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TECNOLÓGICO NACIONAL DE MÉXICO Instituto Tecnológico de Tepic EFECTO DE PLASMA FRÍO EN LA REDUCCIÓN DE OCRATOXINA A EN CAFÉ DE NAYARIT (MÉXICO) TESIS Por: MCA. PALOMA PATRICIA CASAS JUNCO DOCTORADO EN CIENCIAS EN ALIMENTOS Director: Dra. Montserrat Calderón Santoyo Co - director: Dr. Juan Arturo Ragazzo Sánchez Tepic, Nayarit Febrero 2018 RESUMEN Casas-Junco, Paloma Patricia. DCA. Instituto Tecnológico de Tepic. Febrero de 2018. Efecto de plasma frío en la reducción de ocratoxina A en café de Nayarit (México). Directora: Montserrat Calderón Santoyo. La ocratoxina A (OTA) se considera uno de los principales problemas emergentes en la industria del café, dado que el proceso de tostado no asegura su destrucción total. El objetivo de este estudio fue identificar las especies fúngicas productoras de OTA en café tostado de Nayarit, así como evaluar el efecto de plasma frío en la inhibición de esporas de hongos micotoxigénicos, detoxificación de OTA, así como en algunos parámetros de calidad del café. Se aislaron e identificaron hongos micotoxigénicos mediante claves dicotómicas, después se analizó la producción de OTA y aflatoxinas (AFB1, AFB2, AFG2, AFG1) por HPLC con detector de fluorescencia. Las cepas productoras de toxinas se identificaron por PCR utilizando los primers ITS1 e ITS4. Después se aplicó plasma frío en muestras de café tostado inoculadas con hongos micotoxigénicos (A. westerdijikiae, A. steynii, A. niger y A. versicolor) a diferentes tiempos 0, 1, 2, 4, 5, 6, 8, 10, 12, 14, 16 y 18 min, con una potencia de entrada 30 W y un voltaje de salida de 850 voltios y helio publicitario (1.5 L/min). El café tostado fue contaminado artificialmente con hongos micotoxigénicos (1x105 esporas/g) a 27 ºC (21 días). Las muestras fueron tratadas con plasma frío a las mismas condiciones, pero a 0, 1, 4, 8, 12, 16, 20, 24 y 30 min. OTA fue cuantificada por HPLC acoplado a MS. Se realizaron pruebas de toxicidad mediante el bioensayo de Artemia salina. Finalmente se investigó actividad antioxidante, polifenoles solubles totales, perfil aromático y características sensoriales en muestras de café tratadas con plasma frío. Se detectó producción de OTA en las cepas R16, 6N y 11 con una concentración en µg/kg de micelio de 0.33, 1.16 y 0.36, respectivamente. Estas fueron identificadas como A. niger, A. versicolor y Byssochlamys spectabilis. La tecnología emergente de plasma frío mostró una disminución de 4 log en 6 min en los hongos micotoxigénicos. Referente a la detoxificación de OTA, en los tratamientos aplicados por 30 min, se obtuvieron reducciones aprox. del 50 %. Esto se pueda atribuir a las diferentes especies reactivas formadas + + − − + durante el proceso de plasma como son iones (H3O , O , O , OH , N2 ), especies moleculares (O3, H2O2), radicales reactivos (O•, OH•, NO•) e irradiación UV. Mediante análisis por HPLC-MS se determinó que OTA da origen a dos fragmentos principales, el anillo de isocumarina clorada y el grupo amino de la molécula L - b fenilalanina. El café tostado sin tratar se determinó como “tóxico”, mientras que la toxicidad para el café tratado con plasma frío se redujo a “ligeramente tóxico”. El perfil aromático de café tratado con plasma no presentó diferencias significativas (p < 0.05) respecto al café sin tratamiento. De acuerdo a la evaluación sensorial los panelistas calificaron “me gusta moderadamente” en los parámetros de olor, color, sabor y aceptación general, para ambos tratamientos con y sin plasma, sin haber diferencia significativa entre ambos (p<0.05). La diferencia de color ∆E determinada entre café tratado con plasma frío y sin tratar, fue inferior a 1, por lo que es difícilmente perceptible por el ojo humano. En cuanto a los fenoles solubles totales, se determinó una reducción del 12 %, mientras en la actividad antioxidante hubo aumento del 14 %. Por lo tanto, se concluye que el plasma frío es un método prometedor para la destrucción de hongos micotoxigénicos y para la degradación de OTA en café tostado, por lo que podría ser útil en la cadena de procesamiento de este producto alimenticio. Palabras claves: Café tostado, Aspergillus, OTA, plasma frío, Artemia salina. ii CONTENIDO LISTA DE CUADROS ....................................................................................................... v LISTA DE FIGURAS ....................................................................................................... vii INTRODUCCIÓN .............................................................................................................. 1 CAPÍTULO 1. ANTECEDENTES .................................................................................... 4 1.1 Fruto de café (Coffea arábica L.) ............................................................................. 4 1.2 Composición química del café en grano .................................................................. 4 1.3 Micotoxinas ................................................................................................................ 5 1.3.1 Los factores que influyen en el crecimiento fúngico y la producción de micotoxinas .......................................................................................................................... 6 1.4 Hongos productores de ocratoxina A (OTA) .......................................................... 7 1.5 Ocratoxinas .............................................................................................................. 12 1.5.1 Ocratoxina A (OTA) ................................................................................................ 13 1.5.2 Propiedades fisicoquímicas de OTA ...................................................................... 15 1.5.3 Toxicocinética de la OTA ........................................................................................ 16 1.5.3.1 Absorción y distribución .......................................................................... 16 1.5.3.2 Metabolismo .............................................................................................. 17 1.5.3.3 Eliminación ................................................................................................ 17 1.5.4 Toxicodinámica: mecanismo de acción .................................................................. 20 1.5.5 Toxicidad .................................................................................................................. 21 1.5.5.1 Toxicidad aguda ........................................................................................ 22 1.5.5.2 Toxicidad crónica ...................................................................................... 23 1.6 Métodos de análisis para la determinación de ocratoxina (OTA) ...................... 24 1.7 Plasma frío ............................................................................................................... 27 1.7.1 Reactor de plasma .................................................................................................... 29 1.8 Mecanismo de inhibición de la tecnología emergente plasma frío ...................... 30 ii 1.9 Efecto de plasma en micotoxinas ........................................................................... 31 CAPÍTULO 2. JUSTIFICACIÓN ................................................................................... 34 CAPÍTULO 3. HIPÓTESIS ............................................................................................. 36 CAPÍTULO 4. OBJETIVOS ........................................................................................... 38 4.1 Objetivo general ...................................................................................................... 38 4.2 Objetivos específicos ............................................................................................... 38 CAPÍTULO 5. MATERIALES Y MÉTODOS .............................................................. 41 5.1 Metodología .............................................................................................................. 41 5.2 Diseño estadístico y experimental .......................................................................... 43 5.3 Materia prima .......................................................................................................... 44 5.4 Actividad de agua (aw) ............................................................................................ 44 5.5 Aislamiento e identificación de hongos relacionados con muestras de café tostado en grano y café molido. .................................................................................... 44 5.6 Identificación de hongos ......................................................................................... 45 5.7 Determinación de ocratoxina A (OTA) y aflatoxinas totales (AFLA) en hongos micotoxigenéticos ........................................................................................................... 47 5.7.1 Extracción y purificación de ocratoxina A (OTA) y aflatoxinas totales (AFLA) ............................................................................................................................................. 47 5.7.2 Método de cuantificación de ocratoxina A (OTA) y aflatoxinas totales por HPLC ................................................................................................................................. 47 5.7.3 Curva de calibración para la cuantificación de OTA y AFLA ............................ 48 5.8 Análisis molecular ..................................................................................................
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  • A Thesis Submitted in Partial Fulfillment of the Requirements For

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    i Screening of Soil Fungi from Plant Genetic Conservation Project Area, Rajjaprabha Dam, Suratthani Province which Produced Antimicrobial Substances Kawitsara Borwornwiriyapan A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Microbiology Prince of Songkla University 2013 Copyright of Prince of Songkla University ii Thesis Title Screening of Soil Fungi from Plant Genetic Conservation Project Area, Rajjaprabha Dam, Suratthani Province which Produced Antimicrobial Substances Author Miss Kawitsara Borwornwiriyapan Major Program Master of Science in Microbiology Major Advisor: Examining Committee: ………………………………………… ..……………………………Chairperson (Assoc. Prof. Dr. Souwalak Phongpaichit) (Asst. Prof. Dr. Youwalak Dissara) Co-advisor: ............................................................... (Assoc. Prof. Dr. Souwalak Phongpaichit) ………………………………………… ………………………………………… (Dr. Jariya Sakayaroj) (Dr. Jariya Sakayaroj) ………………………………………….. (Dr. Pawika Boonyapipat) The Graduate School, Prince of Songkla University, has approved this thesis as partial fulfillment of the requirements for the Master of Science Degree in Microbiology. ………………………………….. (Assoc. Prof. Dr. Teerapol Srichana) Dean of Graduate School iii This is to certify that the work here submitted is the result of the candidate’s own investigations. Due acknowledgement has been made of any assistance received. ...………………………………Signature (Assoc. Prof. Dr. Souwalak Phongpaichit) Major advisor ...………………………………Signature (Miss Kawitsara Borwornwiriyapan)
  • Phylogeny and Nomenclature of the Genus Talaromyces and Taxa Accommodated in Penicillium Subgenus Biverticillium

    Phylogeny and Nomenclature of the Genus Talaromyces and Taxa Accommodated in Penicillium Subgenus Biverticillium

    available online at www.studiesinmycology.org StudieS in Mycology 70: 159–183. 2011. doi:10.3114/sim.2011.70.04 Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium subgenus Biverticillium R.A. Samson1, N. Yilmaz1,6, J. Houbraken1,6, H. Spierenburg1, K.A. Seifert2, S.W. Peterson3, J. Varga4 and J.C. Frisvad5 1CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 2Biodiversity (Mycology), Eastern Cereal and Oilseed Research Centre, Agriculture & Agri-Food Canada, 960 Carling Ave., Ottawa, Ontario, K1A 0C6, Canada, 3Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, IL 61604, U.S.A., 4Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Közép fasor 52, Hungary, 5Department of Systems Biology, Building 221, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark; 6Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. *Correspondence: R.A. Samson, [email protected] Abstract: The taxonomic history of anamorphic species attributed to Penicillium subgenus Biverticillium is reviewed, along with evidence supporting their relationship with teleomorphic species classified inTalaromyces. To supplement previous conclusions based on ITS, SSU and/or LSU sequencing that Talaromyces and subgenus Biverticillium comprise a monophyletic group that is distinct from Penicillium at the generic level, the phylogenetic relationships of these two groups with other genera of Trichocomaceae was further studied by sequencing a part of the RPB1 (RNA polymerase II largest subunit) gene. Talaromyces species and most species of Penicillium subgenus Biverticillium sensu Pitt reside in a monophyletic clade distant from species of other subgenera of Penicillium.