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Predictive Mycology and Use of Natural Antifungals to Prevent the Mycotoxin Food Hazard

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Predictive Mycology and Use of Natural Antifungals to Prevent the Mycotoxin Food Hazard Predictive mycology and use of natural antifungals to prevent the mycotoxin food hazard Daiana García Predictive mycology and use of natural antifungals to prevent the mycotoxin food hazard Thesis submitted by Daiana García To fulfil the requerimets of the degree of Doctor Thesis Directors: Sonia Marín Sillué Antonio J. Ramos Girona October 2012 Dissertation presented by Daiana García to obtain the PhD degree from the University of Lleida. This work was carried out under the supervision of Sonia Marín Sillüe and Antonio J. Ramos. The present work has been carried out in the Mycology Research Group in the Food Technology Department and is included in the “Ciéncia i Tecnologia Agraria i Alimentaria” doctorate program. The work is part of the following projects: a- Presencia simultánea de micotoxinas en alimentos. Evaluación del peligro potencial y real. (AGL2007-66416-C05-03). MECI- Ministerio de Educación y Ciencia. b- New integrated strategies for reducing mycotoxins in the world in food and feed chains.. MYCORED (KBBE-2007-2-5-05 project) Spanish (AGL2010-22182-C04-04 project) MECI- Ministerio de Educación y Ciencia. c- Cambio climático y nuevos hábitos alimentarios: Nuevos escenarios con impacto potencial sobre el riesgo de micotoxinas en España.(AGL2010-22182-C04-04 project). MECI- Ministerio de Educación y Ciencia. The financing of the PhD candidate was through: January 2009 - December 2012 Catalan Doctoral grant FI-DGR 2009. PhD Candidate Daiana García Director Sonia Marín Sillüe, PhD Signature of approval Co-Director Antonio J. Ramos Signature of approval “La verdadera locura quizá no sea otra cosa que la sabiduría misma que, cansada de descubrir las vergüenzas del mundo, ha tomado la inteligente resolución de volverse loca” Heinrich Heine AGRADECIMIENTOS Un 4 de septiembre de 2008 llegué a Lleida sin saber realmente dónde estaba ubicada la ciudad en el mapa, decidida a experimentar las vivencias que me iban a traer los siguientes cuatro años de doctorado. Y a día de hoy, pasado ese tiempo, me doy cuenta que hay mucha gente a la que tengo que agradecer: … y en primer lugar estás vos que fuiste el que me dio las fuerzas necesarias para enfrentar mi último año de doctorado, para mí, el más difícil. Gracias Fran por tu apoyo y compañía diaria; gracias por ponerte en mi lugar y hacerme sentir tan bien cada día y levantarme el ánimo cuando decaía. Gracias mi amor por todo lo que me diste desde Argentina durante este tiempo. ¡Te amo! Gracias a los tres jefes del laboratorio por su total confianza puesta en mí. Gracias al Dr. Vicente Sanchis, Dr. Antonio J. Ramos y en especial a la Dra. Sonia Marín. Gracias Sonia por brindarme tus conocimientos, por tu buena predisposición, y sobre todo por tu paciencia al momento de la escritura de la tesis. Los espero en a los tres Argentina!!! Quiero agradecer también a todo el grupo de Microbiología del Departamento de Tecnología de los Alimentos por los buenos momentos compartidos tanto en el ámbito laboral como las juntadas fueras del laboratorio. Gracias Bernarda, Esther, Amani, Cyndia, Ana, Nuria, Jordi, Arnau, Germán y Montse por la buena onda y por estar presente en todo momento. Montse, te nombro nuevamente porque te merecés un agradecimiento aparte. Gracias por toda la ayuda que me diste en éstos cuatro años. Gracias por acompañarme en las cosas buenas y en las no tan buenas que sólo vos y yo sabemos. Gracias a vos y a toda tu familia por haberme “adoptado” como una más. Te voy a extrañar mucho, pero te espero en mí país así te llevo de recorrida. Al personal del grupo de antioxidantes Alba, María, Xenia, Laura, Aida y Manuel que hicieron que cada día sea un buen día, poniendo pilas para poder vivirlo y trabajarlo. Agradecer en particular a Aida y Germán, mis compañeros de trabajo con los que emprendimos juntos este camino del doctorado y compartimos horas y horas en la sala de análisis, que entre charlas y risas de por medio, hicieron que el trabajo sea más ameno. Nuevamente agradecer a Aida por su amistar y por abrirme las puertas de su casa y familia, ya sea para compartir una tarde normal, en familia o unas Navidades. Fueron 4 años trabajando en el mismo lugar y me llevo muchos recuerdos… sobre todo los viajecitos ;-) ¡Gracias amiga!. Ahora ya sé a donde van a ser mis próximas vacaciones!!! Al grupo de investigación de Micología del departamento de Microbiología e Inmunología de la Universidad Nacional de Río Cuarto, especialmente a Sofía Chulze y Germán Barros que desde que empecé la carrera de microbióloga estuvieron presentes. Gracias además por permitirme realizar la estancia de investigación durante marzo-abril de 2010. Gracias Germán por toda tu ayuda y por la buena onda que me tiraste desde mis comienzos del doctorado. Al grupo de investigación del departamento de Microbiología III, en la Facultad de Ciencias Biológicas de la Universidad Complutense, en especial a la Dra. Belén Patiño por brindarme sus conocimientos durante mi estadía de investigación en Madrid. I want to thanks to Mycology research group in Istituto di Scienze delle Produzioni Alimentari from Bari, especially to Dr. Giancarlo Perrone, Dra. Antonia Gallo, Dra. Filomena Epifani and Vincenzo Achille. Thanks Giancarlo for letting me work on your research group and thanks for the knowledge provided. I hope see you again, but this time in my country. Gracias a mis “hermanitos del alma” Mikel, Diego, Raziel, Ruymán y Addy por los buenos momentos y la compañía. Gracias por formar parte de mi familia en el extranjero. Los voy a extrañar, pero más me van a extrañar a mi ;-) A mis compis del gym Montse, Belén, Juanjo y Adrián por los buenos momentos compartidos dentro del gimnasio, en la pista de balón mano, y durante las cenas donde el lema siempre fue: “No hay mañana”!!! Aunque al otro día “el mañana” sí existía. No se olviden del triatlón Europa-Argentina. Los espero con las bicicletas en Buenos Aires. Quiero agradecer también a la compañía de teatro Talía por incorporarme como su “demonieta” en las actuaciones de Navidad. Es duro pasar dichas fiestas solo y lejos de tu familia, pero ustedes también participaron en disminuir la soledad de las fechas. A Ingrid y Sergi por abrirme las puertas no sólo de su casa sino también de su corazón. Por tenerme en cuenta en todo momento, por el tiempo compartido, por brindarme su confianza y amistad. “LOS RE-QUIERO”. A mi familia que a pesar de los miles kilómetros de distancia estuvieron en cada momento. Gracias papá y mamá, abuela, y gracias a mis hermanas Meli y Eli. Les prometo que ya no me van a tener tan lejos y los asados en familia van a pasar a ser semanales y no anuales. Los quiero mucho!!!. A mis amigos de Argentina tanto a los de Río Cuarto como a los de E. V. Bunge. Gracias Naty, Negro, Nacho, Anahí y especialmente a vos, mi amiga de la infancia, Mari, que desde que nos hicimos amiga no dejamos de regalarnos eso tan lindo llamado amistad. Por suerte, a partir de unos días volveremos a vernos y las juntadas dejarán de ser una vez al año para pasar a ser más frecuentes. Los quiero mucho. Y a todas aquellas personas que de alguna manera estuvieron presentes y caminamos juntos en alguna etapa de la vida, a aquellas que se marcharon antes de tiempo, pero que siempre serán parte de buenos recuerdos...y a los que vendrán... Summary Filamentous moulds may cause spoilage in raw materials, foods and feeds. Some of them synthesize mycotoxins which are a risk for human and animal health. From the food safety point of view, regarding foodborne moulds only mycotoxins, as chemical hazards, are relevant. Nevertheless, despite the absence of a direct correlation between mould growth and mycotoxins production, the prevention of fungal growth in raw materials and foods leads invariably to the prevention of mycotoxins presence. Due to the fact that moulds can contaminate foods from raw materials till end products, different strategies could be used at the different steps in the food chain. Preharvest strategies include the use of resistant varieties, crop rotation, soil preparation, optimal irrigation, fertilizer, herbicides, insecticides and chemical and biological agents application. Post-harvest strategies include improved drying and storage conditions, together with the use of natural and chemical agents. Predictive models may be used as a strategy to predict and prevent mycotoxigenjc fungal growth and mycotoxins accumulation. The present PhD work focused in two main strategies: a) The use of antifungals of natural origin to prevent mycotoxigenic fungi and mycotoxins. Equisetum arvense and Stevia rebaudiana extracts were analized as possible natural agents to inhibit growth and mycotoxin accumulation in in vitro and in vivo experiments. Both extracts were effective against mycotoxigenic moulds and the mycotoxigenic Aspergillus and Fusarium isolates studied were completely inhibited by E. arvense extract at 3%. However, the effect decreased in the in vivo test. In the last case, Equisetum was effective against Aspergillus section Flavi and Fusarium section Liseola growth at high water activity levels and with high infection levels, but mycotoxins levels were not significantly affected. b) The assessment of the usefulness of predictive models to manage the mycotoxin problem. In an initial experiment, four particular points which deserved in depth study to assess the viability of predictive microbiology in the moulds field were identified: 1) models should be developed for longer time periods; 2) food and raw materials prone to mycotoxin contamination are usually stored under marginal conditions for mould growth, thus performance of models should be checked under such conditions; 3) the impact of the inoculum size in the performance of the models; and 4) the impact of the potential intraspecies variability among isolates in prediction performance.
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