VI Workshop Red de Excelencia MICOFOOD

ISSN: 2444-3158 Editado en Valencia por: Red Nacional sobre las micotoxinas y hongos toxigénicos y de sus procesos de descontaminación.

Jorge Calpe

Micofood 2021

Contenido COMITÉ CIENTÍFICO Y ORGANIZADOR

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PROYECTO MICOFOOD

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PROGRAMA

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ÍNDICE DE ABSTRACTS

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PRESENTACIONES ORALES

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POSTERS

36

Comité científico Jordi Mañes Vinuesa (Universitat de València) Giuseppe Meca (Universitat de València) Misericordia Jiménez Escamilla (Universitat de València) Mar Rodríguez Jovita (Universidad de Extremadura) Vicente Sanchis Almenar (Universitat de Lleida) Agustín Ariño Moneva (Universidad de Zaragoza) Covadonga Vázquez Estévez (Universidad Complutense de Madrid) Elena González-Peñas (Universidad de Navarra) Adela López de Cerain (Universidad de Navarra) Luis González Candelas (Consejo Superior de Investigaciones Científicas) Alberto Cepeda Sáez (Universidad de Santiago de Compostela) Francisco Javier Cabañes Saenz (Universitat Autònoma de Barcelona) Ana María García Campaña (Universidad de Granada)

Comité organizador Jorge Calpe Ruano (Universitat de València) Covadonga Vázquez Estévez (Universidad Complutense de Madrid) Belén Patiño Álvarez (Universidad Complutense de Madrid) Jéssica Gil Serna (Universidad Complutense de Madrid) Marta García Díaz (Universidad Complutense de Madrid) Carolina Gómez Albarrán (Universidad Complutense de Madrid) Clara Melguizo Ávila (Universidad Complutense de Madrid) Mari Cruz Moreno Bondi (Universidad Complutense de Madrid) Elena Benito Peña (Universidad Complutense de Madrid) María Luisa Fernández Cruz (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria) Raquel Torrijos Caparrós (Universitat de València) Tiago de Melo Nazareth (Universitat de València) Alessandra Cimbalo (Universitat de València) Victor D’Opazo Taberner (Universitat de València) Pilar Vila Donat (Universitat de València)

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Proyecto Micofood El objetivo de la red de excelencia trata de profundizar en la evaluación del riesgo, mejora de la calidad y seguridad alimentaria, y en definitiva proteger y promover la salud de la población, focalizándose en el estudio de distintos aspectos relativos a los hongos toxigénicos y a sus metabolitos las micotoxinas (MTs) con los siguientes objetivos concretos: − Caracterizar las especies productoras de micotoxinas más relevantes de los géneros Aspergillus, Fusarium y Penicillium, en materias primas destinadas a la alimentación humana y animal. − Establecer métodos rápidos, específicos y fiables para la detección de los hongos micotoxigénicos. − Desarrollar métodos de análisis multi-micotoxinas en alimentos y fluidos biológicos. − Evaluar la exposición de la población a través de los valores de contaminación de los alimentos de la dieta y los compuestos presentes en fluidos biológicos humanos − Comprobar el empleo de diferentes tratamientos térmicos en la estabilidad y contenido de micotoxinas durante la fabricación y/o el procesado de alimentos y/o su posterior almacenamiento. − Emplear compuestos naturales para reducir la presencia de hongos toxigénicos y sus respectivas micotoxinas en materias primas destinadas a la alimentación humana y animal. − Caracterizar el peligro de las combinaciones de micotoxinas más frecuentemente encontradas en los estudios de exposición. − Caracterizar las rutas de síntesis y su regulación genética y ambiental con el fin de evitar la contaminación en origen.

Ajustado a los objetivos del Programa Marco para la Investigación y la Innovación “Horizonte 2020”, MICOFOOD pretende profundizar la relación entre los investigadores, la industria alimentaria y la administración sanitaria con objeto de abordar y en lo posible minimizar los problemas provocados por la presencia de los hongos toxigénicos en los alimentos, así como favorecer la implantación y actuaciones de las medidas de gestión de la calidad.

El coordinador de la red: Dr. Jordi Mañes Vinuesa

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Programa científico

JUEVES 4 DE NOVIEMBRE DE 2021 9:30- 9.45 – INAUGURACIÓN DEL WORKSHOP

9:45-10:45 - CONFERENCIA INAUGURAL Ángel Medina. Working with rural communities in Ethiopia to fight mycotoxins: what we have learnt.

Applied Mycology Group. Cranfield Soil and AgriFood Institute. Cranfield University (UK).

Sesión I: TOXICOLOGY Moderan Jordi Mañes i Vinuesa y María Luisa Fernández Cruz

10:45 -11:00

THE ROLE OF PUMPKIN EXTRACT AND FERMENTED WHEY AGAINST AFB1 AND OTA-INDUCED IMMUNOTOXICITY IN VITRO M. Frangiamone, M. Lozano, A. Cimbalo, G. Font, L. Manyes

11:00 -11:15

ENHANCED CYTOTOXICITY PRODUCED BY THE CO-EXPOSURE OF MYCOTOXINS IN THE RTGILL-W1 FISH CELL LINE E. Bernal, L. Martínez-Ortega, A. Valdehita, M.L. Fernández-Cruz

11:15-11:30

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, Universitat de València, Burjassot, Spain.

Department of Environment and Agronomy, National Institute for Agricultural and Food Research and Technology, CSIC, Madrid, Spain.

OCHRATOXIN A EXPOSURE INDUCES BEHAVIORAL CHANGES IN MICE AFTER ORAL AND IP ADMINISTRATION M. Serrano1, E. Beraza1, M. Izco2, E. González-Peñas3, A. López de Cerain1,3, A. Vettorazzi1, 3, L. Álvarez-Erviti2

Department of Pharmacology and Toxicology, Research Group MITOX, School of Pharmacy and Nutrition, Universidad de Navarra, Pamplona, Spain.

2 Laboratory of Molecular Neurobiology, Center for Biomedical Research of La Rioja (CIBIR), Logroño, Spain.

3 Department of Pharmaceutical Technology and Chemistry, Research Group MITOX, School of Pharmacy and Nutrition, Universidad de Navarra, Pamplona, Spain.

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11:30-12:00

Coffee Break

12:00-12:15

AFLATOXIN B1 AND OCHRATOXIN A DISRUPT NEURONAL DIFFERENTIATION IN SHSY5Y CELLS M. Alonso-Garrido, M. Frangiamone, C. Carfagnini, G. Font, L. Manyes Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Burjassot, Spain.

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SYSTEMATIC REVIEW OF NEUROTOXIC EFFECTS OF OCHRATOXIN A E. Beraza1, M. Serrano1, L. Álvarez-Erviti2, A. Vettorazzi1, 3

12:15-12:30

Department of Pharmacology and Toxicology, Research Group MITOX, School of Pharmacy and Nutrition, Universidad de Navarra, Pamplona, Spain.

2 Laboratory of Molecular Neurobiology, Center for Biomedical Research of La Rioja (CIBIR), Logroño, Spain.

3 IdiSNA, Navarra Institute for Health Research, Pamplona, Spain.

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12:30-12:45

REDUCTION OF AFB1 AND OTA BIOACCESSIBILITY IN BREAD ENRICHED WITH MILK WHEY AND PUMPKIN P. Vila-Donat, F. Agahi, J. Mañes, G. Meca, L. Manyes, L. Escrivá

12:45-13:00

BENEFICIAL EFFECT OF MILK FERMENTED WHEY AND PUMPKIN EXTRACT AGAINST AFB1 AND OTA IN VITRO A. Cimbalo, M. Frangiamone, M. Lozano, L. Escrivá, P. Vila-Donat, L. Manyes

13:00-13:30 13:30-15:00

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Burjassot, València, Spain

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, Universitat de València, Bujassot.

Spain.

Stannek-Goebel, Lorena. Probiotics in animal nutrition and their potential to degrade mycotoxins.

EVONIK. PAUSA COMIDA

Sesión II: DETECCIÓN DE MICOTOXINAS Y HONGOS TOXÍGENOS Moderan Ángel Medina y Mari Cruz Moreno-Bondi

15:00-15:15

ION MOBILITY-MASS SPECTROMETRY TO EXTEND ANALYTICAL PERFORMANCE IN THE DETERMINATION OF ERGOT ALKALOIDS IN CEREAL SAMPLES L. Carbonell-Rozas1,2, M. Hernández-Mesa1,2, L. Righetti3, F. Monteau2, F.J. Lara1, L. Gámiz-Gracia1, B. Le Bizec2, C. Dall’Asta3, A.M. García-Campaña1, G. Dervilly2 Department of Analytical Chemistry, University of Granada, Granada, Spain.

Oniris, INRAE, LABERCA, 44300 Nantes, France.

3 Department of Food and Drug, University of Parma, Parco Area delle Scienze, 43124 Parma, Italy.

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2

15:15-15:30

CLASSIFICATION OF INDIVIDUAL WHEAT KERNELS ACCORDING TO FUSARIUM SYMPTOMS AND DEOXYNIVALENOL BY NEAR INFRARED HYPERSPECTRAL IMAGING A. Femenias, A.J. Ramos, V. Sanchis, S. Marín

Applied Mycology Unit, Food Technology Department, University of Lleida, AGROTECNIO-CERCA Center, Lleida, Spain.

15:30-15:45

DISPERSIVE SOLID PHASE EXTRACTION USING MAGNETIC NANOCOMPOSITE FOR THE DETERMINATION OF EMERGENT MYCOTOXINS M. García-Nicolás, R. Peñalver, N. Arroyo-Manzanares, N. Campillo, P. Viñas Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Murcia, Spain.

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15:45-16:00

MOLECULARLY IMPRINTED POLYMERS FOR THE SIMULTANEOUS ANALYSIS OF ZEARALENONE AND ALTERNARIOL MYCOTOXINS IN OIL SAMPLES T. Moya-Cavas,1 J. Urraca,1 Luis A. Serrano-González,2 M.C. Moreno-Bondi1

Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, Madrid, Spain.

2 Department of Organic Chemistry, Faculty of Chemistry, Complutense University of Madrid, Madrid, Spain.

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APPLICATION OF NEAR-INFRARED SPECTROSCOPY IN THE DIFFERENTIATION OF OTA AND NON-OTA-PRODUCING MOULDS IN DRY-CURED HAM MODEL E. Cebrián1, A. González-Mohino2, F. Núñez1, M. Rodríguez1, S. Grassi3

16:00-16:15

16:15-16:30

Food Hygiene and Safety, Meat and Meat Products Research Institute (IProCar), Faculty of Veterinary Science, University of Extremadura, Cáceres, Spain.

2 Food Technology, Meat and Meat Products Research Institute (IProCar), Faculty of Veterinary Science, University of Extremadura, Cáceres, Spain.

3 Department of Food, Environmental, and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Milan, Italy.

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LIQUID-CHROMATOGRAPHIC DETERMINATION OF AFLATOXINS IN COCOA AND CHOCOLATE PRODUCTS G. Salas1, M. Herrera1, S. Lorán1, T. Juan1, C. Guiral1, A. Sejas1, J.J. Carramiñana1, C. Yagüe2, A. Herrera1, A. Ariño1 Instituto Agroalimentario de Aragón-IA2, Universidad de Zaragoza-CITA, Veterinary Faculty, Zaragoza, Spain.

2 Faculty of Health and Sports Science, Universidad de Zaragoza, Huesca, Spain.

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16:30-16:45

DETERMINATION OF ENNIATINS AND BEAUVERICIN BY NON-AQUEOUS CAPILLARY ELECTROPHORESIS–TIME OF FLIGHT MASS SPECTROMETRY IN ALL IONS MODE M.M. Delgado-Povedano, Francisco J. Lara, Emilio Borrego-Marín, Laura GámizGracia, Ana M. García-Campaña Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain.

16:45-17:00 17:00-18:00

Descanso

Reunión de coordinación de la red

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VIERNES 5 DE NOVIEMBRE DE 2021 Sesión IIIa: BIOLOGICAL APPROACHES TO COUNTERACT MYCOTOXINS AND MYCOTOXIGENIC FUNGI Moderan Ana-Rosa Ballester y Covadonga Vázquez

09:30-09:45

09:45-10:00

ANTIFUNGAL VOLATILE COMPOUNDS PRODUCED BY Debaryomyces hansenii AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES M. Álvarez, J. Delgado, E. Roncero, F. Gómez, M. J. Andrade

University of Extremadura, Institute of Meat and Meat Products, Faculty of Veterinary Science, Food Hygiene and Safety, Cáceres, Spain.

EMPLEO DE LOS MEDIOS FERMENTADOS DE HARINA Y SALVADO DE MOSTAZA AMARILLA POR BACTERIAS ÁCIDO-LÁCTICAS PARA LA BIOPRESERVACIÓN DE TOMATES T. M. Nazareth, R. Torrijos, J. Mañes, G. Meca

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

10:00-10:15

AISLAMIENTO DE MICROORGANISMOS COMO POTENCIALES AGENTES DE BIOCONTROL DE HONGOS PRODUCTORES DE MICOTOXINAS EN VIÑEDO ECOLÓGICO P. de la Huerta Bengoechea, J. Gil-Serna, B. Patiño Álvarez, C. Vázquez

Departamento de Genética, Fisiología y Microbiología, Universidad Complutense de Madrid, Madrid, España.

10:15-10:30

REDUCCIÓN DEL CRECIMIENTO FUNGICO EN QUESO MEDIANTE EL USO DE UN RECUBRIMIENTO NATURAL BASADO EN SUERO DE LECHE FERMENTADO M. Vitali, P. Vila. F. Illueca, J. Calpe, J.M. Quiles, G. Meca

10:30-10:45

CHARACTERIZATION OF Penicillium expansum MUTANTS DEFECTIVE IN PATULIN PRODUCTION AS POTENTIAL BIOCONTROL AGENTS B. Llobregat, L. González-Candelas, A.R. Ballester

10:45-11:00

USO DE HIDROLIZADOS DE CASEINAS EN LA BIO-PRESERVACIÓN DEL PAN CONTRA HONGOS TOXIGÉNICOS V. D’ Opazo, C. Luz, J. Calpe, J.M. Quiles, F. Illueca, M. Vitali, G. Meca

11:00-11:15

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

Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Paterna, Valencia, Spain.

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

EFFECT OF INTERACTIONS AMONG STRAINS OF BLACK ASPERGILLI ON OCHRATOXIN A PRODUCTION G. Castellá, M.R. Bragulat, F.J. Cabañes

Veterinary Mycology Group, Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.

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11:15-11:30

LA BIOPRESERVACIÓN DE MASAS DE PANADERÍA Y PANES MEDIANTE EL EMPLEO DE BACTERIAS ÁCIDO LÁCTICAS F. Illueca, V. D'Opazo, J.M. Quiles, C. Luz, M. Vitali, J. Calpe, G. Meca

11:30-12:00

Coffee break

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

Sesión IIIb: BIOLOGICAL APPROACHES TO COUNTERACT MYCOTOXINS AND MYCOTOXIGENIC FUNGI Moderan Elena Benito-Peña y Belén Patiño

12:00-12:15

EVALUATION OF THE ANTIFUNGAL ACTIVITY OF CLOVE ESSENTIAL OIL AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES E. Roncero, M.J. Andrade, M.M. Álvarez, E. Cebrián, J.C. Pulido, J. Delgado

12:15-12:30

SCREENING OF ISOLATED BACTERIA FOR ANTIFUNGAL ACTIVITY AGAINST MYCOTOXIGENIC FUNGI OF CITRUS FRUITS C. Stracquadanio, J. Calpe, C. Luz, G. Meca

12:30-13:00

13:00-13:30 13:30-14:00

Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary Science, University of Extremadura, Cáceres, Spain.

Departamento de Medicina Preventiva, Facultat de Framacia, Universitat de Valencia, Burjassot, Valencia, Spain.

Nieves Aparicio. Granos ancestrales, una apuesta por la diversificación de cultivos.

Unidad de Herbáceos. Instituto Tecnológico Agrario de Castilla y León (ITACyL).

Angélica Pérez. Un caso empresarial: Procesado de nuevas especies y demanda. Área de Calidad y Seguridad Alimentaria. Harinera Emilio Esteban S.L.

CEREMONIA DE CLAUSURA DEL WORKSHOP

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Índice de abstracts PRESENTACIONES ORALES

WORKING WITH RURAL COMMUNITIES IN ETHIOPIA TO FIGHT MYCOTOXINS: WHAT WE HAVE LEARNT THE ROLE OF PUMPKIN EXTRACT AND FERMENTED WHEY AGAINST AFB1 AND OTAINDUCED IMMUNOTOXICITY IN VITRO 12 ENHANCED CYTOTOXICITY PRODUCED BY THE CO-EXPOSURE OF MYCOTOXINS IN THE RTGILL-W1 FISH CELL LINE 13 OCHRATOXIN A EXPOSURE INDUCES BEHAVIORAL CHANGES IN MICE AFTER ORAL AND IP ADMINISTRATION 14 AFLATOXIN B1 AND OCHRATOXIN A DISRUPT NEURONAL DIFFERENTIATION IN SHSY5Y CELLS 15 SYSTEMATIC REVIEW OF NEUROTOXIC EFFECTS OF OCHRATOXIN A

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REDUCTION OF AFB1 AND OTA BIOACCESSIBILITY IN BREAD ENRICHED WITH MILK WHEY AND PUMPKIN 17 BENEFICIAL EFFECT OF MILK FERMENTED WHEY AND PUMPKIN EXTRACT AGAINST AFB1 AND OTA IN VITRO 18 PROBIOTICS IN ANIMAL NUTRITION AND THEIR POTENTIAL TO DEGRADE MYCOTOXINS ION MOBILITY-MASS SPECTROMETRY TO EXTEND ANALYTICAL PERFORMANCE IN THE DETERMINATION OF ERGOT ALKALOIDS IN CEREAL SAMPLES 19 CLASSIFICATION OF INDIVIDUAL WHEAT KERNELS ACCORDING TO FUSARIUM SYMPTOMS AND DEOXYNIVALENOL BY NEAR INFRARED HYPERSPECTRAL IMAGING 20 DISPERSIVE SOLID PHASE EXTRACTION USING MAGNETIC NANOCOMPOSITE FOR THE DETERMINATION OF EMERGENT MYCOTOXINS 21 MOLECULARLY IMPRINTED POLYMERS FOR THE SIMULTANEOUS ANALYSIS OF ZEARALENONE AND ALTERNARIOL MYCOTOXINS IN OIL SAMPLES 22 APPLICATION OF NEAR-INFRARED SPECTROSCOPY IN THE DIFFERENTIATION OF OTA AND NON-OTA-PRODUCING MOULDS IN DRY-CURED HAM MODEL 23 LIQUID-CHROMATOGRAPHIC DETERMINATION OF AFLATOXINS IN COCOA AND CHOCOLATE PRODUCTS 24 DETERMINATION OF ENNIATINS AND BEAUVERICIN BY NON-AQUEOUS CAPILLARY ELECTROPHORESIS–TIME OF FLIGHT MASS SPECTROMETRY IN ALL IONS MODE 25 8

ANTIFUNGAL VOLATILE COMPOUNDS PRODUCED BY Debaryomyces hansenii AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES 26 EMPLEO DE LOS MEDIOS FERMENTADOS DE HARINA Y SALVADO DE MOSTAZA AMARILLA POR BACTERIAS ÁCIDO-LÁCTICAS PARA LA BIOPRESERVACIÓN DE TOMATES 27 AISLAMIENTO DE MICROORGANISMOS COMO POTENCIALES AGENTES DE BIOCONTROL DE HONGOS PRODUCTORES DE MICOTOXINAS EN VIÑEDO ECOLÓGICO 28 REDUCCIÓN DEL CRECIMIENTO FUNGICO EN QUESO MEDIANTE EL USO DE UN RECUBRIMIENTO NATURAL BASADO EN SUERO DE LECHE FERMENTADO 29 CHARACTERIZATION OF Penicillium expansum MUTANTS DEFECTIVE IN PATULIN PRODUCTION AS POTENTIAL BIOCONTROL AGENTS 30 USO DE HIDROLIZADOS DE CASEINAS EN LA BIO-PRESERVACIÓN DEL PAN CONTRA HONGOS TOXIGÉNICOS 31 EFFECT OF INTERACTIONS OCHRATOXIN A PRODUCTION

AMONG

STRAINS

OF

BLACK

ASPERGILLI

ON 32

LA BIOPRESERVACIÓN DE MASAS DE PANADERÍA Y PANES MEDIANTE EL EMPLEO DE BACTERIAS ÁCIDO LÁCTICAS 33 EVALUATION OF THE ANTIFUNGAL ACTIVITY OF CLOVE ESSENTIAL OIL AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES 34 SCREENING OF ISOLATED BACTERIA FOR ANTIFUNGAL ACTIVITY AGAINST MYCOTOXIGENIC FUNGI OF CITRUS FRUITS 35

GRANOS ANCESTRALES, UNA APUESTA POR LA DIVERSIFICACIÓN DE CULTIVOS UN CASO EMPRESARIAL: PROCESADO DE NUEVAS ESPECIES Y DEMANDA POSTERS

ACTIVIDAD ANTIFÚNGICA DEL ÁCIDO PERACÉTICO CONTRA HONGOS TOXIGÉNICOS EN MAÍZ Y LA CEBADA 37 DEVELOPMENT OF A HIGH RESOLUTION MASS SPECTROMETRY METHODOLOGY FOR MULTI-MYCOTOXIN ANALYSIS OF MARKETED DARK CHOCOLATE 38 MECANISMO IMPLICADO EN LA DETOXIFICACIÓN DE MICOTOXINAS POR Hanseniaspora uvarum U1, Kazachstania unispora KC19_L5 y Debaryomyces hansenii KCAR20_L1 39 DESARROLLO DE UN MÉTODO RÁPIDO MICROSATÉLITES DE Hanseniaspora uvarum

PARA

COMPARAR

PERFILES

DE 40

DETERMINATION OF PATULIN IN COMMERCIAL STRAWBERRY JAM THROUGH UHPLC Q-EXACTIVE ORBITRAP HRMS 41 9

DETERMINATION OF MULTIPLE MYCOTOXINS IN LIVER USING SALTING-OUT LIQUIDLIQUID EXTRACTION FOLLOWED BY DISPERSIVE LIQUID-LIQUID MICROEXTRACTION AND HPLC-MS/MS 42 OCHRATOXIN-A´S CONJUGATES AS POTENTIAL BIOMARKERS OF EXPOSURE

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

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CYTOPROTECTION ASSESSMENT AGAINST MYCOTOXINS ON SH-SY5Y CELLS BY BEET ROOT EXTRACTS 45 EFFECT OF LACTIC ACID BACTERIA ON AFB1 AND OTA REDUCTION AFTER INCUBATION IN CULTURE MEDIA 46 AFLATOXIN M1 IN MILK OF ASSAF DAIRY EWES RECEIVING DIFFERENT ADSORBENTS IN FEED CONTAMINATED WITH AFLATOXIN B1 47 METODOLOGIA APLICADA A LA EVALUACION DEL EFECTO DE LOS SECUESTRANTES DE MICOTOXINAS EN LA CALIDAD NUTRICIONAL DE LOS PIENSOS PARA LA ACUICULTURA 48 RESPONSE SURFACE MODELS IN THE PREDICTION OF OCHRATOXIN A PRODUCTION IN WHEAT BY Aspergillus steynii 49 PREDICTION OF THE EFFECT OF CLIMATOLOGICAL VARIABLES ON THE PRODUCTION OF TYPE A TRICHOTHECENES BY Fusarium sporotrichioides USING NEURAL NETWORKS 50 ASSESSMENT OF THE RISK OF INTAKE OF T-2 AND HT-2 TOXINS THROUGH THE CONSUMPTION OF DIFFERENT CEREAL KINDS CONTAMINATED WITH Fusarium sporotrichioides 51

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Presentaciones orales

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THE ROLE OF PUMPKIN EXTRACT AND FERMENTED WHEY AGAINST AFB1 AND OTA-INDUCED IMMUNOTOXICITY IN VITRO M. Frangiamone, M. Lozano, A. Cimbalo, G. Font, L. Manyes Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, Universitat de València, Burjassot, Spain.

Massimo2.frangiamone@uv.es Mycotoxins are considered a major risk factor affecting human and animal health being the most dangerous contaminants of foodstuffs. Among them, AFB1 and OTA are the most toxic and studied.

Thus, the aim of the study was to evaluate through a transcriptomic approach the effect of AFB1 and OTA-immunotoxicity on Jurkat cells. Moreover, the possible protective role of functional ingredients, pumpkin and fermented whey, was evaluated. Cells were treated during 7 days with AFB1 and OTA, individually and in combination, at the concentration of 100nM in 0.1% of DMSO. This solvent concentration was used as control. Moreover, cells were exposed for 7 days to bread extracts, obtained from a simulated human digestion in vitro, which contained pumpkin (20% w/w) and fermented whey, along with mycotoxins (AFB1:196 nM, OTA:1037 nM, AFB1+OTA:187+837 nM). Extracted RNA was sequenced by NextSeq500 (Illumina) and the quality control (QC) of reads was performed by FastQC software v0.11. The trimming was performed with FASTX-Toolkit v0.1, discarding the low-quality bases from 5’- and 3’- extremes. The trimmed reads alignment was performed by STAR software v2, using as reference the GRCh38 genome version. The Sequence Alignment Map (SAM) files were transformed in their binary version (BAM), ordered and indexed using SAM tools software v1.1. BAM files obtained with STAR was used to generate an expression matrix in R software while normalization and statistical analyses were performed with edgeR package. Regarding standard mycotoxin-exposure, the treatment that altered in a greater extent the gene expression, considering a p-value<0.05 and log FC|0.5|, was AFB1+OTA-exposure with 3236 2

differentially expressed genes (DEGs) (981 down and 2255 up-regulated). Conversely, in mycotoxin-exposure with functional ingredients, it has been obtained 6882 DEGs (3183 down and 3699 up-regulated) upon OTA-administration, 7314 DEGs (3072 down and 4242 up-regulated) in mixed-exposure and 214 DEGs (43 down and 171 up-regulated) in AFB1-treatment.

Acknowledgements: Spanish Ministry of Science and Innovation Project (PID2019-108070RB-I00ALI) and the Generalitat Valenciana (PROMETEO/2018/126).

Keywords: Jurkat, transcriptomics, RNA-seq, mycotoxins.

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ENHANCED CYTOTOXICITY PRODUCED BY THE CO-EXPOSURE OF MYCOTOXINS IN THE RTGILL-W1 FISH CELL LINE E. Bernal, A. Valdehita, M.L. Fernández-Cruz Department of Environment and Agronomy, (National Institute for Agricultural and Food Research and Technology, CSIC) 28040 Madrid, Spain fcruz@inia.es Mycotoxins are frequently present in cereals and feed and a high level of co-occurrences in the same sample are described. In the last decades the aquaculture industry has introduced plant-based ingredients as a source of protein for fish feeds. This has led to mycotoxin contaminations which can represent a hazard for fish and humans. A previous research conducted in the fish cell line RTgill1

W1 indicated that most of the fifteen assayed mycotoxins exerted a pronounced acute effect.

The objective of the present work was to study the cytotoxicity of the co-occurrences of mycotoxins most frequently described in the literature in cereals and in different feeds (including fish feeds. The RTgill-W1 was exposed during 24 hours to a range of a mycotoxin concentrations (0.012-100 µg/mL) in co-exposure with the concentrations of another mycotoxin which decrease the viability by 10% or 50% (EC10 and EC50). The co-occurrences studied were deoxynivalenol (DON) + zearalenone (ZEA), DON + beauvericin (BEA); ZEA + BEA, DON + fumonisin B1 or B2; DON + enniatins A or A1 or B or B1. The cytotoxicity was evaluated with a triple cytotoxicity assay (AlamarBlue, CFDA-AM, and Neutral Red Uptake), which measures the cell metabolism at the mitochondrial level and the plasma lysosome membrane integrities, respectively.

An enhanced toxicity was observed for almost all the co-exposures studied except for DON+ZEA and DON+FB2. This result points to the need to include an additional safety factor in the establishment of permissible levels of mycotoxins in fish feeds to avoid the risks of the frequent coexposures.

Bernal-Algaba, E.; Pulgarín-Alfaro, M.; Fernández-Cruz, M.L. Cytotoxicity of Mycotoxins

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Frequently Present in Aquafeeds to the Fish Cell Line RTGill-W1. Toxins 2021, 13, 581.

Keywords: mycotoxin; cytotoxicity; co-occurrence; RTgill-W1; fish

13

OCHRATOXIN A EXPOSURE INDUCES BEHAVIORAL CHANGES IN MICE AFTER ORAL AND IP ADMINISTRATION M. Serrano E. Beraza , M. Izco , E. González-Peñas , A. López de Cerain , A. Vettorazzi , L. Álvarez-Erviti 1

1

,

2

3

1,3

1, 3

2

Department of Pharmacology and Toxicology, Research Group MITOX, School of Pharmacy and Nutrition, Universidad de Navarra, Pamplona, Spain Laboratory of Molecular Neurobiology, Center for Biomedical Research of La Rioja (CIBIR), Logroño, Spain Department of Pharmaceutical Technology and Chemistry, Research Group MITOX, School of Pharmacy and Nutrition, Universidad de Navarra, Pamplona, Spain IdiSNA, Navarra Institute for Health Research, Pamplona, Spain 1

2

3

4

avettora@unav.es Ochratoxin A (OTA) is a mycotoxin that contaminates a great variety of crops. The main concern is its genotoxicity/carcinogenicity. Although poorly explored, OTA has also showed some neurotoxic effects. On the other hand, etiological factors for Parkinson’s disease (PD) are still unknown. PD is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies which contain aggregates of alpha-synuclein (α-syn). There is increasing evidence that the transmission of the pathology between neurons plays a central role in disease progression. Braak hypothesis proposes that Lewy body pathology may arise in the periphery/enteric nervous system, possibly in the gastrointestinal (GI) tract, and transfer to the brain stem via the glossopharyngeal and vagus nerves. The aim of the present study was to evaluate if OTA induced behavioral changes in mice and to check if the observations are dependent of the route of administration. The oral route was selected to mimic human e

xposure to OTA, while intraperitoneal (ip) was selected in order

to avoid the GI tract. Animals were administered daily for 28 days either with vehicle or with two different OTA doses (0.21 or 0.5 mg/kg bw). Behavioral tests (wire hang, negative geotaxis and rotarod) were carried out during the administration and for 6 months after the last administration.

Clinical changes, body weight and OTA levels were also monitored. OTA induced behavioral alterations with both routes of administration, although with some differences in the timeframe. No clinical signs of toxicity were observed during the studies. After oral administration, OTA was detectable after the 28 days of administration in plasma and brain but was undetectable in both tissues 6 months after the last administration. OTA analysis is still ongoing for the ip administration.

Our results point to a potential neurodegenerative effect of OTA. Funding: European Regional Development Fund (FEDER) "A way to make Europe" (6FRSABC008), “Ministerio de Economia, Industria y Competitividad, Agencia Estatal de Investigacion” of the Spanish Government (AGL2017-85732-R) (MINECO/AEI/FEDER, UE) and ¨Gobierno de Navarra” (2019-project 43).

Keywords: Ochratoxin A, neurodegeneration, neurotoxicity, Parkinson´s disease, mice 14

AFLATOXIN B1 AND OCHRATOXIN A DISRUPT NEURONAL DIFFERENTIATION IN SH-SY5Y CELLS M. Alonso-garrido, M. Frangiamone, C. Carfagnini, G. Font, L. Manyes Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Ave. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain manuel.alonso-garrido@uv.es Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are well known to human health as the most toxic secondary metabolites produced by fungi. They are a risk to public health due to their occurrence in food and feed, being contaminated wheat one of the main concerns in food safety. On the other side, it has been shown that both fermented whey and pumpkin extract can interact with these molecules significantly reducing their occurrence. They can cross the blood brain barrier via paracellular and could trigger different molecular responses associated to a wide range of neurodegenerative pathologies. Four different types of bread (bread (B); pumpkin (1%) bread (PB); fermented whey (1%) bread (FWB); fermented whey (1%) and pumpkin bread (FWPB) (1%)) were elaborated with the same concentrations of mycotoxins. This study is focused in the alteration of neuronal cell differentiation by these different bread extracts previous simulated stomach digestion, using SHSY5Y cells in vitro. Cell cycle by flow cytometry and dopamine and β-tubulin III by immunofluorescence were analyzed as markers of neuronal differentiation. For both experiments, cells were cultivated for seven days and treated with the bread extracts at 2% (AFB1 60 nM; OTA 240 nM; AFB1 (60 nM) + OTA (240 nM)) and retinoic acid (0.1%). For cell cycle analysis 20000 cells per well were seed in 48-well plates and for dopamine and β-tubulin III detection 300000 cells per well were inoculated in 6-well plates. Flow cytometry analysis reported significantly less differentiation for B-OTA, FW-OTA, FW-AFB1-OTA, FWPB-AFB1, FWPB-OTA, FWPB-AFB1OTA and significantly higher differentiation rate for PB-AFB1 and FWB-AFB1 respect to their correspondent controls. Immunofluorescence showed lower significant concentrations of β-tubulin III and dopamine for all treatments versus their control. In conclusion, even low concentrations of mycotoxins can change neuronal differentiation in vitro. Interaction between pumpkin, fermented whey and mycotoxins needs further research.

Acknowledgements: Spanish Ministry of Science and Innovation project (PID2019-108070RB-I00ALI) and PhD grant (BES-2017-081328).

Keywords: whey, pumpkin, flow cytometry, immunofluorescence, in vitro 15

SYSTEMATIC REVIEW OF NEUROTOXIC EFFECTS OF OCHRATOXIN A E. Beraza , M. Serrano , L. Álvarez-Erviti , A. Vettorazzi 1

1

2

1, 3

Department of Pharmacology and Toxicology, Research Group MITOX, School of Pharmacy and Nutrition, Universidad de Navarra, Pamplona, Spain Laboratory of Molecular Neurobiology, Center for Biomedical Research of La Rioja (CIBIR), Logroño, Spain IdiSNA, Navarra Institute for Health Research, Pamplona, Spain 1

2

3

eberaza@alumni.unav.es Ochratoxin A (OTA) is a naturally occurring mycotoxin that has been widely studied as a nephrotoxic and carcinogenic agent. However, not many studies have been conducted in order to assess its potential neurotoxic effects. The current review focuses on OTA as a possible neurotoxic compound

and

the

underlaying

mechanisms

that

may

lead

to

neurodegeneration

or

neurodevelopmental toxicity. A systematic literature search was performed in Pubmed database using the following keywords: “(Parkinson OR neurotoxicity OR neurodegeneration OR alzheimer OR neurological OR autism OR neurodevelopmental OR lateral amyotrophic sclerosis OR dementia with Lewy bodies OR pure autonomic failure OR multiple system atrophy OR neuron) AND ochratoxin”. The scientific articles retrieved from the search were filtered out using the following exclusion criteria: 1) Aim of the article was to evaluate mycotoxins occurrence in food/feed or human/animal exposure, 2) articles focusing on the analytical development of methods for mycotoxins decontamination, detection or quantification, 3) toxicological endpoint not focused on/applicable to neurotoxicity or neurodegeneration, 4) in silico articles and 5) review articles.

A total of 25 articles were selected. Among them, 13 were in vitro studies, 8 were in vivo studies, 2 combined both types of studies and 2 were clinical trials. Data extraction was made separately for in vitro, in vivo and clinical trial articles, classifying as well in vitro and in vivo papers by their main endpoint (neurotoxicity or neurodevelopmental toxicity). Results emphasize the difficulty to compare conclusions obtained from in vitro articles, even cytotoxicity data, due to the differences in experimental designs. Moreover, further in vivo studies are needed to get to know the long-term effects of low doses of OTA, as humans are exposed to it mainly by diet, but the majority of published works are related to the effect of acute doses of OTA. Funding: ¨Gobierno de Navarra” (2019-project 43).

Keywords: Ochratoxin, neurodegeneration, neurodevelopmental, neurotoxicity.

16

REDUCTION OF AFB1 AND OTA BIOACCESSIBILITY IN BREAD ENRICHED WITH MILK WHEY AND PUMPKIN

P. Vila-Donat, F. Agahi, J. Mañes, G. Meca, L. Manyes, L. Escrivá Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, València, Spain Pilar.vila@uv.es The presence of mycotoxins in cereals and cereal products remains a significant issue. Bread and bakery products are the main foodstuff consumed around the world and are particularly important as a source of carbohydrates, proteins and vitamins, however they can be manufactured from flour containing mycotoxins. The use of conventional chemical preservatives has several drawbacks, urging the development of clean-label alternatives. The use of natural ingredients such as pumpkin and whey which contains bioactive compounds like carotenoids and bioactive peptides could reduce the growth of mycotoxigenic fungi or counteract the toxic effects produced by mycotoxins. The aim of the present work was to study the bioaccessibility of AFB1 and OTA mycotoxins in bread using an in vitro digestion model as well as to evaluate the effect of milk whey (with and without lactic acid bacteria fermentation) and pumpkin as natural ingredients added to bread on reducing mycotoxins bioaccessibility. Gastric and intestinal extracts were analysed by HPLC-MS/qTOF and mycotoxins bioaccessibility was calculated. All tested ingredients significantly reduced mycotoxins bioaccessibility. Pumpkin extract demonstrated to be the most effective treatment applied in loaf bread with significant reductions of AFB1 and OTA bioaccessibility of 74 and 34%, respectively.

Whey, fermented whey and the combination of pumpkin-fermented whey showed bioaccessibility reductions between 57-68% for AFB1, and between 11-20% for OTA. These results pointed to pumpkin and milk whey as potential ingredients that may have promising applications in bakery industry as natural preservatives extending the product self-life and enriching their nutritional value.

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

Keywords: bioaccessibility, mycotoxins, bread, whey, pumpkin

17

BENEFICIAL EFFECT OF MILK FERMENTED WHEY AND PUMPKIN EXTRACT AGAINST AFB1 AND OTA IN VITRO A. Cimbalo, M. Frangiamone, M. Lozano, L. Escrivá, P. Vila-Donat, L. Manyes Laboratory of Food Chemistry and Toxicology. Faculty of Pharmacy. Universitat de València.

Carrer Vicent Andrés Estellés s/n. 46100 Bujassot. Spain. alscim@uv.es Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are mycotoxins which widely contaminate cereals and cereal-based products. However, it has been demonstrated that the use of functional ingredients can reduce mycotoxins absorption and toxicity in both humans and animals. The aim of the study was to evaluate the effect of milk fermented whey (WF) and pumpkin rich in carotenoids against AFB1 and OTA cytotoxicity in Jurkat T cells through a proteomic approach. For this purpose, cells were exposed to intestinal digests of bread contaminated with mycotoxins and enriched with 20% of pumpkin and WF, during 7 days. Moreover, a standard condition with AFB1 and OTA (100 nM) dissolved in organic solvent (DMSO) was used as control. A gel-free shotgun proteomic approach was employed to identify features across QTOF-LC/MS system. Data were analyzed with Spectrum Mill software and the differentially expressed proteins have been statistically evaluated and filtered by abundance through Mass Professional Profiler software (Agilent) (p<0.05). Bioinformatic analysis using DAVID platform showed the identification of proteins involved in several metabolic pathways, mainly in gluconeogenesis, antioxidant activity and nucleosome assembly. More specifically, histones’ expression implicated in nucleosome assembly (H2A, H2B, H2C, H3 and H4) was increased when exposing cells to functional ingredients. Furthermore, repression of cyclin A2 associated to limiting the growth of carcinogenic cells, confirmed the preventive effect of these functional ingredients.

Keywords: mycotoxin, proteomics, bioactive compounds, carotenoids, QTOF-LC/MS Acknowledgements: This work was supported by the Spanish Ministry of Science and Innovation (PID2019-108070RB-I00-ALI) and the Generalitat Valenciana (PROMETEO/2018/126).

18

ION MOBILITY-MASS SPECTROMETRY TO EXTEND ANALYTICAL PERFORMANCE IN THE DETERMINATION OF ERGOT ALKALOIDS IN CEREAL SAMPLES L. Carbonell-Rozas , M. Hernández-Mesa , L. Righetti , F. Monteau , F.J. Lara , L. Gámiz-Gracia , B. Le Bizec , C. Dall’Asta , A.M. García-Campaña , G. Dervilly 1,2

1,2

2

3

3

2

1

1

1

2

Department of Analytical Chemistry, University of Granada, Campus Fuentenueva, 18071 Granada, Spain.

Oniris, INRAE, LABERCA, 44300 Nantes, France.

Department of Food and Drug, University of Parma, Parco Area delle Scienze, 43124 Parma, Italy 1

2 3

rozas@ugr.es This work evaluates the potential of travelling wave ion mobility spectrometry (TWIMS) to improve the analytical performance, in terms of selectivity and concentration sensitivity, of liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determination of ergot alkaloids (EAs). EAs are mycotoxins produced mainly by fungi of the Claviceps genus, prevalent in cereals whose ingestion can cause ergotism in humans and animals. The simultaneous determination of EAs with current LC-MS methods has some limitations since several of these compounds are epimers and show similar retention times and an identical mass-to-charge ratio (m/z). In this regard, taking advantage of the third dimension offered by TWIMS, which provides the measurement of the rotationally averaged collision cross section (CSS), we report the first CCS database for the main EAs to support their unequivocal identification. The generated CCS database was inter-laboratory cross-validated and compared with CCS values predicted by machine-learning models. In this context, slight differences were observed in the experimental CCS values obtained for ergotamine, ergosine and ergocristine and their corresponding epimers (ΔCCS/CCS between 3.3 and 4 %), which were sufficient to achieve peak-to-peak resolution in the TWIMS dimension. In addition, a LCTWIM-MS method was applied to the analysis of the main EAs in cereal samples. The integration of TWIMS in the LC-MS workflow improved the signal-to-noise ratio (S/N) between 2.5 and 4-fold; therefore, signal sensitivity was improved. Furthermore, cleaner mass spectra were observed as EAs were also separated from the chemical background in the TWIMS dimension. The analysis of cereal samples resulted in the finding of positive samples in EAs with concentration levels between 8.3 and 36.8 μg kg-1 in barley samples, and between 5.2 and 65.0 μg kg-1 in wheat samples.

Acknowledgements: Research Project RTI2018-097043-B-I00 financed by MCIN/AEI /10.13039/501100011033/ FEDER “Una manera de hacer Europa” and Junta de Andalucía-Programa Operativo FEDER (B-AGR-202-UGR20).

Keywords: travelling wave ion mobility-mass spectrometry, ergot alkaloids, cereal samples, collision cross section.

19

CLASSIFICATION OF INDIVIDUAL WHEAT KERNELS ACCORDING TO FUSARIUM SYMPTOMS AND DEOXYNIVALENOL BY NEAR INFRARED HYPERSPECTRAL IMAGING A. Femenias, A.J. Ramos, V. Sanchis, S. Marín Applied Mycology Unit, Food Technology Department, University of Lleida, AGROTECNIOCERCA Center, Av. Rovira Roure 191, 25198, Lleida, Spain antoni.femenias@udl.cat Farmers, importers and cereal processors remain interested on a rapid and cost-effective technology able to detect Fusarium damage and associated mycotoxins, as deoxynivalenol (DON). Near infrared hyperspectral imaging (HS-NIR) not only has the faculty of detecting chemical changes of cereal batches, but also the ability to analyze individualized areas within the sample. This ability permits to look beyond the traditional cereal bulk analysis to focus on the individual kernel management. In this way, HSI-NIR would be able to detect, by individual kernel analysis, the high-contaminated fraction of a batch as a cereal sorting strategy. Keeping this idea in mind, the main purpose of this work was to build classification models based on multivariate analysis able to discriminate between healthy and Fusarium and DON contaminated kernels by using NIR data. To reach this aim, NIR spectra of 300 wheat kernels were extracted by the imaging system prior to UHPLC analysis. Several spectral pretreatments were applied to NIR data to highlight valuable attributes and to reduce noise and scatter effects. For kernel Fusarium damage classification, the most accurate discrimination precision reached was 85.8 % by the use of Artificial Neural Networks (ANN) classifier and the absorbance spectra. Otherwise, for DON contaminated kernels discrimination, the precision was reduced to 76.9 % of correctness for Standard Normal Variate pretreated spectrum, probably due to the low correlation observed between fungal symptoms and mycotoxin production. The achievements demonstrated the potential of HSI-NIR to analyze physical and chemical variations of wheat kernels for fungal damage and mycotoxin management.

Acknowledgements: This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO/AEI/FEDER, UE, project AGL2017- 87755-R and PID2020114836RB-I00). Antoni Femenias acknowledges the financial support of the University of Lleida (predoctoral grant).

Keywords: Hyperspectral imaging; near infrared; single wheat kernels; Fusarium; deoxynivalenol

20

DISPERSIVE SOLID PHASE EXTRACTION USING MAGNETIC NANOCOMPOSITE FOR THE DETERMINATION OF EMERGENT MYCOTOXINS M. García-Nicolás, R. Peñalver, N. Arroyo-Manzanares, N. Campillo, P. Viñas Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, E-30100 Murcia, Spain.

1

maria.garcia66@um.es Dispersive magnetic solid-phase extraction (DMSPE) has received growing attention for sample treatment preconcentration prior to the separation of analytes. In the present work, the potential of DMSPE for the determination of emergent mycotoxins (enniatins A, A1, B and B1 and beauvericin) is investigated for the first time in both clinical and food samples. Different magnetic nanoparticles were tested and magnetic multiwalled carbon nanotube (Fe O @MWCNT) and cellulose-ferrite 3

4

(Cellulose@Fe O ) nanocomposites were selected for the extraction and preconcentration of the five 3

4

target mycotoxins before their analysis by ultrahigh performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). Both nanocomposites were characterized using field emission scanning electron microscopy and energy dispersive X-ray spectrometry in terms of morphology and elemental composition. Several parameters affecting the adsorption and desorption of DMSPE steps were optimized and the methods were fully validated. Due to matrix effect, matrixmatched calibration curves were necessary to carry out quantification. In this way, limits of quantification were between 0.04 and 0.1 μg/L for urine and between 9.5 and 9.9 μg/kg for paprika samples. RSD values lower than 12% and recoveries between 89.3 and 98.9% were obtained. The analysis of 26 paprika samples, including conventional and organic, demonstrated the presence of ENNB1 at 11.2 μg/kg in one of the samples studied. However, non-mycotoxins were detected in the human urine samples.

Acknowledgments: The authors thank the Comunidad Autónoma de la Región de Murcia (CARM, Fundación Séneca, Project 19888/GERM/15) and the Spanish MICINN (PGC2018-098363-B-I00).

M.G.N. acknowledges a fellowship 21464/FPI/20 from Fundación Séneca.

Keywords: emergent mycotoxins, nanocomposite, DMSPE, urine, paprika.

21

MOLECULARLY IMPRINTED POLYMERS FOR THE SIMULTANEOUS ANALYSIS OF ZEARALENONE AND ALTERNARIOL MYCOTOXINS IN OIL SAMPLES T. Moya-Cavas,1 J. Urraca,1 Luis A. Serrano-González,2 M.C. Moreno-Bondi1 1

Departments of Analytical and 2 Organic Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040 Madrid, Spain tammoya@ucm.es

Molecularly imprinted polymers are tailor made materials containing specific recognition cavities with a predetermined selectivity for a specific analyte or group of similar analytes. In this work, we report the development of MIPs for the simultaneous preconcentration of zearalenone (ZON) and alternariol (AOH) in oil samples using molecularly imprinted solid phase extraction (MISPE). The polymers were prepared with N-(2-aminoethyl) methacrylamide as functional monomer, methacrylamide as co-monomer and ethylene glycol dimethacrylate as cross-linker. Two template molecules,

namely,

3,8,9-trihydroxy-6H-dibenzo[b,d]pyran-6-one

and

cyclododecyl

2,4-

dihydroxybenzoate, were used as subrogates of AOH and ZON, respectively, for MIP synthesis. The MISPE method was optimized using a chemometric approach that maximized the selective retention of both mycotoxins in the MIP vs the NIP. . In the optimized conditions the cartridges, filled with 150 mg of a mixture (50:50, w/w) of the AOH/ZON selective MIPs, were loaded with 30 mL of sample, followed by a washing step with 2 mL of ACN/water (20/80, v/v) and elution with 2.5 mL of trifluoroacetic acid/MeOH (3/97, v/v). The extracts were analyzed by HPLC coupled to a fluorescence detector (FLD). No cross-reactivity was observed in the presence of mycotoxins from other families. The optimized MISPE-HPLC-FLD method, has been applied to the analysis of the mycotoxins in maize oil samples (2 g) with a limit of detection of 2 and 5 μg kg-1 for AOH and ZON, respectively. Recoveries in the range of 94% to 113% (RSD < 5%, n = 6) for AOH and 92% to 108% (RSD < 6%, n = 6) for ZON were obtained in the analysis of spiked maize oil samples. The method has been validated based on the European Commission Decision 2002/657/EC. The results have been confirmed by HPLC-MS/MS. Acknowledgements. Work funded by the Spanish MCIN (grant RTI2018-096410-B-C21). TMC thanks the MCIN por a predoctoral grant.

Keywords: molecularly imprinted polymers, mycotoxin, alternariol, zearalenone, food analysis

22

APPLICATION OF NEAR-INFRARED SPECTROSCOPY IN THE DIFFERENTIATION OF OTA AND NON-OTA-PRODUCING MOULDS IN DRY-CURED HAM MODEL E. Cebrián1, A. González-Mohino2, I. Martín1, F. Núñez1, M. Rodríguez1, S. Grassi3 1

Food Hygiene and Safety, Meat and Meat Products Research Institute (IProCar), Faculty of Veterinary Science, University of Extremadura. Cáceres, Spain; 2 Food Technology, Meat and Meat Products Research Institute (IProCar), Faculty of Veterinary Science, University of Extremadura. Cáceres, Spain 3 Department of Food, Environmental, and Nutritional Sciences (DeFENS), Università degli Studi di Milano, Milan, Italy. evcebrianc@unex.es Moulds commonly grow on the surface of dry-cured meat products. Some of them are able of producing ochratoxin A (OTA), posing a food safety concern. Therefore, the detection of the OTAproducing moulds before OTA synthesis is convenient. To the aim, this work evaluated the potential of near infrared spectroscopy (NIRS) as a rapid and non-destructive method, for the discrimination between OTA-producing (P. nordicum, P. verrucosum and A. westerdijkiae) and non-producing mould (P. commune and P. polonicum) species on dry-cured ham-based substrate. Cultures were incubated at 12 and 25 ºC for 32 days. Four NIRS measurements (5, 13, 25 and 32 days after inoculation) were taken in quintuplicate at five locations for each plate at the two study temperatures.

The collected spectra were used to develop Support Vector Machines–Discriminant Analysis (SVMDA) models by a hierarchical approach. First a SVM-DA model was developed to classify into OTA and non-OTA producing moulds. Then, two models were tested to discriminate species among the ochratoxigenic and non-ochratoxigenic moulds. The SVM-DA model could discriminate OTA and non-OTA producers with 85% of sensitivity and 86% of specificity in prediction. The following models were able to differentiate among non-ochratoxigenic species (95% of sensitivity and specificity) and among OTA-producing species (69% of sensitivity and 90% of specificity). This preliminary in vitro study is promising and the use of a portable NIRS system could be a rapid and non-destructive tool to monitor the presence of ochratoxigenic moulds on dry-cured ham surfaces.

Therefore, further assays have been started on dry-cured ham to test its effectiveness.

Acknowledgements. This work has been financed by the Spanish Ministry of Science and Innovation, Government of Extremadura and FEDER (PID2019-104260GB-100, GR18056). E. Cebrián is recipient of a pre-doctoral fellowship from the Spanish Ministry of Science and Innovation (PRE2020-093605).

Keywords: toxigenic moulds; ochratoxin A (OTA); near-infrared spectroscopy (NIRS); portable device 23

LIQUID-CHROMATOGRAPHIC DETERMINATION OF AFLATOXINS IN COCOA AND CHOCOLATE PRODUCTS G. Salas1, M. Herrera1, S. Lorán1, T. Juan1, C. Guiral1, A. Sejas1, J.J. Carramiñana1, C. Yagüe2, A. Herrera1, A. Ariño1 1

Instituto Agroalimentario de Aragón-IA2 (Universidad de Zaragoza-CITA), Veterinary Faculty, 50013 Zaragoza, Spain; 2Faculty of Health and Sports Science (Universidad de Zaragoza), 22002 Huesca, Spain. herremar@unizar.es The aflatoxins B1, B2, G1, and G2 are mycotoxins classified by IARC as Group 1 (carcinogenic to humans). These toxic metabolites are produced by toxigenic strains of the fungi Aspergillus flavus and Aspergillus parasiticus, which prevail in areas with a hot and humid climate. Aflatoxins contaminate foodstuffs as a result of both pre- and post-harvest fungal contamination. In the recent EFSA risk assessment of aflatoxins in foods, cocoa and derived products made one of the largest contributions to the dietary exposure to aflatoxin B1 in all age classes. On that basis, this study was addressed to evaluate the occurrence of aflatoxins B1, B2, G1 and G2 in 71 samples of cocoa powder and 57 chocolate bars (32 milk and 25 dark chocolates). Aflatoxins were extracted with metanol: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 targeted mycotoxins. 37 out of 71 cocoa powder samples (52.1%) and 20 out of 57 chocolate bars (35.1%) were positive for at least one aflatoxin at concentrations between 0.02 - 3.33 µg/kg and 0.02 - 0.09 µg/kg, respectively. The most prevalent aflatoxin in the analyzed samples was AFB1, both in cocoa powder (27 samples, 38.0%) and in chocolate bars (19 samples, 33.3%). A correlation was observed between the occurrence of aflatoxin and the percentage of cocoa in the samples as indicated on their labeling.

Confirming this trend, 28.1% of milk chocolate bars versus 44.0% of those with dark chocolate were contaminated with aflatoxins. In addition, these results represent a contribution to provide reliable data on the aflatoxins contamination rates in order to establish maximum contents for aflatoxins in cocoa and derived products to protect the public health.

Acknowledgements. The authors thank the financial support of the Spanish State Research Agency (PID2019-106877RA-I00) and the Government of Aragón (Grant Grupo A06_20R).

Keywords: aflatoxins, cocoa, chocolate, HPLC

24

DETERMINATION OF ENNIATINS AND BEAUVERICIN BY NON-AQUEOUS CAPILLARY ELECTROPHORESIS–TIME OF FLIGHT MASS SPECTROMETRY IN ALL IONS MODE M.M. Delgado-Povedano, Francisco J. Lara, Emilio Borrego-Marín, Laura Gámiz-Gracia, Ana M. García-Campaña Avda. de Fuente Nueva s/n, 18071, Granada (University of Granada, Faculty of Sciences, Department of Analytical Chemistry) Spain mmdelgado@ugr.es Enniatins (ENN) and beauvericin (BEA) are considered emerging mycotoxins that have been traditionally determined by liquid chromatography coupled to tandem mass spectrometry (MS/MS).

However, to the best of our knowledge, no analytical methods based on capillary electrophoresis (CE)-MS/MS has been reported so far. Despite the absence of easily ionizable groups, the ionophoric character of these compounds makes them compatible with CE separation. Due to their apolar nature, in this work, a non-aqueous CE (NACE) method coupled to QTOF-MS is proposed for the first time to identify and quantify these mycotoxins. Separation was achieved in 4 min under optimum conditions: 40 mM ammonium acetate in 80:20 (v/v) MeCN-MeOH (buffer), 30 kV (voltage), 80 cm (capillary length), 20 ºC (capillary temperature) and 50 mbar × 20 s (injection). Higher selectivity can be achieved when compared with LC due to the formation of exclusive CE adducts such as [M+CH CH NH ] . The “All ions” acquisition mode was selected as it allows the quantification of the 3

2

3

+

usual enniatins, for which standards are available, and the identification of unusual enniatins, for which standards are not available. The method was validated for wheat samples, obtaining LOQs between 5 and 10 μg/kg, recovery values >87.3%, and intra- and inter-day precision values (RSDs) <15.2% in all cases. The method was applied to 29 wheat samples, finding 25 positives for ENNB (16.1-1480 μg/kg), 19 for ENNB1 (19.9-550 μg/kg), 7 for ENNA (10-55μg/kg), 4 for ENNA1 (12.677 μg/kg) and 4 for BEA (12.3-16.4 μg/kg) and two other ENNs (tentatively identity confirmed).

Acknowledgement: Financial support of the Spanish Ministry of Science, Innovation and Universities (RTI2018-097043-B-I00) and the Junta de Andalucía - Programa Operativo FEDER (BAGR-202-UGR20). M.M.D.P. is grateful for a postdoctoral contract to the Junta de Andalucía (DOC_00230).

Keywords: enniatins, NACE–QTOF MS/MS, beauvericin, all-ions, wheat.

25

ANTIFUNGAL VOLATILE COMPOUNDS PRODUCED BY Debaryomyces hansenii AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES M. Álvarez , J. Delgado , E. Roncero , F. Gómez , M. J. Andrade 1

1

1

1

1

University of Extremadura, Institute of Meat and Meat Products, Faculty of Veterinary Science, Food Hygiene and Safety. Cáceres, Spain.

1

maalvarezr@unex.es Debaryomyces hansenii FHSCC 253H has been reported as an efficient biocontrol agent against toxigenic moulds, including Penicillium nordicum, the main ochratoxin A (OTA) producer in drycured meat products. Within the yeast’s antifungal mode of action, the production of volatile antifungal compounds (VOCs) has been reported. The aim of this study was to evaluate the involvement of the VOCs produced by D. hansenii in the OTA reduction during the ripening of drycured fermented sausages. For this, a batch containing 10 cells/g of D. hansenii in the meat dough 6

before stuffing was manufactured. A non-inoculated batch was also made. Both batches were then superficially inoculated with P. nordicum. After their ripening using the usual conditions in industries, OTA was extracted by QuEChERS and measured through uHPLC-MS/MS-QqQ. The VOCs were extracted by SPME using a DVB/CAR/PDMS fibre and analysed by GS-MS. D. hansenii significantly decreased the OTA production at the same time it significantly increased the concentration of acetic acid, 3-methylbutanol and 2-phenyletanol respect to the non-inoculated batch.

These compounds have previously shown antifungal activity against Penicillium and Aspergillus spp. (de Souza et al., 2018; Hua et al., 2014; Jin et al., 2021; Núñez et al., 2015; Quiao et al., 2020). Other compounds were only detected in the batch containing the yeast, such as 2-methylpropanol, 3methylbutanal and acetoin, which have also demonstrated antifungal effect (Delgado et al., 2021; Gao et al., 2018; Gergolet Diaz et al., 2021). Therefore, the production of specific VOCs seems to take part in the antifungal mode of action of D. hansenii, although more studies are necessary to fully elucidate their target site and the possibility of other involved mechanisms.

Acknowledgement: This research was funded by Junta de Extremadura-Consejería de Economía, Ciencia y Agenda Digital-, Fondo Europeo de Desarrollo Regional-“Una manera de hacer Europa” [IB16045 project and GR18056 grant]. M. Álvarez is recipient of a fellowship from the Spanish Ministerio de Economía, Industria y Competitividad [BES-2017-081340].

Keywords: Debaryomyces hansenii, Penicillium nordicum, volatile compounds, dry-cured fermented sausages 26

EMPLEO DE LOS MEDIOS FERMENTADOS DE HARINA Y SALVADO DE MOSTAZA AMARILLA POR BACTERIAS ÁCIDO-LÁCTICAS PARA LA BIOPRESERVACIÓN DE TOMATES Nazareth TM 1, Torrijos R 1, Mañes J 1, Meca G 1 1

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Ave. Vicent Andrés Estellés s/n, 46100, Burjassot. raquel.torrijos@uv.es La contaminación fúngica postcosecha de hortofrutícolas supone importantes pérdidas económicas y un riesgo por la síntesis de micotoxinas. Actualmente, en la Industria Agroalimentaria existe una creciente demanda por reemplazar los fungicidas sintéticos por alternativas vegetales para garantizar la seguridad de los consumidores. El empleo de bacterias ácido-lácticas (BAL) representa una estrategia prometedora en la bio-preservación de alimentos. En este estudio se evaluó la actividad antifúngica cualitativa de los medios fermentados de harina de mostaza amarilla (YMF) y salvado de mostaza amarilla (YMB) con BAL frente a hongos de los géneros Aspergillus, Penicillium, Fusarium y Botrytis. Se seleccionaron Lactobacillus plantarum 5H1 y 5L1 por su mayor potencial antifúngico, determinándose su Concentración Mínima Inhibitoria (MIC) y la Concentración Fungicida Mínima (MFC) frente a los hongos. El medio YMF mostró una actividad antifúngica mayor, obteniéndose valores de MFC entre 25 y 100 g/L en las cepas más sensibles, Penicillium verrucosum VTT D-01847, Fusarium verticillioides ISPA 12052 y Botrytis cinerea CECT 20973.

Además, se determinaron el ácido láctico y el ácido feniláctico (PLA), entre otros metabolitos antifúngicos, en los medios fermentados. La aplicación del medio liofilizado de 1% YMF fermentado por Lactobacillus plantarum 5H1 y 5L1 evidenció su efectividad frente al crecimiento de F. verticillioides ISPA 12052 y B. cinerea CECT 20973 en tomates, obteniéndose una reducción fúngica de 5,04 log UFC/g y 3,13 log UFC/g, así como una inhibición completa de la producción de Fumonisina B1 por F. verticillioides ISPA 12052 y de Ocratoxina A por P. verrucosum VTT D01847 en tomates.

Keywords: Bacterias ácido-lácticas, Lactobacillus plantarum, harina de mostaza amarilla, actividad antifúngica, tomate

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AISLAMIENTO DE MICROORGANISMOS COMO POTENCIALES AGENTES DE BIOCONTROL DE HONGOS PRODUCTORES DE MICOTOXINAS EN VIÑEDO ECOLÓGICO P. de la Huerta Bengoechea1, J. Gil-Serna1, B. Patiño Álvarez1, C. Vázquez1.

1

Departamento de Genética, Fisiología y Microbiología, Universidad Complutense de Madrid, Madrid, España pauladeh@ucm.es La presencia en viñedos de hongos del género Aspergillus productores de micotoxinas, como la ocratoxina A, supone un problema para la seguridad alimentaria y la economía. El uso de microorganismos como agentes de control biológico (ACB) es una de las estrategias más prometedoras para evitar el crecimiento fúngico y la producción de toxinas en estos cultivos. En este trabajo, se aislaron 542 microorganismos procedentes de suelos de viñedos ecológicos de diferentes regiones de España. Se identificaron 11 hongos entre los que había importantes productores de micotoxinas y un posible ACB (Trichoderma sp.). Con las 480 bacterias y 33 levaduras aisladas se realizó un cribado secuencial para seleccionar aquellas con las características más adecuadas para ser utilizadas como ACB. Los 16 aislamientos que cumplieron todos los requisitos se identificaron mediante secuenciación. Se descartaron todas las levaduras por pertenecer al género Cryptococcus y se seleccionaron 9 aislamientos de 4 especies bacterianas para ensayar su potencial para controlar a los tres principales hongos toxígenos en uvas: A. carbonarius, A. welwitschiae y A. flavus.

Arthrobacter sp., Rhodococcus sp. y Mycetocola sp. destacaron por su capacidad de reducir el crecimiento de los hongos indicados.

Keywords: micotoxinas, agentes de biocontrol, Aspergillus, actinobacterias, viñedos.

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REDUCCIÓN DEL CRECIMIENTO FUNGICO EN QUESO MEDIANTE EL USO DE UN RECUBRIMIENTO NATURAL BASADO EN SUERO DE LECHE FERMENTADO M. Vitali, P. Vila. F. Illueca, J. Calpe, J.M. Quiles, 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. mattvi@alumni.uv.es Este trabajo muestra como la reutilización de suero lácteo como medio fermentable por bacterias acido lácticas (BAL) puede ser una alternativa para biopreservar el queso. Para ello se aislaron BAL de derivados lácteos y se comprobó de forma cualitativa la actividad antifúngica de cada BAL y de forma semicuantitativa la actividad antifúngica de los liofilizados de caldo MRS y suero lácteo fermentado por estas BAL frente a cepas fúngicas del género Penicillium. Se determinó cuantitativamente la Concentración Mínima Inhibitoria (MIC) y la Concentración Mínima Fungicida (MFC) de los liofilizados de suero lácteo fermentado. Con el objetivo de estudiar la viabilidad de P. commune CECT 20767 en un recubrimiento formado por hidroxipropilmetilcelulosa (HPMC) y liofilizado de suero lácteo fermentado, en el ensayo de MIC y MFC se seleccionó el liofilizado de suero lácteo fermentado con mayor actividad antifúngica frente a este hongo. En el ensayo de MIC y MFC los liofilizados de suero lácteo fermentado por KK , KB , KB y KB fueron los que mayor 13

2

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actividad antifúngica presentaron. El liofilizado de suero lácteo fermentado por KB3 fue seleccionado por presentar la menor MFC, concretamente, 15.23 mg/mL frente a P. commune CECT 20767. Los recubrimientos de HPMC y 100 mg/mL de liofilizado de suero lácteo fermentado por KB3 inhibieron el crecimiento de P. commune CECT 20767.

Keywords: bacterias acido lácticas, actividad antifúngica, Penicillium, suero lácteo.

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CHARACTERIZATION OF Penicillium expansum MUTANTS DEFECTIVE IN PATULIN PRODUCTION AS POTENTIAL BIOCONTROL AGENTS B. Llobregat, L. González-Candelas, A.R. Ballester* Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC). Calle Catedrático Agustín Escardino 7, Paterna 46980. Spain. ballesterar@iata.csic.es Due to its high pathogenicity on harvested fruits, Penicillium expansum is one of the most studied molds in the genus Penicillium. It causes the common postharvest disease known as blue mold rot.

Patulin is the most researched and well-documented mycotoxin among the many secondary metabolites (SM) produced by P. expansum. This polyketide-derived mycotoxin has a wide range of toxic effects, including cytotoxicity or genotoxicity. This fact has generated intense concern in the society, particularly in recent years, due to the health and economic risks it represents. The gene cluster responsible for patulin biosynthesis in P. expansum is composed of 15 genes (patA - patO); among them is the patK gene, which encodes 6-methylsalicylic acid synthase, the first enzyme of the patulin pathway. In fungi, both pathway-specific gene clusters and global regulatory factors are known to regulate secondary metabolism. In particular, the VELVET family of regulatory proteins (VeA, VelB, LaeA, among others) work to coordinate fungal growth and SM production. In the present study, we have characterized the competitive ability of P. expansum veA- and patK-gene deleted mutants against the wild-type strain and their effect on patulin production. The knockout mutants displaced the wild-type strain during in vitro growth and, consequently, there was a reduction in patulin levels. This preliminary study raises the possibility of using non-mycotoxigenic strains of P. expansum as biocontrol agents.

Funding: RTI2018-093392-A-I00 (AEI/FEDER, UE), JAEIntro2019-CSIC (JAEINT_19_01896), PRE2019-089326 y RYC-2017-22009.

Keywords: Penicillium expansum, patulin, competitive ability, patK, veA

30

USO DE HIDROLIZADOS DE CASEINAS EN LA BIO-PRESERVACIÓN DEL PAN CONTRA HONGOS TOXIGÉNICOS D’ Opazo, V., Luz, C., Calpe J., Quiles, J.M., Illueca, F., Vitali, M., Meca G. Laboratory of Food Chemistry and Toxicology. Faculty of Pharmacy. University of Valencia. Spain victor.dopazo@uv.es En la actualidad los hongos son una de las mayores preocupaciones para la industria alimentaria debido a su capacidad de contaminar alimentos y de afectar la salud humana. Una de las industrias más afectadas por este tipo de contaminación es la industria panadera, la cual sufre cuantiosas pérdidas anualmente causadas por estos microorganismos. La respuesta común a este tipo de contaminaciones es el uso de antifúngicos sintéticos. Pero ante el miedo del consumidor a este tipo de preservantes la industria busca nuevas alternativas, como el uso de péptidos bioactivos. Siguiendo esta tendencia en este trabajo se evaluó la capacidad antifúngica de la caseína hidrolizada con pancreatina y tripsina. Para ello también se estudió el grado de hidrolisis de estas proteínas en contacto con estas enzimas y su potencial como antioxidantes. Finalmente, se realizaron panes con las caseínas como ingrediente donde se determinaron las propiedades tecnológicas de las masas y panes, además de, su capacidad de ser usadas para la bio-preservación de panes frente a hongos del género Aspergillus, Penicillium y Fusarium. Los ensayos de actividad antifúngica demostraron inhibición del crecimiento de hongos pertenecientes al género Aspergillus y un retraso en la esporulación de todos los hongos testados. A mayor tiempo en contacto con la enzima aumentó el grado de proteólisis. La caseína hidrolizada por tripsina evidenció el mayor aumento de la actividad antioxidante de todos los tratamientos estudiados. Finalmente, los panes con caseína como ingrediente no presentaron diferencias significativas en las propiedades tecnológicas respecto a los controles, no obstante, se observó una reducción en el crecimiento de los hongos en los panes con un 2 % de caseína y con un 0.5 % de caseína hidrolizada con pancreatina.

Keywords: caseínas, hidrólisis enzimática, hongos, pan, enzimas, péptidos antifúngicos

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EFFECT OF INTERACTIONS AMONG STRAINS OF BLACK ASPERGILLI ON OCHRATOXIN A PRODUCTION G. Castellá, M.R. Bragulat, F.J. Cabañes Veterinary Mycology Group, Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.

Gemma.Castella@uab.cat Ochratoxin A (OTA) is a nephrotoxic mycotoxin that can be found in a variety of common foods and beverages. After cereals, grapes and their derivatives are the second most contaminated products with OTA, being Aspergillus carbonarius the main source of this mycotoxin. In previous studies, we isolated and characterized three atypical and unique non-OTA producing strains of A. carbonarius [1, 2, 3, 4]. Biological control using microbial antagonists has emerged as a promising approach for control of mycotoxins in both pre- and post-harvested crops. As an example, one of the more promising strategies for control of aflatoxins in crops involves the use of atoxic strains of A. flavus as biological control agents [5]. In this study, in vitro interactions between ochratoxigenic strains of A. carbonarius and A. niger and non-ochratoxigenic strains of A. carbonarius and A. tubingensis were assessed in order to evaluate their potential for controlling OTA production. Two non-OTAproducing strains, A. carbonarius (Ac-) and A. tubingensis (At), used as potential biocontrol agents, were screened for their ability to inhibit or reduce OTA production in co-inoculation with an OTAproducing strain of A. carbonarius (Ac+) and an OTA-producing strain of A. niger (An+). The effect of the potential biological agent on OTA production was evaluated using different mixed spore suspensions of OTA-producing strain:non-OTA-producing strain ratios. In all cases of coinoculation, the presence of Ac- and At decreased the production of OTA at different percentages, depending on their inoculum load. Of the two non-ochratoxigenic species assayed, the non-OTAproducing strain of A. carbonarius gave the best control resulting in practically complete inhibition of OTA production in co-inoculation with an ochratoxigenic strain of A. niger and high percentages of OTA reduction (74%) with an ochratoxigenic strain of A. carbonarius. This study opens new possibilities for using non-ochratoxigenic strains of A. carbonarius as biocontrol agents in grapes.

1. Cabañes FJ, Bragulat MR Castellá G. Characterization of nonochratoxigenic strains of Aspergillus carbonarius from grapes. Food Microbiol 36: 135-141. 2013.

2. Cabañes, F. J., Sanseverino, W., Castellá, G., Bragulat, M. R., Cigliano, R. A., Sanchez, A., 2015. Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius. Sci. Rep. 5, 9086.

3. Castellá, G., Bragulat, M.R., Puig, L., Sanseverino, W., Cabañes, F.J., 2018. Genomic diversity in ochratoxigenic and non ochratoxigenic strains of Aspergillus carbonarius. Sci. Rep. 8, 5439.

4. Castellá, G., Bragulat, M.R., Cigliano, R.A., Cabañes, F.J., 2020. Transcriptome analysis of non-ochratoxigenic Aspergillus carbonarius strains and interactions between some black aspergilli species. Int. J. Food Microbiol. 317, 108498.

5. Ehrlich, K.C. 2014. Non-aflatoxigenic Aspergillus flavus to prevent aflatoxin contamination in crops: advantages and limitations. Frontiers in Microbiology 5, 50.

Acknowledgments: This research was supported by the Ministerio de Economía y Competitividad of the Spanish Government (AGL2014-52516-R).

Keywords: Aspergillus carbonarius, ochratoxin A, fungal interactions, biological control agents.

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LA BIOPRESERVACIÓN DE MASAS DE PANADERÍA Y PANES MEDIANTE EL EMPLEO DE BACTERIAS ÁCIDO LÁCTICAS F. Illueca, V. D'Opazo, JM Quiles, C.Luz, M.Vitali, J.Calpe, G.Meca Universitat de València. Facultat de Farmàcia. Departamento de Medicina Preventiva y Salud Pública, Ciencias de la Alimentación, Toxicología y Medicina Legal. Av. Vicente Andrés Estellés s/n 46100 Burjassot, Valencia, España. franillueca_31@hotmail.com Currently, one of the most studied fields in food bioconservation is the application of lactic acid bacteria (LAB). In this work, the antifungal capacity of eight strains of Lactobacillus plantarum against fungi contaminating bread of the genera Botrytis, Alternaria, Aspergillus and Penicillium was studied. In this way, the antifungal activity of different types of fermented milk serum (SLF) was characterized by BAL using qualitative methods, such as agar diffusion and subsequently quantitative, such as the minimum inhibitory concentration and minimum fungicidal concentration (MIC-MFC) method. After that, the antioxidant capacity was also studied. Finally, the WF with the BAL that had shown the highest efficacy against fungal growth was used as a treatment in bread.

Thus, the impact of the treatment on the properties of the food and the bioconservation capacity against Aspergillus flavus and Penicillium verrucosum was studied. The results obtained in the agar diffusion showed that all the fermented media showed activity against fungi, especially against Aspergillus and Penicillium. The MIC-MFC highlighted that SL100 fermented by L. plantarum 5L1 presented the highest antifungal activity and the fungi most sensitive to the treatment were A. flavus and P. verrucosum. The FW presented a low antioxidant activity. Finally, the treatment used on the bread did not generate great changes in its properties, but it did extend the useful life and reduce the fungal load in A. flavus and more clearly in P. verrucosum.

Keywords: Lactic acid bacteria, fermented whey, bread, Aspergillus and Penicillium.

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EVALUATION OF THE ANTIFUNGAL ACTIVITY OF CLOVE ESSENTIAL OIL AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES E. Roncero , M.J. Andrade , M.M. Álvarez , E. Cebrián , J.C. Pulido , J. Delgado 1

1

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Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary Science, University of Extremadura, Avda. de las Ciencias s/n. 10003 Cáceres, Spain

1

eroncerob@unex.es Microbial contamination occurs during the processing of dry-cured meat products, being one of the main concerns for the meat industry. Focusing on products such as dry-cured fermented sausages, moulds predominate in the final stages of their processing, during which the most worrying hazard is the contamination with ochratoxin A (OTA)-producing species, such as Penicillium nordicum and Penicillium verrucosum. As an alternative to the strategies used in the industry to control their mycotoxin production, the application of plant extracts, such as essential oils, is being analysed. The objective of this study was to evaluate the antifungal activity of clove essential oil (CEO) against three strains of P. nordicum (Pn92, Pn15 and Pn856) in two meat products. Sterile portions of raw chorizo and salchichón were inoculated with each of the three moulds together with the CEO (250 μL/mL). A negative control without antifungal agent, and two positive controls based on a commercial antifungal at subinhibitory level (AF1: 150 μL and AF2: 50 μL) were also prepared.

After 15 days under similar conditions to those of the curing process, OTA was quantified using a Qexactive mass spectrometer. In the negative control, it was observed that salchichón was more favorable for OTA production than chorizo for the three strains. AF2 did not affect OTA production, whereas AF1 significantly increased OTA production in Pn92. A significant reduction of OTA levels was obtained for the three strains in salchichón in the presence of CEO, which was less effective in reducing OTA in chorizo. Pn15 and Pn856 similarly behaved in both substrates in response to the applied treatments. Consequently, CEO could be proposed as a potential effective antifungal agent in salchichón.

Acknowledgments: Research funded by Junta de Extremadura and FEDER “Una manera de hacer Europa” [IB16045 project; GR18056 grant]. Q-Exactive mass spectrometer was funded by MINECO (Ref. UNEX-AE-3394).

Keywords: Ochratoxin A, mould, clove essential oil, dry-cured fermented sausages

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SCREENING OF ISOLATED BACTERIA FOR ANTIFUNGAL ACTIVITY AGAINST MYCOTOXIGENIC FUNGI OF CITRUS FRUITS C. Stracquadanio , J. Calpe , C. Luz , G. Meca 1

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Departemento de Medicina Preventiva, Facultat de Framacia, Universitat de Valencia, Av. Vincent Andrés Estellés s/n, 46100, Burjassot, Valencia, Spain.

1

claudia.stracquadanio@uv.es Fungal rots are the leading cause of postharvest losses (about 30%) of citrus and can greatly reduce its shelf life. Some postharvest fungal pathogens of citrus fruits produce mycotoxins that can pass through and be found in juices. The aims of this study were to screen bacteria from different matrices, explored their antagonistic properties against the main mycotoxigenic pathogens of citrus fruits, belonging to Aspergillus sp., Penicillium sp., Fusarium oxysporum and Alternaria alternata. Isolated bacteria were tested against the pathogens with overlay assay that showed inhibition by direct contact. Active bacteria were identified based on their protein and peptide fingerprints by MALDI-TOF/MS and fermented in MRS broth to produce secondary metabolites with antifungal activity. Preliminarily, the 13 cell-free supernatants (CFS) were evaluated by agar diffusion assay in which the activity was established by the presence of the inhibition halo.

Subsequently, the minimum inhibition concentration and the minimum fungicidal concentration (MIC & MFC) were determined. In addition, volatile organic compounds (VOCs) present in CFS were identified by GC-MS. A total of 30 strains were isolated of which 13 isolates, identified belonging to the genera Pediococcus, Lactobacillus and Leuconostoc, showed clear zones of inhibition on the tested pathogens. The cell-free supernatants (CFS) of 13 isolates inhibited the growth of most tested pathogens, in particular three CFS of isolates 5H1, 5L1 and N2B2 showed good activity with MIC values between 15.6-125 mg/mL and MFC values between 15.6-250 mg/mL. Keywords: antifungal activity, isolated bacteria, mycotoxigenic pathogen, fermentation

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Posters

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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 (CH COOH, O y H O) 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.

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

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DEVELOPMENT OF A HIGH RESOLUTION MASS SPECTROMETRY METHODOLOGY FOR MULTI-MYCOTOXIN ANALYSIS OF MARKETED DARK CHOCOLATE A. Narváez , L. Izzo , S. Lombardi , L. Castaldo , Y. Rodríguez-Carrasco , A. Ritieni 1,2

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

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*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 (r > 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.

2

Keywords: Orbitrap; ultra-high performance liquid chromatography; food safety; emerging Fusarium toxins Acknowledgement: PID2020-115871RB-100

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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 1

Departamento 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. Izzo , A. Narváez , L. Castaldo , A. Gaspari , M. Grosso , Y. Rodríguez-Carrasco , A. Ritieni 1*

1

1

1

2,3

4

1,5

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

1

2

3

4

5

* 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. Sacco , I. Aquila , P. Ricci , P. Llorens , J.C. Moltó , C. Juan 1*

1

1

2

2

2

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

1

2

*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.

2.

3.

4.

5.

6.

7.

Coronel B., et al. (2011). Food and Chemical Toxicology, 49, (6) 1436-1442.

Dekant R., et al. (2021). Toxins 13, 587.

Sueck F., et al. (2019). Mycotoxin Res. 36, 1–10.

Heyndrickx Ll., et al. (2015). Environment International, 84, 82-89.

Wallin S., et al. (2015). Food and Chemical Toxicology, 83, 133-139.

Silva L. J.G., et al. (2020). Food and Chemical Toxicology. 135, 110883.

Hana Z., et al. (2013). Journal of Chromatography B. 925, 46– 53.

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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.

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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]-2,5diphenyltetrazolium 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.

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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

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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 1

Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), 50059 Zaragoza, Spain; Instituto 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 2

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

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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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