Empleo del psyllium para el desarrollo de nuevos productos a base de cereales.

PROGRAMA DE DOCTORADO EN CIENCIA E INGENIERÍA AGROALIMENTARIA Y DE BIOSISTEMAS TESIS DOCTORAL EMPLEO DEL PSYLLIUM PARA EL DESARROLLO DE NUEVOS PRODUCTOS A BASE DE CEREALES Mayara Lopes da Silva Belorio Dirigida por: Manuel Gómez Pallarés “El saber es el único espacio de libertad del ser.” Michel Foucault AGRADECIMIENTOS Agradecimientos Los últimos años fueron, sin duda, de mucho aprendizaje y crecimiento, tanto a nivel profesional como personal. Fue una gran decisión dejar mi país de origen (Brasil), mi antiguo trabajo, mi familia y mis amigos, para trasladarme a España con el objetivo de centrarme en mi crecimiento profesional y hacer la tesis de doctorado. Me siento muy feliz al decir que he podido vencer grandes obstáculos y que fui capaz de superar mis propias expectativas durante estos años. Como resultado de mucha dedicación, esfuerzo, fuerza de voluntad y compromiso, puedo afirmar con mucha alegría que ¡concluí la tesis de doctorado! Quiero aprovechar la oportunidad para agradecer al Fondo Europeo de Desarrollo Regional por el soporte a través del proyecto TRANSCOLAB (0612_TRANS_CO_LAB_2_P) y también a la Fundación General de la Universidad de Valladolid (beca de formación) que hicieron posible la realización de la presente tesis doctoral gracias a su apoyo y financiación. Gracias a mi director de tesis, Manolo, por estar siempre dispuesto a ayudar y creer en mi potencial y mi capacidad como profesional. Muchas gracias por el tiempo dedicado, por la confianza y por todo el soporte a lo largo de estos años. A mis compañeros de doctorado y de despacho al contribuir en mi aprendizaje y mi adaptación en Palencia. No hubiera sido lo mismo sin vosotros. Quiero agradecer por toda la paciencia, ayuda y por los momentos de risas que compartimos en nuestros cafés y almuerzos. Agradezco a todos: Ángela, Marta S., María, Vicky, Felipe, Blas, Candela, Marta T., Cristina, Juan y Priscila. AGRADECIMIENTOS A Marta Sahagún, por la amistad que ha nacido entre nosotras y por darme siempre soporte, tanto en el laboratorio como en la vida personal. Gracias por la motivación y por siempre creer en mí. A todos mis amigos que me ayudaron en los momentos difíciles y que me proporcionaron momentos de mucha alegría en Palencia. Vosotros siempre estuvisteis a mi lado y sois mi “familia Palentina”. Gracias a Léo, Danilo, Dago, Samanta y Estefanía. A mis amigos de Brasil que siempre creyeron en mi capacidad y me incentivaron a hacer la tesis. Sois grandes: Clara Lis, Cléo, Gabi. A Jaime, por estar siempre a mi lado para ayudarme y por incentivar el desarrollo de lo mejor que hay en mí. Gracias por tu paciencia, tu apoyo y por estar siempre a mi lado. ¡Eres parte fundamental de este logro de mi vida! A mi familia dedico un agradecimiento muy especial, porque siempre estuvieron a mi lado y, a pesar de la distancia, ¡siempre han estado, están y estarán presentes en mi vida! Mi padre João, mi madre Gisele, mis hermanos Gisiane y João, mis sobrinas Anna y Catarina, ¡quiero que sepan que vosotros sois parte fundamental de mí! Gracias por las palabras de motivación y por la fuerza que me hicieron seguir adelante y concluir la tesis. ¡Gracias a todos! ÍNDICE Índice Resumen .................................................................................................................................................................... I Abstract .................................................................................................................................................................. IV Lista de los artículos ...................................................................................................................................... VII Introducción .......................................................................................................................................................... 2 Objetivos .............................................................................................................................................................. 50 Estructura ............................................................................................................................................................ 54 Capítulo 1: Propiedades funcionales del psyllium ........................................................................ 60 ▪ Influence of psyllium versus xanthan gum in starch properties .......................................... 60 Capítulo 2: Influencia del psyllium en la elaboración de panes sin gluten ...................... 88 ▪ Effect of hydration on gluten-free breads made with hydroxypropyl methylcellulose in comparison with psyllium and xanthan gum ............................................................................... 88 Capítulo 3: Empleo del psyllium como sustituto de grasa en bizcochos ....................... 115 ▪ Psyllium as a fat replacer in layer cakes: batter characteristics and cake quality. ..... 115 Capítulo 4: Estudio de la calidad y la reducción de grasa en galletas de maíz .......... 141 ▪ Influence of flour particle size distribution on the quality of maize gluten-free cookies. ........................................................................................................................................................................ 141 ▪ Assessing psyllium as a fat replacer in wheat and gluten-free cookies. ......................... 165 Conclusiones ................................................................................................................................................... 190 Anexo .................................................................................................................................................................. 194 TABLE OF CONTENTS Table of contents Resumen .................................................................................................................................................................... I Abstract .................................................................................................................................................................. IV List of original papers ................................................................................................................................... VII Introduction ........................................................................................................................................................... 2 Objectives ............................................................................................................................................................ 50 Structure .............................................................................................................................................................. 54 Chapter 1: Functional properties of psyllium ................................................................................ 60 ▪ Influence of psyllium versus xanthan gum in starch properties .......................................... 60 Chapter 2: Influence of psyllium in the elaboration of gluten-free breads ................... 88 ▪ Effect of hydration on gluten-free breads made with hydroxypropyl methylcellulose in comparison with psyllium and xanthan gum ............................................................................... 88 Chapter 3: The use of psyllium as fat replacer in cakes ......................................................... 115 Psyllium as a fat replacer in layer cakes: batter characteristics and cake quality. ............... 115 Chapter 4: Study of the quality and fat reduction in maize cookies ................................ 141 ▪ Influence of flour particle size distribution on the quality of maize gluten-free cookies. ........................................................................................................................................................................ 141 ▪ Assessing psyllium as a fat replacer in wheat and gluten-free cookies. ......................... 165 Conclusions ..................................................................................................................................................... 190 Annex .................................................................................................................................................................. 194 RESUMEN I Resumen Los hidrocoloides son ingredientes muy utilizados en la industria alimentaria debido principalmente a dos características: su solubilidad y su viscosidad. Sin embargo, los hidrocoloides son considerados aditivos y, en vista de la demanda actual por productos “clean label”, es necesario buscar alternativas naturales que tengan propiedades similares. El psyllium es un ingrediente natural, con propiedades funcionales similares a de la goma xantana, que presenta grandes ventajas nutricionales, debido a su alto contenido en fibras. Así se ha demostrado su acción positiva sobre diversas enfermedades o disfunciones del organismo, como el estreñimiento, la diarrea, el síndrome de intestino irritable (SII), el cáncer de colon, la diabetes y la hipercolesterolemia. Además, el psyllium es uno de los sustitutos del gluten más utilizados en la elaboración de panes comerciales sin gluten a nivel mundial, junto con el HPMC, la goma xantana y la goma guar. Sin embargo, los efectos de la incorporación del psyllium en productos horneados están poco estudiados. Se sabe que el psyllium tiene interesantes propiedades gelificantes y espesantes, por ello, la presente tesis evalúa el efecto del psyllium en las propiedades del almidón de maíz, ya que el almidón es una de las alternativas a la harina de trigo más utilizadas en la elaboración de panes comerciales, y otros productos sin gluten. A partir de este estudio y mediante una mejor comprensión de las propiedades funcionales del psyllium, se observó que su uso en la elaboración de panes sin gluten podría afectar a la hidratación de dichas masas, lo que alteraría los efectos de este hidrocoloide en la masa y en la calidad final de los panes. Por lo tanto, esta tesis también evalúa el efecto del psyllium sobre la hidratación de los panes sin gluten, INTRODUCCIÓN _ INTRODUCCIÓN 2 Introducción 1. HIDROCOLLOIDES: ASPECTOS GENERALES Los hidrocoloides son un grupo grande y heterogéneo de sustancias poliméricas que incluyen principalmente algunas proteínas (gelatina) y polisacáridos cuya composición puede ser derivada de la unión de las mismas unidades de glucosa (celulosa y almidón), de dos monómeros distintos (alginato) o por un mayor número de monómeros (goma arábica) (BeMiller, 2008). Los hidrocoloides poseen una gran cantidad de grupos hidroxilos, lo que aumenta su afinidad para unirse a las moléculas de agua, al convertirlos en compuestos hidrofílicos. Además, son capaces de producir una dispersión que es intermedia entre una solución verdadera y una suspensión, y así exhiben propiedades de un coloide. Teniendo en cuenta estas dos propiedades, se comprende mejor la nomenclatura de hidrocoloide (Saha y Bhattacharya, 2010). El uso de los hidrocoloides en la industria de alimentos está muy relacionado con su solubilidad, debido a los grupos hidroxilos presentes en su estructura y a su efecto sobre la viscosidad, que cambia de acuerdo con su concentración, peso molecular o estructura (lineal, muy ramificada, o ligeramente ramificada) (AlAssaf y Phillips, 2015). Los hidrocoloides pueden clasificarse de acuerdo con su origen (vegetal, animal o sintético) y son divididos en: gomas naturalmente encontradas en la naturaleza, gomas modificadas obtenidas por cambios químicos de las gomas naturales y gomas sintéticas que son obtenidas por síntesis química, o por el empleo de microorganismos (Nussinovitch y Hirashima, 2014). En general, entre los hidrocoloides es común utilizar el término “goma” en su nomenclatura, por ejemplo, _ INTRODUCCIÓN 3 la goma xantana o la goma garrofín. Este nombre está relacionado con la capacidad de estos hidrocoloides en disolverse o dispersarse en agua, generando una solución viscosa o una dispersión. A su vez, el termino mucilago también es muy utilizado y está asociado a los materiales viscosos encontrados en las cáscaras de las semillas de algunas plantas, como es el caso del psyllium (Li y Nie, 2016). El uso de los hidrocoloides como ingredientes en la industria de alimentos suele estar muy relacionado con su influencia en las propiedades reológicas de los productos. Las distintas funcionalidades de los hidrocoloides se muestran en la figura 1. Figura 1: Funcionalidades de los hidrocoloides. Adaptado de Li y Nie (2016). Varios hidrocoloides pertenecen a la categoría de aditivos alimentarios permitidos en muchos países. Diversas formulaciones de alimentos como sopas, caldos y salsas para ensaladas utilizan hidrocoloides como aditivos para lograr una viscosidad y “sensación en la boca” agradable al consumidor. Los hidrocoloides también son utilizados en otros productos para crear la textura deseada, como por ejemplo en panes, helados, mermeladas, postres lácteos, bizcochos y caramelos (Milani y Maleki, 2012). _ INTRODUCCIÓN 4 En los productos de panadería, los hidrocoloides se utilizan para mejorar la tolerancia a un exceso de amasado, las características del producto final y la calidad sensorial. A menudo se añaden a las formulaciones para actuar sobre la textura de las migas y reducir el endurecimiento del pan durante el almacenamiento. También pueden utilizarse en la preparación de masas congeladas (para evitar daños por congelación) o con fines nutricionales, ya que son fibras (Saha y Bhattachrya, 2010). Teniendo en cuenta su función en el ajuste de la viscosidad y la textura de los alimentos, se han realizado numerosos estudios sobre la aplicación de hidrocoloides en diversos sistemas alimentarios. Entre estos estudios destacan los centrados en la elaboración de productos sin gluten, pues en estos es necesario utilizar un “sustituto del gluten”. Los panes elaborados con harina de trigo presentan una red de gluten, la cual está formada por las proteínas de la harina (principalmente la glutenina y la gliadina) y se desarrolla al hidratar las proteínas y someterlas a trabajo mecánico. Esta red es la responsable de las propiedades viscoelásticas y de la extensibilidad de la masa, así como de la retención del gas producido durante la fermentación. Algunos autores han evaluado la combinación de distintos hidrocoloides como sustitutos del gluten, analizando como modifican las características y la calidad de distintos productos sin gluten, como, panes, bizcochos o galletas (Anton y Artfield, 2008; Gao et al., 2017; Román et al., 2019). 2. PSYLLIUM: ORIGEN, OBTENCIÓN Y ESTRUCTURA El psyllium es el ingrediente obtenido de la cáscara que recubre las semillas de la planta del género Plantago genus (figura 2). Las especies Plantago ovata y Plantago psyllium (o Plantago asiática) son las más importantes y son muy usadas en las _ INTRODUCCIÓN 5 industrias farmacéutica, cosmética y, de manera más reciente en la de los alimentos (Board, 2003; Gupta, 1991; Panda, 2002). La cáscara de Plantago psyllium se obtiene al separarse de la semilla durante la molienda. La trituración de la semilla suele realizarse con un molino que dispone de un sistema de aspiración neumática. Así, la cáscara es separada del resto de las semillas mediante una ligera presión mecánica. Mientras las semillas siguen el proceso de molturación, el subproducto aspirado (cáscara y semillas residuales) es tamizado, para eliminar todas las semillas restantes. Las cáscaras, en este punto ya totalmente separadas, son molidas para la reducción del tamaño de partícula y obtención del psyllium en polvo (Ajit Patel, 2013). La cáscara es una membrana mucilaginosa inodora, insípida, translúcida y de color marrón claro, que constituye aproximadamente el 30% del peso de la semilla (Gupta, 1991). Este producto es rico en fibras, las cuales son responsables de las ventajas nutricionales, medicinales y funcionales del psyllium. Figura 2: Planta de Plantago psyllium y sus semillas. Fuente: Nie et al. (2018). La mayor parte de la fibra soluble que compone el psyllium está compuesta por un polisacárido, del tipo arabinoxilano, similar a los que se encuentran en la mayoría de los cereales (Izydorczyk y Biliaderis, 1995). Diferentes estudios describieron el _ INTRODUCCIÓN 6 psyllium como un arabinoxilano compuesto por diferentes monosacáridos, como la xilosa, arabinosa, galactosa, ramnosa, glucosa y manosa. La diferente proporción molar y composición de los monosacáridos entre estos polisacáridos puede estar relacionada con los métodos de separación o purificación que pueden utilizarse para obtener el psyllium (Zangh et al., 2019; Nie et al., 2018). La figura 3 representa la estructura propuesta por Nie et al. (2018) para el polisacárido obtenido de las semillas de Plantago asiática, tras distintas cromatografías y procesos de caracterización. Figura 3: Estructura primaria obtenida para el polisacárido obtenido de las semillas de Plantago asiática tras distintos procesos de separación y caracterización (Adaptado de Nie et al., 2018). La fibra tanto del psyllium como del salvado de trigo está compuesta en gran parte por arabinoxilanos muy ramificados, que consisten en espinas dorsales lineales de xilopiranosa (Xylp) unidas por enlaces β-D-(1-->4). A estas cadenas se adhieren unidades de α-L-arabinofuranosa (Araf) como residuos, a través de los enlaces α(13) y α(1-2). La fibra de psyllium posee una estructura más compleja que la fibra del salvado de trigo, con una amplia variación en las cadenas laterales y en los _ INTRODUCCIÓN 7 sustituyentes unidos en las posiciones O-2 y/o O-3 (Edwards et al., 2003; Fischer et al., 2004). En el psyllium es posible distinguir muchos tipos de arabinoxilanos, similares en su composición monomérica pero que varían en su conformación polimérica (Yin et al., 2012a; Yu et al., 2017). En la extracción del mucílago influirá la temperatura del agua. Así el mucílago extraído en agua fría tiene una alta proporción de ramnosa y ácido galacturónico, y un bajo contenido de arabinosa y xilosa, en comparación con el mucílago obtenido en condiciones extremas (calor y alcalinidad). El mucílago extraído en frío también tiene un mayor peso molecular y un radio hidrodinámico menor que el resto (Yu et al., 2017). Estas características son responsables de algunas propiedades de absorción que hacen del psyllium un ingrediente muy útil para ser empleado en la elaboración de una gran variedad de productos (Ziemichód et al., 2018). La India es el mayor productor de Plantago psyllium y también el mayor exportador, con más del 90% de su producción exportada a Estados Unidos, Alemania y el Reino Unido (Golkar et al., 2017). El mercado del psyllium está en crecimiento y las ventas seguirán impulsadas por el aumento del uso de este ingrediente en productos medicinales como, por ejemplo, en laxantes de origen natural para el estreñimiento crónico. Otro importante empleo del psyllium está relacionado a su inclusión en productos ricos en fibra, que presentan un gran potencial para la reducción del colesterol. El empleo del psyllium como aditivo alimentario es todavía minoritario frente a otros hidrocoloides. Sin embargo, se espera que se incremente en los próximos años debido a los estudios que se están desarrollando y por el mayor acceso de la industria agroalimentaria a este ingrediente (figura 4). _ INTRODUCCIÓN 8 Figura 4: Mercado global de produtos elaborados com psyllium. Adaptado de XPloreMR, 2019. 3. VENTAJAS NUTRICIONALES Y BENEFICIOS PARA LA SALUD DEL PSYLLIUM Los beneficios para la salud del psyllium están relacionados a su viscosidad y han sido discutidos en múltiples artículos científicos. Entre estos beneficios se incluyen sus efectos sobre el estreñimiento, la diarrea, el síndrome de intestino irritable (SII), la enfermedad inflamatoria intestinal (colitis ulcerosa), el cáncer de colon, la diabetes y el hipercolesterolemia (Singh, 2007; Wärnberg et al., 2009). Estos efectos y las ventajas nutricionales del psyllium están relacionados con algunas de sus propiedades que, por lo tanto, serán discutidas brevemente a continuación. • Control glucémico y del colesterol: El carácter viscoso del psyllium tiene una gran influencia en el transito gastrointestinal. Se sabe que el consumo de fibras viscosas puede alterar la viscosidad de la digestión en el tracto gastrointestinal, inhibiendo así la absorción de nutrientes, en particular de la glucosa y el colesterol (Dikeman y Fahey, 2006), lo que directamente influye en el control de estas sustancias en los organismos. Así, los efectos sobre la reducción del colesterol y la mejora del control glucémico están relacionados con las modificaciones que provoca el psyllium en el intestino delgado _ INTRODUCCIÓN 15 concentraciones más altas del polisacárido redujeron la dependencia de la viscosidad aparente frente a la frecuencia, y los valores de G' se incrementaron. Al calentar las mezclas, la viscosidad aparente y su dependencia frente a la frecuencia disminuyeron, mientras que se promovió una reducción de los valores de G' y G". De hecho, a partir de 60 °C los valores de G" superan los de G'. No obstante, hay que tener en cuenta que el método de extracción de estos polisacáridos puede afectar a su funcionalidad, al igual que lo hace a su estructura. Con una extracción en frío, el mucílago de psyllium presentó un comportamiento de fluido viscoelástico (tg δ > 1) comparado con los resultados obtenidos en condiciones de extracción en caliente o alcalinas, en las cuales el comportamiento fue de gel (tg δ < 1) (Yu et al., 2017). El mismo estudio demostró que la G' y la G" del mucílago extraído en frío eran altamente dependientes de la frecuencia, y este efecto se redujo al aumentar la fuerza de la extracción. Los valores de G' disminuyeron con el aumento de la temperatura y se acentuó alrededor de 70-80 °C, lo que coincide con la temperatura de termogelatinización de la hidroxipropilmetilcelulosa (HPMC). Por este motivo Haque et al. (1993) propusieron su uso combinado como sustituto del gluten en productos de panadería. Se sabe que la presencia de iones de calcio influye en gran medida en las propiedades reológicas del psyllium. Así, Guo et al. (2009) confirmaron que la presencia de calcio aumentaba drásticamente los valores de G' de los geles debido a una estructura más densa y con capas lineales más espesas. De hecho, la eliminación del calcio de los polisacáridos obtenidos de Plantago asiatica generó una reducción de la viscosidad intrínseca, del radio hidrodinámico y del peso molecular (Yin et al., 2015), así como de la estabilidad térmica (Yin et al., 2012b). Los valores de G' de _ INTRODUCCIÓN 16 estos geles también aumentaron en presencia de iones de sodio, pero el efecto fue menos pronunciado que el obtenido con iones de calcio (Yin et al., 2016). Las propiedades reológicas de los geles de psyllium también pueden verse afectadas por otros ingredientes presentes en la solución. Se ha encontrado que la presencia de goma arábiga o Z-trim (biofibra de maíz) reduce la viscosidad de las soluciones con psyllium, mientras que las maltodextrinas no producen ningún efecto. Sin embargo, la presencia de goma garrofín provoca una ligera reducción de la viscosidad inicial, seguida de un aumento no significativo (Kale et al., 2016). La reología de los geles de psyllium también está influenciada por el pH. Los valores de G' se reducen a pH ácido (2,5), lo que Farahnaky et al. (2010) atribuyeron al hecho de que el psyllium es un polisacárido aniónico. La extensión e interacción entre las cadenas moleculares son influenciadas por repulsiones electrostáticas que generan enlaces intermoleculares, lo que provoca la gelificación. Sin embargo, las repulsiones electrostáticas y la interacción entre las moléculas disminuyen con la reducción del pH. Otra posible explicación para los cambios en el comportamiento reológico del psyllium en condiciones ácidas está relacionada con la reducción del área superficial de sus partículas (Cheng et al., 2009). De hecho, la capacidad de retención de agua y de hinchamiento del psyllium se reducen después de estos tratamientos, por lo que los geles obtenidos son más débiles y menos adhesivos. No obstante, las propiedades reológicas de los geles de psyllium no presentan diferencias en los pH habituales en las formulaciones de los alimentos (entre 4,7 y 10) o estas diferencias son muy limitadas (Farahnaky et al., 2010). Un efecto similar al obtenido con tratamientos ácidos se puede lograr con tratamientos enzimáticos. Así, Yu et al. (2003) redujeron la capacidad de absorción _ INTRODUCCIÓN 17 de agua del psyllium y la dureza de sus geles al pretratarlos con una combinación enzimática que incluía celulasas, hemicelulasas, xilanasas, arabinosas y βglucanasas. Estos autores observaron una reducción en el área superficial y una superficie menos espesa en las partículas de psyllium tras el tratamiento enzimático. Entre estas enzimas, las que actúan específicamente sobre los xilanos son las más eficaces, pero se podría obtener un mejor resultado con una combinación de ellas (Yu y Perret, 2003a, 2003b). Esta hidrólisis enzimática modifica las propiedades funcionales del psyllium, pero no reduce sus efectos hipolipidémicos (Allen et al., 2004). Modificaciones químicas del psyllium, como la sulfatación, la hidroxipropilación y la succinilación, también redujeron al mínimo la dureza y la adhesividad de los geles y la capacidad de hinchamiento del psyllium (“swelling capacity”), pero aumentaron su capacidad de unirse a los ácidos biliares. Esto es importante porque la unión de los ácidos biliares a los polímeros puede mejorar su eliminación, lo que promueve la conversión del colesterol existente en el hígado en ácidos biliares, y así, puede reducir los niveles de colesterol total y del LDL en el plasma y, por último, reducir el riesgo de enfermedades cardiovasculares (Niu et al., 2013). 5. APLICACIÓN DEL PSYLLIUM EN PRODUCTOS DE PANADERÍA • Panes con gluten: La calidad del pan con gluten está muy relacionada a la presencia de la red de gluten, que confiere características viscoelásticas importantes a la masa. Los hidrocoloides no juegan un papel tan importante en los panes con gluten como en los panes sin gluten, pero pueden ayudar a mejorar su calidad final. _ INTRODUCCIÓN 18 En los panes con gluten, la incorporación de hidrocoloides permite un aumento de la hidratación de la masa y puede promover una mayor estabilidad durante la fermentación y una mejor retención de la humedad durante la cocción, así como mejorar el volumen específico de los panes finales (Rosell et al., 2001). Se ha comprobado que el uso de psyllium con otros hidrocoloides mejora la absorción de agua de la masa y el tiempo de amasado, lo que produce masas más fuertes (Czuchajowska et al., 1992). Farbo et al. 2020 evaluaron la incorporación del psyllium en masas elaboradas con una variedad antigua de trigo y compararon los resultados entre diferentes hidrocoloides. El estudio demostró que el psyllium aumenta la extensibilidad y la capacidad de retención de gases de la masa, cuyo efecto es similar al de la goma xantana. De la misma manera, el psyllium puede reducir la pérdida de humedad durante el almacenamiento, previniendo el envejecimiento y retrasando el endurecimiento de la miga (Davidou et al., 1996; Guarda et al., 2004). Este efecto antienvejecimiento también se observó en los panes precocidos (Bárcenas y Rosell, 2007) en los cuales para obtener estos resultados solo es necesaria una pequeña cantidad de hidrocoloides, menos del 0,5%, dependiendo del tipo de hidrocoloide empleado. Aunque los efectos del psyllium en el volumen del pan no son uniformes y dependen de la formulación o del tipo de harina utilizada, diferentes estudios admiten que el uso de psyllium produce panes con mayor humedad, menos secos y con una textura más blanda (Czuchajowska et al., 1992; Park et al., 1997). Este efecto también ha sido comprobado en panes cocidos al vapor (“steamed breads”) (Sim et al., 2015). Por el contrario, Jensen et al. (2015) observaron que el psyllium redujo significativamente el volumen de los panes con harina de tapioca y aumentó su dureza. Es importante destacar que en este estudio no se modificó la hidratación de _ INTRODUCCIÓN 19 las masas en función de análisis previos con un farinógrafo o mixógrafo. Esto muestra la enorme importancia de hacer esta corrección, debido a la influencia que el psyllium tiene sobre la absorción de agua. En general, los estudios sobre el uso de psyllium se ven limitados por su uso en pequeños porcentajes, no superiores al 1%. Es conveniente utilizar una mayor cantidad de psyllium para lograr sus beneficios nutricionales en los productos finales. Existen pocos estudios que hayan analizado la incorporación de altas cantidades de psyllium, aunque Man et al. (2017) comprobó que al aumentar el porcentaje de incorporación de psyllium (15%) se eleva la humedad de los panes con gluten en más de un 25%, pero se reduce el volumen específico en un 37%. Es importante mencionar que el estudio no especificó la formulación, ni las características de la harina de trigo utilizada. Por su parte, Pejcz et al. (2018) observaron que la incorporación de un 8% de psyllium llevó a un aumento en el volumen del pan sin diferencias significativas en la aceptabilidad entre estos panes y el control. Estos autores utilizaron masas con una consistencia de 300 FU Brabender, que es menor que la de otros estudios (500 FU), lo que indica la necesidad de realizar nuevas investigaciones para comprobar estas diferencias. Otra oportunidad de investigación está relacionada al porcentaje correcto de psyllium que es necesario para lograr una reducción efectiva del índice glucémico de los panes. Ray et al. (2018) reemplazó harina de trigo del pan parotta (pan típico del Sur de India), por una combinación de psyllium (7,5% del peso total de la harina), harina de garbanzos y semilla de fenogreco (“fenugreek”) en polvo. Ellos demostraron que con el 25% de sustitución se obtiene un incremento del almidón _ INTRODUCCIÓN 20 de lenta digestión y del almidón resistente y, en consecuencia, reducción significativa del almidón de rápida digestión. • Panes sin gluten: En las formulaciones de panes sin gluten es común la incorporación de gomas e hidrocoloides como sustitutos del gluten, con el objetivo de obtener productos con volumen y textura similares a los de los panes de trigo (Anton y Artfield, 2008; Sciarini et al., 2010). Entre estos sustitutos el HPMC es el más utilizado, tanto en los panes comerciales como en la investigación científica, seguido de la goma xantana (Masure et al., 2016, Mir et al., 2016; Román et al., 2019). El HPMC tiene la ventaja de generar panes con mayor volumen que otros hidrocoloides (Sabanis y Tzia, 2011), pero estos panes tienen una textura más seca y más desmenuzable (Liu et al., 2018). Por esta razón, es común combinar el HPMC con otros hidrocoloides que poseen mayor capacidad de retención de agua, como las gomas guar o xantana (Horstmann et al., 2018). El mayor empleo del psyllium en la industria alimentaria se da en los productos de panadería, pero especialmente en los panes sin gluten. Un estudio de 228 panes comerciales sin gluten, producidos en diferentes países, mostró que el 16% de esos productos utilizaban psyllium como principal sustituto del gluten, y en el 34% el psyllium estaba presente en las formulaciones, siendo el cuarto hidrocoloide más utilizado, sólo por detrás del HPMC, la goma xantana y la goma guar (Román et al., 2019). El psyllium es una alternativa natural a otros hidrocoloides, y tiene beneficios nutricionales con propiedades reológicas similares a las de la goma xantana, como lo demuestran Haque et al. (1993). Estos autores propusieron el uso tanto del HPMC _ INTRODUCCIÓN 21 como del psyllium para la elaboración de panes sin gluten, porque la G' de las masas con psyllium disminuyen a 70-80 °C, lo que coincide con la termogelación del HPMC, por lo que ambos efectos podrían ser compensados durante la cocción. Sin embargo, el uso de psyllium en los panes sin gluten se ha estudiado en menor medida que otros hidrocoloides, y se limita a su combinación con el HPMC (Haque y Morris, 1994; Mancebo et al., 2015) o con otras gomas y fibras (Aprodu y Banu, 2015; Cappa et al., 2013; Collar et al., 2015; Tubili et al., 2016). Cuando se mezclan dos hidrocoloides, ellos no se comportan de la misma manera que lo harían en solitario, por lo que es importante estudiarlos individualmente para evaluar como sus interacciones pueden afectar en la elaboración de los productos (Gao et al., 2017). Solo dos estudios han analizado el uso del psyllium como único sustituto del gluten. Así, Zandonadi et al. (2009) elaboraron panes sin gluten con psyllium y obtuvieron productos con buenas características organolépticas, pero otras características tecnológicas, como el volumen o la textura no fueron evaluadas. Fratelli et al. (2018) también utilizaron psyllium en panes sin gluten y demostraron su efecto en la reducción de la respuesta glucémica de estos productos. En general, la cantidad de psyllium utilizada en estos estudios no superó el 2%. Así, Fratelli et al. (2018) establecieron un óptimo cercano al 2% para la calidad organoléptica de los panes e incorporaron casi un 17% de psyllium para reducir la respuesta glucémica. La adicción de psyllium a las masas de panes sin gluten también afecta a la reología como observaron Collar et al. (2015). Estos autores atribuyeron el aumento de los valores de G' y G" a la gran capacidad espesante y de absorción de agua del psyllium. Este efecto es muy importante, ya que se ha demostrado que cuanto menor es la consistencia de estas masas mayor es el volumen específico de los panes obtenidos, _ INTRODUCCIÓN 22 hasta cierto punto en el que las masas son excesivamente débiles y no pueden soportar su estructura durante la fermentación o la cocción (Mancebo et al., 2017). Algunos estudios también han considerado la hidratación de las masas con psyllium (Fratelli et al., 2018; Mancebo et al., 2015). Estos estudios demostraron que es necesario aumentar la hidratación de la masa de pan sin gluten, cuando se incorpora psyllium en la formulación, para lograr mejores resultados de volumen y una reducción de la dureza en los panes finales. Mariotti et al. (2009) propusieron un cambio en la hidratación de la masa a través del análisis con el farinográfo, al evaluar las masas de pan sin gluten con y sin psyllium. Estos autores demostraron que cuando se modificó la hidratación, en base al análisis farinográfico, los valores de G' se igualaron, pero los de G" seguían siendo más elevados en el caso de las masas con psyllium. En cualquier caso, los resultados sobre el efecto del psyllium en el volumen específico y la textura de los panes dependen de si se modifica la hidratación para cada formulación, y de cómo se realiza esta modificación. Además del empleo del psyllium en polvo, que es la forma más habitual y disponible de este ingrediente, algunos estudios evaluaron el empleo de las semillas de psyllium en la elaboración de panes. Así, la incorporación de la semilla entera (natural o molida) de Plantago psyllium en la elaboración de panes sin gluten también ha sido estudiada. Los panes obtenidos con semilla de psyllium presentaron un volumen específico menor (menos de 2 cm³/g) en todos los casos, pero el uso de estas semillas mejoró la textura del pan (menor dureza, mayor elasticidad y cohesividad) y al aumentar la hidratación de la masa se logró un mayor rendimiento (Ziemichód et al., 2018). En este caso, se utilizó un 5% de semillas, como proporción del total de la harina utilizada, siendo este valor mayor que el de otros estudios que utilizaron cáscara o goma de psyllium. Por su parte, Pejcz et al. (2018) demostraron _ INTRODUCCIÓN 23 que el uso de semillas de psyllium molidas, en lugar de la cáscara, redujo el volumen y la humedad final de los panes, al contrario de lo observado en estudios con fibra o cáscara de psyllium, ya que este efecto se relaciona con la alta capacidad de absorción de agua de la goma de psyllium que se encuentra en la cáscara. La mayor ventaja de las semillas de psyllium es su mayor nivel de polifenoles y capacidad antioxidante, por lo que al utilizar las semillas es posible mejorar estos factores en el producto final (Li et al., 2005). • Bizcochos: Los estudios sobre el uso de psyllium en masas batidas, como las de bizcochos, son escasos y, en general, afirman que el aire incorporado y el volumen específico de los bizcochos disminuye, las migas de los bizcochos se oscurecen y la dureza aumenta cuando se emplea más del 5% de psyllium en la formulación, lo que puede estar relacionado con una menor expansión durante la cocción (Beikzadeh et al., 2016; Bhise y Kaur, 2015). El uso de gomas con características similares a las del psyllium, como la goma xantana, en pequeños porcentajes (1%), pueden mejorar las propiedades organolépticas de los bizcochos y minimizar la pérdida de humedad y la dureza durante el almacenamiento (Gómez et al., 2007). El uso de pequeños porcentajes (1%) de hidrocoloides, como la goma xantana, también puede reducir el enranciamiento de los bizcochos (Beikzadeh et al., 2017). Estos resultados, así como los estudios sobre el empleo de algunos hidrocoloides con propiedades similares al psyllium, como es el caso de la goma xantana o de los β-glucanos, para la reducción de grasa en bizcochos, indica el potencial del psyllium para la mejora de bizcochos (Kalinga y Mishra, 2009; Lee et al., 2005). _ INTRODUCCIÓN 24 • Galletas: El empleo del psyllium también ha sido considerado en formulaciones de galletas. Algunos autores han propuesto el uso de 3%-20% de psyllium en las recetas de galletas (Fradinho et al., 2015; Krystyjan et al., 2018; Raymundo et al., 2014). En general, cuanto más alto sea el contenido de psyllium en la fórmula, mayores serán los valores de G', G" y la dureza de la masa, lo cual está relacionado con el poder espesante del psyllium. El uso de psyllium en galletas elaboradas con harina de trigo incrementa el índice de expansión (“spread factor”), lo que puede estar relacionado con la competencia entre el psyllium y el gluten por la cantidad de agua disponible, reduciendo la formación de la red de gluten, ya que un gluten fuerte disminuye la expansión de las galletas durante la cocción (Pareyt y Delcour, 2008). También se ha observado que las galletas con psyllium son más oscuras, mientras que los resultados de textura son inconsistentes. Al incorporarse hasta 9% de psyllium a la formulación de galletas, Fradinho et al. (2015) encontraron un claro aumento de la dureza. Por su parte, Raymundo et al. (2014) confirmaron que hasta un 10% de incorporación de psyllium llevaría a una disminución de la dureza y obtuvieron valores similares al control con porcentajes más altos. Krystyjan et al. (2018) también encontraron una reducción en la dureza de las galletas con psyllium. 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Effect of psyllium and gum Arabic biopolymers on the survival rate and storage stability in yogurt of Enterococcus durans IW3 encapsulated in alginate. Food Science & Nutrition, 5, 554-563. Nie, S., Cui, S. W., & Xie, M. (2018). Psyllium Polysaccharide. In Bioactive Polysaccharides (p. 395-443). Elsevier. https://doi.org/10.1016/B978-0-12809418-1.00008-3. Niu, Y. G., Xie, Z. H., Zhang, H., Sheng, Y., & Yu, L. L. (2013). Effects of structural modifications on physicochemical and bile acid-binding properties of psyllium. Journal of Agricultural and Food Chemistry, 61, 596-601. Nussinovitch, A., & Hirashima, M. (2014). Cooking innovations: Using hydrocolloids for thickening, gelling, and emulsification. Boca Raton: Taylor & Francis/CRC Press. Olson, B.H., Anderson, S. M., Becker, M. P., Anderson, J. W., Hunninghake, D. B., Jenkins, D. J., LaRosa, J. C., Rippe, J. M., Roberts, D. C., Stoy, D. B. (1997). Psylliumenriched cereals lower blood total cholesterol and LDL cholesterol, but not HDL cholesterol, in hypercholesterolemic adults: Results of a meta-analysis. Journal of Nutrition, 127, 1973–1980. _ INTRODUCCIÓN 47 Ziemichód, A., Wójcik, M., & Rózyło. R. (2018). Seeds of Plantago psyllium and Plantago ovata: Mineral composition, grinding, and use for gluten-free bread as substitutes for hydrocolloids. Journal of Food Process and Engineering, 42, e12931. OBJETIVOS OBJETIVOS 50 Objetivos Esta tesis doctoral tiene como objetivo principal estudiar las propiedades funcionales básicas del psyllium y evaluar su empleo en la elaboración de productos a base de cereales (galletas, bizcochos y panes), y como afecta a sus características reológicas, físicas y aceptabilidad. Así, para lograr el objetivo general de la tesis, se han marcado unos objetivos específicos: I. Estudiar el efecto del uso del psyllium en las propiedades funcionales del almidón de maíz. II. Analizar el efecto sobre la hidratación óptima y las características finales de panes sin gluten elaborados con diferentes fuentes de almidón (harina de arroz y almidón de maíz), comparando el uso de psyllium con otros hidrocoloides (HPMC y goma xantana). III. Estudiar el uso del psyllium como sustituto del aceite en la formulación de bizcochos para la elaboración de productos más saludables. IV. Analizar cómo influye la sustitución de grasa por pastas de psyllium en la calidad de galletas de trigo y libres de gluten. ESTRUCTURA ESTRUCTURA 54 Estructura La presente tesis doctoral está estructurada en base a las diversas publicaciones científicas obtenidas a partir de las investigaciones desarrolladas a lo largo de la tesis. Los diferentes capítulos se distinguen en base al tipo de producto en el que se contempla la incorporación de psyllium. Capítulo 1: Propiedades funcionales del psyllium 1. Belorio, M., Marcondes, G., & Gómez, M. (2020). Influence of psyllium versus xanthan gum in starch properties. Food Hydrocolloids, 105, 105843. https://doi.org/10.1016/j.foodhyd.2020.105843 Capítulo 2: Influencia del psyllium en la elaboración de panes sin gluten. 2. Belorio, M., & Gómez, M. (2020). Effect of hydration on gluten-free breads made with hydroxypropyl methylcellulose in comparison with psyllium and xanthan gum. *Enviado al Journal of Food Engineering en 06 de julio de 2020. Capítulo 3: Empleo del psyllium como sustituto de grasa en bizcochos. 3. Belorio, M., Sahagún, M., & Gómez, M. (2019). Psyllium as a fat replacer in layer cakes: batter characteristics and cake quality. Food and Bioprocess Technology, 12, 2085-2092. https://doi.org/10.1007/s11947-019-02362-3. Capítulo 4: Estudio de la calidad y la reducción de grasa en galletas de maíz. 4. Belorio, M., Sahagún, M., & Gómez, M. (2019). Influence of flour particle size distribution on the quality of maize gluten-free cookies. Foods, 8, 83. https://doi.org/ 10.3390/foods8020083. 5. Belorio, M., Moralejo, C., & Gómez, M. (2020). Assessing psyllium as a fat replacer in wheat and gluten-free cookies. *Enviado a Food Science and Technology International en 15 de junio de 2020. CAPÍTULO 1: Propiedades funcionales del psyllium CAPÍTULO 1: Propiedades funcionales del psyllium 63 using substitutions of 0%, 2%, 5% and 10%, with respect to the starch weight. All the tests were carried out in duplicate. 2.2.1. Hydration properties Water-binding capacity (WBC), which is the amount of water retained by the sample after centrifugation, was evaluated according to the American Association of Cereal Chemists (AACC) method 56-30.01(AACC, 2012). Water-holding capacity (WHC) and swelling volume (SV) were obtained using a total of 5 g of solids to which 100 mL of distilled water was added. The samples were kept at room temperature (25 °C) for 24 h. After the amount of water in excess was carefully removed, the amount of water retained by the sample, without any stress, was obtained from the ratio of the difference between the hydrated sample and the dried sample relative to the weight of the hydrated sample. The SV was calculated by dividing the total volume of the swollen sample by the original dry weight of the sample. The water absorbance index (WAI) evaluates the amount of water retained by the sample after heating and centrifugation. A total of 2.5 g of solids was dispersed in 30 mL of water and centrifuged (600 rpm) at 90 °C for 15 min, followed by centrifugation at 3000 rpm for 10 min, without heating. The water in excess was carefully removed, and the hydrated sample was weighed to calculate the WAI as the ratio between the hydrated sample and the dried sample. 2.2.2. Pasting properties The pasting properties of maize starch and maize starch with different percentages of weight substitution (0%, 2%, 5%, 10% and 100%) using psyllium and xanthan gum, respectively, were evaluated using an RVA (RVA-4C, Newport Scientific Pty. CAPÍTULO 1: Propiedades funcionales del psyllium 64 Ltd., Warriewood, Australia). A suspension was prepared by dispersing 3.5 g of solids in 25 g of distilled water. The sample was maintained at 50 °C for 1 min, then heated until 95 °C and held at this temperature for 2.5 min. Afterwards, it was cooled to 50 °C and held at this temperature for 2 min under a rotation of 160 rpm, then cooled to 30 °C and held at 35 °C for 2.5 min to assist the gelation process of the hydrocolloids. The curve for each analysis was obtained. 2.2.3. Rheological properties Pastes obtained from the RVA were analysed in a rheometer (Haake RheoStress 1, Thermo Fischer Scientific, Scheverte, Germany) installed with a titanium, parallel, serrated plate geometry sensor PP60 Ti (60 mm diameter) and a 3 mm gap. Once the rheometer was stabilised at 30 °C, the sample was placed on the plate and covered with Vaseline oil to avoid drying. Before initiating the test, the sample rested for 500 s. A dynamic oscillatory test was performed through first executing deformation sweeps (0.1–100 Pa) at a constant frequency (1 Hz) to determine the maximum deformation achieved by the sample in the linear viscoelastic range. Subsequently, a frequency sweep test, was commenced using a strain value within the linear viscoelastic region over a frequency range of 10–0.1 Hz. The parameters obtained were storage modulus (G′ [Pa]), loss modulus (G′′ [Pa]) and loss tangent (tan  = G′′/G′) as a function of frequency. 2.2.4. Gel texture Gels were prepared using the pastes obtained from the RVA, which were placed into cylindric plastic recipients (100 mm in diameter by 20 mm height) and cooled in a fridge at 4 °C for 24 h. After, the gels rested at room temperature (25 °C) for 30 min before carrying out the texture analysis using a TA.XT2i texture analyser (Stable CAPÍTULO 1: Propiedades funcionales del psyllium 65 Micro Systems Ltd., Surrey, UK) equipped with Texture Expert version 1 software for Windows. A 5 kg load cell was applied to force calibration and a 50 mm-diameter cylindrical probe was used for compression cycle which was conducted at a constant velocity of 10 mm s–¹ to a sample depth of 10 mm, followed by a return to the original position. A curve force versus time was obtained and used to calculate the values of the peak force obtained in the compression cycle (hardness). 2.2.5. Gel colour The gel colour was measured in the CIE L*a*b* colour space using a Minolta CN-508i chromameter (Minolta Co., Ltd., Osaka, Japan) under a standard D65 lamp and 2° standard observer. 2.2.6. Statistical analysis A one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test (p < 0.05) was performed to differentiate between the medians. Statistical analyses were completed using Statgraphics Centurion XVI software (StatPoint Technologies, Inc., Warrenton, VA, USA). 3. Results and discussion 3.1. Hydration properties As observed in Table 1, as single ingredients, both psyllium and xanthan gum presented hydration properties (WHC, WBC and SV) under cold conditions that were very similar to each other but markedly higher than those of maize starch. CAPÍTULO 1: Propiedades funcionales del psyllium 66 Table 1. Hydration properties. Sample WHC (g water/g solid) SV (mL/g) WBC (g water/g solid) WAI (g water/g solid) Control 0.90 ± 0.16 a 1.60 ± 0.00 a 0.75 ± 0.01 a 5.13 ± 0.04 b PSY 2% 1.49 ± 0.15 b 2.80 ± 0.01 b 1.51 ± 0.01 b 6.23 ± 0.04 c PSY 5% 1.79 ± 0.02 bc 3.19 ± 0.00 c 2.73 ± 0.01 c 5.21 ± 0.07 b PSY 10% 1.63 ± 0.10 b 3.20 ± 0.00 c 3.96 ± 0.07 e 7.08 ± 0.04 d PSY 100% 3.42 ± 0.06 d 5.20 ± 0.01 e 4.82 ± 0.01 f 11.99 ± 0.02 e XAN 2% 1.81 ± 0.01 bc 2.80 ± 0.01 b 1.41 ± 0.16 b 2.59 ± 0.88 a XAN 5% 2.08 ± 0.16 c 3.20 ± 0.01 c 3.07 ± 0.32 d 2.73 ± 0.06 a XAN 10% 2.12 ± 0.16 c 3.20 ± 0.01 c 5.00 ± 0.00 f 11.98 ± 0.01 e XAN 100% 3.64 ± 0.38 d 4.00 ± 0.00 d 4.98 ± 0.01 f 11.99 ± 0.01 e Data are expressed as means ± SD of duplicate assays. Values with the same letter in the same column do not present significant differences (p < 0.05). PSY: psyllium. XAN: xanthan gum. WHC: water-holding capacity. SV: swelling volume. WBC: water-binding capacity. WAI: water absorbance index. Similarly, Dello Staffolo et al. (2017) and Sandhu, Simsek, and Manthey (2015) observed a greater water absorption capacity of psyllium and xanthan gum, respectively, relative to starch. These comparatively high hydration and swelling capacity properties of psyllium and xanthan gum increased the hydration properties of their respective mixtures with starch. The effect of both hydrocolloids on the WHC and SV was very similar, although the WBC was slightly higher for xanthan gum than CAPÍTULO 1: Propiedades funcionales del psyllium 67 psyllium when greater percentages (5% and 10%) were used. For all the parameters evaluated, the mixes of starch and psyllium or xanthan exhibited greater values than those of the single ingredients, considering each percentage. Thus, a synergistic effect was observed, and this was most apparent for the WHC and SV at the smallest percentage of starch substitution (2%), and less so at the higher percentages. The WBC increased progressively as the percentage of substitution was increased. For xanthan gum, the values obtained when it reached 10% of starch substitution were equal to those achieved with this hydrocolloid alone. This ability of hydrocolloids to increase starch hydration in the cold has already been proved in other studies with agar or carrageenan (Martínez et al., 2015), and with basil seed gum (Matia-Merino et al., 2019). Gularte and Rosell (2011) found this effect was more predominant for starch added with xanthan gum than with other hydrocolloids, such as guar gum, HPMC, carboxyl methylcellulose or pectin. According to Christianson, Hodge, Osborne, and Detroy (1981), the addition of hydrocolloids can considerably increase the shear forces exerted on the swollen granules in the centrifuged phase compared with the forces present in starch–water suspensions. These increased forces can enhance water uptake (increasing swelling), granule breakdown and the amount of material exuded into the continuous phase. Unlike the results of the hydration properties in cold conditions, there were no great differences in this parameter between the starch and the hydrocolloids after heating, even if the WAI values of the hydrocolloids were more than double that of the starch. The reason is because of the starch gelatinisation during the process, which increases the water absorption capacity when compared with the CAPÍTULO 1: Propiedades funcionales del psyllium 68 ungelatinised starch. Accordingly, flours in which the starch is pre-gelatinised, have higher water absorption capacity relative to the non-treated flours (Martínez et al., 2015). Both hydrocolloids showed similar WAI values. However, psyllium progressively increased the WAI values of starch, except at 5% of starch substitution, while there was a marked difference in the WAI between the smallest percentages (2% and 5%) of added xanthan gum. Consequently, the addition of xanthan gum lowered the WAI values of starch, such that the 10% mixed suspension had a WAI equal to the value of xanthan gum alone. Matía-Merino et al. (2019) observed increasing values of WAI even with comparatively smaller doses of added hydrocolloid. In general, an increase in water retention is expected because of the presence of hydroxyl groups in the hydrocolloids, which bind to the water molecules. However, the competitive activity for water between starch and hydrocolloid, their interactions and the variations in the hydration conditions can generate the differences observed. 3.2. Pasting properties Figure 1 shows how the pasting properties of starch are affected by the incorporation of psyllium (Fig. 1a) and xanthan gum (Fig. 1b). The smallest percentage of psyllium (2% of starch substitution) slightly increased the viscosity of the samples over the whole curve obtained from the starch gelatinisation, similarly to the behaviour with 5%. This effect coincides with the amylograph data published by Buksa and Krystyjan (2019), who incorporated arabinoxylans into a rye starch dough. It also corroborates the findings of Collar et al. (2015), who mixed psyllium with maize starch and rice flour, and those of Manceo et al. (2015) when psyllium was added to a mix of rice flour and HPMC. However, an irregular curve was CAPÍTULO 1: Propiedades funcionales del psyllium 69 generated for the mixture with 5% psyllium after starch gelatinisation, which indicates the formation of broken structures (gel-like) and a great setback. (a) (b) Figure 1: Pasting properties from starch due to different percentages of psyllium (a) and xanthan gum (b). CAPÍTULO 1: Propiedades funcionales del psyllium 70 This effect was higher with 10% psyllium, and this percentage also promoted a strong increase in the viscosity, and a delay in the gelatinisation temperature, which can be related to the low availability of water, such as found for other hydrocolloids (BeMiller, 2011). Although there are still far too few studies of psyllium, Mancebo et al. (2015) also observed a dramatic increase in the setback with an increasing amount of psyllium. This trend could be associated with arabinoxylans that increase starch retrogradation, as observed by Gudmundsson et al. (1991) with the derivates from wheat or rye. Likewise, it could explain the nature and activity of the complexes formed between these products and amylopectin (Michniewicz & Jankiewicz, 1988). Xanthan gum delayed the gelatinisation temperature in all the samples evaluated, but there was no increase in the viscosity after heating, considering all the percentages studied. This increase in the gelatinisation temperature has been noticed by other authors using different starches (Chaisawang & Suphantharika, 2006; Zhang et al., 2018). The effect was attributed to the low availability of water to the starch because of the higher water absorption capacity of xanthan gum (BeMiller, 2011). However, the viscosity in cold conditions progressively increased as the amount of xanthan gum increased because of both its thickener property and its water absorption capacity. In earlier studies about the interaction between xanthan gum and other hydrocolloids, the peak viscosity results are contradictory. For instance, Chaisawang and Suphantharika (2006) showed this viscosity increased with cassava starch but decreased with anionic cassava starch. Kim and BeMiller (2012) observed differences to the viscosity analysis of pea starch when guar gum was used and not to the other hydrocolloids studied. Lee et al. (2002) found that hydrocolloids decreased the peak viscosity of sweet potato starch, and Korus et al. (2004) related an increase with triticale. Zhang et al. (2018) observed CAPÍTULO 1: Propiedades funcionales del psyllium 71 that the presence of xanthan gum reduced the extent of starch swelling and maintained the integrity of the grain, suggesting that it could stabilise the grains by acting as a lubricant and a barrier. Regarding the effects of hydrocolloids on maize starch, both an increase (Alloncle & Doublier, 1991) and a decrease in the peak viscosity (Song, Kim, & Shin, 2008; Weber et al., 2009) have been reported, which coincides with the results found in this study. However, the effect of the viscosity on the RVA curve depends on the type of starch used and the starch:hydrocolloid:water ratio, which usually differs between each test, as does the sample preparation method (Mandala & Bayas, 2004). Moreover, in this study, the concentration of the hydrocolloids is lower when compared with other studies. It must be emphasised that for great amounts of xanthan gum, differently from what happens with psyllium, a reduction in the setback is obtained. This phenomenon was previously observed by Chantaro and Pongsawatmanit (2010) with cassava starch, and by Weber et al. (2009) with maize starch. 3.3. Rheology As shown in Table 2, both hydrocolloids influenced, in a slightly different way, the rheology of the gels obtained in the RVA. A significant correlation at 95% was observed between the values of WHC, WBC and G′, with r equals to 0.56 and 0.60 respectively. The small values of r, and the lack of correlation with the G′′ values, indicate that the water absorption capacity cannot explain all the changes observed in the rheology of the pastes. Thus, the incorporation of psyllium did not modify the values of G′ but progressively increased G′′ with increasing amounts of psyllium in the samples. On the contrary, the xanthan gum only increased G′ when added at the smallest percentage and, even it increased the values of G′′, independently of the CAPÍTULO 1: Propiedades funcionales del psyllium 72 xanthan concentration. As a consequence of these changes, tan  increased as the amount of added hydrocolloid increased, but the increase was greater with the addition of psyllium than xanthan gum. The results obtained with xanthan gum are surprising. Previous research affirmed that when xanthan gum is incorporated with distinct starches, it increases G′′ and, more so G′, and decreases the tan  values, which are influenced by the increase in the amount of xanthan (Alloncle & Doublier, 1991; Kim & Yoo, 2006; Ptaszek et al., 2009). However, in these studies, pastes were elaborated with very small percentages of starch substitution by hydrocolloids, less than 5%. By contrast, when the concentration of solids increased, similarly as proposed in our study, the values of G′ and G′′ hardly varied (Biliaderis et al., 1997) or even reduced, in the case of G′′ (Aguirre-Cruz, Mendez-Montealvo, Solorza-Feria, & Bello-Perez, 2005). Thus, it seems that the increase in the values of the rheological parameters is related to the availability of free water. Our experiments confirmed this hypothesis because a slight increase in the rheological parameters occurred with the addition of the smallest amount of xanthan gum, but when the water absorption capacity of the samples was higher, with the largest concentration of this hydrocolloid, the rate of increase was reduced, and no differences were observed relative to the control. In the case of psyllium, despite only a few previous studies, the same behaviour could be expected. 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CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten EFFECT OF HYDRATION ON GLUTEN-FREE BREADS MADE WITH HYDROXYPROPYL METHYLCELLULOSE IN COMPARISON WITH PSYLLIUM AND XANTHAN GUM Mayara Belorio, Manuel Gómez Food Technology Area, College of Agricultural Engineering, University of Valladolid, 34071, Palencia, Spain. Enviado al Journal of Food Engineering en 06 de julio de 2020. _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 88 Effect of hydration on gluten-free breads made with hydroxypropyl methylcellulose in comparison with psyllium and xanthan gum Abstract The use of hydrocolloids in gluten-free breads is a strategy to improve their quality and obtain products with acceptable structural and textural properties. Hydration level (HL) optimization is important to maximize the hydrocolloids effects on dough and bread quality. This study evaluated the optimum hydration level (OHL) for gluten-free breads prepared with different starch sources (rice flour or maize starch) and hydroxypropyl methylcellulose (HPMC) in comparison with psyllium and xanthan gum. Breads with the same final volume and the maximum HL were evaluated. Breads made with HPMC had high specific volume and greater dependence on the HL, especially for elaborations with maize starch. Psyllium had similar behaviour to xanthan gum with respect to specific volume and weight loss. Breads elaborated with maize starch and HPMC had low hardness due to their specific volume; however, the combined decreased hydration and similar specific volume generated a harder bread than the use of psyllium or xanthan. 1. Introduction Gluten plays an important role in bread formulation. The gluten network is formed by wheat proteins that with correct hydration and mechanical work, form a cohesive, extensible and elastic dough, which is able to retain the gas formed during fermentation and baking (Delcour et al., 2012). To elaborate gluten-free breads, it is necessary to resort to starches and gluten-free flours, but it is also important to replace gluten with another ingredient. However, a functionally equivalent ingredient has not yet been found that allows for the full replacement of gluten. The _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 95 which bread volume decreases during baking. In fact, Mir et al. (2016) affirmed that the internal viscosity of doughs should not be too low to avoid the release of bubbles during baking. Encina-Zelada et al. (2019) also observed that the specific volume of breads made with xanthan gum or guar gum increased with increasing hydration. In this case, a relation with dough rheology was also mentioned, and it was shown that for high levels of xanthan, it was necessary to add more water. (a) _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 96 (b) Figure 1: Variation of specific volume at different hydration levels for each gluten-free bread formulation and hydrocolloid: a) Rice flour (RF) b) Maize starch (MS). In the case of psyllium, doughs with greater than 100% hydration, although they grew during fermentation, dropped over the edges of the moulds during baking. It is possible that the viscosity was reduced during the early stages of baking (before gelatinization) because of the increase in temperature, which promoted excessively liquid doughs with a weak structure that dropped over the edges of the mould. Thus, over 100% hydration, it was not possible to obtain properly baked rice bread containing 2% of psyllium (Figure 1). The specific volume of breads made with psyllium was similar to those made with xanthan gum. This could be related to the rheological properties of psyllium, which are very similar to those of xanthan gum (Haque et al., 1993). _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 97 Among breads made with MS, those with HPMC had the highest specific volume at the optimum hydration of 80% (Figure 1), and their volume gradually decreased with increasing hydration. This optimum is similar to the results obtained by Sahagún and Gómez (2018) using a very similar formulation. Breads with xanthan gum presented an OHL at 110% hydration, because at 120% the volume of the dough decreased during fermentation and increased again during baking, but it was not larger than that obtained with 110%, and there was no significant difference between the two hydration levels. MS breads with psyllium increased in specific volume up to 90% hydration; however, at this level, breads were completely hollow, which indicated that the dough structure was too weak, and during baking the interior matrix sunk, while at the external surface, a thin crust was formed because of drying that occurred at the beginning of baking. Thus, it was not possible to measure these breads because of their weak structures. MS breads behaved similarly to RF breads, because formulations with HPMC at the OHL had nearly double the specific volume of those breads elaborated with psyllium or xanthan gum; the differences between the latter two were small. It is important to highlight that breads made with MS had a higher specific volume than those made with RF, considering all hydration levels and the different hydrocolloids used. All hydrocolloids (HPMC, psyllium and xanthan gum) increased the final specific volume of breads by almost 50%, considering the maximum specific volume obtained for each of them. The highest specific volumes were previously found with the use of HPMC in comparisons of breads elaborated with MS to those with RF (Martínez and Gómez, 2017; Mancebo et al., 2015a). Martínez and Gómez (2017) attributed these differences to the higher consistency of doughs made with RF, but as observed in Figure 1, all the doughs elaborated with MS had _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 98 large volumes for all hydration levels and hydrocolloids. These authors also suggested another possible explanation, which is based on the presence of a protein layer that covers the starch grains of the flour, modifying the pasting behaviour and increasing the pasting temperature. With respect to the OHL, breads made with HPMC clearly had lower OHL in the presence of MS than with RF, but in the case of xanthan gum this value was higher with MS. In the case of psyllium, it was not possible to compare values of OHL, since they were not determined by considering the maximum specific volume but because of structural problems discovered in case of high hydration levels. However, considering the use of psyllium, the OHL was larger with MS (sinking of the internal structure) than RF (dough dropped outside of the mould during baking). 3.2 Gluten-free bread properties Texture and volume were analysed by using breads with the same final volume to avoid the influence of volume on texture measurements. For this purpose, the amount of dough added to the moulds was re-calculated on the basis of specific volume. In this way, breads made with MS and HPMC were used as references, because they presented similar final volumes to commercial wheat-based breads. Thus, it was necessary to increase the amount of dough in the moulds for other formulations (with psyllium or xanthan). Nevertheless, in some optimally hydrated elaborations, the dough exceeded the height of the mould and dropped over the edges due to the weakness of the dough. These hydrations were disregarded and reduced for each formulation, until the level was reached at which the dough had a sufficiently strong structure to rise over the edges of the mould. Table 1 shows the optimum hydration, specific volume and weight loss for each formulation. The final _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 99 breads are shown in Figure 2. Breads with MS allowed the same OHL (80%) for all the hydrocolloids, even though elaborations with psyllium or xanthan gum generated breads with high specific volumes at high hydration levels. However, breads made with RF and psyllium or xanthan were elaborated with 90% hydration, which was a similar level (100%) to that obtained for the largest specific volume for these hydrocolloids. On the other hand, breads with HPMC were elaborated with only 70% hydration to obtain a final dough with a desirable structure. This hydration was lower than the optimum, and the final specific volumes of these breads were less than one third of the maximum volume obtained, but similar to that of breads made with psyllium or xanthan. Figure 2: Variation of specific volume at different hydration levels for each gluten-free bread formulation and hydrocolloid: a) Rice flour (RF) b) Maize starch (MS). _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 100 Table 1: Variation of specific volume at different hydration levels for each gluten-free bread formulation and hydrocolloid: a) Rice flour (RF) b) Maize starch (MS). OHL (%) Specific Volume (cm³/g) Weight Loss (g/100g) RF HPMC 70 1.33 ± 0.01a 0.0933 ± 0.0088a RF Psyllium 90 1.44 ± 0.02ab 0.0989 ± 0.0018a RF Xanthan 90 1.48 ± 0.03b 0.0976 ± 0.0018a MS HPMC 80 7.58 ± 0.04d 0.2820 ± 0.0012c MS Psyllium 80 2.37 ± 0.08c 0.1660 ± 0.0086b MS Xanthan 80 2.25 ± 0.08c 0.1750 ± 0.0150b Data are expressed as means ± SD of duplicate assays. Values with the same letter in the same column do not present significant differences (p < 0.05). OHL: optimum hydration level. RF: rice flour. MS: maize starch. Table 2 shows the values of texture parameters for the different formulations. Breads elaborated with psyllium or xanthan gum and MS presented greater hardness than those formulated with HPMC. This could be related to the high specific volume of these breads. In fact, this relation between specific volume and hardness was indicated in other studies (Gallagher et al., 2003; Mancebo et al., 2017; Martínez and Gómez, 2017). Xanthan gum and psyllium breads showed similar water losses during baking. However, breads with HPMC lost more water. Other studies found that breads with a high specific volume, such as those elaborated with HPMC, tended to lose more water during baking (Mancebo et al., 2017), in agreement with the results found in this study. Nevertheless, these papers _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 101 considered breads of different final volumes, as they used the same amount of dough in each mould, and water loss is related to the exchange surface. In this study, it was not possible to justify changes in hydration with differences in external surface area, because all breads had the same final volume. Thus, the greater weight loss observed in breads made with HPMC may be related to the low retention capacity of this hydrocolloid compared with xanthan (Horstmann et al., 2018). With respect to the other parameters, there were no significant differences in springiness among the different hydrocolloids. Breads made with xanthan were less cohesive and resilient, although there were no significant differences in resilience between these breads and those made with HPMC. Table 2: Texture parameters of gluten-free breads made with RF or MS for each hydrocolloid. Hardness (N) Springiness Cohesiveness Resilience RF HPMC 42.44 ± 0.21d 0.796 ± 0.004a 0.656 ± 0.023ab 0.383 ± 0.009a RF Psyllium 14.98 ± 0.60c 0.891 ± 0.025b 0.748 ± 0.037c 0.479 ± 0.041bc RF Xanthan 9.04 ± 3.00b 0.922 ± 0.043bc 0.807 ± 0.024c 0.501 ± 0.013bc MS HPMC 1.44 ± 0.12a 1.011 ± 0.023d 0.754 ± 0.030c 0.493 ± 0.034bc MS Psyllium 19.51 ± 3.40c 0.974 ± 0.004cd 0.733 ± 0.037bc 0.550 ± 0.052c MS Xanthan 19.58 ± 1.55c 0.964 ± 0.002cd 0.606 ± 0.037a 0.420 ± 0.047ab _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 102 Data are expressed as means ± SD of duplicate assays. Values with the same letter in the same column are not significantly different (p < 0.05). RF: rice flour. MS: maize starch. Breads made with the different hydrocolloids and RF did not differ significantly among them with respect to weight loss or specific volume. Breads made with HPMC were expected to lose the least amount of weight, because of its high capacity to retain water. However, this effect could be compensated by the high HPMC+flour/water ratio, since these breads had low hydration. We emphasize that breads made with RF lost less weight than those made with MS, which may be related to the lower water retention of starches compared with flours (MatiaMerino et al., 2019), which is probably due to the high protein content of the latter. In contrast to the results obtained for breads with MS, the use of HPMC in RF breads gave greater hardness than other hydrocolloids, while the use xanthan gum produced the softest breads. Various studies found an increase in the hardness of gluten-free breads with the use of xanthan gum when compared with other hydrocolloids (Lazaridou et al., 2007; Schober et al., 2007). The high hardness of breads made with HPMC is a novel finding and is due to the correct hydration and the consequent specific volume of the final breads; in this respect, there were no differences between hydrocolloids. In previous studies, breads with HPMC presented high specific volumes and low hardness (Sabanis and Tzia, 2011). However, rather than being soft, those breads were drier and had a crumblier texture (Liu et al., 2018). Thus, the greater hardness of breads with HPMC found in this study may be attributed to the gels reverting to a weakly entangled form upon cooling, which reduced crumb firmness after baking (Crockett et al., 2011; Grover, 1982). With respect to the differences between breads _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 103 elaborated with MS or RF, those made with HPMC presented the highest specific volumes and the lowest hardness. Nevertheless, despite the higher specific volume of breads elaborated with MS, breads made with psyllium showed a similar hardness, and those made with xanthan gum were even harder. These differences can be explained by the distinct effect of xanthan gum on the pasting properties of the starch, which includes retrogradation between native starches or flours (MatiaMerino et al., 2019). Thus, it seems that maize starch generates harder breads than rice flour. This coincides with the findings of Mancebo et al. (2015b), who reported that the texture of breads made with starch was inferior to that of breads made with rice flour. Regarding other texture parameters, formulations with RF and HPMC presented low values for springiness, cohesiveness and resilience, while those made with psyllium and xanthan had similar values between them. These differences confirmed the distinct behaviour of gels made with HPMC after baking. In comparisons of crust colour (Table 3), breads made with xanthan gum were the darkest (small values of L*), and no significant differences were observed between breads made with HPMC and psyllium. Neither were significant differences observed between breads elaborated with RF and MS, despite of the higher protein content of RF, which could influence Maillard reactions. Values of a* and b* had small significant differences, and no clear tendency was observed. However, breads made with xanthan gum had the largest values of a* in elaborations containing RF and the smallest values of b* among those made with MS. Breads containing HPMC presented the highest values of a* and b* among elaborations with MS. The crust colour of breads is related to the Maillard reaction, which occurs between amino acids and reducing sugars, as well as sugar caramelization (Purlis and Salvadori, 2009). Originally, differences in sugar content and amino acids should not exist _________CAPÍTULO 2: Influencia del psyllium en la elaboración de panes sin gluten 104 between breads elaborated with different hydrocolloids. However, water activity can vary depending on the hydrocolloid and hydration of the doughs; this in turn can affect Maillard reactions favouring the mobility of reactants (Gonzales et al., 2010). In fact, Sabanis and Tzia (2011) also found significant differences among the crust colours of breads made with distinct hydrocolloids. Table 3: Crust colour parameters of gluten-free breads. L* a* b* RF HPMC 81.68 ± 3.05c 1.64 ± 0.25bc 17.15 ± 0.68bc RF Psyllium 79.92 ± 4.67bc 1.23 ± 0.39b 15.36 ± 0.18b RF Xanthan 75.05 ± 0.83ab 4.48 ± 0.08d 20.25 ± 1.22c MS HPMC 82.09 ± 0.04c 2.64 ± 0.14c 19.32 ± 0.22c MS Psyllium 86.20 ± 2.13c -0.05 ± 1.07a 14.56 ± 2.64b MS Xanthan 71.26 ± 1.92a 0.06 ± 0.09a 9.72 ± 1.93a Data are expressed as means ± SD of duplicate assays. Values with the same letter in the same column are not significantly different (p < 0.05). RF: rice flour. MS: maize starch. 4. Conclusion In general, breads elaborated with HPMC and maize starch showed higher specific volumes than elaborations with other hydrocolloids or rice flour. Nevertheless, the degree of hydration of the dough can change these results. The hydration effect is much more evident in breads prepared with HPMC than in those made with psyllium CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos. PSYLLIUM AS A FAT REPLACER IN LAYER CAKES: BATTER CHARACTERISTICS AND CAKE QUALITY. Mayara Belorio, Marta Sahagún, Manuel Gómez Food Technology Area, College of Agricultural Engineering, University of Valladolid, 34071, Palencia, Spain. Food and Bioprocess Technology (2019) 12:2085-2092 CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 115 Psyllium as a fat replacer in layer cakes: batter characteristics and cake quality. Abstract Consumers are demanding healthier and lower calorific products. In this study, oil was substituted in layer cakes using a combination of psyllium and water. Psyllium was used as oil replacer because of its gelling and emulsifying properties, as well as its beneficial health properties. Substitutions of 25, 50, 75 and 100% were carried out to evaluate batter (density and bubbles distribution) and cake characteristics (specific volume, weight loss, texture and colour). An acceptability test was also made. A higher substitution of oil increased bubbles size, but no significant difference was observed in batter density. Increasing the oil replacement decreased the specific volume of cakes, however there was no significant difference in hardness compared to control cake, even after seven days of storage. There was no significant difference in cohesiveness and springiness when 25% of the oil was replaced, but it did increase with higher substitution levels. Crust colour became lighter with increases in oil replacement, showing smaller values to a* and higher to b*. Cakes replaced with 25% of psyllium:water showed no significant differences in acceptability when compared to control cakes, but those with up to 75% of oil replacement were highly acceptable (7 out of 9 points). 1. Introduction Obesity and heart disease affect many people, and in most cases, they are related to the consumption of large amounts of fat. People have adopted some changes to their eating habits because of both health and lifestyle reasons. In this way, a reduction of CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 116 fat in food would provide new possibilities to those consumers who look for a decrease in fat. Among bakery products, layer cakes have the highest fat content (Matz 1992). Fats or oils facilitate the incorporation of air in batter contributing to an increase in volume during baking and improving the final tenderness of the cake (Psimouli and Oreopoulou 2013). Distinct hydrocolloids and fibres have been used as fat replacers in cookies (Forker et al. 2011; Rodríguez-García et al. 2012a). Several oil and fat replacement ingredients has been proposed in muffins and cakes, such as green banana puree (Oliveira et al. 2018), avocado puree (Othman et al. 2018) or berry pomace (Quiles et al. 2018) and different fibres such as chia mucilage (Felisberto et al. 2015), functional ingredients derived from flaxseeds (Eslava-Zomeño et al. 2016), succinyl chitosan (Rios et al. 2018), cocoa fibres (Karp et al. 2017), resistant maltodextrin or potato fibre (Diez-Sánchez et al. 2018), inulin (Zahn et al. 2010; Rodríguez-García et al. 2012b, 2014; Majzoobi et al. 2018) and guar and xanthan gums (Zambrano et al. 2004) . All these studies found that batter with lower fat content showed changes in rheology and greater bubbles size, resulting in harder cakes and, in most cases, smaller volumes. Moreover, no one of these studies showed a fat reduction of more than 30% without changing the sensory properties and acceptability of the cakes. Psyllium is a natural fibre obtained from a tropical plant known as Plantago genus. This fibre has similar properties to xanthan gum (Haque 1993) exhibiting good gelling and emulsifying properties when applied as fat replacers (Yu 2008). Psyllium has also been used extensively both as a pharmacological supplement and in processed food to aid weight control, to regulate glucose control for diabetic patients and to reduce serum lipid levels in hyperlipidaemic (Singh 2007). CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 117 The objective of this work is to replace oil in layer cake formulations (oil substitutions of 25, 50, 75 and 100%) by hydrated psyllium (one tenth psyllium and nine tenths water). The density and microscopy of the cake batters were evaluated. Cake characteristics, such as specific volume, weight loss, texture (fresh cakes and seven-day old cakes) and colour (crust and crumb) were also measured. In addition, a sensory analysis was carried out to evaluate cake acceptability. 2. Materials and Methods 2.1 Materials Wheat flour (10.9g/100g moisture; 8.98g/100g protein) and psyllium used in the layer cakes preparation was supplied by Harina Castellana S.A (Valladolid, Spain) and Rettenmaier Ibérica (Barcelona, Spain), respectively. The other ingredients used were liquid whole milk (Lactalis Food Service Iberia, Madrid, Spain), liquid pasteurised eggs (Alvarez Camacho Sevilla, Spain), refined sunflower oil (Urzante, Navarra, Spain), baking powder (Puratos, Gerona, Spain), and white sugar (AB Azucarera Iberia, Valladolid, Spain. 2.2 Methods 2.2.1 Cake Elaboration Layer cakes were prepared according to the following formulation: 350g wheat flour, 315g white sugar, 210g milk, 175g liquid pasteurised eggs, 105g sunflower oil and 10.5g baking powder. All ingredients were mixed for 10 minutes (speed 4 for 1 minute and speed 6 for 9 minutes) using a professional mixer (Kitchen Aid, St. Joseph, Michigan, USA. The reduced fat cakes were made by replacing the sunflower oil by a mixture of psyllium and water (ratio of 1:9) in different proportions (25, 50, CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 118 75 and 100%). The proportion of psyllium:water was determined in preview tests to maintain batter viscosity similar to the final viscosity of the control batter. All the formulations are shown in Table 1. The cake batter (185g) was placed into an oil-coated aluminium pan (127 x 98 x 33mm) and baked at 190 °C for 25 minutes. After baking, the cakes were left to cool at room temperature for 60 minutes, then placed in plastic bags to avoid drying out and put in a chamber at 20 °C until analysis. All the cakes were made in duplicate. 2.2.2 Batter Characteristics Batter density was evaluated after the mixing process using an Elcometer 1800 pycnometer (Manchester, UK). Batter microstructure was evaluated at 20 times magnification using a DM750 microscope (Leica Microsystems, Wetzlar, Germany) and the images were captured using LAS-EZ software (Leica Microsystems, Wetzlar, Germany). Before being submitted for analysis, a sample of the batter was placed on a glass slide covered with a lip. To obtain a uniform thickness, a weight of 1kg was supported on the cover. All the analyses were performed in duplicate. CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 119 Table 1: Composition of cakes formulations (g/100g wheat flour) evaluated. Ingredients Control WF25 WF50 WF75 WF100 WF 100 100 100 100 100 White sugar 90 90 90 90 90 Milk 60 60 60 60 60 Liquid pasteurized egg 50 50 50 50 50 Sunflower oil 30 22.5 15 7.5 - Baking powder 3 3 3 3 3 Psyllium - 0.75 1.50 2.25 3 Water - 6.75 13.50 20.25 27 WF: wheat flour. WF25: layer cake with 25% of oil replacement. WF 50: layer cake with 50% of oil replacement. WF75: layer cake with 75% of oil replacement. WF100: layer cake with 100% of oil replacement. To maintain similar final viscosities, the viscosity of the batters was evaluated using a Rapid Visco Analyser (RVA-4) (Newport Scientific model 4-SA, Warriewood, Australia) with 25g of cake batter (Sahagún et al. 2018). The final viscosity obtained for all the samples was equal to the control (3257 cp) with a margin of ± 2%. Analysis was carried out at 30 °C after stirring for 3 minutes at 160 rpm. 2.2.3 Cake Specific Volume and Weight Loss Specific volume was obtained using the ratio between the volume of the cakes and their weight. Cake volume was determined by a Volscan Profiler volume analyser CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 120 (Stable Microsystems, Surrey, UK). Weight loss was calculated by considering the difference between the weight of the batter placed in the pan and the weight of the cakes after baking. The analyses were carried out in duplicate on each batch at 24 hours after baking, being cakes stored in plastic bags in a chamber at 20 °C until the measurement. 2.2.4. Texture Analysis Crumb texture was evaluated using a 25mm diameter cylindrical aluminium probe to execute a Texture Profile Analysis (TPA) with a double compression test at 50% of depth penetration, a test speed of 2 mm/s and a delay of 30 seconds between the first and second compression. The tests were carried out using a TA-XT2 texture analyser (Stable Microsystems, Surrey, UK). Two central slices (each 2cm thick) of two cakes from each batch (2x2x2) were evaluated. Hardness, cohesiveness and springiness values were obtained from the TPA graphic (Gómez et al. 2007). Texture parameters were evaluated on cakes stored after 24 hours and seven days in plastic bags in a chamber at 20 °C. 2.2.5 Crust and Crumb Colour Crust colour was measured at four different points of the cake surface in four cakes of each batch (4x4x2). Crumb colour was evaluated at the central point of four different slices obtained from two cakes of each batch (4x2x2). The analyses were carried out using a Minolta CN-508i spectrophotometer (Minolta Co., Ltd., Osaka, Japan) and a D65 illuminant with the 2° standard observer. Values were expressed in the CIE L* a* b* colour space and the parameter ∆E*, which refers to the total colour difference between the control and other cake samples with different levels of oil substitutions. It was calculated according to the equation: CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 127 The values with the same letter in the same column do not present significant differences (p<0.05). WF25: layer cake with 25% of oil replacement. WF 50: layer cake with 50% of oil replacement. WF75: layer cake with 75% of oil replacement. WF100: layer cake with 100% of oil replacement. Regarding the rest of parameters, there was an increase in cohesiveness in those cakes with higher levels of fat substitution, with significant differences in substitutions of above 50%. However, there was no clear trend in the springiness of the cake. Some authors did not find significant differences of cohesiveness when using various levels of fat replacements (Zahn et al. 2010; Rodríguez-García et al. 2012b; Román et al. 2015; Eslava-Zomeño et al. 2016; Diez-Sánchez et al. 2018). Some studies showed increasing springiness values with higher fat substitutions (Zahn et al. 2010; Rodríguez-García et al. 2014; Diez-Sanchéz et al. 2018) while others reported decreasing values (Román et al. 2015) or no differences (EslavaZomeño et al. 2016) when the fat was replaced. These results indicate that texture parameters differ depending on the fat substitute used. During storage, hardness increased and cohesiveness was reduced for all cakes. These changes were independent of the differences in the percentage of oil replacement. As it was observed in a previous study, there was correlation between initial hardness and its evolution over time (Gómez 2008), which confirms our observations. The results obtained for the hardness evolution coincided with those reported by Román et al. (2015) for cakes with extruded wheat flour which indicated that these type of oil replacements do not modify the texture of the cakes during storage. CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 128 Colour parameters are shown in Table 4. The crust of the cakes made with oil replacer showed increasing L* values, which means they were lighter than the others, but significant differences were only found in substitution levels above 75%. Samples WF75 and WF100, besides showing significant differences to ∆E*, they also presented significant differences in a* and b* values, which decreased and increased, respectively. High darkness of the crust was expected because of caramelisation and Maillard reactions which occurs during the baking process (Purlis 2010). However, the quantities of sugar and protein were constant for all samples, so this does not explain the changes in crust colour. The influence of lipids on this type of reaction could explained the changes of colour observed, since a derivate substance coming from their oxidation modify products colour while present (Hidalgo and Zamora 2000). Table 4. Crust and crumb colour of layer cakes with reduced fat content. CONTROL WF25 WF50 WF75 WF100 Crust ∆E* 0,00 ± 0,00a 2,47 ±1,78a 3,25 ± 0,33a 15,82 ± 0,33b 17,89 ± 3,13b L* 51,99 ± 0,83ab 50,77 ± 1,05a 53,92 ± 0,85b 64,31 ± 0,86c 65,30 ± 1,84c a* 13,72 ± 0,47b 13,36 ± 0,47b 13,03 ± 0,47b 8,21 ± 0,47a 8,40 ± 0,47a b* 15,45 ± 2,12a 14,72 ± 0,59a 17,96 ± 1,80a 23,67 ± 1,29b 26,13 ± 0,77b Crumb ∆E* 0,00 ± 0,00a 0,88 ± 0,88ab 1,02 ± 0,63ab 0,66 ± 0,11a 2,97 ± 1,53b L* 75,71 ± 1,46a 76,36 ± 2,45a 76,29 ± 2,34a 75,37 ± 1,60a 72,88 ± 2,98a a* -0,135 ± 0,06a -0,27 ± 0,28a -0,11 ± 0,23a -0,045 ± 0,26ab 0,64 ± 0,40b b* 15,74 ± 0,43a 15,91 ± 1,01a 15,86 ± 1,39a 15,73 ± 1,19a 15,755 ± 1,04a CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 129 The values with the same letter in the same column do not present significant differences (p<0.05). WF25: layer cake with 25% of oil replacement. WF 50: layer cake with 50% of oil replacement. WF75: layer cake with 75% of oil replacement. WF100: layer cake with 100% of oil replacement. No significant differences of L* and b* values were observed in the crumb colour of those cakes made with oil replacement compared with control cakes and Figure 2 shows the crumbs for each formulation. However, greater fat substitutions presented significant differences to ∆E* compared to the control. High a* values were only observed in cakes with total oil substitution, whereas some studies of fat or oil replacement found major differences. Felisberto et al. (2015) obtained darker crumbs when using chia mucilage gel and lighter ones when incorporating inulin in the cakes (Rodríguez-García et al. 2012b). The temperature inside the cakes does not exceed 100 °C, so Maillard reactions and caramelisation cannot occur. In this way, the crumb colour depends mainly on the colour of the ingredients. Therefore, the differences found in these studies depends on the oil employed and the oil replacer. Fradinho et al. (2015) observed that cookies with psyllium incorporated had increased values of L* which was attributed to the darker colour of the psyllium. However, it must be noted that in our study the quantity of water added to the cake formulations was important as it diluted this effect. CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 130 Figure 2: Cellular structure from the crumb of the cakes. 3.3. Consumer Test Results of sensory evaluation are shown in Table 5. In general, the control and cakes with 25% of the oil replaced presented higher values for all the sensory parameters, with significant differences in taste, texture and overall acceptability compared to the rest samples. The cakes with 50 and 75% of oil replacement did not show any significant differences either in all sensory parameters but produced minor differences in the values for taste and texture compared to the control cakes, which reduced their global acceptability. However, all the cakes obtained a high evaluation (nearer to 7 and above 9) with differences that did not exceed 0.5 points between CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 131 the control cakes and those with 75% of oil substitution. The cakes with 100% of oil replacement produced worse values than the control cakes in all aspects evaluated and were worse than all the other samples in aspects including odour, texture and overall acceptability. It must be considered that these cakes had a lower specific volume and lighter crust colour which could have negatively influenced the evaluations of the consumers. The texture of the cakes substituted with oil were given poor evaluations by the consumers, except to the cakes with 25% of substitution. Other studies also found differences in the acceptability of cakes with fat replacement. Cakes made with green banana puree were less acceptable due to their darker colour (Oliveira de Souza et al. 2018). Diez-Sánchez et al. (2018) found that using potato fibre as a substitute produced lower acceptability levels when 30% of fat was substituted which was attributed to a deterioration in their texture. When using inulin as a substitute, Rodríguez-García et al. (2012) observed minor levels of acceptability of cakes with 100% of fat replacement, which was explained by the crumbling and irregular crumb cell structure. In general, oil or fat substitution by fibres or other ingredients reduced consumers’ acceptability of the cakes, but in the case of psyllium, this reduction was very small, and the products received a good global evaluation. Therefore, it might be possible to replace oil in cakes up to 25% with hydrated psyllium without reducing their acceptability and up to 75% with only a minimum decrease in sensorial evaluation. CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 132 Table 5: Sensorial acceptability of layer cakes with reduced fat content. Sample Appearance Odour Taste Texture Overall acceptability CONTROL 7.27 ± 1.24b 7.36 ± 1.30b 7.33 ± 1.34 c 7.15 ± 1.31c 7.45 ± 1.10c WF25 7.23 ± 1.25b 7.27 ± 1.26b 7.14 ± 1.41bc 7.14 ± 1.28c 7.39 ± 1.01c WF50 7.41 ± 1.08b 7.03 ± 1.32b 6.78 ± 1.28b 6.60 ± 1.33ab 7.00 ± 1.08b WF75 7.17 ± 1.35b 6.97 ± 1.30b 6.74 ± 1.56ab 6.70 ± 1.41b 6.99 ± 1.33b WF100 6.08 ± 1.96a 6.44 ± 1.54a 6.32 ± 1.64a 6.20 ± 1.52a 6.34 ± 1.51a The values with the same letter in the same column do not present significant differences (p<0.05). WF25: layer cake with 25% of oil replacement. WF 50: layer cake with 50% of oil replacement. WF75: layer cake with 75% of oil replacement. WF100: layer cake with 100% of oil replacement. 4. Conclusion The incorporation of pre-hydrated psyllium can be a convenient alternative for reducing the oil percentage in cake formulations. The higher oil replacement developed an irregular batter structure with greater bubbles and less stability, which results in cakes with lower specific volume. However, these volume changes did not produce changes in hardness for any substitution level although the cohesiveness increases slightly. For its part, crust colour gets lighter as the oil content in the formulation decreases. Despite these changes, cakes with 25% of oil substitution did not produce significant differences in consumer’s acceptability compared to the control cakes. In addition, cakes with 75% of replaced oil obtained acceptability scores higher than 6 points by consumers. Therefore, it is possible to CAPÍTULO 3: Empleo del psyllium como sustituto de grasa en bizcochos 133 produce cakes with a lower lipid content and higher consumer acceptability by using psyllium. 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