POSTERS

36

ACTIVIDAD ANTIFÚNGICA DEL ÁCIDO PERACÉTICO CONTRA HONGOS TOXIGÉNICOS EN MAÍZ Y LA CEBADA

J.M. Quiles, P. Vila. F. Illueca, J. Calpe, G. Meca

Laboratorio de Toxicología, Departamento de Medicina Preventiva y Salud Pública, Ciencias de la Alimentación, Toxicología y Medicina Legal, Facultat de Farmàcia, Universitat de València, España.

juan.quiles@uv.es

Este trabajo presenta la importancia de evitar las pérdidas postcosecha de los granos de maíz especialmente durante la etapa de almacenamiento. Describe como las relaciones que se producen entre diversas especies fúngicas y otros factores son las responsables de la pérdida de calidad del grano almacenado. Las especies de Aspergillus Flavus y Penicillium Verrucosum provocan el deterioro del maíz durante el periodo de almacenamiento y producen micotoxinas que son compuestos tóxicos para humanos y animales. El ácido peracético es utilizado en la industria alimentaria para la desinfección de alimentos y superficies en contacto con alimentos. No deja residuos tóxicos y sus productos de descomposición (CH3COOH, O2 y H2O) son compatibles con el medio ambiente. Con el objetivo de intentar aplicar el ácido peracético para conservar los granos de maíz durante la etapa de almacenamiento, se determinó cuantitativamente la Concentración Mínima Inhibitoria (MIC) y la Concentración Fungicida Mínima (MFC) del ácido peracético frente a estas dos especies fúngicas en medio liquido PDB (contacto directo) y utilizando métodos volátiles. En ambos casos el ácido peracético presentó actividad antifúngica. Finalmente, se estudió a escala de laboratorio la posible aplicación de un gel antifúngico y antimicotoxigénico a partir de ácido peracético y gel de hidroxietil celulosa. Los granos de maíz y el gel se introdujeron en tarros de 1 L herméticos para simular un silo, comprobándose cualitativamente cómo el gel con una dosis de 200 mg/L de ácido peracético presentó actividad antifungica frente a A. Flavus ITEM 8111.

Keywords: Ácido peracético, actividad antifúngica, Aspergillus, maíz, Penicillium.

37

DEVELOPMENT OF A HIGH RESOLUTION MASS SPECTROMETRY METHODOLOGY FOR MULTI-MYCOTOXIN ANALYSIS OF MARKETED DARK CHOCOLATE

A. Narváez1,2, L. Izzo1, S. Lombardi1, L. Castaldo1, Y. Rodríguez-Carrasco2, A. Ritieni1

1Via D. Montesano, 49 - 80131 Napoli. Università di Napoli Federico II, Department of Pharmacy, Italy; 2Av/ Vicent A. Estellés, s/n, 46100, Burjassot, Valencia. University of Valencia, Department of Food Chemistry and Toxicology, Spain.

*alfonso.narvaezsimon@unina.it

Dark chocolate stands as the preferred typology of chocolate by Italian consumers, partly due to the interest of sustainable and healthier alternatives. Therefore, the high consumption rates, even for children, force to apply strict quality controls in order to ensure a safe consumption. Mycotoxins have been previously detected in chocolate, but analytical methodologies are mainly focused on aflatoxins (AFs) and/or ochratoxin A (OTA). Hence, the aim of this study was to develop and validate an analytical methodology based on ultra-high performance liquid chromatography coupled to high resolution Q-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-HRMS) for simultaneously assessing the presence of the main AFs, OTA, enniatins (ENNs) A, A1, B, B1, beauvericin (BEA), neosolaniol (NEO), deoxynivalenol (DON) and T-2 and HT-2 toxins. Then, the validated methodology was applied to marketed dark chocolate samples (n = 15). Extraction procedure was performed using acetonitrile acidified with acetic acid 4% as solvent of extraction, whereas no cleanup stage was required. Validation was successfully conducted according to the in-force legislation. Good linearity (r2 > 0.999) was observed in calibration curves, with limits of quantification (LOQs) ranging from 0.2 to 12.5 ng/g. Signal suppression/enhancement effect values (74-120%) remarked a non-negligible to moderate matrix interference, whereas spiking experiments at 50, 25 and 2 ng/g showed suitable recoveries (71-96%) with high inter- and intra-day precision (RSD < 14%). Analysis of marketed chocolate showed the presence BEA in all samples at a mean concentration of 4.94 ng/g whereas ENNB was quantified in 80% of samples at a mean concentration of 0.71 ng/g. AFB1 was also detected in one sample below the LOQ value (0.2 ng/g). The developed procedure is proposed as a powerful analytical tool to evaluate the mycotoxin profile in chocolate products, whereas more evidence about the extensive presence of emerging Fusarium toxins BEA and ENNs in foodstuffs is provided.

Keywords: Orbitrap; ultra-high performance liquid chromatography; food safety; emerging Fusarium toxins

Acknowledgement: PID2020-115871RB-100

38

MECANISMO IMPLICADO EN LA DETOXIFICACIÓN DE MICOTOXINAS POR

Hanseniaspora uvarum U1, Kazachstania unispora KC19_L5 y Debaryomyces hansenii KCAR20_L1

C. Gómez-Albarrán1, B. Patiño1, C. Vázquez1, J. Gil-Serna1 1 Departamento de Genética, Fisiología y Microbiología, Facultad de Biología, Universidad Complutense de Madrid, Madrid, España.

caroli13@ucm.es

La presencia de micotoxinas en alimentos y piensos supone una gran amenaza para la economía y la salud humana y animal. La coexistencia de varias micotoxinas en un mismo producto es de gran relevancia debido al efecto acumulativo y sinérgico en la toxicidad y carcinogenicidad. Entre ellas destacan la aflatoxina B1 (AFB1), la fumonisina B1 (FB1) y la ocratoxina A (OTA). La detoxificación biológica se posiciona como una de las estrategias más prometedoras para reducir la concentración de estas micotoxinas. Estudios anteriores llevados a cabo en este laboratorio han

demostrado que distintas levaduras aisladas de productos probióticos y uvas son capaces de reducir de forma significativa la concentración inicial de AFB1, OTA y FB1. A partir de estos resultados, se seleccionaron Hanseniaspora uvarum U1, aislada de uva, Kazachstania unispora KC19_L5 y Debaryomyces hansenii KCAR20_L1, aisladas de kéfir, para determinar cuál en el mecanismo implicado en la detoxificación de estas micotoxinas. Para ello, se ha valorado la reducción de AFB1, OTA y FB1 en un extracto utilizando células viables (VC) o inactivadas térmicamente (HIC) y a diferentes pH. Los resultados de este estudio parecen indicar que tanto H. uvarum U1 como K. unispora KC19_L5 detoxifican las micotoxinas por adsorción a la pared celular y por un mecanismo activo. En el caso de D. hansenii KCAR20_L1 el mecanismo más probable es la adsorción debido a que se observa un mayor porcentaje de eliminación en las células inactivadas. Estos resultados muestran el gran potencial de H. uvarum U1, K. unispora KC19_L5 y D. hansenii KCAR20_L1 como agentes de detoxificación biológica de micotoxinas.

Agradecimientos: Trabajo financiado por el MICINN (RTI 2018-097593-B-C21). Carolina Gómez Albarrán es beneficiaria de una beca FPI del MICINN (PRE 2019-087768).

Palabras clave: aflatoxina B1, fumonisina B1, ocratoxina A, detoxificación biológica, levaduras

39

DESARROLLO DE UN MÉTODO RÁPIDO PARA COMPARAR PERFILES DE MICROSATÉLITES DE Hanseniaspora uvarum

C. Melguizo, J. Gil-Serna1 , C. Vázquez, B. Patiño1

1Departamento de Genética, Fisiología y Microbiología, Facultad de Biología, Universidad Complutense de Madrid, Madrid, España

claramel@ucm.es

Las micotoxinas suponen un gran problema a nivel económico por su gran impacto sobre las cosechas. Tradicionalmente, el control de micotoxinas se ha llevado a cabo mediante la aplicación de fungicidas, pero la generación de resistencias y la actual demanda social de productos más sostenibles ha favorecido el desarrollo de otros métodos como el control biológico. En trabajos previos llevados a cabo en nuestro laboratorio, se caracterizó el potencial como agente de control biológico de la levadura Hanseniaspora uvarum U1 aislada de uva sobre diversos hongos toxígenos productores de aflatoxinas, ocratoxina A y fumonisinas. Según el Reglamento de la Comisión Europea 1107/2009 que regula la comercialización de productos fitosanitarios, es necesario poder seguir el establecimiento en el medio de los agentes de control biológico tras su aplicación. Por ello, en este trabajo se utilizó el método de tipado por microsatélites descrito por otros autores para genotipar cepas de Hanseniaspora uvarum, entre las que se encuentra H. uvarum U1. Con el fin de agilizar el procesado de comparación de perfiles, se desarrolló un código en lenguaje de programación Python que permitió la rápida comparación entre 115 perfiles de microsatélites distintos de H. uvarum. Tras el estudio de los 6 nuevos perfiles de microsatélites de aislados de uvas de esta especie de levadura se pudo determinar que dos de los aislados pertenecían a una misma cepa y que el perfil de microsatélites de H. uvarum U1 era único. Estos resultados muestran que el método de tipado utilizado es capaz de discriminar a H. uvarum U1 de otras 120 cepas comparadas permitiendo comprobar y seguir su implantación en el medio tras su aplicación como agente de control biológico.

Agradecimientos: Este trabajo fue financiado por el MICINN (RTI 2018-097593-B-C21)

Palabras clave: Hanseniaspora uvarum, tipado, microsatélites, agente de control biológico, micotoxinas

40

DETERMINATION OF PATULIN IN COMMERCIAL STRAWBERRY JAM THROUGH UHPLC Q-EXACTIVE ORBITRAP HRMS

L. Izzo1*, A. Narváez1, L. Castaldo1, A. Gaspari1, M. Grosso2,3, Y. Rodríguez-Carrasco4, A. Ritieni1,5

1 Department of Pharmacy, University of Naples “Federico II”, Via Domenico Montesano 49, 80131 Naples, Italy; 2 Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples “Federico II”; 3 CEINGE-Biotecnologie Avanzate, 80131 Naples, Italy; 4 Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Valencia, Spain; 5 Health Education and Sustainable Development, Federico II University, 80131 Naples, Italy.

* luana.izzo@unina.it

Patulin (PAT) is a common mycotoxin frequently occurring in products intended for human consumption, especially fruits. Strawberries is one of the most perishable fruit subjected to several postharvest losses, including fungal deterioration. The goal of this work was to develop a rapid method for the determination of patulin in commercial strawberry jam using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC Q-Exactive Orbitrap HRMS). The proposed methodology showed satisfactory results in terms of linearity (r2> 0.999), matrix effect (97.7%), recovery (range 87.2-104%), precision (RSDr ≤14%; RSDR ≤10%) and sensitivity (LOQ 1.56 ng/g), in line with the regulation in force, and was successfully applied to 17 strawberry jam samples from different brands commercialized in Campania region (Italy). The incidence of patulin was 82.3% (14/17) with a concentration of up to 17.879 µg/kg. Overall, samples did not exceed the maximum limit of 50 µg/kg set by the EU regulation, but 4 samples surpassed the maximum limit established for foods intended for infants and young children. The risk associated with the dietary exposure to patulin was assessed, being infants the most exposed population group. Hence, a continuous monitoring of mycotoxins is needed and the here developed methodology is proposed as an alternative analytical tool being able to determine patulin in 8 min analytical runtime.

Acknowledgement: PID2020-115871RB-100

Keywords: patulin; strawberry jam; risk characterization; UHPLC-Q-Orbitrap HRMS

41

DETERMINATION OF MULTIPLE MYCOTOXINS IN LIVER USING SALTING-OUT LIQUID-LIQUID EXTRACTION FOLLOWED BY DISPERSIVE LIQUID-LIQUID MICROEXTRACTION AND HPLC-MS/MS

A. Castell1 , N. Arroyo-Manzanares1 , N. Campillo1 , J. Fenoll2 , P. Viñas1 1 Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, E-30100, Murcia, Spain 2 Sustainability and Quality Group of Fruit and Vegetable Products, Murcia Institute of Agri-Food Research and Development, C/ Mayor s/n. La Alberca, 30150, Murcia, Spain

ana.castell@um.es

Mycotoxins are mainly found in agricultural products and their ingestion can cause permanent damage to the animal and human organisms. Up to date, most studies on the detection of mycotoxins in humans have focused on biological fluid samples, being plasma and urine the most studied; however, there are few studies based on organs and human tissues. In this work, high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) is proposed for the determination of a total of 13 mycotoxins belonging to the genera Aspergillus [aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2)] and Fusarium [zearalenone (ZAN), T-2 and HT-2 toxin, deoxynivalenol (DON), enniatin A (ENNA), A1 (ENNA1), B (ENNB), B1 (ENNB1) and beauvericin (BEA)] in human and animal liver samples. For this purpose, the mycotoxins extraction was carried out by salting-out liquid-liquid extraction (SALLE) followed by dispersive liquid-liquid microextraction (DLLME) achieving suitable limits of detection (from 0.001 to 30 μg kg-1 ) and quantification (from 0.01 to 100 μg kg-1 ) for each mycotoxin. In order to check the suitability of the proposed method, repeatability and intermediate precision were evaluated, obtaining values lower than 9%. Furthermore, ENNB and ENNB1 were determined in all human liver samples analysed, ENNB and ENNA1 in pig liver samples, BEA and ENNA1 in calf liver, ENNB in chicken liver and ENNB, BEA, ENNB1, ENNA1 and ENNA in lamb liver. Therefore, the proposed method is presented as an efficient approach to determine the mycotoxins occurrence in human organs as liver in order to study its accumulation and toxicity.

Acknowledgements: The authors acknowledge the financial support of the Comunidad Autónoma de la Región de Murcia (CARM, Fundación Séneca, Project 19888/GERM/15) and the Spanish MICINN (PGC2018-098363-B-100).

Keywords: mycotoxins; liver; liquid chromatography; tandem mass spectrometry

42

OCHRATOXIN-A´S CONJUGATES AS POTENTIAL BIOMARKERS OF EXPOSURE

M.A. Sacco1*, I. Aquila1, P. Ricci1, P.Llorens2, J.C.Moltó 2, C. Juan 2

1 Institute of Legal Medicine (“Magna Graecia” University of Catanzaro, Department of Medical and Surgical Sciences) Italy; 2 Laboratory of Food Chemistry and Toxicology (University of Valencia Faculty of Pharmacy) Spain.

*matteoantoniosacco@gmail.com

Estimating OTA-exposure is a public health problem and requires sensitive biomarkers. A number of OTA metabolites have been suggested as biomarkers of OTA exposure. Numerous in vitro and in vivo experiments have shown that OTA and OTalpha are metabolized through phase-II reactions with glucuronic acid and these glucuronate metabolites could be potential biomarkers of OTAexposure (1). However, Sueck et al. (2019) recently reported detection of a mercapturic acid (Nacetyl-cysteine; NAC) conjugate of OTB (OTB-NAC) in human urine samples. This putative metabolite, the chlorine atom of OTA is replaced by N-acetyl-cysteine, was suggested to be formed by direct catalysis by glutathione-S-transferases (2, 3). For qualitative analysis of OTA metabolites, radioactivity, thin layer chromatography (TLC), nuclear magnetic resonance (NMR) and liquid chromatography based analytical methods were also established. However, the metabolites purified from the biomatrices or the commercial standards were needed in these previous studies, thus certainly making the procedures tedious and expensive. For glucoronate metabolites is necessary to treat with ß-glucuronidase and an indirect analysis is carried out. OTA´s increase after this enzymatic hydrolysis in urinary and blood samples has demonstrated their circulation. In this review, that metabolites have been detected in urine, plasma and liver. Coronel et al., (2011) signs that the range detection in human blood was from 0.0306 ng/mL in Turkey to 37 ng/mL in Czech Republic. In human urine, recent studies have detected glucoronate OTA in Spain 0.057 to 0.562 ng/mL (1); in Belgium 0.0027 to 0.368 ng/mL (4); in Sweden 0.90 ± 0.50ng/mL (5); and Portugal 0.019 to 0.052 ng/mL (6). Recently, liquid chromatography coupled with time of flight mass spectrometry (LC–TOF-MS) has been proposed as a powerful approach for identification of unknown compounds (7). Failure to directly identify these metabolites suggests that will require further investigation in the future.

Keywords: (ochratoxin A, mycotoxins, glucuronide coniugates, OTA-metabolites)

1. Coronel B., et al. (2011). Food and Chemical Toxicology, 49, (6) 1436-1442. 2. Dekant R., et al. (2021). Toxins 13, 587. 3. Sueck F., et al. (2019). Mycotoxin Res. 36, 1–10. 4. Heyndrickx Ll., et al. (2015). Environment International, 84, 82-89. 5. Wallin S., et al. (2015). Food and Chemical Toxicology, 83, 133-139. 6. Silva L. J.G., et al. (2020). Food and Chemical Toxicology. 135, 110883. 7. Hana Z., et al. (2013). Journal of Chromatography B. 925, 46– 53.

43

BIOACCESSIBILITY ASSESSMENT OF AFB 1 , OTA AND ZEN IN BEETROOT BREAD

Llorens P.*, Moltó J.C., Mañes J., Juan C.

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia.

paullo3@alumni.uv.es

Red beet (Beta vulgaris) is a very nutritious tuber, with antioxidant activity, anti-inflammatory properties, antimicrobial and antiviral effect. Its incorporation into bread, one of the most consumed foods and susceptible to the action of molds, can increase the half-life of the product and be a source of antioxidants. Mycotoxins are contaminants of cereals and derivatives, with economic and toxic effects, highlighting aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEN). Controlling the storage conditions, as well as those preservatives or natural ingredients that are integrated into the food, can reduce their presence. The evaluation of its bioavailability and bioaccessibility are decisive to know the amount of mycotoxin ingested that reaches the organism and produces toxic effects. The components can interfere with the bioaccessibility of both nutrients and contaminants. The objective was to study the bioaccessibility of AFB1, OTA and ZEN in 10% beetroot bread through in vitro digestion (Minekus et al., 2014). This digestion consisted of a salivary phase (5 min at 37oC), a gastric phase (2h), and finally the small intestine phase (2h at 37oC). The bread made with lyophilized beetroot (10%) was fortified with AFB1, OTA and ZEN (10 μg / g) single and combined. After digestion, they were extracted with ethyl acetate and analyzed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS / MS). Comparing beetroot bread versus wheat bread, in its two forms (individual and combined), it can be concluded that OTA, AFB1 and ZEN have a different bioaccessibility when they are fortified simultaneously in beetroot bread at 10 ng/ g (87±1.1%, 25±0.6%, 107±19%, respectively). According to the results obtained, ZEN was highly bioaccessible mycotoxin in this experimental test.

Acknowledgement. The authors thank the Ministry of Science and Innovation for funding with the

PID2019-108070RB-I00ALI.

Keywords: beetroot, bread, mycotoxin, in vitro, bioaccessibility.

Bibliography: Minekus M, et al. (2014). Food Funct. 5 (6): 1113-1124.

44

CYTOPROTECTION ASSESSMENT AGAINST MYCOTOXINS ON SH-SY5Y CELLS BY BEET ROOT EXTRACTS

R. Penalva, M. Fernández-Franzón and A. Juan-García

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Avda. Vicent Andrés Estellés, s/n, 46100- Burjassot- Valencia, Spain

rapeol@alumni.uv.es

Fumonisin B1 (FB1) is a secondary metabolite of Fusarium moniliforme and F. proliferatum, mostly

present in corn as a natural contaminant; whereas Ochratoxin A (OTA) is produced by species of the

genera Aspergillus and Penicillium, its presence is more remarkable in cereal grains. Both FB1 and

OTA have been classified by the IARC as carcinogenic to humans (Group 2B). Beetroot is

commonly used as natural dye and is a source of dietary fiber, folic acid, and vitamin C. Also, some

studies have suggested an antioxidant activity of beetroot. Therefore, in this work it is presented the

cytoprotective effect of beetroot extract (BRE) on a neuroblastoma cell line (SH-SY5Y cells) against

FB1 and OTA- Cytotoxicity was studied by the MTT ([3-4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide) assay, for 24 h and 48. Simultaneous treatment, and pre-treatment

strategies at the 1:512-1:2 and 1:0 dilutions of BRE and at the concentration range from 0.4 to 100

μM for FB1 and from 0.04 to 10 μM for OTA were tested. Individual IC50 values were detected at

all times assayed for OTA (>0.16 μM) and no cytotoxic effect was detected at the assayed

concentrations for FB1. When the simultaneous strategy of BRE + OTA was performed,

cytoprotection with increases of viability was observed. Lastly, in the pre-treatment strategy, where

the cells were 24 h previously exposed to the BRE, better protection than the one shown in the

simultaneous assay was observed.

Keywords: FB1; OTA; Beetroot; cytoprotection; SH-SY5Y cells.

45

EFFECT OF LACTIC ACID BACTERIA ON AFB1 AND OTA REDUCTION AFTER INCUBATION IN CULTURE MEDIA

L. Escrivá, F. Agahi, G. Font, G. Meca, L. Manyes, P. Vila-Donat

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, València, Spain

laura.escriva@uv.es

Biological detoxification exhibits high potential to decontaminate mould food spoilage, as well as mycotoxins contamination on a cost-effective and large scale. The use of lactic acid bacteria (LAB) as an alternative strategy for mycotoxins decontamination is increasing interest since many LAB have shown mould growth inhibition and the potential to interact with mycotoxins. The aim of the present study was to evaluate the effect of several LAB on reducing OTA and AFB1 content in culture media. Twelve LAB (B1, B2, B3, B4, B5, B6, B7, B9, B10, BS4, BS6, BS7), as well as a control without LAB, were incubated in triplicate with OTA (500µg/L) and AFB1 (100µg/L) in MRS broth (37ºC) for 72h. Aliquots at different time points (0, 2, 4, 6, 24, 48, 72h) were collected and analysed by HPLC-MS/qTOF. The percentage of mycotoxin reduction with respect the control was calculated and the most effective LAB were selected for further experiments. Significant OTA reductions (p<0.05) were observed increasing over the incubation time, reaching 11% (2h), 16% (4h), 29% (6h), and up to 40% at 24, 48 and 72h. Nine of the twelve studied LAB significative reduce OTA concentration after 72h in a range of 12-40% compared to the control, with the highest reductions for B3, B10, and BS7. AFB1 reductions higher than 10% were mainly observed after 24, 48 and 72h incubation. Five LAB significative reduced AFB1 in a range of 11-35% after 72h, with B3 showing the highest reductions. Three LAB (B3, B10 and BS7) were selected as the most efficient ones by reaching reductions higher than 25% for both mycotoxins after 72h incubation. Selected LAB will be applied as ingredient in bread formulation to evaluate their potential on reducing mycotoxins in the final food product through both LAB fermentation and during a simulated in vitro digestion process.

Acknowledgements: Spanish Ministry of Science and Innovation project (PID2019-108070RB100).

Keywords: biopreservative, lactic acid bacteria, fermentation, mycotoxins, reduction

46

AFLATOXIN M1 IN MILK OF ASSAF DAIRY EWES RECEIVING DIFFERENT ADSORBENTS IN FEED CONTAMINATED WITH AFLATOXIN B1

T. Juan1, 2, R. Bodas3, M. Herrera2, J.R. Bertolín1,2 ,S. Lorán2, J.J. Carramiñana2, C. Yagüe4, A. Ariño2, F.J. Giráldez5, J.J. García-García3, S. Olmedo3

1Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), 50059 Zaragoza, Spain; 2Instituto Agroalimentario de Aragón-IA2 (Universidad de Zaragoza-CITA), Veterinary Faculty, 50013 Zaragoza, Spain 3Instituto Tecnológico Agrario de Castilla y León. Subdirección de Investigación y Tecnología, Área de Investigación Ganadera, Línea de Rumiantes. Spain; 4Faculty of Health and Sports Science, 22002 Huesca, Spain; 5Instituto de Ganadería de Montaña (CSICUniversidad de León), Finca Marzanas, 24346 Grulleros, León, Spain

tjuan@cita-aragon.es

A strategy to counteract the effects of mycotoxicosis in animals and to reduce the presence of their metabolites in milk is the use of adsorbents that can reduce the absorption and promote the excretion of mycotoxins. Mineral adsorbents are naturally abundant and inexpensive whereas organic ones (which include organic ingredients such as yeasts) entail a longer and expensive manufacturing process. This study aimed to investigate the effects of two different feed adsorbents (mineral and organic) on the presence of AFM1 in milk from dairy sheep exposed to an 11-day AFB1 challenge in feed. Thirty Assaf ewes in mid-lactation, individually penned, milked (8 a.m.) and fed (9 a.m.) received 120 µg AFB1 daily from day 1 to 11. Ewes were divided into three experimental groups: C (no adsorbent); A (organic adsorbent) and S (inorganic adsorbent). Five grams of the corresponding adsorbent were added to the daily diet from day 4 to 11. Milk samples were collected at 1, 2, 3, 4, 5 and 11 days. After IAC cleanup, AFM1 in milk was analyzed by UPLC coupled to fluorescence detector, with a limit of detection of 0.92 ng/L. AFM1 was detected in the three groups from day 2 to 11 at concentration always exceeding the EU maximum level (50 ng/L). From day 2 to 11, average AFM1 excretion pattern (277 ng/day) and carry-over rate from feed to milk (0.23 %) were similar in the three groups (p>0.10), regardless the type of adsorbent added to the daily ration. The carry-over rate was within the range observed by our research group in previous studies carried out with dairy Assaf sheep receiving lower doses of AFB1 in the diet.

Acknowledgements: The authors thank the financial support of the Ministry of Science and Innovation (INIA RTA 2017-00085-C2) and the Government of Aragón (Grant Grupo A06_20R).

Keywords: adsorbent, aflatoxin M1, milk, sheep

47

METODOLOGIA APLICADA A LA EVALUACION DEL EFECTO DE LOS SECUESTRANTES DE MICOTOXINAS EN LA CALIDAD NUTRICIONAL DE LOS PIENSOS PARA LA ACUICULTURA

X. Pascari, I. Teixido, F. Molino, S. Marín, V. Sanchis, A.J. Ramos

Applied Mycology Unit, Food Technology Department, University of Lleida, UTPV-XaRTA, Agrotecnio, Av. Rovira Roure 191, 25198 Lleida, Spain

xenia.pascari@udl.cat

La utilización de la harina de pescado en la alimentación de los peces carnívoros supone un coste muy elevado para la industria, limitando también su sostenibilidad. Varios estudios han buscado sustituir la harina de pescado por una fuente de origen vegetal, pero reemplazarla por completo afectaría la productividad debido a la presencia de componentes anti-nutritivos que disminuyen la asimilación de los nutrientes esenciales. Este problema se ha solucionado suplementando los piensos con complejos polivitamínicos, aminoácidos y minerales. Un peligro omnipresente relacionado con las materias primas de origen vegetal son las micotoxinas. Los adsorbentes de origen mineral son una estrategia ampliamente utilizada en la mitigación del efecto tóxico de las micotoxinas, existiendo un secuestrante a base de montmorillonita aprobado por la Unión Europea para su uso contra la aflatoxina B1. Nuestro trabajo actual analiza la capacidad de los secuestrantes de no solo adsorber las micotoxinas, sino también algunos nutrientes esenciales para el desarrollo de las especies acuícolas. Hemos desarrollado y validado dos métodos HPLC-FD/DAD para el análisis de tres vitaminas hidrosolubles (B5, B7 y B12) y dos aminoácidos (metionina y lisina), y un método fotométrico para la evaluación del fósforo (a partir de fosfato sódico) en un medio in vitro. Los métodos analíticos desarrollados permiten, con modificaciones mínimas, cuantificar el porcentaje de adsorción tanto en tampón con pH=5, como en jugos gástrico (pH=2) e intestinal (pH=8) simulado.

Agradecimientos: Ministerio de Ciencia e Innovación, Proyecto RTC2019-007143-2 (cofinanciado por la UE a través de FEDER-Una manera de hacer Europa). X. Pascari agradece al Ministerio de Ciencia e Innovación su contrato postdoctoral.

Keywords: Micotoxinas, HPLC, Aminoácidos, Vitaminas, Acuicultura.

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RESPONSE SURFACE MODELS IN THE PREDICTION OF OCHRATOXIN A PRODUCTION IN WHEAT BY Aspergillus steynii

E.M. Mateo1, A. Tarazona2, J.V. Gimeno-Adelantado3, F. Mateo4

1 Dep. de Microbiología, Facultad de Medicina y Odontología, Universitat de València, Spain; 2 Dep. de Microbiología y Ecología, Universitat de València, Spain; 3 Dep. de Química Analítica, Universitat de València, Spain; 4 Dep. de Ingeniería Electrónica, ETSE, Universitat de València, Spain

Fernando.mateo@uv.es

Ochratoxin A (OTA) is produced by some species of the genera Aspergillus and Penicillium. Aspergillus steynii stands out among all of them, both for the levels of mycotoxin produced and for its frequency in cereals. OTA is nephrotoxic, hepatotoxic, teratogenic, immunotoxic and possible carcinogenetic in humans. The possible foods that can contain OTA are very diverse, but the highest levels have been generally found in cereals and their by–products. OTA ingestion through cereal consumption can account for up to 54% of the total intake of the toxin in the diet. Predictive models, such as response surface models (RSM), have been applied in microbiology to forecast microbial growth under certain environmental conditions. There is little information concerning the development of models to describe the effect of those factors on the production of mycotoxins. The parameters involved in microbial growth can be obtained from a sigmoidal function commonly used to relate growth and time. The Baranyi model can estimate specific growth rate, lag time and maximal population density. The related equations might be used to forecast mycotoxin production in cultures. The aim of this work was to study the influence of water activity, temperature, spore concentration and time on OTA production of by A. steynii in wheat grain. According to the Barany model, it was observed that OTA accumulation in wheat grain by A. steynii depends on all the studied factors. The accuracy of the model with regards to OTA accumulation was low because the mycotoxin level varied during the final stage of the process depending on the different conditions. The models obtained by RSM should not be generalized to all the isolates of A. steynii but they may be used to estimate the behavior of the fungus in different media and under different conditions.

Acknowledgements: Funding by project RTI2018-097593-B-C22 from Spanish Ministry of Economy and Competitiveness and ERDF is acknowledged.

Keywords: Ochratoxin A; wheat; response surface methodology; Aspergillus steyni

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PREDICTION OF THE EFFECT OF CLIMATOLOGICAL VARIABLES ON THE PRODUCTION OF TYPE A TRICHOTHECENES BY Fusarium sporotrichioides USING NEURAL NETWORKS

F. Mateo1, A. Tarazona2, E.M. Mateo3

1 Dep. de Ingeniería Electrónica, ETSE, Universitat de Valéncia, Spain; 2 Dep. de Microbiología y Ecología, Universitat de València, Spain; 3 Dep. de Microbiología, Facultad de Medicina y Odontología, Universitat de València, Spain

Fernando.mateo@uv.es

Type A trichothecenes are produced by various Fusarium species such as F. sporotrichioides that contaminate cereal crops worldwide. The most concerning type A trichothecenes are T-2 toxin (T2) and HT-2 toxin (HT2) because of their haematotoxicity and immunotoxicity. Accurate prediction of the accumulation of both toxins in cereals would be interesting in the food safety field. However, it is a difficult task because many factors influence fungal development and mycotoxin production. Predictive models can aid to forecast the levels that mycotoxins can reach in food/feed. Neural networks (NN) may be useful in the field of predictive mycology. This study was performed to explore the possibility of using NN to predict T2+HT2 accumulation over time in wheat seeds contaminated with F. sporotrichioides. The inputs were temperature, water activity (aw), time, and the diameters of cylindrical plugs from a culture of a F. sporotrichioides strain used to inoculate the seeds. The outputs were toxin concentrations. Wheat grains showing undetectable toxin levels were inoculated and incubated under different temperature/aw regimes. At selected times cultures were analyzed by HPLC for toxins. A data set of inputs-outputs was obtained. The criterion for model optimization was minimizing the mean square error of prediction for a test data subset. Generally, toxins were not detected during the first incubation days. After a rapid/short phase the sum of both toxins remained nearly constant. Toxin accumulation depended on the four input variables which are needed to design a predictive model. NNs were able to predict the accumulation of T2+HT2 in seeds. The prediction accuracy depended on the NN type, and the training algorithms used affected the best architecture. A radial-basis function network provided the best model giving a R2 -value for the plot of predicted vs observed levels >0.96. Hence, accurate predictability for T2+HT2 in wheat seeds can be reached using NN modeling.

Acknowledgements: Funding by project RTI2018-097593-B-C22 from Spanish Ministry of Economy and Competitiveness and ERDF is acknowledged.

Keywords: Type A trichothecenes; wheat; Fusarium sporotrichioides; neural networks; predictive mycology

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ASSESSMENT OF THE RISK OF INTAKE OF T-2 AND HT-2 TOXINS THROUGH THE CONSUMPTION OF DIFFERENT CEREAL KINDS CONTAMINATED WITH Fusarium sporotrichioides

E.M. Mateo1, A. Tarazona2, F. Mateo3

1 Dep. de Microbiología, Facultad de Medicina y Odontología, Universitat de València, Spain 2 Dep. de Microbiología y Ecología, Universitat de València, Spain; 3 Dep. de Ingeniería Electrónica, ETSE, Universitat de València, Spain

Eva.mateo@uv.es

Cereal crops are prone to Fusarium spp. infection which, generally, reduces grain yield and/or contaminates grain with mycotoxins such as the type A and type B trichothecenes. T-2 toxin (T2) and HT2 toxin (HT2) are the most toxic type A trichothecenes. They can inhibit DNA, RNA and protein synthesis and induce DNA fragmentation characteristic of apoptosis. High T2 and HT2 levels were found in cereals in Nordic countries and the UK. These toxins are not regulated in the EC, but maximum values for their sum are recommended. Among the cereals, T2 and HT2 usually have higher incidence and concentration in oats. Fusarium sporotrichioides is the major producer of these toxins in cereal grains in Southern Europe. However, there are few data available regarding the host sensitivity and the effect of ecological variables linked to weather and agro-climatic regions on the biosynthesis of these toxins by F. sporotrichioides. Our aim was to know the effect of the cereal species (host) and environmental conditions on T2 and HT2 production by three F. sporotrichioides isolates. Toxin production was significantly affected by four factors: cereal type, isolate, aw and temperature. HT2 levels were higher than T2 levels and the order was oats>barley>wheat>corn>sorghum>rice. The highest levels of both toxins were reached in oat grains at 25 ºC/0.98 aw, which were the optimal conditions for their production. Oats constitutes an excellent substrate for the production of these mycotoxins by F. sporotrichioides, which explains the general higher incidence and concentrations of T2 and HT2 in oats among the cereal species. The results of this research may be useful to avoid/minimize accumulation of these toxins in cereal grains, to explain the variable concentration of T2 and HT2 in different cereals and different agroclimatic regions and to predict the climate change effects.

Acknowledgements: Funding by project RTI2018-097593-B-C22 from Spanish Ministry of Economy and Competitiveness and ERDF is acknowledged.

Keywords: T-2 toxin; HT-2 toxin; cereals; Fusarium sporotrichioides

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