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Corrigiendo Tesis Doctorado Paloma Casas Junco 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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