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Alternaria Spp. and Their Mycotoxins in Tomatoes. a Scientific Approach from Field to Food Industry Núria Estiarte Piñol

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Alternaria Spp. and Their Mycotoxins in Tomatoes. a Scientific Approach from Field to Food Industry Núria Estiarte Piñol Alternaria spp. and their mycotoxins in tomatoes. A scientific approach from field to food industry Núria Estiarte Piñol http://hdl.handle.net/10803/390957 ADVERTIMENT. L'accés als continguts d'aquesta tesi doctoral i la seva utilització ha de respectar els drets de la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials d'investigació i docència en els termes establerts a l'art. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix l'autorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No s'autoritza la seva reproducció o altres formes d'explotació efectuades amb finalitats de lucre ni la seva comunicació pública des d'un lloc aliè al servei TDX. Tampoc s'autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs. ADVERTENCIA. El acceso a los contenidos de esta tesis doctoral y su utilización debe respetar los derechos de la persona autora. Puede ser utilizada para consulta o estudio personal, así como en actividades o materiales de investigación y docencia en los términos establecidos en el art. 32 del Texto Refundido de la Ley de Propiedad Intelectual (RDL 1/1996). Para otros usos se requiere la autorización previa y expresa de la persona autora. En cualquier caso, en la utilización de sus contenidos se deberá indicar de forma clara el nombre y apellidos de la persona autora y el título de la tesis doctoral. No se autoriza su reproducción u otras formas de explotación efectuadas con fines lucrativos ni su comunicación pública desde un sitio ajeno al servicio TDR. Tampoco se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR (framing). Esta reserva de derechos afecta tanto al contenido de la tesis como a sus resúmenes e índices. WARNING. Access to the contents of this doctoral thesis and its use must respect the rights of the author. It can be used for reference or private study, as well as research and learning activities or materials in the terms established by the 32nd article of the Spanish Consolidated Copyright Act (RDL 1/1996). Express and previous authorization of the author is required for any other uses. In any case, when using its content, full name of the author and title of the thesis must be clearly indicated. Reproduction or other forms of for profit use or public communication from outside TDX service is not allowed. Presentation of its content in a window or frame external to TDX (framing) is not authorized either. These rights affect both the content of the thesis and its abstracts and indexes. Alternaria spp. and their mycotoxins in tomatoes. A scientific approach from field to food industry. DISSERTATION to obtain the degree of Doctor by the University of Lleida in the Agricultural and Food Science and Technology Program by Núria Estiarte Piñol Supervisors: Dr. Antonio J. Ramos Girona Dra. Ana Crespo Sempere July 2016 El Dr. ANTONIO JAVIER RAMOS GIRONA, Catedrático de Universidad del Departamento de Tecnología de Alimentos de la Universitat de Lleida, y la Dra. ANA CRESPO SEMPERE, investigadora de ValGenetics, S.L. HACEN CONSTAR Que, bajo su dirección, la Sra. Nuria Estiarte Piñol ha realizado el trabajo de investigación titulado “Alternaria spp. and their mycotoxins in tomatoes. A scientific approach from field to food industry”, que presenta para optar al grado de Doctora por la Universitat de Lleida. Considerando que el trabajo realizado constituye tema de Tesis Doctoral, autorizan su exposición y defensa en la Universitat de Lleida. Y para que así conste, se expide el presente a 1 de junio de 2016. Dr. Antonio J. Ramos Girona Dra. Ana Crespo Sempere Assessment committee Membres del Tribunal Dra. Inmaculada Viñas Almenar Universitat de Lleida (Spain) Dr. Naresh Magan Cranfield University (United Kingdom) Dra. Belén Patiño Álvarez Universidad Complutense de Madrid (Spain) “The only way to do great work is to love the work you do” Steve Jobs Als meus pares i als padrins. Al Jaume. LIST OF ABBREVIATIONS 1-MCP 1-methylcyclopropene AA Amino acid adenylation domain AAL TA1, TA2 A. alternata f. spp. lycopersici toxins ACP Acyl carrier protein AF Aflatoxin AFM1 Aflatoxin M1 ALT Altenuene AltR Alternariol regulator AME Alternariol monomethyl ether AOH Alternariol AT Acyltransferase ATX-I, -II, -III Altertoxin I, -II, -III BLAST Basic local alignment search tool CCP Critical control point CD Condensation domain CS Chalon-and Stilben-Synthase (N)/(C) DFMO Difluoromethylornithine DH Dehydratase DMI Sterol-demethylation inhibitor DON Deoxynivalenol EFSA European Food Safety Authority ER Enoyl reductase FB Fumonisin B1, B2 HPLC High-performance liquid chromatography HT-2 HT-2 toxin IARC International agency of research on cancer KR Ketreductase KS β-ketoacyl synthase MeT Methyltransferase NAD NAD binding domain I NIV Nivalenol NLX Norlichexanthone ODC Ornithine decarboxylase OTA Ochratoxin A PAT Patulin PCR Polymerase chain reaction PKS Polyketide synthases PMA Propidium monoazide PTI Polyamine transport inhibitor QoI Quinone outside inhibitor qPCR Quantitative polymerase chain reaction RCP Representative concentration pathway STX III Stemphyltoxin III T-2 T-2 toxin TeA Tenuazonic acid TEN Tentoxin UDG Uracil DNA Glycolase Superfamily UV Ultraviolet ZEN Zearalenone II RESUM La Comissió Europea va demanar a l’Autoritat Europea en Seguretat Alimentària (EFSA) avaluar el risc de les toxines produïdes per les espècies d’Alternaria, sovint presents en productes alimentaris i pinsos, amb el propòsit que la pròpia Comissió Europea i les autoritats competents dels diferents estats membres poguessin considerar la possibilitat d’establir mesures de control i, a la vegada, omplir els buits de coneixement existents. Com a resultat, al 2011, l’EFSA va emetre un comunicat científic titulat “Opinió científica sobre el risc existent per animals i la salut pública en relació a la presència de toxines d’Alternaria en productes alimentaris i pinsos”. Va ser arran de la publicació d’aquest comunicat que va sorgir la idea de desenvolupar la present Tesi Doctoral. Per iniciar aquesta Tesi Doctoral, en primer lloc, es va avaluar l’estat de la qüestió. Amb aquest objectiu es van escollir dues empreses alimentàries de la província de Lleida i es va iniciar la recol·lecta de mostres de tomata durant dues campanyes de collita compreses entre el 2012 i el 2014. D’aquestes mostres de tomata es va determinar la presència d’espècies viables d’Alternaria mitjançant la tècnica PMA- qPCR. A més, també es va analitzar la presència de dues micotoxines d’Alternaria, l’alternariol (AOH) i l’alternariol monometil èter (AME). Els resultats van demostrar la presència d’Alternaria en tomates fresques destinades per a la producció de derivats de tomata en ambdues industries alimentàries. Curiosament, es va observar que els nivells d’Alternaria augmentaven en les mostres preses dins de la planta de producció. Aquest fet, probablement era degut a què les tomates fresques es submergien en banys d’aigua, els quals probablement estaven altament contaminats amb Alternaria. En relació a la presència d’AOH i AME al llarg del processat industrial, els resultats van demostrar que els nivells d’ambdues toxines disminuïen a mesura que avançava el procés, exceptuant les mostres de concentrat de tomata, en les quals aproximadament el 50% de les mostres estaven contaminades en un interval comprés entre 22.6 i 137.4 g/kg de tomata. III En segon lloc, es va intentar abordar el tema des d’una perspectiva més global, del camp a la taula. Primerament, es van estudiar dos components de la família de proteïnes Velvet (VeA i LaeA) i, a la vegada, es va indagar en el metabolisme de les poliamines en Alternaria alternata. L’objectiu d’ambdós estudis era trobar noves diana útils per al disseny de nous fungicides, ja que s’ha observat l’aparició de fongs resistents a determinats fungicides comunament emprats comercialment. Els resultats derivats d’aquests estudis van demostrar que, tant LaeA com VeA, estaven involucrats en funcions essencials d’A. alternata, incloent el creixement fúngic, el desenvolupament asexual i la biosíntesi d’AOH i AME. Per tant, seria convenient explorar més a fons ambdós components per tal d’utilitzar-los, en un futur, com a possibles diana antifúngics. En relació al metabolisme de les poliamines, va ser interessant observar que dos anàlegs de les poliamines (AMXT- 2455 i AMXT-3016) van obtenir resultats prometedors a l’hora de controlar la infecció fúngica provocada per A. alternata, ja sigui en tomateres o en tomates fresques, tot i que en aquest darrer cas caldria optimitzar el tractament. El següent pas dut a terme, ja en un context de postcollita, va ser analitzar els efectes de l’1- metilciclopropè (1-MCP) en A. alternata infectant tomates en relació al creixement fúngic i a la producció d’AOH i AME. Els resultats van demostrar que la infecció per Alternaria era superior en aquelles tomates tractades amb 1-MCP quan les condiciones d’emmagatzematge eren favorables al creixement d’Alternaria. En canvi, el tractament amb 1-MCP de les tomates inoculades amb A. alteranta no va afectar la producció d’AOH i AME en dues de les tres varietats de tomata emprades. Finalment, es va estudiar l’estabilitat de l’AOH i l’AME durant el procés industrial de productes derivats de tomata. Els resultats van demostrar que ambdues micotoxines són altament estables al llarg del processat industrial. Es va observar que el rentat de les tomates, especialment si s’utilitza aigua clorada, és l’etapa industrial que aconsegueix reduir la contaminació d’AOH i AME més eficientment.
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