Ruiz
Laboratory of Toxicology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain * Correspondence: vezin@uv.es Mitochondria are key cellular organelles known to guarantee many physiological processes, such as energy production through the oxidative phosphorylation process (OX-PHOS). In in vitro conditions, cell lines are metabolically adapted to grow rapidly, and, for this reason, they derive most of their energy from glycolysis rather than OXPHOS, a phenomenon known as the Crabtree effect. The substitution of galactose for glucose in the culture medium is an expeditious way to reverse the Crabtree effect and determine mitochondrial toxicity. The aim of the present study was to evaluate the role of mitochondria in the toxicity induced by the mycotoxin sterigmatocystin (STE) on human neuroblastoma SH-SY5Y cells. Cells were cultured in the presence of glucose (25 mM) or galactose (10 mM) as the only sugar available. The effects on cell viability were evaluated by MTT assay. The change in the fuel source caused a cell viability decrease on galactose-grown cells compared to glucose-grown cells, suggesting that STE exhibited an increased level of toxicity in SH-SY5Y cells following the switch to OXPHOS. Furthermore, considering the crucial importance of a functional electron transport chain (ETC) in OXPHOS conditions, we also compared the effect of STE exposure in the presence or not of known ETC inhibitors (antimycin A and rotenone) in cells grown in a galactose-supplemented medium. Treatment with STE and rotenone, a selective inhibitor of the complex I, resulted in a further significant decrease in cell viability with respect to cells only exposed to the mycotoxin, while no effect was observed in the presence of antimycin A, an inhibitor of the complex III. These data highlight that STE might affect complex I, whereas the complex III of the ETC seems not to be involved in STE toxicity. Taken together, our results suggest that the etiology of STE cytotoxicity may depend on mitochondrial impairment. Human biomonitoring has been considered an efficient way to assess human exposure to mycotoxins. Adequate and validated analytical methods are crucial for this purpose. Furthermore, it is necessary to select good biomarkers in biological fluids [1], and plasma seems to be a suitable matrix to perform these studies.

Keywords
The aim of this study is to assess mycotoxin exposure through the analysis of 19 compounds in plasma samples from a region of northern Spain. The impact of some factors (age and gender) on this exposure has also been evaluated. Plasma from healthy adults (n = 438, aged 19-68 years) was analyzed using an LC-MS/MS validated method [2]. Sample preparation was carried out by deproteinization and cleanup using Captiva EMR ® -lipid (3 mL) cartridges. In order to study the presence of some Phase II metabolites, plasma samples were treated with a mixture of β-glucuronidase/arylsulfatase enzymes.
The most prevalent mycotoxin was ochratoxin A (OTA), with an incidence of 97.3% and positive samples are in the range of 0.4 to 45.7 ng/mL (0.4-23.3 ng/mL after enzymatic treatment) with a mean value of 2.87 ng/mL (2.40 ng/mL after enzymatic treatment). Ochratoxin B has also been detected (10% of the samples), and its presence can be related to that of OTA. Sterigmatocystin was detected in 85.8% of the samples, only after enzymatic hydrolysis, supporting conjugation as a pathway of its metabolism in humans. None of the other studied mycotoxins (aflatoxins B1, B2, G1, G2, and M1; T-2 and HT-2 toxins; deoxynivalenol, deepoxy-deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol; zearalenone; nivalenol; fusarenon-X; neosolaniol; and diacetoxyscirpenol) were detected in any of the samples, neither before nor after enzymatic treatment. To the best of our knowledge, this is the first study carried out in Spain to determine multi-mycotoxin exposure through their presence in human plasma [3].
The present biomonitoring study generates reliable and critical data regarding the exposure of the Spanish population to mycotoxins. Human SH-SY5Y neuroblastoma cells represent a suitable in vitro model to investigate toxicity in the brain and reproduce accurately neurodegenerative diseases. Although SH-SY5Y cells are widely used in neuronal research, they are epithelial cells with no neuronal proprieties unless they are treated with retinoic acid (RA) and differentiated into dopaminergic neurons. Thus, neuronal features, discriminating undifferentiated and RA-differentiated SH-SY5Y cells and showing significant differences between these cell models, will be characterized. In this regard, two different techniques, flow cytometry, and microscopy are being implemented to highlight morphological and functional changes induced by differentiation. Cells will be exposed to several mycotoxins concentrations, individually and in combination, and the exposure will start at different differentiation time points in order to assess the risk of exposure to these food contaminants. Flow cytometry experiment allows outlining differences in the cell cycle since differentiated cells featured a significant decrease in the proliferation rates, mainly associated with a decrease in S phase in combination with an arrest in G2-M. In this case, DNA composition will be analyzed using propidium iodide. Instead, optical microscopy enables analysis of the cell morphology since RA-differentiated cells showed an increased neurite density, suggesting a change from epithelial to a stellate neuronal morphology. Moreover, immunofluorescence microscopy permits detection of the dopamine content, potentiated by RA-differentiation. Neurites density and dopamine reactivity will be evaluated using anti-bIII tubulin and antidopamine antibodies, respectively, followed by incubation with their Alexa 488-conjugated antibodies and nuclear dye Hoechst, thus achieving random images with a fluorescence microscope. Hence, through three different experiments: Cell cycle and cellular growth, dopamine immunoreactivity, and neurite density differentiation, alterations induced by the presence of mycotoxins will be investigated.

Fojan Agahi *, Ana Juan-García and Cristina Juan
Laboratory of Toxicology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain * Correspondence: fojan@alumni.uv.es Beauvericin (BEA), α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL) are produced by several Fusarium species that contaminate cereal grains. These mycotoxins can cause cytotoxicity and genotoxicity in various cell lines, and they are also capable of produce oxidative stress at the molecular level. However, mammalian cells are equipped with a protective endogenous antioxidant system formed by non-enzymatic antioxidant and enzymatic protective systems such as glutathione peroxidase (GPx), glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT).
The aim of this study was evaluating the effects of α-ZEL, β-ZEL, and BEA, on enzymatic GPx, GST, SOD, and CAT activity in human neuroblastoma cells using the SH-SY5Y cell line, over 24 h and 48 h with individual treatment at the concentration range from 1.56 to 12.5 µM for α-ZEL and β-ZEL, from 0.39 to 2.5 µM for BEA, from 1.87 to 25 µM for binary combinations and from 3.43 to 27.5 µM for tertiary combination.
Our results revealed a significant increase in GPx activity after 24 h of exposure in all treatments except for tertiary combination, which decreased notably; while after 48 h, only BEA and triple mixture increased GPx activity considerably. GST activity in SH-SY5Y cells decreased significantly after exposing them to α-ZEL and β-ZEL, while in combinations increased notably after 24 h, except for β-ZEL + BEA, where a considerable decrease at lowest concentrations and increase at highest concentrations was detected. After 48 h, a significant increase in β-ZEL and BEA, whereas a decrease in α-ZEL + β-ZEL combination was observed. CAT activity decreased significantly in all treatments after 24 h except in β-ZEL + BEA, which revealed an increase. For SOD, no changes were observed after 24 h, although a significant increase was observed in binary combinations α-ZEL + BEA and β-ZEL + BEA after 48h, respectively. Enniatins (ENs) are hexadepsipeptides produced by Fusarium fungi, which can act as ionophores, disturbing membrane homeostasis. In this study, a proteomic analysis to determine the acute response of rat's liver to ENs exposure at different concentrations was carried out. A total of 14 female 2 months old Wistar rats were employed, divided into 3 groups. Five of the treated ones were intoxicated with medium concentrations: Single dose of ENA 256, ENA1 353, ENB 540, ENB1 296 µg/mL; and other five with the higher ones: Single dose of ENA 513, ENA1 706, ENB 1021, ENB1 593 µg/mL for 8 hours exposure. Protein extraction was performed using 10 mg of powdered liver tissue in an 8M Urea/2M Thiourea/50mM Tris-HCl lysis buffer. Protein concentration was determined by using a spectrophotometer NanoDrop™ 2000 and subsequently standardized to 1 mg/mL. Samples were mixed with dithiotreitol and iodoacetamide for alkylation of cysteine residues and digested with the addition of trypsin (1:40) overnight. Peptides were dried on a vacuum concentrator and eluted in 0.1 % acetic acid: Acetonitrile (98:2 v/v) to a final concentration of 100 µg/µL. Samples were analyzed using an LC system coupled with quadrupole time of flight (Q-TOF), and the obtained chromatograms were aligned with Mass Hunter Professional software (Agilent). Peptides identification was carried out by Spectrum Mill software and statistically filtered by abundance using Mass Professional Profiler software (Agilent). Results reported a total of 57 differentially expressed proteins in both medium and high treated animals when compared to the control. DAVID gene ontology analysis revealed acetylation, nucleotide phosphate-binding region:NAD, and catalytic activity as the most represented terms in the bioinformatics analysis. Moreover, 13 of these proteins were found in the mitochondrion, and 12 were related to oxidoreductase activity. Regarding reactome overrepresentation test results, metabolism was both the most significant pathway and the most enriched.

Manuel Alonso-Garrido *, Massimo Frangiamone, Guillermina Font and Lara Manyes
Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain * Correspondence: Manuel.alonso-garrido@uv.es Aflatoxins and ochratoxin neurotoxicity is a field of interest for food science researchers, as they are able to cross the blood-brain barrier, triggering mechanisms such as altered gene expression and oxidative stress, which are related to neurodegenerative disorders. On the contrary, carotenoids are known for their antioxidant capacity, and they have also been found in the brain. Thus, a method using SH-SY5Y neuroblastoma cells and low concentrations of mycotoxins (100 nM) and carotenoids (500 nM) is being developed simulating a real scenario in the human body. First, the effect of these mycotoxins on SH-SY5Y differentiation, individually and combined, will be assessed through optical, immunofluorescence, and confocal microscopy as well as flow cytometry. Optical microscopy will be used to visually discriminate non-differentiated cells, due to diverse morphology. Immunofluorescence will allow estimating neurite density through β-III tubulin detection, a marker for neuronal differentiation. Confocal microscopy will serve to detect dopamine immunoreactivity, which is also characteristic of neuronal cell activity. Second, Next Generation Sequencing (NGS) will be performed to find the most altered genes by the different cell exposures on an Illumina sequencer by RNA-seq technique. Data analysis will be performed on different bioinformatics tools for Differential Gene Expression (DEGs) and altered pathways. Third, validation of NGS will be done on the most affected genes by qPCR to confirm DEGs. Fourth, proteomics will be carried out in order to analyze if the profile is also modified, and these results will be compared with DEGs. Mass spectrometry technique (LC/Q-TOF MS) will allow to quantify the proteome followed by bioinformatics for peptide, protein identification and pathways involved.

Biomonitoring Study of Citrinin and its Metabolite Dihydrocitrinone in Human Urine
Alfonso Narváez 1, *, Yelko Rodríguez-Carrasco 2 , Carmen Martínez Alonso 2 , Luana Izzo 1 and Alberto Ritieni 1 Citrinin is a mycotoxin produced by Penicillium and Aspergillus spp. with nephrotoxicity and genotoxicity attributed in human cell lines. Therefore, the European Food Safety Authority set a threshold of toxicological concern for citrinin at 0.2 µg/kg b.w., but the lack of data on food contamination hampers a proper exposure assessment. To overcome this, strategies based on the biomonitoring of citrinin in biological samples have become an alternative in order to assess human exposure. Since the toxins occur at very low concentrations in human samples, selective and sensitive methodologies are required for accurate measurements. Hence, the aim of this study was to evaluate the presence of citrinin and its metabolite dihydrocitrinone for the first time in 300 human urine samples from South Italy through an ultrahigh-performance liquid chromatography high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) methodology. Citrinin was quantified in 47% (n = 300) of samples with concentrations ranging from below the limit of quantification (LOQ = 0.012 ng/mL) to 4.003 ng/mL (mean value = 0.286 ng/mL), and dihydrocitrinone was detected in 21% of samples at levels from below the LOQ (0.012 ng/mL) up to 2.481 ng/mL (mean value = 0.386 ng/mL). These results are in accordance with previous works where the metabolite reflects higher average levels than the parental compound. Heavier contamination was observed when compared to other European countries, with a 6-to-10-fold increase of mean values, which could be explained by a higher intake of cereals within the Italian population. Statistical analysis revealed differences according to the age of the volunteers, with citrinin being significantly more present in the population from 30 to 60 years old that may be due to different dietary habits. These data reflect a high exposure to citrinin within the Italian population, supporting further toxicological and food safety investigations for a better understanding of its impact.
Keywords: citrinin; exposure assessment; biomonitoring; urine; Orbitrap Acknowledgments: This research was funded by the project grant fiven by the Generalitat Valenciana (Spain) GV/2020/020 (Conselleria d'Innovacio, Universitats, Ciencia I Societat Digital, Generalitat Valenciana). The impact of climate change on the presence of mycotoxins in food and feed is a topic of great concern. Mycotoxins are ubiquitously present in feeding stuff, being to date, Fusarium mycotoxins (deoxynivalenol, DON; zearalenone, ZEN; and fumonisins, FBs) prevalent in areas of temperate climates such as Europe and Aspergillus mycotoxins contamination more frequently in hot climates. Every mold species has its own optimum conditions of temperature and activity water or the growth and formation of mycotoxins, thus environmental factors such as high temperatures, high moisture levels, and insect damage contribute to the presence of mycotoxins in feeds. The aim of this work was to review recent data reported on the mycotoxin's occurrence patterns in Europe because of environmental changes.

Pilar Vila-Donat *, Josefa Tolosa, María José Ruiz and Houda Berrada
In the data obtained from the available literature, the evidence shows A. flavus infection and aflatoxin (AF) contamination, previously uncommon in Europe, would become increasingly important. Over the last decade, several hot seasons have led to severe A. flavus infections in maize in several European countries (Italy, Romania, Serbia, and Spain). AF outbreaks have been reported in some regions of South Europe, such as infection of maize by AFB1 in Italy from 2003 as a result of a hot and dry growing season. It is even being detected AFM1 in cow's milk samples, in which differences depending on the season of sampling have been observed.
On the other hand, the prevalence of Fusarium graminearum (species adapted to hot conditions) and the main producer of DON in cereal grain, has already increased in Central Europe and is likely to increase in North Europe due to the expected changes in weather conditions. These facts point out that occurrence patterns of mycotoxins in Europe are changing as a consequence of rising average temperatures and, consequently, a potential increase in consumer health risk in Europe.

Keywords: mycotoxins; climate change; occurrence patterns; Europe
Acknowledgments: Authors are grateful to Regional Government to fund GV/2020/020 project. Mycotoxins produced by Fusarium genus may exert harmful effects on human health and, therefore, represent a hazard to public health and a challenge to ensure food safety. For this reason, the exposure assessment to deoxynivalenol (DON) and enniatin B (ENNB) through cereal-based food (e.g., bread, cookies, and pasta, among others) has been conducted. Consumption data have been gathered from surveys conducted in Catalonia during 2019, contamination data has been obtained from the analysis of the samples also collected in Catalonia. The data have been combined using three types of statistical approaches: A deterministic estimation, non-parametric estimation, and parametric estimation. In the case of infant exposure through baby formula, a deterministic estimation has been conducted due to a large number of censored samples. The results of these estimations (using the limit of detection to substitute censored data) showed the exposure of babies would be an average of 75 ng/kg bw/day for DON and 31 ng/kg bw/day for ENNB for the estimation through composite, man would be exposed to 102 ng/kg bw/day for DON and 83 ng/kg bw/day for ENNB, woman would be exposed to 100 ng/kg bw/day for DON and 82 ng/kg bw/day for ENN, and elderly would be exposed to 93 ng/kg bw/day for DON and 76 ng/kg bw/day for ENNB. The current exposure estimates for DON appeared to be similar to the previous assessment conducted in 2009, with the exception of a decrease among babies and a slight increase among elders, in any case far from the tolerable daily intake of 1000 ng/kg bw/day. OTA and AFM1 were the most present. The global mean and range detected were greatly different between continents and countries. A high incidence was observed in Tanzania, Iran, Jordan, and Turkey. However, the highest values were observed in Egypt, Sudan, and Serbia, being higher than the EU maximum limits (25 ng/kg for AFM1 and 500 ng/kg for OTA) (Regulation (EC) No 1881/2006). A provisional estimated daily intake (PDI) was calculated using the mean observed and an approximation of daily consumption of breast milk from 630 g/day to 890 g/day. EFSA (2006) established a tolerable daily intake (TDI) only for OTA with 14 ng/kg/day and non-intake for AFM1. It should be noted that the AFM1 values ranged from <1 ng/L to 7100 ng/L (Brazil and Egypt, respectively), then PDI was from 0.5 and 595 ng/kg bw/day. On the other hand, the range of OTA s means were 4 to 1990 ng/L (Brazil and Iran) with an PDI from 0.38 to117 ng/kg bw/day, values that represent the 3 to 1194% of the TDI. The review indicates that more controls on raw materials should be applied in Egypt, Iran, and Turkey. Tomato fruit rot is a serious disease caused by the Fusarium species. The aim of this study is to evaluate the ability of 2 strains of Trichoderma (T. asperellum and T. atroviride) to inhibit Fusarium spp.

Keywords
The inhibitory effect of Trichoderma extract with ethylacetate (EtOAc) was studied in vitro and in vivo. Trichoderma was grown in PDB at 30 • C for 30 days. The cultured filtrate was extracted with EtOAc. The extract of T. asperellum (TaE) and T. atroviride (TatE) were tested in vitro against 6 species of Fusarium (in 96-well plates), obtaining the minimum inhibitory concentration and the minimum fungicidal concentration (MIC and MFC).
Starting from MFC, three concentrations were tested in vivo on tomato fruits. Tomato fruits were inoculated with F. verticillioides, treated with the extracts at 3 different concentrations (ranges 0.78-3.12 mg/mL), and incubated at 4 and 11 days to room temperature.
The extracts showed MIC values ranging between 0.19 and 0.78 and MFC values ranging between 0.78 and 1.56 mg/mL for TaE and TatE, respectively.
In vivo, both extracts (at 3.12 mg/mL) showed efficacy in inhibiting the growth of F. verticillioides with a significant difference (p < 0.05) compared to control (untreated). At 4 days after treatment, infection rates (IR%) of 7.5% and 23%, and Log CFU/g of 2.9 and 5.2 were for TaE and TatE, respectively. However, only TatE showed persistent efficacy at 11 days with an IR% of 19% and 4.8 Log CFU/g. Keywords: antifungal activity; Trichoderma asperellum; Trichoderma atroviride; tomato fruit rot

Bernat Borràs-Vallverdú *, Antonio J. Ramos, Sonia Marín, Vicente Sanchis and Juan José Rodríguez-Bencomo
Food Technology Department, Agrotecnio Center, University of Lleida, 25198 Lleida, Spain * Correspondence: bernatborrasvallverdu@gmail.com Deoxynivalenol (DON) is a mycotoxin frequently found in cereals grains like wheat, barley, oats, or maize. DON is mainly produced by Fusarium graminearum and Fusarium culmorum, both of which can cause Fusarium Head Blight disease (FHB) in cereal crops. Ingestion of DON can result in chronic and acute toxic effects, the former being the most common. Chronic toxic effects comprise altered nutritional efficiency, weight loss, and anorexia, while acute effects include nausea, vomiting, and food rejection. Because of its toxicity, most countries have regulated DON maximum levels in different foods and commodities. Recommended maximum limits have also been established for animal feed. In this study, we developed a method for degrading DON on wheat kernels by exposing them to ammonia (NH 3 ) vapors. Under optimal conditions (90 • C and 2 h treatment), more than 75% of the toxin was degraded in wheat kernels contaminated with 2000 µg DON/kg. No influence of the DON concentration was observed on the toxin degradation extent (concentrations of 200, 500, and 2000 µg/kg were tested). The proposed methodology for decontaminating DON in wheat kernels could be easily scaled up in the industry without the need for complex and expensive facilities. Four degradation products derived from the reaction between DON and NH 3 were tentatively identified. Their toxicity and biological activities were in silico evaluated, and in general, lower potential negative effects were observed in comparison to the parental mycotoxin.

Victor D'Opazo *, Carlos Luz, Francisco Lluenca, María Paz, Jordi Mañes and Giuseppe Meca
Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain * Correspondence: dotavic@uv.es Filamentous fungi infection is the principal cause of fruit and vegetal losses in the field. Chemical pesticides are the regular response to those infections. Nevertheless, the exaggerated use of these pesticides as a normal response those these contaminations brought several environmental and health problems. New and safer methods are being tested, among them, the use of microorganisms (MO) as biopreservative strategies to become an alternative to regular pesticides. In this study, the antifungal potential of 33 MO isolated from red grape was studied. First, a Gram stain was performed to identify the MO. Then, the characterization of the antifungal activity was performed using the agar diffusion method and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) method against fungi from Aspergillus, Alternaria, Botrytis, Fusarium, and Penicillium genera. The antifungal agent tested was a cell-free supernatant (CFS) from MRS medium fermented by the MO. Gram stain revealed that the MO was yeast and grampositive coccus bacteria. The Agar diffusion method showed that overall, the bacterial CFS was more active against the fungi than the CFS made by yeast. The CFS fermented by UTA6 bacteria exhibited the greatest fungal inhibition. The MIC-MFC exposed similar results, CFS fermented by bacteria had higher antifungal activity, especially UTA6 reaching MIC's from 6.3 to 50 g/L and MFC's from 6.3 to 100 g/L. Future investigations will focus on the identification and quantification of the compounds with antifungal activity from the CFS and the possible application as a biopreservative on red grape.

Clara Melguizo *, Jéssica Gil-Serna, Covadonga Vázquez and Belén Patiño
Department of Genetics, Physiology and Microbiology, Faculty of Biology, Complutense University of Madrid, 28040 Madrid, Spain * Correspondence: claramel@ucm.es Mycotoxins are toxic secondary metabolites produced by some species of filamentous fungi, which represent an important threat for human and animal health and suppose large economic losses to the agri-food sector. The development of new control methods to prevent fungal growth and mycotoxin production in foodstuffs is essential, and biocontrol has been revealed as a promising alternative to conventional fungicides. In a previous work carried out in our group, we isolated a highly predominant yeast from a vineyard soil, and it was identified as Hanseniaspora uvarum. In this project, we tested this yeast as a potential biocontrol agent against aflatoxin-(A. flavus and A. parasiticus), ochratoxin A-(OTA) (A. steynii and A. westerdijkiae), and fumonisin-(F. proliferatum and F. verticillioides) producing fungi. CYA plates were supplemented by a final concentration of 103 cells/mL of H. uvarum and a spot of spores of mycotoxigenic fungi was deposited on the center of the plates. All of the fungi studied reduced their growth rate compared to the control in the presence of the potential biocontrol agent reaching inhibition percentages of 20% and 25 % in the case of A. flavus and A. westerdijkiae, respectively. Besides, mycotoxin production was evaluated by TLC in the case of OTA and aflatoxin producers and using ELISA for fumonisin-producing Fusarium species. In all cases, a clear reduction in OTA and aflatoxin concentration was observed in TLC plates, whereas this decrease was estimated at 50% in the case of fumonisin concentration detected in CYA plates. These initial results are promising and show the potential of H. uvarum as a biocontrol agent to be used in integrated control approaches to avoid mycotoxins in different crops, including those produced using organic management.

Keywords: biocontrol agent; fumonisins; ochratoxin A; aflatoxins; Hanseniaspora uvarum
Acknowledgments: The authors thank the AgroBank-UdL Chair "Quality and Innovation in the Agri-Food Sector" for economic aid for the realization of this work.

Juan José Rodríguez-Bencomo *, Vicente Sanchis, Inmaculada Viñas, Olga Martín-Belloso and Robert Soliva-Fortuny
Food Technology Department, Agrotecnio Center, University of Lleida, 25198 Lleida, Spain * Correspondence: jrbencomo@gmail.com Apple products are considered to be by far the most significant dietary source of patulin. Its origin is related to the contamination of the raw materials by molds (mainly by Penicillium expansum). In humans, the unhealthy effects of this mycotoxin include gastrointestinal disorders, nausea, and vomiting, thus that its contents in apple-based products are regulated by the food safety authorities. The removal or degradation of this mycotoxin in contaminated apple juices has been studied with different approaches with uneven effectiveness. An innovative approach for patulin degradation/detoxification could be a chemical degradation process based on the formation of specific chemical compounds. In this way, the target compounds could be those generated from the patulin cell detoxification process, namely patulin-glutathione conjugates. In this study, the reaction of patulin and glutathione (GSH) induced by pulsed light and catalyzed by ferrous ions (Fe 2+ ) was evaluated in apple juice. Four processing parameters (GSH and Fe 2+ concentrations, number of light pulses, and the thickness of the liquid phase) were studied and optimized through a central composite experimental design and a surface response analysis. In addi-tion, the type of patulin-glutathione conjugates formed was tentatively identified. Results showed effective reductions of patulin contents (up to around 60%) with an optimum dose of GSH of 150 mg/L, the minimum thickness of liquid phase assayed, and a pulsed light energy dose of 1.2 J/cm2·mL. The catalytic effect of the ferrous ions was adequate for a molar ratio GSH/Fe +2 = 5. Mono-substituted patulin-glutathione adducts were identified as the main type of generated conjugates. A plus of this degradation strategy is the use of non-thermal processing of the juice to promote the chemical reaction would also allow keeping the food organoleptic/nutritional characteristics. However, more research is necessary to evaluate the stability and behavior of these adducts in the body after ingestion. Enniatins are secondary fungal metabolites and worldwide natural contaminants of several food and feed products. A 28-day repeated dose preliminary assay, using enniatin A naturally contaminated feed through microbial fermentation by a Fusarium tricinctum strain, was carried out employing 2-month-old female Wistar rats. In order to simulate a physiological test of a toxic compound naturally produced by fungi, 5 treated animals were fed for 28 days with fermented feed. As a control group, 5 rats were fed with standard feed. The estimated amount of enniatin A in serum were: 22.43, 29.02, and 36.80 µg on the 2nd, 3rd, and 4th week, respectively; and enniatin A blood concentrations obtained were: 0.97, 1.25, and 2.70 µg/mL on the 2nd, 3rd and 4th week, respectively. Previous results revealed that the relative number of lymphocytes T cytotoxic cells in the treated rats was inhibited significantly with respect to the control ones (p < 0.001), while lymphocytes T helper cells increased significantly (p < 0.001). In this study, the epigenetic alterations are evaluated in the ovaries of the treated and control rats described, in particular relative telomere length and mitochondrial DNA copy number by real-time PCR and DNA methylation by pyrosequencing. Results regarding telomere length were 1040.29 (1375.40-637.06) for enniatin A exposed rats and 1212.23 (1596.76-674.74) for control (p = 0.55). The mitochondrial DNA copy number showed a media of 303.73 (611.99-20.86) for treated rats and 501.97 (952.01-66.92) for control (p = 0.51). Results from DNA methylation by pyrosequencing are still being processed. Even if non-significant results were obtained, a decreasing tendency in mitochondrial DNA copy number and telomere length was observed, thus in the future, a larger number of samples is recommended to be analyzed in order to obtain more concluding results.

Josefa Tolosa *, Pilar Vila-Donat, María José Ruiz and Emilia Ferrer
Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain * Correspondence: josefa.tolosa@uv.es Mycotoxins are common contaminants in raw materials and feedstuffs intended for livestock. Thus, when animals are fed contaminated feeds, the mycotoxin carry-over into animal organs, edible tissues, and by-products (milk, egg, etc.) can occur. From the public health standpoint regarding mycotoxin occurrence in animal source foods (ASF), the mycotoxins considered to be of the outermost importance are aflatoxin M1 (AFM1) in milk and Ochratoxin A (OTA) in meat products, both classified as possibly carcinogenic to humans (group 2B) by the International Agency for Research on Cancer (IARC). Maximum Levels (MLs) for AFM1 in milk have been set by the European Commission (0.05 µg/kg). However, no MLs have been set in Europe for OTA in meat or meat byproducts. Notwithstanding, some countries have enforced MLs of OTA concentrations, and other countries have developed national guidelines for OTA levels. The aim of this work was to review recent data reported on the AFM1 occurrence in milk and OTA in meat by-products. On the one hand, AFM1 has been widely assayed in milk samples, exhibiting differences depending on the animal species and the season of sampling, showing high contents in cow milk collected during winter season and levels reaching up to 4.2 µg/kg, exceeding the MLs, have been reported. On the other hand, regarding OTA, different surveys reported their occurrence, especially in dry-cured meat products made from pork tissues, mainly sausages, ham, and different types of salami and prosciutto. High incidence and contents have been reported ranging from 0.06 to 14.7 µg/kg, although high values (up to 691 µg/kg) have been detected in salami samples. This fact highlights the need to establish an ML for OTA in these products to protect human health and to constantly monitoring mycotoxin occurrence in animal by-products. The European Commission has established regulatory levels for aflatoxin (AF) B1 and ergot sclerotia and guidelines for deoxynivalenol (DON), zearalenone (ZEA), ochratoxin A (OTA), fumonisin B1 and B2 (FB1 and FB2), T-2 and HT-2 toxins in raw materials and feed, considered as the first link in the food chain. Moreover, the European Food Safety Authority has published different scientific opinions about the risks to animal health related to the presence of some emerging mycotoxins as enniatins (ENN) and beauvericin (BEA).
As a summary, the majority of the samples were in accordance with EU regulations (which do not address emerging mycotoxins), although 3.1% of samples showed contents of ZEA above the recommended levels. However, the high co-occurrence should be a matter of concern, as synergistic or additive effects could increase the toxicity of mycotoxins.

Noelia Pallarés *, Houda Berrada, Mónica Fernández-Franzón and Emilia Ferrer
Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, 46100 Burjassot, Valencia, Spain * Correspondence: noelia.pallares@uv.es Nowadays, people are seeking freshness, high vitamin content, minerals, and lowcalorie products (Mandappa et al., 2018). This fact encouraged the implementation of nonthermal food processing techniques, such as high-pressure processing (HPP) and pulsed electric fields (PEF), with low impact on food nutritional components (Picart-Palmade et al., 2018). These technologies are explored for mycotoxin reduction or elimination without producing toxic residues (Gavahian et al., 2020). Aflatoxins (AFs) constituted one of the most investigated mycotoxins and were reported in food commodities such as groundnuts, sesame seeds, millet, maize, rice, wheat, fig, spices, and cocoa (Mahato et al., 2019). AFs are carcinogenic, mutagenic, and immuno-suppressive compounds that have been correlated with liver cancer (Marín et al., 2013). The European Commission (EC) has set maximal concentrations of AFB1, and the sum of AFB1, AFB2, AFG1, and AFG2 in certain foodstuffs, but maximum levels of AFs have not been set in juices (EC, 2006). The aim of this study is to investigate the effect of HPP and PEF technologies on AFB2 reduction in grape juice. Grape juice samples were spiked with AFB2 at a concentration of 100 µg/L and treated by HPP and PEF technologies. PEF treatment was carried out under conditions of the field strength of 3 Kv/cm and specific energy of 500 KJ/kg. HPP treatment conditions were set in pressure at 500 MPa for 5 minutes. After both treatments, AFB2 was extracted employing dispersive liquid-liquid microextraction method (DLLME) and determined by liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS-IT). The reductions observed were about 72% after PEF treatment and 14% after HPP. Moreover, an AFB2 degradation product with m/z 355.0711 has been identified by quadrupole time of flight mass spectrometry detector (qTOF-MS) after PEF treatment. Mycotoxins are present in stored grain, and co-exposure through the diet is very common. Although several studies have been done in vitro and in vivo, the toxic effects associated with metabolites generated once ingested are still unknown and difficult to study. The present study defines the metabolomics profile of all three mycotoxins (zearalenone (ZEA), α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL)) and explores the prediction of their toxic effects proposing an in silico workflow by using three programs of predictions: MetaTox, SwissADME, and PASS online. Metabolomic profile was also defined and toxic effect evaluated for all metabolite products from Phase I and II reaction (a total of 15 compounds).

Keywords
Results revealed that products describing metabolomics profile were: From O-glucuronidation (1z and 2z for ZEA and 1ab, 2ab, and 3ab for ZEA s metabolites), S-sulfation (3z and 4z for ZEA and 4ab, 5ab, and 6ab for ZEA s metabolites), and hydrolysis (5z and 7ab for ZEA s metabolites, respectively). Lipinsky s rule-of-five was followed by all compounds except those coming from O-glucuronidation (HBA > 10). Metabolite products had better properties to reach the blood-brain barrier than initial mycotoxins. According to Pa values (probability of activation) order of toxic effects studied was carcinogenicity > nephrotoxic > hepatotoxic > endocrine disruptor > mutagenic (AMES TEST) > genotoxic. Prediction of inhibition, induction, and substrate function on different isoforms of Cytochrome P450 (CYP1A1, CYP1A2, CYP2C9, and CYP3A4) varied for each compound analyzed; similarly, for activation of caspases 3 and 8. Relying on our findings, the metabolomics profile of ZEA, α-ZEL, and β-ZEL analyzed by in silico programs predicted alteration of systems/pathways/mechanisms that end up causing several toxic effects, giving an excellent sight and direct studies before starting in vitro or in vivo assays contributing to the 3Rs principle. However, confirmation can only be demonstrated by performing those assays. Fungal spoilage causes important economic losses in the food industry and is a challenge in food safety due to the production of mycotoxins, highly toxic compounds. In the past years, multiple strategies have been evaluated using natural compounds to prevent fungal spoilage. The aim of the study was to evaluate the antifungal properties of Yellow Mustard Seed (YMS) and Yellow Mustard Bran (YMB) extracts against toxigenic fungi of the Aspergillus, Penicillium and Fusarium genera. For this, a qualitative evaluation test on PDA plates was performed. YMB evidenced the highest antifungal activity, and, for this reason, the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) were established in vitro. The Penicillium genera evidenced the lowest MIC and MFC values, ranging from 0.3 to 4.7 g/L. Then, the use of YMB was studied to increase the shelf life of bread contaminated with P. commune CECT 20767. For this, different amounts of YMB were tested (2.5, 5, 7.5, and 10 g/Kg). In addition, a commercial treatment with sodium propionate (E-281) and a control treatment without preservatives was performed. The use of 10 g/Kg increased the shelf life in comparison to the control by 3 days and, in addition, equated the reduction of the fungal population as the commercial treatment. These results suggest the promising employment of YMB as an antifungal additive in the food industry because they satisfy the consumers' demand for natural additives. The presence of aflatoxin M1 (AFM1) in sheep milk is becoming a concern for farmers, industry, and consumers. In order to examine the presence of AFM1 in ovine milk, bulk tank milk samples were collected from 51 dairy sheep farms from Castilla and León (Spain) in autumn 2019. Each farmer was interviewed and a questionnaire was filled to characterize farm management and productivity at the time of sample collection: Number of total and milking ewes, daily milk production, type of milking parlour, number of farm workers, bedding frequency, use of total mixed rations, use of adsorbents, and previous mycotoxicoses. Aflatoxin M1 was determined by two methods: (a) A rapid test (lateral flow immunoassay, LFI) with a limit of detection (LOD) of 8 ng/L and (b) ultrahigh-performance liquid chromatography with a fluorescence detector (UPLC-FLD) with a LOD of 0.92 ng/L. Samples below LOD were considered as negative and assigned a value of zero. Both methods detected a different number of positive samples; thus, 37% of samples were positive by LFI and 88% by UPLC-FLD. The average values observed in the positive samples were 11.2 and 3.8 ng AFM1/L milk for LFI and UPLC-FLD, respectively. The preliminary analyses did not allow to link the occurrence of AFM1 in milk with any of the studied farm characteristics. Therefore, albeit AFM1 could be found in ovine milk, it must be highlighted that the observed concentrations were far below the EU maximum level (50 ng/L). Aflatoxins are mycotoxins contaminating raw materials, feed and food, and known for their genotoxic and carcinogenic effects on various animal species and humans (Group 1 by IARC). Raw milk is contaminated with the hydroxy-metabolite aflatoxin M1, following exposure of lactating animals to aflatoxin B1 present in feedstuffs. Most studies on the carry-over of aflatoxins present in feedstuff into milk have been reported for cows, whereas research on lactating ewes is very scarce despite the great importance of the latter in the Mediterranean area. This study aimed to investigate the aflatoxin carry-over in dairy sheep. Thirty Assaf ewes in mid-lactation, individually penned, fed, and milked were divided into 3 experimental groups, each animal receiving a different daily dose of AFB1 over 13 days: 0 (Control); 40 µg (L) and 80 µg (H). Milk samples were collected at 0, 1, 2, 3, 6, 13, 14, 16, and 17 days. After IAC cleanup, AFM1 was analyzed by UPLC coupled to fluorescence detector, with a limit of detection of 0.92 ng/L. AFM1 was detected in both L and H groups from day 1 to 14, AFM1 concentration in group H (69 ng/L) exceeding the EU maximum level (50 ng/L) on day 1. AFM1 excretion pattern was similar in L and H groups, with an increase until days 3-5, and then a gradual decline until reaching a stable concentration. AFM1 was not detected in milk from the 2nd day after removal of the contaminated feed. The carry-over rate of aflatoxins from feed to milk was nearly 0.2% on average (lower than the 1-2% reported for cows). It was found that the carry-over rate was higher in L group than in H group (p < 0.05).
Keywords: aflatoxins; dairy sheep; carry-over; feed; milk Mycotoxins were extracted with methanol:water (80:20) followed by cleanup using immunoaffinity columns. Finally, the determination was made by HPLC coupled to photochemical (PHRED) and fluorescence (FLD) detectors, with a limit of detection of 0.02 µg/kg for each of the aflatoxins. There were 37 out of 71 samples positive for total aflatoxins, with levels ranging from 0.02 µg/kg to 3.33 µg/kg. The presence of the different aflatoxins was: B1 (27 samples), G1 (15 samples) and B2 (9 samples); no aflatoxin G2 was detected. Aflatoxins B1, B2, and G1 were detected simultaneously in four samples, B1 and G1 in six samples, while B1 and B2 coexisted in eight samples. The incidence of total aflatoxins was similar in organic (46%) and conventional (53%) cocoa samples. The appropriateness of setting a maximum level for aflatoxins in cocoa and derived products should be considered in the light of public health risks. In the last decades, the demand for fish as a protein source for human consumption has increased, leading to some adaptations in the aquaculture industry. One of the most important changes is the introduction of vegetables as a source of protein in feeds, giving rise to new concerns about its contamination with mycotoxins. Mycotoxins in aquafeeds can represent an ecological, health, and economic problem, and it is necessary to for better knowledge about the occurrence of mycotoxins in aquafeeds and their toxicity or bioaccumulation in fish.
The objective of this study was to screen in vitro the toxicity of 15 mycotoxins to the fish. The RTgill-W1 fish cell line was exposed to a range of mycotoxins concentrations (0.012-100 µg/mL) during 24 hours. The cytotoxicity was evaluated with triple assay (AlamarBlue, CFDA-AM, and Neutral Red Uptake), which measures the cell metabolism at the mitochondrial level and the plasma lysosome membrane integrities, respectively.
The results indicate that most of the mycotoxins assayed exert a high acute effect in the fish cell line, indicating a possible concern for the aquaculture industry and for the consumers. Further research is needed to study the toxic and bioaccumulation profile of these mycotoxins in the fish. The occurrence of mycotoxins in maize kernels (Zea mays L.) is of great concern worldwide and is often associated with mycotoxicosis in livestock and in humans. In Spain, maize is produced all over the country but mainly in the regions Castile and Leon, Aragon, and Extremadura, followed by Catalonia, Castile-La Mancha, Navarre, Andalusia, and Galicia. The most prominent mycotoxins in cereals are aflatoxins B1, B2, G1, and G2 (AFB1, AFB2, AFG1, and AFG2), fumonisins B1 and B2 (FB1 and FB2), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), 3-and 15-acetyl-deoxynivalenol (3-and 15-ADON), and T-2 and HT-2 toxins. The goal of the present study was to investigate the occurrence of these mycotoxins in maize kernels commercialized and consumed in Spain. For this purpose, a sensitive, rapid, and reliable UPLC-(ESI+)-MS/MS multi-mycotoxin method using matrix-matched calibration for the determination of target mycotoxins in maize kernels was validated and applied to study the distribution of these mycotoxins in 98 maize kernels samples collected in 26 stores across different Spanish regions for a 5-year period (2015-2019). FB1, FB2, DON, 3-ADON, ZEA, AFB1, AFB2, AFG2, T-2 and HT-2 were quantified in 71.4%, 56.1%, 31.6%, 5.1%, 24.5%, 9.2%, 9.2%, 2%, 5.1%, and 5.1% of samples, respectively. The maximum EU limits for FB1 + FB2, ZEA, AFB1, and the sum of aflatoxins, were exceeded in 20.4%, 5.1%, 3%, and 3% of the analyzed samples, respectively. OTA and AFG1 were not detected, and the sum of T-2 + HT-2 was below the EU recommended limit. Various mycotoxins (two to seven) co-occurred at levels ≥ LOQ in about 34% of the samples, which may increase toxicity due to possible additive or synergistic effects. The results obtained in this study contribute to increase the knowledge on mycotoxin contamination of maize and warn about the need to assess mycotoxin levels in food and feed derived from corn.
Keywords: maize; mycotoxins; UPLC-(ESI+)-MS/MS method; co-occurrence; fumonisins Toxigenic fungi have very negative consequences on human society. These fungi and their mycotoxins cause devastating effects on agricultural crops, the economy, food security, and human and animal health. T-2 and HT-2 toxins (T-2 and HT-2) are considered two of the most relevant mycotoxins in cereals in Europe, especially in oats. In Southern countries with warmer weather, the species Fusarium sporotrichioides is considered the most relevant species concerning T-2 and HT-2 production in cereals. These mycotoxins inhibit DNA, RNA, and protein synthesis and induce DNA fragmentation characteristic of apoptosis. There are currently various approaches to reduce toxigenic fungi and mycotoxins, but the problems associated with T-2 and HT-2 in cereals in pre-and post-harvest have not been resolved. The development of active antifungal films containing pure components of essential oils (EOC) (GRAS compounds) is of great interest in food microbiology and technology. Likewise, predictive models based on machine learning (ML) algorithms might be innovative tools for the appropriate management of toxigenic fungi and mycotoxins in food. The aims of this study were: (a) To evaluate the potential of ethylene-vinyl alcohol copolymer (EVOH) containing EOC in the control of F. sporotrichioides growth in oats under different environmental conditions and in the control of T-2 and HT-2 production, and (b) to assess the ability of various ML methods to predict the growth rates of this fungus and T-2/HT-2 accumulation under the assay conditions. Mycotoxins were determined by UPLC-MS/MS. Effective doses of EVOH films containing cinnamaldehyde (CINHO), isoeugenol (IEG), citral (CIT), or linalool (LIN) were calculated. Four ML methods (neural networks (NN), random forest (RF), support vector machines (SVM), and extreme gradient boosted trees (XGBoost) models were evaluated for the first time for modeling growth and T-2 and HT-2 production by F. sporotrichioides. The most effective films were EVOH-CIT, EVOH-IEG, and EVOH-CINHO and XGBoost and RF provided the best performance as predictive models.
Keywords: T-2 and HT-2 toxins; physicochemical conditions; F. sporotrichioides; bioactive films machine learning * Correspondence: Eva.mateo@uv.es Oats (Avena sativa) is a cereal belonging to the Poaceae grass family of plants. Although traditionally used for animal feed, oat products' beneficial nutritional and physiological effects have generated an increased demand for oats in human nutrition. Oats have good taste, dietetic properties, high beta-glucan content, and anticarcinogenic effects. Betaglucan is known as a prebiotic, stimulating the growth of some beneficial residential colon microorganisms, such as bifidobacteria. However, oats can be contaminated by toxigenic fungi and their associated mycotoxins in the field or during storage. The European Commission (EC) has established Maximum Levels (MLs) in oats for a selected number of mycotoxins. They are aflatoxin B1 (AFB1), the sum of aflatoxins (AFB1 + AFB2 + AFG + AFG2), deoxynivalenol (DON), zearalenone (ZEA), and ochratoxin A (OTA). MLs for the sum of T-2 and HT-2 toxins (T-2 and HT-2) in oats and other cereals have been recommended by the EC. Several LC-MS/MS methods have been reported for the determination of mycotoxins in cereals and cereal-based products, mostly for rice, maize, wheat, and barley. However, only a limited number of reports have focused on the occurrence of the mycotoxins in oats. The present study was aimed at investigating the occurrence of these and other mycotoxins in oats collected and commercialized in different Spanish regions for a five-year period (2015-2019).
One hundred samples of oat grains were collected in stores located throughout the country and analyzed for mycotoxins using a validated multi-mycotoxin UPLC-MS/MS method. AFB1, AFB2, AFG2, DON, 3-acetyl-DON, ZEA, T-2, HT-2, fumonisin B1, fumonisin B2, and OTA were detected, and their respective quantification limits were exceeded in some samples. The most frequently detected mycotoxins were ZEA, HT-2, and DON. The Fungal spoilage is not only a global food quality concern but also presents serious health problems due to the production of mycotoxins, some of which present considerable challenges to food safety and generate large economic losses. Chemical preservatives are successful in retarding microbial growth, yet the growing demand for clean label products requires manufacturers to find natural alternatives to replace chemical ingredients. Lactic acid bacteria (LAB) are generally recognized as safe (GRAS), therefore, they are considered a good candidate for their use as a natural preservative in food to control fungal growth and subsequent mycotoxin production, as well as to improve shelf life. The objectives of this study were to identify and characterize LAB isolated from dry-cured meats with the potential to inhibit the growth of five toxigenic fungi (Aspergillus flavus ITEM 8111, Cladosporium oxysporum CECT 20421, Penicillium nordicum CECT 2320, Penicillium verrucosum VTT 47, and Penicillium griseofulvum CECT 2605). The A. flavus proved to be more resistant against all the LAB strains tested. From a total of 90 LAB strains isolated, 7 were selected for their high growth inhibitory effect in direct contact with the fungus by overlay technique, and another 7 were selected for their capacity to inhibit fungal growth by halo diffusion assay. In addition, the ability of the bacteria to hydrolyze meat proteins was also considered. For further studies, we propose the application of fermented swine loin extracts produced by these bacteria in casings to manufacture dry-cured meat products. The food packaging industry is facing a series of challenges as consumers and the rest of the world deal with changes related to COVID-19.
Customer demand has shifted drastically-the pandemic shut down restaurants and food-service outlets. Consumers have moved to buy grocery purchases, for which the use of packaging has risen.
Consumers' wishes to stockpile and their panic purchases of food, beverages, and homecare necessities have accentuated this trend.
Single-use, disposable food packaging appears to have made a comeback rising on the coattails of the COVID-19 pandemic, as many consumers believe this to be safer and/or more hygienic, but is this true?
In the food packaging challenges in the age of COVID 19 presentation, we will review, which the food packaging challenge are and how the sector can solve them. Keywords:food packaging; active; edible 6.2. COVID 19: It Is Not a Food Crisis, But It is an Important Challenge for The Food Industry

Roberto Ortuño
AINIA Technology Centre, Technology Park, 46980 Paterna, Valencia, Spain; rortuno@ainia.es COVID-19 is not considered a food transmission disease. The European Food Safety Agency (EFSA) is closely monitoring the information generated in the context of this crisis and has reported that there is no evidence that food is a COVID-19 source or route. The same view has been expressed by the FDA (Food and Drug Administration of the United States) and WHO.
However, the food sector has a triple challenge in this pandemic, meeting unusual demand, safeguarding worker's health, and ensuring food safety of products. To achieve this triple objective (focusing on measures that impact food safety and worker health), it is important to strengthen hygiene practices in processing and handling operations. The food sector currently has robust food safety management systems, like APPCC (hazard analysis and critical control points), including the adoption of correct hygiene practices in mandatory form or BRC, IFS, and ISO 22.000 as non-mandatory systems.
Therefore, we can ensure that the food sector has the necessary tools to ensure food safety, but it is important to take measures to strengthen food hygiene practices and monitoring.
In the first weeks of the pandemic, we considered that it could be useful to develop the "COVID Manual. Strengthening food hygiene measures in the productive environment" in order to provide food industries with guidelines to deal with this special situation.
This manual includes measures concerning plant staff, visits, and other operations, such as maintenance, cleaning, and disinfection, etc. It has received more than 3000 downloads and has been presented in 2 webinars with more than 1000 attendees.

Amparo Devesa
Importaco, S.A.U, 46469 Beniparrell, Valencia, Spain; adevesa@importaco.com Nuts are suitable substrates for fungal growth if production and trade conditions are appropriate. It is well-known that some of these molds can be mycotoxigenic, thus several mycotoxins may be present in our products. According to the Food Agriculture Organization (FAO), around 25% of crops are affected by mycotoxigenic moulds worldwide and, it has been estimated that over 1000 million tons are lost each year due to this reason. Besides, according to the annual report of the Rapid Alert System for Food and Feed, nuts were two of the most affected food categories by mycotoxins. Aflatoxins were the primary mycotoxins associated with the notifications, but more and more, other types and emergent mycotoxins are also of relative concern for the food industry. Therefore, this is one of the main concerns in our enterprise.
Traditionally, food safety control in the food industry consisted in determining the levels of mycotoxins in acquired goods according to legislated levels. This is a one-side control mechanism that does not provide complete information considering the heterogeneous distribution of mycotoxins. However, in our enterprise, we designed a 360 • model based on a multifactorial point of view. This model is intended to ensure food safety and quality control throughout the entire food production chain, namely from farm to fork. This way, we are capable of gathering data from several key points that enable us to work in a preventive approach instead of a corrective mode. Our final aim is to achieve a predictive model that guarantees no presence of mycotoxins in our goods from the first beginning of the chain.

Acknowledgments
The Vth Workshop of the Spanish National Network on Mycotoxins and Toxigenic Fungi and Their Decontamination Processes (MICOFOOD) has been supported by the Spanish Ministry of Economy, Industry and Competitiveness (Project AGL2017-90787-REDT).
The Faculty of Pharmacy (University of Valencia, Spain), AIMPLAS (Plastic Technology Centre), AINIA Technology Centre, IATA (Institute of Agrochemistry and Food Technology, CSIC) and IMPORTACO are also acknowledged for their support in the organization of this workshop.