Desarrollo de sistemas de envasado activo mediante la ... - RUA [PDF]

DESARROLLO DE SISTEMAS DE ENVASADO ACTIVO MEDIANTE LA FORMULACIÓN DE MATRICES POLIMÉRICAS Y NANOCOMPUESTOS CON AGENTES ANTIOXIDANTES Y ANTIMICROBIANOS DE ORIGEN NATURAL

Marina Ramos Santonja

Departamento de Química Analítica, Nutrición y Bromatología Departament de Química Analítica, Nutrició i Bromatologia

DESARROLLO DE SISTEMAS DE ENVASADO ACTIVO MEDIANTE LA FORMULACIÓN DE MATRICES POLIMÉRICAS Y NANOCOMPUESTOS CON AGENTES ANTIOXIDANTES Y ANTIMICROBIANOS DE ORIGEN NATURAL

Marina Ramos Santonja Programa de Doctorado en Química Tesis presentada para aspirar al grado de DOCTORA POR LA DE ALICANTE MENCIÓN DE DOCTORA INTERNACIONAL

Dirigida por: Profesor Alfonso Jiménez Migallón Profesora María del Carmen Garrigós Selva Doctora Mercedes Ana Peltzer

Departamento de Química Analítica, Nutrición y Bromatología Departament de Química Analítica, Nutrició i Bromatologia

Dña. MARÍA SOLEDAD PRATS MOYA Directora del Departamento de Química Analítica, Nutrición y Bromatología de la Facultad de Ciencias de la Universidad de Alicante Certifica: Que Dña. MARINA RAMOS SANTONJA ha realizado bajo la dirección de los profesores Dr. D. ALFONSO JIMÉNEZ MIGALLÓN y Dra. Dña. MARIA DEL CARMEN GARRIGÓS SELVA de la Universidad de Alicante, el trabajo bibliográfico y experimental correspondiente a la obtención del Grado de Doctor en Química sobre el tema: ”Desarrollo de sistemas de envasado activo mediante la formulación de matrices poliméricas y nanocompuestos con agentes antioxidantes y antimicrobianos de origen natural”. Alicante, Enero 2016

Fdo. Dra. María Soledad Prats Moya

Departamento de Química Analítica, Nutrición y Bromatología Departament de Química Analítica, Nutrició i Bromatologia

Los profesores Dr. D. ALFONSO JIMÉNEZ MIGALLÓN y Dra. Dña. MARIA DEL CARMEN GARRIGÓS SELVA del Departamento de Química Analítica, Nutrición y Bromatología de la Universidad de Alicante, en calidad de Directores de la Tesis Doctoral presentada por Dña. MARINA RAMOS SANTONJA, con el título: “Desarrollo de sistemas de envasado activo mediante la formulación de matrices poliméricas y nanocompuestos con agentes antioxidantes y antimicrobianos de origen natural”. Certifican: Que la citada Tesis Doctoral se ha realizado en el Dpto. de Química Analítica, Nutrición y Bromatología de la Universidad de Alicante, y en los centros “Materials Science and Technology Center, Department of Civil and Environmental Engineering, Universidad de Perugia (Italia)”, y “School of Food & Nutritional Sciences, Universidad de Cork (Irlanda)”; y que, a su juicio, reúne los requisitos necesarios y exigidos en este tipo de trabajos. Alicante, Enero 2016

Fdo. Dr. Alfonso Jiménez Migallón

Fdo. Dra. María del Carmen Garrigós Selva

Agradecimientos Llegado este punto, queda terminar con la última parte de este trabajo, o mejor dicho una de las aventuras hasta el momento más importantes en mi carrera profesional. Podría decirse que la culminación de muchos años de esfuerzo y sacrificio pero que han estado cargados de momentos para el recuerdo, dulces, otros más amargos, felices, bonitos, inolvidables pero también tristes, así como situaciones irrepetibles que quedarán en mi memoria y que muchas veces me harán sonreír. Mucha gente ha participado en este camino desde que comencé mi carrera en 2002 hasta este momento, y de una forma u otra siempre han estado para poner una piedrecita en este tortuoso camino para ayudarme a avanzar y conseguir mi meta. ¡A tod@s gracias! A mis directores, el Profesor Alfonso Jiménez Migallón, la Profesora María del Carmen Garrigós Selva y la Doctora Mercedes Ana Peltzer por brindarme la oportunidad de poder formarme como investigadora y sobre todo como persona, transmitiéndome toda la pasión e ilusión por lo que hacemos. Por su paciencia, dedicación y sus palabras de aliento en momentos difíciles. Y sobre todo por la confianza que depositaron en mí sin apenas conocerme. Porque gracias a ellos me encuentro escribiendo estas líneas. ¡Muchísimas gracias! A mi grupo de investigación “Análisis de Polímeros y Nanomateriales”, porque somos un pequeña gran familia. A mis envasologas poliméricas: Arancha, Nuria y Cris. Chicas muchas gracias por haberme acompañado en esta aventura. Nos hemos reído, hemos llorado, hemos soñado y hemos disfrutado del día a día juntas. Sin vosotras muchas partes de este trabajo no tendrían sentido, porque siempre me habéis ayudado. Y en momentos de bajón, siempre ha habido una palabra que me ha hecho cambiar el rumbo. Al Departamento de Química Analítica, Nutrición y Bromatología y a todos mis compañeros por la ayuda ofrecida. A la Universidad de Alicante por la concesión de la beca predoctoral y al Ministerio de Economía y Competitividad por la financiación económica suministrada a través de la concesión de los Proyectos de Investigación MAT2011-28468-C02-01 y MAT2014-59242C2-2-R. A mis compañer@s del departamento, por los momentos durante las comidas y las “charretas” por los pasillos o en nuestra sala de becarios, que de una forma u de otra me han ayudado a disfrutar de mi trabajo. Así mismo, hago extensivos estos agradecimientos al Profesor José María Kenny del “Dipartamento di Ingegneria Civile e Ambiantale” de la Universidad de Perugia (Italia) por

haberme brindado la oportunidad de realizar parte del trabajo experimental en el grupo de investigación que él mismo dirige y de este modo ayudarme a ampliar mis conocimientos. En especial a la Doctora Elena Fortunati a quien admiro como persona y profesional e hizo que mi estancia en Italia fuera inolvidable. Al Profesor Joseph Kerry del “Food Science Department” de la Universidad de Cork (Irlanda) por haberme dado la oportunidad de trabajar con su grupo de investigación adquiriendo interesantes conocimientos. En especial a los Doctores Stefano Molinaro y Malco Cruz por su ayuda y paciencia durante toda la estancia. A los Profesores Juan López Martínez y Rafael Balart Gimeno y los compañeros del “Instituto de Tecnología de Materiales (ITM)” de la Universidad Politécnica de Valencia (UPV) por su colaboración y ayuda en el trabajo realizado. A Artur J. M. Valente por su colaboración y ayuda en el trabajo realizado. A Ana porque contigo empecé. Te armaste de paciencia y me enseñaste lo que yo hoy intento enseñar. ¡Siempre serás una gran amiga! A mis amigas y amigos que entre risas comenzaron a entender la importancia de los envases, y que en momentos inolvidables siempre era, “Marina que trabaja en polímeros, con polipropileno y compuestos del orégano, ¿es que no sabes que es un envase activo…?”. A Teresa por ser mi amiga y hermana. A mi hermano por sus consejos y aguantarme en días malos. Por todos esos momentos de risas y aunque en estos últimos años hayamos estado de aquí para allá, siempre te he sentido a mi lado, nuestra complicidad nos hace únicos. ¡Gracias por estar siempre! A mis padres, pilar fundamental en mi vida. Gracias por todo vuestro apoyo por alentarme y estar siempre a mi lado. Por enseñarme a ser fuerte y a luchar por los sueños que uno mismo persigue. Porque desde la humildad siempre me habéis enseñado a ser mejor persona y ver siempre el lado bueno de las cosas. ¡Muchas gracias por todo vuestro apoyo y estar siempre a mi lado! A Juan, porque tu has hecho especial todos estos momentos. Me has apoyado desde el principio, y nunca me has dejado tirar la toalla. Comencé este camino a tu lado y siempre nos hemos adaptado a los cambios. Has comprendido este trabajo y soy muy feliz de poderlo compartir contigo. ¡Muchísimas gracias! ¡A Olga porque cuando sea mayor entenderá el significado de estas palabras para su mamá!

Resumen1

1En el resumen no se han incluido referencias.

Resumen

1. Introducción El sector del envasado representa uno de los principales sectores en la industria del plástico a nivel mundial y ha experimentado en los últimos años un continuo crecimiento debido a la diversidad de productos y aplicaciones que comprende. Los plásticos utilizados en envasado alimentario deben contribuir al mantenimiento de la calidad y seguridad de los alimentos desde su procesado hasta su consumo, incluyendo en este ciclo el almacenamiento y el transporte. Para ello, los envases alimentarios deben proporcionar protección mecánica, óptica y térmica a los alimentos envasados, además de preservarlos contra factores o condiciones de degradación,

tales

como

microorganismos,

oxígeno,

humedad,

contaminantes químicos, radiación y elevadas temperaturas. Los polímeros convencionales han sido tradicionalmente muy utilizados en este sector, en especial los polímeros termoplásticos derivados del petróleo cuya introducción se produjo en las décadas de 1950 y 1960. Su uso se extendió rápidamente debido a su gran versatilidad que comprende un amplio abanico de propiedades que los hace adecuados para aplicaciones específicas en el ámbito de la alimentación. Entre dichas propiedades cabe destacar su alta disponibilidad a un coste relativamente bajo y su buen comportamiento físico-químico en condiciones de tracción y resistencia al desgarro; además de la buena barrera al oxígeno, vapor de agua, CO2 y aromas que ofrecen, así como la buena capacidad de procesamiento. Sin embargo, estos polímeros presentan el doble inconveniente de su origen no renovable y su baja capacidad de degradación en condiciones naturales, por lo que en los últimos años son cada vez más las voces que abogan por el uso de biopolímeros en sistemas de envasado, materiales

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que se caracterizan por su biodegradabilidad y su origen a partir de fuentes renovables. Su desarrollo en las dos últimas décadas los hace merecedores de ser considerados una alternativa válida para reducir significativamente el impacto medioambiental de los plásticos convencionales, considerando la problemática relacionada con su eliminación, aliviando, a su vez, la dependencia del petróleo y otras fuentes no renovables. El uso de estos biopolímeros, como pueden ser el poli(ácido láctico) (PLA) o el almidón termoplástico (TPS), está cada vez más introduciéndose en el cada vez más competitivo mercado del envase alimentario. Por otra parte, la idea de mejorar la calidad y ampliar la vida útil de los alimentos, mientras se mantienen sus propiedades organolépticas y nutritivas, está generando un importante interés en el entorno científico y de la industria alimentaria. De hecho, el concepto tradicional de envase alimentario que basa su función en contener y preservar al alimento sin que se produzca ninguna interacción entre éste y el material de envase, ha evolucionado con el fin de permitir interacciones entre ellos, así como con el medioambiente. Este nuevo concepto es el de envase activo, y se basa en favorecer el proceso de transferencia de masa e interacción entre los materiales de envase y los alimentos. El objetivo principal de los sistemas de envasado activo es alargar la vida útil de los productos envasados, minimizando o suprimiendo los efectos negativos producidos por agentes del entorno que producen los procesos de descomposición del alimento. Entre dichos procesos destacan la presencia de microorganismos y las oxidaciones de lípidos, que causan pérdidas inaceptables en la calidad del alimento, haciéndolo inviable para su consumo en un corto tiempo. El creciente interés por este tipo de envases y la demanda de los consumidores en lo que se refiere a productos naturales, seguros, frescos y de larga vida útil ha incrementado el estudio del uso de aditivos de origen

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Resumen

natural, como extractos de plantas y aceites esenciales, en sistemas de envasado. Estos productos se clasifican como “Generally Recognized as Safe” (GRAS) por la “Food and Drug Administration” (FDA) de Estados Unidos, así como por la Legislación Europea actual para materiales destinados a estar en contacto con alimentos. Los aditivos naturales pueden ser obtenidos de diferentes fuentes, incluyendo plantas, animales, bacterias, algas u hongos, además de subproductos generados en la industria alimentaria como pueden ser, por ejemplo, la cáscara de la almendra, deshechos de salvia o de cascara de naranja. Los aceites esenciales y sus principales compuestos constituyentes son algunos de los agentes activos más estudiados para ser incorporados como aditivos activos en formulaciones poliméricas, sean estos de origen natural o sintético, ya que son aditivos naturales que provienen de fuentes renovables, son fáciles de obtener y presentan un elevado carácter antimicrobiano y/o antioxidante. Entre ellos, destacan el carvacrol (5isopropil-2-metilfenol)

y

timol

(2-isopropil-5-metilfenol,

dos

monoterpenos fenólicos que se caracterizan por ser dos de los aditivos naturales más estudiados debido a sus excelentes propiedades antioxidantes y antimicrobianas, descritas ampliamente en la literatura científica. Estas propiedades, que les confieren un amplio abanico de posibilidades en el área de los materiales para envasado activo, se deben a la presencia de grupos hidroxilo, altamente reactivos, en su estructura. Ambos compuestos son isómeros obtenidos a partir de diferentes plantas aromáticas y aceites esenciales de la familia de las Labiatae, incluyendo las especies Origanum, Satureja, Thymbra, Thymus y Corydothymus. Por otra parte, la aplicación de los conceptos básicos de la nanotecnología a la ciencia de materiales ha constituido una revolución en los paradigmas

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de dicha ciencia, ya que se han desarrollado nuevos nanomateriales con resultados novedosos y sorprendentes. En el área del envasado de alimentos estos nanomateriales han supuesto un cambio considerable debido a las nuevas propiedades y funciones que aportan, que han hecho que el uso de nanocargas haya ganado importancia como aditivos en materiales de envasado. Investigaciones recientes centradas en el uso de nanopartículas han permitido crear, entender, caracterizar y usar estos nanocompuestos debido a su acción en la mejora de algunas de sus propiedades clave tales como la resistencia mecánica, propiedades de barrera a gases o estabilidad térmica. Asimismo, un aspecto de alta relevancia para este trabajo es que la adición de nanopartículas con propiedades

antimicrobianas

y/o

antioxidantes

resulta

en

una

combinación excelente con los aditivos activos para conseguir la máxima protección en los alimentos envasados. El uso de nanomateriales en combinación con sistemas de envasado activo ha permitido mejorar la integridad estructural y las propiedades de barrera de las matrices poliméricas debido a la adición de los nanomateriales (ya sea nanoarcillas o nanopartículas metálicas), así como la mejora en el comportamiento antimicrobiano y/o antioxidante por la acción de los aditivos activos, como los aceites esenciales de orégano o clavo o bien compuestos con actividad intrínseca como el timol, carvacrol, tocoferoles o hidroxitirosol. Las nanopartículas de cobre, zinc, titanio, oro y plata, así como algunos de sus óxidos metálicos, también han sido propuestas como aditivos activos con el fin de extender la vida útil de los alimentos y proporcionar estrategias innovadoras, aceptables y seguras para desarrollar nuevos nanocompuestos activos. Una vez desarrollado el nanocompuesto activo su efecto sobre el alimento debe ser evaluado ya que dependerá de la actividad de cada aditivo y de

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Resumen

sus propiedades, así como del tipo de alimento considerado, su composición y contenidos en grasa y agua. Se ha indicado que los alimentos lipídicos tienden a oxidarse más fácilmente que los acuosos, así como que los alimentos procesados pueden tener una mayor tendencia a sufrir una contaminación cruzada que conlleve al deterioro del producto. En este sentido, el polímero desempeña el papel más importante en la acción de liberación de los aditivos mediante el control de la difusión de los componentes activos en el seno de la matriz polimérica y la distribución homogénea de las nanocargas en su estructura. La aplicación de los sistemas de envasado activo, en especial aquellos que comprenden el uso de nanocompuestos, debe ser evaluada de forma que se pueda determinar de forma inequívoca la seguridad para el consumidor de estos materiales en contacto con alimentos, siempre considerando las normativas internacionales que limitan, prohíben y/o autorizan el uso de este tipo de aditivos. En la actualidad, se están desarrollado un número elevado de investigaciones para evaluar la posibilidad de desarrollo y utilización de nanocompuestos con propiedades activas mediante la combinación

de

diferentes

tipos

de

nanocargas

(nanoarcillas,

nanocelulosas, nanopartículas de plata, etc.) con aditivos de origen natural con propiedades antimicrobianas y/o antioxidantes en matrices poliméricas. Los principales esfuerzos en el desarrollo de estos materiales se han centrado en estudiar sus propiedades funcionales mediante el uso de métodos de determinación del comportamiento antimicrobiano y antioxidante, estudios de desintegración, toxicológicos y de migración.

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Resumen

2. Objetivos El objetivo general de este trabajo es el desarrollo de nuevos sistemas activos de envasado con capacidad de protección de las características organolépticas y nutricionales de los alimentos y prolongada vida útil. Para ello, en el presente trabajo se han desarrollo dos líneas básicas de investigación: (1) sistemas de envasado activo utilizando polipropileno (PP) como polímero base en combinación con timol y/o carvacrol; y (2) nanocompuestos activos utilizando PLA como matriz biodegradable en combinación con timol y dos tipos de nano-refuerzos comerciales, una nanoarcilla (Dellite®43B, D43B) y nanopartículas de plata (Ag-NPs). Para satisfacer el objetivo principal del presente trabajo se han planteado una serie de objetivos específicos que se enumeran a continuación: i.

Desarrollo y caracterización de películas basadas en PP con carvacrol y timol. Se han estudiado diferentes propiedades físicoquímicas de las películas activas obtenidas (morfología, comportamiento mecánico, propiedades térmicas y propiedades de barrera). La actividad antimicrobiana proporcionada por los aditivos a estos sistemas de envasado fue evaluada frente a una bacteria Gram positiva (Staphylococcus aureus y otra Gram negativa (Escherichia coli). También se estudió la liberación de timol y carvacrol desde la matriz polimérica hacia distintos simulantes alimentarios; incluyendo un estudio cinético para modelar el comportamiento de migración de estos dos compuestos en diversos medios. También se ha evaluado la actividad antioxidante de los extractos obtenidos. Por último, la eficiencia de las nuevas películas activas fue evaluada estudiando el aumento de la vida útil de dos muestras de alimentos (fresas y pan de molde) almacenados en diferentes condiciones.

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Resumen

ii.

Desarrollo y caracterización de nanocompuestos activos en una matriz de PLA y usando timol como aditivo activo. En este punto, dos formulaciones diferentes fueron propuestas. a.

Nanocompuestos activos en base PLA con timol y una nanoarcilla

comercial

orgánicamente

modificada

(Dellite®43B, D43B). La caracterización de estos nanocompuestos se llevó a cabo estudiando sus propiedades morfológicas, mecánicas, térmicas, ópticas y de barrera a gases. También se llevó a cabo la evaluación de la velocidad de desintegración en condiciones de compostaje, así como el estudio de la liberación de los compuestos

activos

en

condiciones

controladas

utilizando un simulante alimentario acuoso y la actividad antimicrobiana y antioxidante de las nuevas películas activas. b. Nanocompuestos activos utilizando PLA, timol y nanopartículas de plata, procesando dos tipos de morfologías: películas y probetas. Se llevó a cabo una caracterización completa de las nuevas formulaciones, en lo referente a sus propiedades morfológicas, mecánicas, térmicas, ópticas y de barrera a oxígeno y vapor de agua. La desintegración de estos nanocompuestos fue evaluada en condiciones de compostaje. Se llevaron a cabo estudios de liberación en un simulante alimentario acuoso. Por último, se estudió la actividad antioxidante y la actividad antimicrobiana de estos nanocompuestos activos.

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3. Resultados y discusión 3.1. Capítulo 1 El timol y el carvacrol han sido seleccionados para ser adicionados a las nuevas formulaciones activas en base PP debido a sus características antimicrobianas y antioxidantes y su origen natural. Un total de nueve formulaciones activas se han obtenido con 4, 6 y 8 % en peso de timol y/o carvacrol, que fueron utilizadas para obtener las diferentes películas activas. Una vez obtenidas las películas mediante mezclado en fundido seguido de moldeo por compresión se caracterizaron evaluando sus propiedades térmicas, morfológicas, mecánicas y funcionales. Los resultados obtenidos en este capítulo mostraron que la presencia de los aditivos no afectó a la estabilidad térmica de la matriz de PP, pero sí que produjo una disminución significativa de la cristalinidad del material debido a las interacciones entre la matriz polimérica y los aditivos. Esta disminución en la estructura cristalina del material tiene una importante influencia sobre los ensayos de tracción, donde se observó un significativo descenso en el módulo elástico y por tanto en la rigidez del material. Estos resultados además indicaron que la presencia de timol y carvacrol tuvieron un ligero efecto plastificante sobre la matriz polimérica. Las imágenes obtenidas de estos materiales mediante microscopía electrónica de barrido (SEM) mostraron superficies homogéneas en todas las películas, pero una cierta porosidad supericial en las de mayor concentración de aditivos debido probablemente a una evaporación parcial del timol y/o carvacrol durante el procesado, aunque en los estudios mediante análisis termogravimétrico (TGA) se comprobó que una fracción importante de dichos aditivos permaneció en la matriz tras el procesado.

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Los resultados obtenidos para los parámetros de inducción a la oxidación demostraron que la adición de timol y carvacrol favoreció el incremento de la estabilidad ante la oxidación a temperaturas altas, demostrando con ello la actividad antioxidante de ambos aditivos. Además, el estudio antimicrobiano demostró que las formulaciones a concentraciones elevadas de timol (8 % en peso) presentaron una mayor actividad antimicrobiana. Se evaluó la capacidad de liberación de las nuevas películas mediante estudios de migración. Para ello, se seleccionaron las formulaciones con mayor concentración de aditivo (8 % en peso) y la migración de carvacrol y timol se determinó utilizando procedimientos analíticos rápidos y fiables, desarrollados y validados en este mismo trabajo, para la determinación de los extractos obtenidos en diferentes simulantes alimentarios: agua destilada (A), ácido acético 3 % (p/v) (B), y etanol 10 % (v/v) (C) como simulantes acuosos y etanol 95 % (v/v) e iso-octano como simulantes grasos. Para los extractos obtenidos a partir de los simulantes acuosos se llevó a cabo un proceso de extracción en fase sólida (SPE) del timol y carvacrol migrado seguido de determinación mediante cromatografía de gases acoplada a espectrometría de masas (GC/MS). En el caso de los extractos de migración obtenidos a partir de los simulantes grasos ambos compuestos fueron analizados directamente mediante GC/MS y también usando cromatografía de líquidos de alta resolución con detección por espectrofotometría ultravioleta-visible (HPLC-UV) para iso-octano y etanol 95 % (v/v), respectivamente. Los resultados obtenidos en estos ensayos demostraron que la liberación de timol y/o carvacrol desde la matriz polimérica era dependiente del tipo de aditivo y del simulante alimentario utilizado. En concreto, los niveles más elevados de migración se obtuvieron para ambos aditivos en iso-

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octano, mostrando el timol una migración superior a 1000 mg por kg de simulante. La cinética de liberación de timol y carvacrol en las películas obtenidas siguieron un comportamiento de acuerdo con las leyes de Fick para la difusión de componentes de bajo peso molecular en matrices poliméricas, con unos valores del coeficiente de difusión que oscilaron entre 1 hasta 2 x 10-14 m2 s-1; aunque la difusión en iso-octano fue entre 4 y 6 veces superior a estos valores. La actividad antioxidante de los extractos de migración fue confirmada por el método de formación del complejo coloreado DPPH (1,1-difenil-2picril-hidrazilo), demostrando que el timol posee una mayor actividad antioxidante especialmente en iso-octano con un 42,2 % de inhibición. Por último, se estudió la liberación de timol y carvacrol en dos alimentos reales utilizando la micro-extracción en fase sólida para evaluar la cantidad de timol o carvacrol liberados en el espacio de cabeza. Se llevó a cabo una evaluación visual que permitió comprobar que la degradación organoléptica de las fresas y el pan de molde se retrasó, permitiendo un aumento en la vida útil de varios días en ambos alimentos. También se identificó la presencia de sustancias liberadas relacionadas con la degradación de los alimentos y se observó la aparición de estas sustancias a tiempos más largos en los sistemas de envasado activo.

3.2. Capitulo 2 En la primera parte de este capítulo se utilizó el PLA como matriz biopolimérica, y D43B como nanoarcilla con el fin de obtener nuevas formulaciones activas con timol. Uno de los objetivos que se persiguieron con la incorporación de la nanoarcilla era mejorar las propiedades mecánicas del nanocompuesto, debido a su dispersión entre las cadenas

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poliméricas y por tanto al efecto de refuerzo que proporciona a la matriz debido a su estructura laminar. Se comprobó que la incorporación de D43B condujo a un aumento en el módulo elástico y una disminución de la elongación a ruptura. En cambio, la adición de timol provocó un descenso alrededor del 15 % en los valores del módulo elástico respecto a los del PLA puro. Estos resultados son concordantes con el ligero efecto plastificante observado mediante calorimetría diferencial de barrido (DSC) en los nanocompuestos activos, con una disminución en la temperatra de transición vítrea (Tg) de aproximadamente 13 °C para las formulaciones con timol. Sin embargo, los resultados obtenidos mediante TGA demostraron que la estabilidad térmica del PLA no se vio afectada significativamente por la adición de timol ni por la presencia de la nanoarcilla. El análisis estructural de estos nanocompuestos activos demostró la intercalación de la nanoarcilla laminar entre las cadenas poliméricas, ya que en los espectros de difracción por rayos X (XRD) se observó un desplazamiento del pico característico de difracción desde 19,2 Å para la nanoarcilla pura hasta 35,6 Å para los nanocompuestos. Asimismo, las imágenes obtenidas mediante microscopía electrónica de transmisión (TEM) mostraron una dispersión parcial, e incluso en algunas zonas se pudo apreciar cierta exfoliación de las láminas de nanoarcilla en el seno de la matriz polimérica. La cantidad remanente de timol determinada en estas formulaciones tras el procesado mediante HPLC-UV fue 5,57 ± 0,01 % en peso para la formulación con 8 % en peso de timol, y 5,99 ± 0,03 % en peso y 5,78 ± 0,02 % en peso para las formulaciones con timol y 2,5 y 5 % en peso de D43B, respectivamente. De este modo, queda demostrada la presencia de timol tras el procesado, así como un efecto de protección de la nanoarcilla

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Resumen

sobre el timol cuando ambos se encuentran dispersos en la matriz polimérica impidiendo su pérdida durante el procesado. A la hora de evaluar el carácter biodegradable de los nanocompuestos desarrollados en este capítulo se llevó a cabo un estudio de desintegración bajo condiciones de compostaje y según la normativa vigente para este tipo de ensayos. Los resultados mostraron que únicamente son necesarios 35 días para lograr un porcentaje de desintegración superior al 90 %, cumpliendo de esta forma los requerimientos impuestos por la legislación. El proceso de degradación de estos materiales es debido en gran medida a la degradación hidrolítica de sus cadenas poliméricas, viéndose favorecido el proceso por la presencia de timol debido a los grupos hidroxilo libres en la estructura del nanocompuesto. Las muestras sometidas a estos ensayos fueron también caracterizadas mediante espectroscopia infrarroja por transformada de Fourier (FTIR) y DSC. Se pudo demostrar una relación directa entre los cambios visuales con la progresiva degradación de las formulaciones estudiadas ya que se observó un claro descenso en la intensidad de pico relacionada con el grupo carbonilo (-C=O) de la lactida a 1750 cm-1 y una simultanea aparición de un pico característico de los carbonilos, a causa del grupo ácido carboxílico formado por la ruptura hidrolítica de los ésteres. Asimismo, se observó un descenso del valor de Tg y la aparición de picos endotérmicos de fusión debidos al proceso de degradación relacionado con la cristalinidad. La aplicabilidad de estos nanocompuestos en base PLA a sistemas de envasado activo se evaluó mediante un estudio de liberación utilizando como simulante alimentario etanol 10 % (v/v). El estudio de migración mostró una liberación controlada de timol con el tiempo a través de la matriz polimérica hasta alcanzar el simulante alimentario. Estos resultados sugirieron que era posible controlar la liberación de timol en el sistema

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Resumen

activo mediante la incorporación de D43B, ya que un aumento en la dispersión de la nanoarcilla se tradujo en una disminución de la velocidad del proceso y del coeficiente de difusión. La actividad antioxidante de estos nanocompuestos se evaluó en los extractos obtenidos antes y después de los ensayos de migración. El porcentaje de inhibición confirmó que la cantidad de timol presente al inicio y tras el estudio de migración era suficiente para alcanzar una inhibición de la oxidación superior al 70 % confirmando de este modo la capacidad antioxidante de las nuevas formulaciones. La actividad antimicrobiana de estas formulaciones fue evaluada utilizando dos tipos de bacterias características de los alimentos, Staphylococcus aureus (Gram positiva) y Escherichia coli (Gram negativa). El ensayo se llevó a cabo en condiciones aerobias colocando las películas en contacto con una disolución de una concentración determinada de inoculo durante un tiempo de incubación de 3 y 24 horas a 4, 24 y 37 °C. Los resultados mostraron que las formulaciones con timol y D43B presentaron una capacidad de inhibición a diferentes tiempos y temperaturas superior a las formulaciones que incorporan los aditivos por separado. De este modo, se confirmó lo descrito en la bibliografía referente al timol y su poder antimicrobiano frente a diferentes bacterias y hongos; y a las nanoarcillas, a las cuales se les atribuye un cierto poder antimicrobiano por el modificador orgánico que las forma. La segunda parte del Capítulo 2 se centró en el desarrollo de nuevos nanocompuestos activos basados en PLA, timol (6 y 8 % en peso) y AgNPs (1 % en peso). Las nuevas formulaciones se obtuvieron en dos morfologías, probetas y películas con un espesor aproximado de 40 µm. En ambos casos, se llevó a cabo una caracterización físico-química

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Resumen

completa y las películas también fueron utilizadas para evaluar la actividad antimicrobiana y antioxidante aportada por ambos aditivos. Los resultados de caracterización en las probetas mostraron que la combinación de los dos aditivos tuvo una cierta influencia en las propiedades finales del material, particularmente en su degradación térmica y módulo elástico. Se observó un descenso aproximado de 6-12 °C en el valor de Tg de los nanocompuestos a causa del ya comentado efecto plastificante del timol. En las imágenes obtenidas mediante microscopia electrónico de barrido de emisión de campo (FESEM) se pudo observar una buena distribución de ambos aditivos y superficies homogéneas de los nanocompuestos que ratificaron la buena dispersión de las Ag-NPs en la matriz polimérica. En el caso de las películas se llevó a cabo una extracción sólido-líquido con posterior determinación mediante HPLC-UV para determinar la cantidad de timol remanente tras el procesado, que resultó ser aproximadamente un 70 % del timol adicionado inicialmente antes del procesado. El aspecto visual de las películas permitió apreciar superficies homogéneas y con buena transparencia tras la adición de ambos componentes. Esta propiedad fue estudiada mediante espectrofotometría UV-Vis a una longitud de onda característica de la región del visible, obteniéndose un valor de transmitancia en torno al 90 %. Una vez llevada a cabo la caracterización óptica se procedió al análisis de sus propiedades térmicas mediante TGA y DSC así como de sus propiedades de barrera. Los resultados mostraron una disminución del valor de Tg en las películas debido a la adición del timol. La estabilidad térmica de las películas se vio influenciada por la presencia de ambos aditivos. Los valores de OTR no fueron significativamente diferentes para las formulaciones ensayadas por lo que no se empeoró la permeabilidad al

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Resumen

oxígeno, pero sí que se observó un descenso en la permeabilidad al vapor de agua en las formulaciones con timol, y en particular en las formulaciones que combinan el timol y Ag-NPs, obteniéndose una mejora en las propiedades de barrera al vapor de agua en torno al 36 %. Una vez caracterizadas las nuevas formulaciones se llevó a cabo un estudio de desintegración en compost a escala de laboratorio de acuerdo a la norma estandarizada UNE-EN ISO 20200:2006. Los resultados mostraron que el inherente carácter biodegradable del PLA fue mejorado por la adición de timol y Ag-NPs en el caso de las probetas, obteniéndose una degradación más rápida en aquellas formulaciones con presencia de ambos aditivos. Fueron necesarios únicamente 57 días para conseguir un porcentaje de desintegración superior al 90 %, cumpliendo de esta forma con los requerimientos indicados en la legislación vigente. Sin embargo, en las películas únicamente se necesitaron 14 días para conseguir su degradación completa. Las muestras obtenidas tras el estudio de degradación fueron analizadas mediante FTIR, DSC y FESEM (únicamente en el caso de las muestras obtenidas a partir de las probetas), con el fin de determinar los parámetros afectados en la degradación y los posibles cambios estructurales. Los resultados obtenidos mediante FTIR mostraron una relación directa con los cambios visuales observados a simple vista y con la progresiva degradación de las formulaciones con el tiempo ya que se observó un claro descenso en la intensidad de pico relacionada con el grupo carbonilo (-C=O) de la lactida a 1750 cm-1 y una simultanea aparición de un pico característico de los carbonilos, el ácido carboxílico formado por la ruptura hidrolítica de los ésteres. Las imágenes obtenidas mediante FESEM mostraron importantes diferencias a los 7 y 14 días de ensayo ya que aparecieron fracturas y

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Resumen

cavidades que evidenciaron la degradación de los nanocompuestos activos, en particular la formulación con ambos aditivos en las que las cavidades tenían hasta 2 µm de diámetro. Por último, el análisis térmico demostró, tanto en probetas como en películas, que el incremento de la movilidad de las cadenas poliméricas como consecuencia del proceso de hidrólisis provocó la aparición de cristalinidad observándose picos endotérmicos de fusión. Además, en el segundo calentamiento se pudo observar un descenso en los valores de Tg lo cual se relaciona con la ruptura de cadenas poliméricas y formación de nuevas cadenas de oligómeros con características plastificantes según fue aumentando el tiempo de ensayo. La actividad antimicrobiana de estas formulaciones fue evaluada con los mismos tipos de bacterias ya comentados y los resultados mostraron una elevada inhibición por parte de ambos aditivos, siendo la actividad antibacteriana de las películas con Ag-NPs y timol más elevada en el estudio con Staphylococcus aureus en comparación con los resultados obtenidos con Escherichia coli, lo cual se puede explicar por las diferencias en la membrana celular de los dos tipos de bacterias, siendo las Gram negativas más resistentes al ataque por parte de los compuestos fenólicos. Para evaluar la liberación de timol en contacto con alimentos se llevó a cabo un estudio de migración utilizando etanol 10 % (v/v) como simulante alimentario. Los resultados fueron obtenidos tras aplicar diferentes modelos cinéticos con el fin de describir de la manera más próxima a los resultados experimentales los procesos de difusión del timol a través de la matriz polimérica y su posterior liberación al simulante alimentario. Los modelos aplicados permitieron concluir que dicha liberación de timol se podía describir con una cinética de pseudo-segundo orden. Además, se comprobó que la adición de Ag-NPs limitó la

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Resumen

velocidad de liberación de timol, induciendo procesos más lentos al ocupar parte de los huecos en la estructura polimérica que el timol utilizaba para su difusión a través de la matriz. Tras evaluar la liberación de timol, se procedió a estudiar la actividad antioxidante de los extractos obtenidos mediante el método del DPPH. Los resultados mostraron que el porcentaje de inhibición aumentaba según lo hacía la concentración de timol, es decir el tiempo de migración. Sin embargo, el porcentaje de inhibición fue más elevado en los nanocompuestos activos, en particular, la formulación con 8 % en peso de timol y 1 % en peso de Ag-NPs.

4. Conclusiones De acuerdo con los objetivos propuestos en este trabajo y a la vista de los resultados obtenidos, se pueden extraer las siguientes conclusiones generales: I.

Se han obtenido películas de PP con 4, 6 y 8 % en peso de timol y/o carvacrol mediante mezclado en fundido y moldeo por compresión. La caracterización de estas películas activas fue llevada a cabo con el uso de diferentes técnicas analíticas y permitió demostrar la estabilidad térmica de las películas tras el procesado. Además, se comprobó mediante DSC la disminución de la Tg y el desarrollo de cristalinidad de las películas activas, justificando de este modo un cierto efecto plastificante. Se observó cierta porosidad mediante SEM, sobre todo en las formulaciones con una concentración elevada de aditivo. Debido al efecto plastificante, las propiedades mecánicas de las películas activas fueron modificadas, al igual que las propiedades de barrera

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Resumen

a oxígeno. Por ejemplo, el módulo elástico para las formulaciones activas disminuyó en comparación con el obtenido para el PP puro. La presencia de timol y carvacrol también aumentó la estabilización frente la degradación termo-oxidativa de las películas basadas en PP, obteniéndose parámetros de inducción a la oxidación más altos cuando se utilizó un 8 % en peso de timol y carvacrol, lo que sugirió que cierta cantidad de aditivo quedó en la estructura polimérica tras el procesado a altas temperaturas. II.

Las películas con 8 % en peso de carvacrol y timol mostraron un doble efecto, ya que fueron capaces de liberar de forma controlada ambos aditivos y proteger de esta forma los alimentos de posibles degradaciones oxidativas y microbiológicas. Varios métodos analíticos fueron desarrollados y validados para determinar la cantidad de aditivo migrado en cada uno de los simulantes alimentarios utilizados. Los resultados tras el estudio de liberación de los aditivos mostró que dependía del simulante alimentario y de la cantidad de aditivo incorporada a la matriz polimérica. Se calcularon los coeficientes de difusión y se comprobó un comportamiento ajustado a las leyes de Fick de difusión. Los niveles de migración más elevados se obtuvieron en isooctano como simulante graso, en especial para las películas con timol. La actividad AO aumentó con la liberación controlada de ambos aditivos en función del tiempo. Las películas activas con timol y carvacrol, pero en especial las de timol, mostraron cierta inhibición frente a Staphylococcus aureus y Escherichia coli, en particular frente a la primera (Gram positiva). Las películas activas demostraron tras el estudio de vida útil utilizando fresas y pan de molde que son capaces de alargar la viabilidad y calidad del

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alimento fresco debido a sus propiedades antioxidantes y antimicrobianas. III.

Se utilizaron una montmorillonita organo modificada (2,5 y 5 % en peso), D43B, y timol (8 % en peso) como aditivo activo para obtener nanocompuestos en forma de películas utilizando como polímero base el PLA. Dichas películas se procesaron mediante mezclado en fundido y moldeo por compresión. Alrededor del 70-75 % de timol permaneció en los nanocompuestos activos tras el procesado, garantizando de este modo su aplicabilidad en sistemas activos. Las películas obtenidas mostraron una excelente intercalación de las láminas de la nanoarcilla a través de la matriz polimérica, presentando una exfoliación parcial, sobre todo las formulaciones con 2,5 % en peso de D43B, así como un comportamiento a la tracción diferente cuando se comparó con el PLA puro. Los resultados mostraron una cierta disminución en el módulo elástico debido al ligero efecto plastificante inducido por el timol, que, sin embargo, no afectó significativamente la estabilidad térmica del PLA. La incorporación de ambos aditivos no resultó en una modificación clara de las propiedades de barrera a oxígeno, pero sí que se observaron algunas diferencias en el color de las películas activas debido principalmente al cambio inducido por el color natural de ambos aditivos. La transparencia intrínseca del PLA no se vio afectada por la presencia de los aditivos.

IV.

La liberación de timol se determinó mediante HPLC-UV a diferentes tiempos de migración y se propuso un modelo cinético, lo que sugirió que la liberación de timol estaba influenciada por la presencia de la D43B en la matriz de PLA y se calcularon los

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Resumen

coeficientes de difusión para todas las formulaciones con timol. Esta liberación continua favoreció la actividad antioxidante de las películas, resultando en un elevado porcentaje de inhibición en el ensayo DPPH. Por último, tras el estudio de la actividad antibacteriana, se concluyó que la adición de la D43B produjo una mejora de la actividad de las películas. V.

Los nanocompuestos activos formados por PLA como polímero base, Ag-NPs (1 % en peso) y timol (6 y 8 % en peso) fueron obtenidos por extrusión en forma de películas y probetas moldeadas por inyección. El estudio mediante FESEM mostró una distribución homogénea de los dos aditivos en la matriz de PLA. Estos resultados fueron corroborados por el descenso de la permeabilidad al vapor de agua, debido también en gran medida a la presencia de timol. La Tg sufrió un ligero descenso debido únicamente a la presencia de timol, lo que se tradujo en un cierto efecto plastificante.

VI.

Las películas activas con Ag-NPs y timol mostraron resultados muy satisfactorios en los estudios relacionados con su actividad antibacteriana, inhibiendo los dos tipos de bacterias a diferentes tiempos y temperaturas de incubación, así como elevados porcentajes de inhibición cuando se estudió la actividad antioxidante por el método de DPPH. La liberación de timol y Ag-NPs desde la matriz polimérica fue determinada a los 10 días de estudio siendo la liberación de timol muy superior a la de AgNPs. El estudio cinético sugirió que la liberación de timol estaba influenciada por la presencia de Ag-NPs en la matriz de PLA.

VII.

El estudio de degradación de todos los nanocompuestos activos en condiciones de compostaje demostró el carácter biodegradable

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del PLA, aunque la incorporación de 8 % en peso de timol favoreció la velocidad de desintegración, debido a la presencia del grupo hidroxilo en su estructura. Además, la combinación de timol y Ag-NPs o timol y D43B provocó mayores velocidades de degradación, traduciéndose en ventajas medioambientales. VIII.

En resumen, las películas activas con 2,5 % en peso de D43B y 8 % en peso de timol basadas en PLA; y las películas activas utilizando también PLA con 1 % en peso de Ag-NPs y 8 % en peso de timol pueden ser consideradas como posibles alternativas a envases activos con matrices poliméricas convencionales al presentar carácter biodegradable y propiedades antimicrobianas y antioxidantes.

Por todo ello, y como conclusión general del presente trabajo de investigación, se puede afirmar que la adición de componentes activos con propiedades antimicrobianas y antioxidantes, tales como carvacrol y timol, a polímeros convencionales (PP) o de fuentes renovables y características biodegradables (PLA) en aplicaciones de envasado alimentario muestran un gran potencial para mejorar la calidad y seguridad alimentarias. En particular, la capacidad de liberación, tanto desde matrices de PP como de PLA, ha mostrado el gran potencial de estos sistemas para ser utilizados como envases con características antioxidantes y antimicrobianas para diferentes productos alimenticios con el fin de extender su vida útil y calidad organoléptica y nutricional.

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Table of contents I.

INTRODUCTION ........................................................................................................... 1 1.

Food packaging .............................................................................................................. 3 1.1.

From conventional polymers to bioplastics .................................................. 4

1.1.1.

Poly(lactic acid)............................................................................................11

Properties and applications.............................................................................................12 Production......................................................................................................................14 1.2. 1.2.1. 2.

2.1.

Antimicrobial activity of essential oils ..........................................................29

2.2.

Antioxidant activity of essential oils .............................................................32

2.3.

Carvacrol and Thymol .....................................................................................33

II.

3.1.

Nanoclays ...........................................................................................................43

3.2.

Silver nanoparticles (Ag-NPs) ........................................................................49

3.3.

Nanocomposites in food packaging .............................................................53 Preparation and processing .......................................................................57

Active Nanocomposites..............................................................................................58 4.1.

End-of-life for active nanocomposites ........................................................64

4.2.

Risk assessment and migration in active nanocomposites .......................66

5.

Legislation......................................................................................................................70

6.

References .....................................................................................................................75

OBJECTIVES ....................................................................................................................99

III. 1

Use in packaging materials ........................................................................36

Nanotechnology in the food industry ......................................................................41

3.3.1. 4.

Antimicrobial and antioxidant active packaging ...................................20

Natural additives ..........................................................................................................26

2.3.1. 3.

Active packaging ...............................................................................................17

RESULTS AND DISCUSSION ...................................................................... 103

Chapter 1 ................................................................................................................................. 113 1.

Introduction ............................................................................................................... 115

2.

Experimental .............................................................................................................. 122 2.1.

Materials and chemicals ................................................................................ 122

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2.2.

Films preparation ........................................................................................... 122

2.3.

Films characterization ................................................................................... 123

2.3.1.

Scanning electron microscopy (SEM) .................................................. 123

2.3.2.

Mechanical properties ............................................................................. 124

2.3.3.

Thermal properties................................................................................... 124

Thermogravimetric analysis (TGA) ............................................................................ 124 Differential scanning calorimetry (DSC) ..................................................................... 124 2.3.4. 2.4.

Oxygen transmission rate (OTR) .......................................................... 126 Migration study............................................................................................... 126

2.4.1.

Release tests ............................................................................................... 126

2.4.2.

Migration kinetics ..................................................................................... 127

2.5.

Analysis of released active additives into food simulants ...................... 128

2.5.1.

GC/MS analysis ....................................................................................... 129

2.5.2.

HPLC-UV analysis. .................................................................................. 130

2.5.3.

Determination of antioxidant activity .................................................. 130

2.6.

Antibacterial activity ...................................................................................... 131

2.7. Study of the effectiveness of the active films to preserve perishable food: shelf-life study ....................................................................................................... 132 Food samples............................................................................................................... 132 Food packaging. .......................................................................................................... 132 Shelf-life study. ............................................................................................................ 133 2.8. 3.

Statistical analysis ........................................................................................... 135

Results and discussion .............................................................................................. 136 3.1.

Films characterization ................................................................................... 136

3.1.1.

Scanning electron microscopy (SEM) .................................................. 136

3.1.2.

Mechanical properties. ............................................................................ 137

3.1.3.

Thermogravimetric Analysis (TGA)..................................................... 139

3.1.4.

Differential Scanning Calorimetry (DSC). .......................................... 140

Determination of thermal parameters in inert atmosphere............................................ 140 Evaluation of oxidation induction parameters (OIT and OOT)................................. 142 3.1.5.

Oxygen Transmission Rate (OTR) ....................................................... 144

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Table of content

3.2.

2

3.2.1.

Validation of the developed methods .................................................. 145

3.2.2.

Release of active additives into food simulants ................................. 148

3.2.3.

Antioxidant activity of migration extracts. ......................................... 151

3.2.4.

Release kinetics of thymol and carvacrol from active films ............ 154

3.3.

Antibacterial properties ................................................................................ 161

3.4. food

Study of the effectiveness of the active films to preserve perishable 164

3.4.1.

Observation of fungal growth ............................................................... 164

3.4.2.

Headspace analysis by HS-SPME-GC/MS ........................................ 167

4.

Conclusions ................................................................................................................ 170

5.

References .................................................................................................................. 172

Chapter 2 ................................................................................................................................ 183 1.

3

Migration study .............................................................................................. 145

Introduction ............................................................................................................... 187

Section 2.1. ................................................................................................................................ 193 2.

Experimental .............................................................................................................. 197 2.1.

Materials and chemicals ................................................................................ 197

2.2.

Films preparation ........................................................................................... 197

2.3.

Thymol quantification .................................................................................. 198

2.4.

Films characterization ................................................................................... 199

2.4.1.

Thermal analysis ....................................................................................... 199

2.4.2.

Structural analysis..................................................................................... 199

2.4.3.

Morphological analysis............................................................................ 200

2.4.4.

Mechanical properties ............................................................................. 200

2.4.5.

Oxygen transmission rate (OTR) ......................................................... 200

2.4.6.

Colour tests ............................................................................................... 201

2.5.

Degradation in compost............................................................................... 201

2.6.

Applicability of films for food packaging applications .......................... 203

2.6.1.

Release study ............................................................................................. 203

2.6.2.

Antioxidant activity of released thymol ............................................... 204

2.6.3.

Antibacterial activity ................................................................................ 205

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Table of content

2.7. 3.

4. 4

Statistical analysis ........................................................................................... 206

Results and discussion .............................................................................................. 206 3.1.

Determination of thymol in films ............................................................... 206

3.2.

Films characterization ................................................................................... 208

3.2.1.

Thermal analysis ....................................................................................... 208

3.2.2.

Structural analysis ..................................................................................... 212

3.2.3.

Morphological analysis ............................................................................ 214

3.2.4.

Mechanical properties ............................................................................. 215

3.2.5.

Oxygen transmission rate ....................................................................... 216

3.2.6.

Optical properties..................................................................................... 217

3.3.

Disintegrability under composting conditions ......................................... 219

3.4.

Release study ................................................................................................... 228

3.5.

DPPH radical scavenging ability ................................................................. 236

3.6.

Antibacterial activity ...................................................................................... 238

Conclusions ................................................................................................................ 241

Section 2.2. ................................................................................................................................ 243 5.

Experimental .............................................................................................................. 247 5.1.

Materials ........................................................................................................... 247

5.2.

Active nanocomposites preparation........................................................... 247

5.3.

Active nanocomposites characterization ................................................... 249

5.3.1.

Thermal properties................................................................................... 249

5.3.2.

Field emission scanning electron microscopy (FESEM) ................. 249

5.3.3.

Mechanical properties of injection moulded samples ....................... 250

5.3.4.

Optical properties of films ..................................................................... 250

5.3.5.

Barrier properties of films ...................................................................... 251

5.4.

Quantification of thymol in PLA-based films after processing ............ 252

5.5.

Identification of thymol and Ag-NPs in PLA-based films .................... 252

5.6.

Disintegrability under composting conditions ......................................... 253

5.7.

Release tests from PLA-based films........................................................... 254

5.7.1.

Silver release study ................................................................................... 255

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Table of content

5.7.2.

6.

Thymol release study............................................................................... 255

5.8.

Determination of the antioxidant activity................................................. 256

5.9.

Antibacterial activity of PLA-based films ................................................. 257

5.10.

Statistical analysis ........................................................................................... 258

Results and discussion.............................................................................................. 258 6.1.

Characterization of injection moulded samples ...................................... 258

6.1.1.

Thermal properties .................................................................................. 258

6.1.2.

Morphological characterization............................................................. 262

6.1.3.

Mechanical properties ............................................................................. 263

6.2.

Films characterization ................................................................................... 264

6.2.1.

Thermal properties .................................................................................. 264

6.2.2.

Morphology............................................................................................... 268

6.2.3.

Optical properties .................................................................................... 268

6.2.4.

Barrier properties ..................................................................................... 271

6.3.

Quantification of thymol in PLA-based films after processing ........... 273

6.4.

Identification of thymol and Ag-NPs in PLA-based films ................... 274

6.5.

Disintegrability under composting conditions ......................................... 276

6.5.1.

Disintegrability study for injection moulded samples ...................... 277

Structural analysis ................................................................................................... 280 Morphological analysis .......................................................................................... 281 Thermal analysis ..................................................................................................... 282 6.5.2. 6.6.

IV.

Disintegrability study for films .............................................................. 285 Release tests from PLA-based films .......................................................... 288

6.6.1.

Silver release .............................................................................................. 289

6.6.2.

Thymol release.......................................................................................... 290

6.7.

Antioxidant activity ....................................................................................... 297

6.8.

Antibacterial activity from PLA-based films ............................................ 298

7.

Conclusions ................................................................................................................ 302

8.

References .................................................................................................................. 304

General Conclusions .............................................................................................. 317

~V~

Figures Introduction Figure I.1. European plastics demand in 2013. ..............................................................4 Figure I.2. Life cycle of bioplastics. .................................................................................8 Figure I.3. Classification of plastics. ............................................................................. 10 Figure I.4. Global production of bioplastics and by market segment in 2013. ..... 11 Figure I.5. Chemical structures of LA. ......................................................................... 14 Figure I.6. Production routes of PLA. ......................................................................... 15 Figure I.7. Interaction processes between food packaging materials and the environment. ........................................................................................................... 18 Figure I.8. Mechanisms that can cause a loss of quality in food and active packaging applications. .......................................................................................... 19 Figure I.9. Release of active substance in different applications of active packaging systems: films that allow the release of the active additive (a), (b) and (c); films that do not release the active additive (d).................................. 23 Figure I.10. Compounds with potential activity from natural sources used in food packaging. ................................................................................................................ 27 Figure I.11. Chemical structures of some natural additives incorporated in active food packaging with AO/AM character (CAS numbers are indicated in parentheses)............................................................................................................. 29 Figure I.12. Structure of the bacterial cell wall............................................................ 31 Figure I.13. Reaction between the DPPH• radical and AO to form the DPPH complex. ................................................................................................................... 33 Figure I.14. Nanofillers in food packaging applications. ........................................... 42 Figure I.15. Crystalline structure of smectites, (2:1 layered silicate structure) (T, tetrahedral sheet; O, octahedral sheet; C, intercalated cations; d, interlayer distance). .................................................................................................................. 43 Figure I.16. Polymer-clay structures according to the distribution of layered silicates into the polymer matrix.......................................................................... 46

~ VII ~

Figures and Tables

Figure I.17. (A) SEM micrographs of native Escherichia coli cells (a) and cells treated with 50 μg cm−3 of Ag-NPs in liquid medium for 4 hours (b); (B) EDAX spectra of native Escherichia coli (a) and Escherichia coli treated with 50 μg cm−3 of Ag-NPs in liquid medium for 4 hours (b)....................... 51 Figure I.18. European legislation in food contact materials and general aspects of the Framework Regulation (EC) No 1935/2004 on materials and articles intended to come into contact with food and legislation applied to active packaging (surrounded by green lines)................................................................ 72

Chapter 1 Figure 1.1. General scheme of the experimental work presented in Chapter 1. .114 Figure 1.2. Visual observation of neat PP and active films. ....................................123 Figure 1.3. Experimental assembly used for headspace analysis of whole strawberries by HS-SPME. .................................................................................133 Figure 1.4. SEM micrographs (x500) of the edge surfaces for PP0 and samples with 8 wt% of the studied additives. .................................................................136 Figure 1.5. Cross section micrographs (x300) for PP0 and samples with 8 wt% of the studied additives. ............................................................................................137 Figure 1.6. TGA curves obtained for PP0 and formulations with carvacrol under nitrogen. .................................................................................................................139 Figure 1.7. Radical scavenging activity measured by the DPPH method, expressed as percentage of inhibition for migration extracts (isooctane: 20 °C, 2 days; rest of simulants: 40 °C, 10 days) (mean ± standard deviation, n=3)). Different letters represent significant difference at p < 0.05........................152 Figure 1.8. Mean DPPH inhibition values (%) for PPT8, PPC8 and PPTC8. Different letters represent significant differences at p < 0.05. .....................153 Figure 1.9. Release of thymol from PPT8 into different food simulants over 15 days. (A) Ethanol 10 % (v/v), 40 °C; (B) acetic acid, 40 °C; (C) Ethanol 95 % (v/v), 40 °C; and (D) isooctane, 20 °C. Solid lines were obtained by fitting Equation 1.4 to experimental data. ........................................................155

~ VIII ~

Figures and Tables

Figure 1.10. Release of carvacrol from PPC8 into different food simulants over 15 days. (A) Ethanol 10 % (v/v), 40 °C; (B) acetic acid, 40 °C; (C) Ethanol 95 % (v/v), 40 °C; and (D) isooctane, 20 °C. Solid lines were obtained by fitting Equation 1.4 to experimental data. ....................................................... 156 Figure 1.11. Plots of  1  1  M F ,t     M F , 0 

0.5



1



0.5

versus t 0 .5 for the migration of

thymol (A) and carvacrol (B) from PPT8 and PPC8 films into different food simulants. Isooctane (◊), 20 °C; acetic acid (o), 40 °C; ethanol 10 % (v/v) (□), 40 °C; and ethanol 95 %(v/v) (∆), 40 °C. ..................................... 160 Figure 1.12. Antimicrobial activity of PP films with 8 wt% active additives: (a) Staphylococcus aureus; (b) Escherichia coli................................................................ 161 Figure 1.13. Study of the effectiveness of PP0 and active films containing 8 wt% of thymol (PPT8) to preserve cut bread and strawberries by observation of fungal growth. ....................................................................................................... 164 Figure 1.14. Evaluation of the effectiveness of PP0 and active film containing 8 wt% of thymol (PPT8) to preserve uncut strawberries by observation of fungal growth. ....................................................................................................... 165 Figure 1.15. Release of carvacrol in the headspace of bread slices after 0, 2, 5, 10 and 15 days of storage at room temperature. .................................................. 167

Chapter 2 Figure 2.1. General scheme of the experimental work presented in Section 2.1.196 Figure 2.2. Weight loss (wt%) (a) and DTG (b) curves obtained for PLA-based films. ....................................................................................................................... 209 Figure 2.3. DSC thermograms for PLA-based films for the first heating (a) and the second heating scan (b). ............................................................................... 211 Figure 2.4. WAXS patterns of D43B, neat PLA and nanocomposite films. ....... 213 Figure 2.5. TEM images of PLA/T/D43B2.5 active nanocomposite film. ........ 214 Figure 2.6. Visual observation of neat PLA and nanocomposite films. ............... 218

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Figures and Tables

Figure 2.7. Visual observations of PLA-based films at different times under composting conditions at 58 °C.........................................................................220 Figure 2.8. FTIR spectra of PLA, PLA/T and PLA/T/D43B5 before (0 days) and after different incubation times (7 and 21 days) in composting conditions. ..............................................................................................................223 Figure 2.9. DSC curves (1st heating scan) of PLA-based films after different composting times. .................................................................................................226 Figure 2.10. DSC curves (2nd heating scan) of PLA-based films after different composting times. .................................................................................................227 Figure 2.11. Thymol release profiles of PLA/T, PLA/T/D43B2.5 and PLA/T/D43B5 active nanocomposite films. .................................................229 Figure 2.12. Normalized migration of thymol from different polymer matrices: PLA/T, PLA/T/D43B2.5, and PLA/T/D43B5. ..........................................230

Figure 2.13. Plots of

 1 1 M F ,t         M F ,0 

0.5

0 .5 versus t for the migration of thymol

from: PLA/T ( ), PLA/T/D43B2.5 ( ), and PLA/T/D43B5 ( ), into ethanol 10 % (v/v). .............................................................................................................235 Figure 2.14. AO activity obtained from migration extracts of PLA/T (left axis) and migration of thymol from PLA/T films (right axis) by using DPPH method. ...................................................................................................................238 Figure 2.15. Antibacterial activity of PLA-based films at different temperatures against E. coli RB and S. aureus 8325-A. Cells were incubated on PLA with thymol and D43B for 3 h and 24 h at 4, 24 and 37 °C respectively. Results are expressed on a PLA-basis and are represented as mean ± standard deviation, n=3 .......................................................................................................240 Figure 2.16. General scheme of the experimental work presented in Section 2.2. ..................................................................................................................................246 Figure 2.17. TG (a) and DTG (b) curves of neat PLA and nanocomposite injection moulded samples with Ag-NPs and thymol....................................260

~X~

Figures and Tables

Figure 2.18. DSC thermograms for PLA, PLA/Ag, PLA/T8 and PLA/Ag/T8 injection moulded samples; first heating and cooling scans (a) and second heating scan (b). .................................................................................................... 262 Figure 2.19. FESEM micrographs of the surface of nanocomposite injection moulded. ................................................................................................................ 263 Figure 2.20. Cross section micrographs of PLA/Ag/T6 and PLA/Ag/T8 injection moulded samples after processing. ................................................... 263 Figure 2.21. FESEM surface images of PLA and active nanocomposite films. . 268 Figure 2.22. Visual observation of neat PLA and binary and ternary nanocomposite films. .......................................................................................... 269 Figure 2.23. FTIR (a) and UV-Vis (b) spectra of PLA and active nanocomposite films. ....................................................................................................................... 275 Figure 2.24. WAXS patterns of PLA and active nanocomposite films. ............... 276 Figure 2.25. PLA and PLA nanocomposites processed by injection moulding at different times under composting conditions at 58 °C. ................................ 277 Figure 2.26. Disintegrability (%) of PLA and PLA nanocomposite processed by injection moulding at different times in compost at 58 °C. The line at 90 % represents the goal of disintegrability tests as required by the ISO 20200 Standard. ................................................................................................................ 278 Figure 2.27. FTIR spectra of PLA/Ag/T8 at different times under composting conditions. ............................................................................................................. 281 Figure 2.28. FESEM micrographs of the surface of nanocomposite injection moulded samples before (0 days) and after 14 days of disintegration in compost at 58 °C (500x) and after 14 days with higher zoom (10.00 kx). . 282 Figure 2.29. DSC thermograms obtained for nanocomposites processed by injection moulding at different times under composting conditions at 58 °C (first heating scan (10 °C min-1)). ...................................................................... 284 Figure 2.30. Tg values for nanocomposite submitted to injection moulding at 0 and 21 days of disintegration under composting conditions at 58 °C (second heating scan).......................................................................................................... 285

~ XI ~

Figures and Tables

Figure 2.31. Visual appearance of neat PLA and active nanocomposite films at different testing days under composting conditions at 58 °C. .....................286 Figure 2.32. Disintegrability (%) of neat PLA and nanocomposite films at different times under composting conditions at 58 °C (mean ± SD, n = 3). The line at 90 % represents the goal of disintegrability test as required by the ISO 20200 standard. Different superscripts over different samples at the same time indicate statistically significant different values (p < 0.05).........288 Figure 2.33. Release kinetics of thymol from binary systems (black dots) and ternary systems (white dots) at 6 wt% (a) and 8 wt% (b), at 40 °C. Solid lines were obtained by fitting the Equation (2.15) to the experimental data points. .....................................................................................................................292 Figure 2.34. Representative plot of the fitting of linearized forms of pseudo-first (left yy-axis, white squares, equation 2.18) and pseudo-second (right yy-axis, white dots, equation 2.19) order equations to experimental released amounts of thymol from PLA/T8 to ethanol 10 % (v/v) at 40 °C. ............................296

~ XII ~

Figures and Tables

Tables Introduction Table I.1. General properties of some thermoplastics used in food packaging. ......6 Table I.2. Physical data of some commercial bioplastics. ......................................... 10 Table I.3. Structures and physico-chemical properties of carvacrol and thymol. . 34 Table I.4. Carvacrol and thymol in active food packaging. ...................................... 38 Table I.5. Structural characteristics of common smectites (2:1 layered silicates) (adapted from. ........................................................................................................ 44 Table I.6. Commercial organo-modified montmorillonites...................................... 48 Table I.7. Representative examples of nanocomposites application in food packaging. ................................................................................................................ 55 Table I.8. Comparison between two active nanocomposites based on edible films and S-EO and ZMB-EO. ..................................................................................... 63 Table I.9. Food simulants established by EU Regulation No 10/2011. ................ 69

Chapter 1 Table 1.1. Storage and testing conditions in the headspace study of food by HSSPME-GC/MS. .................................................................................................... 134 Table 1.2. Mechanical properties of samples according to ASTM D882-09. ...... 138 Table 1.3. TGA and DSC parameters obtained for all samples. ............................ 141 Table 1.4. Oxidation induction parameters, oxygen transmission rate obtained for all formulations. .................................................................................................... 143 Table 1.5. Main analytical parameters obtained for the studied active additives using the optimized methods. ............................................................................ 147 Table 1.6. Mean recoveries (%) and RSD values (%) in parentheses obtained for each active additive in aqueous simulants by SPE-GC/MS. Rresults are represented as mean ± standard deviation, n=3. ........................................... 148 Table 1.7. Release of thymol and carvacrol (mg (kg-1 simulant)) obtained from PP films into aqueous and fatty food simulants under conditions in agreement with European Standard EN 13130-2005. ...................................................... 150

~ XIII ~

Figures and Tables

Table 1.8. Diffusion coefficients (D×10−14, m2 s-1) calculated from Equation 1.4 for the release of carvacrol and thymol from PP films into different food simulants (mean ± standard deviation, n=3). ..................................................156 Table 1.9. Inhibition zone against Staphylococcus aureus obtained for all formulations...........................................................................................................162 Table 1.10. Identified compounds present in the headspace of food samples packed with PP0 films after 4 days. ...................................................................169

Chapter 2 Table 2.1. PLA-based films formulated in this study. ..............................................198 Table 2.2. Composition of synthetic bio-waste used to simulate the disintegrability in composting conditions. ...................................................................................202 Table 2.3. Quantification of thymol (HPLC-UV) and thermal parameters (TGA, DSC) obtained for all nanocomposite films and neat PLA. .........................207 Table 2.4. Tensile properties (ASTM D882-09), oxygen transmission rate and CIELab colour parameters obtained for PLA-based formulations. ............216 Table 2.5. Disintegrability values (%) of PLA and nanocomposite films at different times under composting conditions at 58 °C. .................................222 Table 2.6. Characteristic parameters for the release of thymol from PLA-based films to ethanol 10 % (v/v). ...............................................................................233 Table 2.7. Radical scavenging activity of thymol measured by the DPPH method for PLA-based formulations. ..............................................................................237 Table 2.8. PLA active nanocomposites formulated in this study. ..........................248 Table 2.9. Thermal parameters and tensile properties obtained for injection moulded samples (neat PLA and active nanocomposites). ...........................260 Table 2.10. Characterization of neat PLA and active nanocomposite films. .......267 Table 2.11. Optical properties of neat PLA and active nanocomposite films. ....270 Table 2.12. Thymol and Ag-NPs migration (ethanol 10 % (v/v) after 10 days at 40 °C) and DPPH scavenging activity (%) of PLA-based films. .................290

~ XIV ~

Figures and Tables

Table 2.13. Fitting parameters of Equations (2.14), (2.15) and (2.16) to experimental migration data of thymol loaded in binary and ternary systems into ethanol 10% (v/v) at 40 °C. ....................................................................... 293 Table 2.14. Kinetic parameters for migration of thymol from PLA-based films by using Equations (2.18) and (2.19)...................................................................... 297 Table 2.15. Antibacterial activity of neat PLA and nanocomposite films, expressed as antimicrobial viability (%), against S. aureus 8325-4 and E. coli RB strains after 3 and 24 hours of incubation at 4, 24 and 37 °C............... 299

~ XV ~

Abbreviations and symbols

Abbreviations and symbols 2θ ∆E*

Scattering angle (2theta) Total colour difference in CIELAB space

∆Hc

Crystallization enthalpy

∆Hm

Melting enthalpy

εB

Elongation at break

εY

Elongation at yield

ρ

Density

χ (%) a*

Percentage of crystallinity Red-green coordinate in CIELAB space

Ag-NP

Silver nanoparticles

AM

Antimicrobial

AO

Antioxidant

b*

Yellow-blue coordinate in CIELAB space

BHA

Butylated hydroxyanisole

BHI

Brian Heart Infusion

BHT

Butylated hydroxytoluene

C10A

Cloisite®10A

C15A

Closite ®15A

C20A

Cloisite®20A

C30B

Closite ®30B

CEC

Cation exchange capacity

CLE

Cutinase-like enzyme

CFU

Colony-forming unit

CNC

Nanocrystalline cellulose

D

Diffusion coefficients

D43B

Dellite®43B

DSC

Differential scanning calorimetry

DPPH

2,2-diphenyl-1-picrylhydrazyl

DTG

Derivative thermogravimetric analysis

E E. coli RB

Elastic Modulus Escherichia coli RB

EDAX

Energy dispersive X ray analysis

EFSA

European Food Safety Agency

EU

European Union

EO

Essential oil

EtOH

Ethanol

FDA

Food and Drug Administration

FESEM

Field emission scanning electron microscopy

FRAP

Ferric reducing antioxidant power

FTIR

Fourier transform infrared spectroscopy

~ XVI ~

Abbreviations and Symbols

GC-FID

Gas chromatography-flame ionization detector

GC/MS

Gas chromatography–mass spectrometry

GRAS

Generally Recognized as Safe

GTE

Green tea extracts

HDPE

High density polyethylene

HPLC-UV

high performance liquid chromatography-UV detector

HS-SPME

Headspace solid phase microextraction

I%

Percentage of inhibition

ICP-AES

Inductively coupled plasma - atomic emission spectrometer

ICP-MS

Inductively coupled plasma -mass spectrometer

ICP-OES

Inductively coupled plasma - optical emission spectrometer

L*

Lightness in CIELAB space

LA

Lactic acid (2-hydroxy propionic acid)

LB

Luria Bertani Broth

LDPE

Low density polyethylene

LOD

Detection limit

LOQ

Quantification limit

MAP

Modified atmosphere packaging

MC

Methylcellulose

MDT

Mean dissolution time

MMT

Montmorillonite

OMMT

Organo modified montmorillonite

OIT

Oxidation induction time

OOT

Oxidation onset temperature

ORAC

Oxygen-radical antioxidant capacity

OTR

Oxygen transmission rate

PA

Polyamides

PBAT

Poly(butyrate adipate terephthalate) copolymer

PCL

Poly(ε-caprolactone)

PET

Poly(ethylene terephthalate)

PHA

Poly(hydroxyalkanoates)

PLA

Poly(lactic acid)

PLLA

Poly(L-lactic acid)

PDLA

Poly(D-lactic acid)

PDLLA

Poly(DL-lactic acid)

PP

Polypropylene

PS

Polystyrene

R2

Determination coefficients

RH

Relative humidity

RSD

Relative standard deviation

RT

Room temperature

Sy/x

Standard deviation of the residues

~ XVII ~

Abbreviations and symbols

S. aureus 8325-4

Staphylococcus aureus 8325-4

S-EOS

Savory essential oil

SD

Standard deviation

SEM

Scanning electron microscopy

SPE

Solid phase extraction

SPI

Soy protein isolate

SPME

Solid phase microextraction

T

Tallow chain

Tc

Crystallization temperature

Ti

Initial degradation temperature

Tg

Glass transition temperature

Tm

Melting temperature

Tmax

Maximum degradation rate temperature

TBARS

Thiobarbituric acid reactive substances

TBHQ

Propyl gallate or tert-butylhydroquinone

TEAC

Trolox-equivalent antioxidant capacity

TEM

Transmission electron microscopy

TGA

Thermogravimetric analysis

TOSC

Total oxidant scavenging capacity

TS

Tensile strength

TPS

Thermoplastic starch

UV-Vis

Ultraviolet-visible

WAXS

Wide angle X-ray scattering

WVP

water vapor permeability

XPS

X-Ray Photoelectorn Spectroscopy

XRD

X-Ray Diffraction

ZMB-EO

Zataria multiflora Boiss essential oil

~ XVIII ~

I.

Introduction

Introduction

1. Food packaging Plastic industries are one of the main production sectors worldwide and have experienced a continuous growth by widening their portfolio and applications window. The increase in plastics production has been reflected in numerous and important sectors of the World economy. In general terms, the plastic industries can be considered a key enabler of innovation in many products and technologies in important sectors of the global economy, such as healthcare, energy generation, aerospace, automotive, maritime, construction, electronics, packaging or textile. With a continuous growth for the last decades, global plastic production reached 299 million tonnes in 2013, with a 3.9 % increase compared to 2012 (Plastics-Europe, 2015). This increase has been recently observed in emerging countries, but it was not the case in the most developed economies. For instance, the plastics production in Europe is currently stable, with no increase in the last decade and similar figures were observed in 2013 when compared to those in 2002. Packaging, building and construction are the largest application sectors for the current production of plastics. Figure I.1 shows the distribution of the plastics consumption in Europe in 2013. In particular, the packaging sector represents 39.6 % of the total plastics demand and almost half of them are used for food packaging in the form of films, sheets, bottles, cups, tubs, or trays; followed by building and construction with 20.3 % of the total European demand. Automotive is the third sector with a share of 8.5 % and the rest of applications comprise a total of 31.6 % of the European plastics demand (Plastics-Europe, 2015).

~3~

Introduction

20.3% Building and construction

39.6% Packaging 46.3 Million tonnes

8.5% Automative Electrical and Electronics

5.6% 4.3%

Agriculture

21.7% Others

Consumer and household appliances, furniture, sport, health and safety,…

Figure I.1. European plastics demand in 2013; (data taken from (Plastics-Europe, 2015).

Plastics are currently considered one of the four basic materials for food packaging, together with glass, metal and paper. Plastics are adequate to satisfy the main function of food packaging, i.e. to maintain the quality and safety of food products from their processing to consumption, including storage and transportation. Packaging materials should provide mechanical, optical, and thermal protection while preventing unfavourable degradation factors or conditions, such as spoilage microorganisms, oxygen, moisture, chemical contaminants, light, external forces, high temperatures, etc. (Rhim, Park and Ha, 2013).

1.1. From conventional polymers to bioplastics Among

the

basic

food

packaging

materials,

petroleum-based

thermoplastics have been extensively used since their introduction in the

~4~

Introduction

1950-1960 decades. They exhibit a range of properties that make them suitable for specific applications in food packaging, such as their large availability; relative low cost; good physico-chemical performance in tensile and tear strength; good barrier to oxygen, water vapour, CO2 and aromas; good processing capabilities; heat sealability; good aesthetic quality and many others (Siracusa, Rocculi, Romani and Rosa, 2008). The most important thermoplastics used in food packaging include low density polyethylene (LDPE), high density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), polyamides (PA) and poly(ethylene terephthalate) (PET), which exhibit many common properties ideal for their use in packaging, such as light weight; low processing temperature (compared to metal and glass); variable barrier properties; good printability; heat sealability and ease of conversion into different forms (Lim, Auras and Rubino, 2008). Table I.1 shows some general properties of the main thermoplastic polymers with interest in food packaging. There are some key properties that make thermoplastics one of the most favourable material families for food packaging applications. For example, thermal properties provide important information about criteria to be considered during processing and their possible resistance to high temperatures. Mechanical properties provide data on the ability of packaging materials to sustain their integrity under the influence of various external factors occurring during processing, handling, and storage of the packaged food. Other important parameters, such as permeability to oxygen and water vapour, should be also considered and controlled to select the most adequate polymers or composite materials for foodstuff with particular requirements (Bastarrachea, Dhawan and Sablani, 2011).

~5~

Introduction

Table I.1. General properties of some thermoplastics used in food packaging (adapted from (Bastarrachea, Dhawan and Sablani, 2011). Property

LDPE

HDPE

PP

PS

PET

PA

δ (g cm-3)

0.92 - 0.94

0.94 - 0.95

0.90 - 0.91

1.04 - 1.12

1.37

1.05 - 1.14

Tm (°C)

120

137

168

250

256 - 260

185 - 260

Tg (°C)

(-45) - (-15)

(-45) - (-15)

(-32) - (-2)

80 - 100

67 - 81

37 - 70

E (GPa)

0.15 - 0.34

0.98

1.1 - 1.6

2.7 - 3.4

3.5

0.70 - 0.98

εB (%)

300 - 900

20 - 50

200 - 1000

2-3

70

200 - 300

Density (δ) Melting temperature (Tm) Glass transition temperature (Tg) Elastic modulus (E) (25 °C, 65 % relative humidity (RH)) Elongation at break (εB) (20-25 °C, 65 % RH)

Polyolefins, such as LDPE, HDPE and PP, are widely used, with a total percentage of application in this sector close to 37 % of the total production (Plastics-Europe, 2015). Among the wide variety of polyolefins, PP offers overall balanced properties, including flexibility; tensile strength; lightness; stability; moisture and chemical resistance; easy processability in different forms (films, small and high packs, etc.) and low cost; while it is well suited for recycling and reuse. PP is used in different sectors, such as automotive, machinery fabrication, sanitary applications or packaging, where thermal resistance is required due to its high melting point (168 °C), such as for hot-filled and microwavable trays (Fages, Pascual, Fenollar, Garcia Sanoguera and Balart Gimeno, 2011). In addition, these properties can be modified by altering the chain regularity content (tacticity) and distribution, and by the incorporation of modifiers into the polymer matrix as fillers (Gocek and Adanur, 2012; Rawi, Jayaraman and Bhattacharyya, 2014). Despite the multiple advantages provided by conventional thermoplastics in their application to food packaging, the raising societal concerns about

~6~

Introduction

environmental issues and the most recent policies implemented by authorities at the international level have introduced some caution on their massive use and uncontrolled disposal. For instance, in 2012, according to the Association of Plastics Manufacturers in Europe, 25.2 million tonnes of post-consumer plastics ended up in the waste upstream, of which 62 % was removed from the environment through recycling and energy recovery processes while 38 % still went to landfills (Plastics-Europe, 2015). Non-biodegradable conventional thermoplastics cause serious environmental problems since they are not easily degraded in Nature, taking centuries to be decomposed into their simple constituents and absorbed by the environment. This problem has become a global concern, in particular from the beginning of the XXI century, since environmental problems, such as the climate change, CO2 footprint and the noticeable shortage in fossil resources have propelled the search for better concepts and sustainable alternatives to conventional plastics for packaging. Reuse and plastics recycling are the most extended alternatives to disposal, but other possibilities are being studied for immediate implementation in massive production. Novel bio-based and biodegradable plastics for food packaging should be developed by strictly following the guidelines for the efficient use of natural and renewable resources, keeping the properties of conventional thermoplastics to preserve food quality and consumer safety, while reducing waste disposal and CO2 footprint by offering new recovery options. In general terms, novel bio-based and biodegradable plastics could be obtained and modified from raw materials with biological origin, or more precisely from renewable resources, to permit a significant reduction in the environmental impact of packaging materials by reducing the waste disposal problems and alleviate the overdependence from

~7~

Introduction

petroleum and other non-renewable sources (Rhim, Park and Ha, 2013). In addition, they should be treated by various recycling and recovery techniques to get waste streams easy for treating and versatile enough to get efficient processes, such as composting, bio-refineries and others. Among them, the use of novel bio-based and biodegradable packaging materials is recognized as one of the main trending topics in the research for new materials, keeping the guiding principle of the efficient use of renewable resources and their easy conversion into simple constituents to close the natural cycle (Figure I.2).

Energy and organic recovery Reuse

Biotechnology and chemistry

Figure I.2. Life cycle of bioplastics.

According to the European Bioplastics Association, bioplastics are defined as plastics that are either bio-based, biodegradable or both. There are three main groups of bioplastics based on their renewable/nonrenewable origin and biodegradable/non-biodegradable character (Figure I.3):

~8~

Introduction

i.

Bio-based or partly bio-based, non-biodegradable plastics, such as Bio-PE, Bio-PET and Bio-PA. These materials are characterized by their bio-based origin (most of them are obtained from sugarrich fractions in the bio-ethanol production) and their properties are similar to those of the main commodities used in food packaging, such as polyolefins. However, they are nonbiodegradable and their waste disposal is similar to the conventional thermoplastics.

ii.

Biodegradable plastics based on fossil resources, such as poly(butyrate adipate terephthalate) copolymer (PBAT) or poly(εcaprolactone) (PCL). These biopolymers degrade fast under environmental conditions, but they are obtained from petroleum. They show, in general, good properties for food packaging materials but their production costs are high and they are not yet competitive with conventional thermoplastics.

Bio-based and biodegradable plastics, including poly(lactic acid) (PLA), cellulose,

starch-based

plastics,

animal

proteins

and

poly(hydroxyalkanoates) (PHA). These biopolymers are the ideal solution from the environmental point of view, but most of them fail in some of the key properties for food packaging applications, such as thermal resistance and barrier effect, making necessary their modification with additives to improve these characteristics. Table I.2 summarizes the most important commercial bio-based and biodegradable polymers and their main properties. This group of bioplastics has produced great interest by their high potential in becoming a sustainable alternative to conventional thermoplastics. Some of them are already produced at the industrial scale, such as PLA, PHA or starch-based polymers as shown in Figure I.4.

~9~

Introduction

Bio-based Bioplastic

Bioplastic

e.g. Bio-based PE, Bio-based PET, Biobased PP

e.g. PLA, PHA, Starch, Cellulose, Gelatines

Conventional plastics

Bioplastic

Non-biodegradable

Biodegradable

e.g. PBAT, PCL e.g. PE, PP, PET

Fossil-based

Figure I.3. Classification of plastics.

Table I.2. Physical data of some commercial bioplastics (adapted from (Jamshidian, Tehrany, Imran, Jacquot and Desobry, 2010). Property

PLA

PHB

PCL

TPS*

Cellulose

Tm (°C)

130-180

140-180

59-64

110-115

-

Tg (°C)

40-70

0-5

-60

(-20)-43

-

E (MPa)

2050-3500

3500

390-470

400-1000

3000-5000

εB (%)

30-240

5-8

700-1000

580-820

18-55

TS** (MPa)

48-53

25-40

4-28

100

100

*Thermoplastic starch (TPS) **Tensile strength (TS)

The current global production capacities of bioplastics have continuously increased and amounted to about 1.6 million tonnes in 2013 with almost 40 % of the production destined for the packaging market, which is the current largest market segment within the bioplastics industry (Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013; European-BioplasticsAssociation, 2015b). This continuous growth in the bioplastics production for food packaging is also possible by the similarity in machinery and

~ 10 ~

Introduction

processing conditions to those traditionally used with conventional thermoplastics. Indeed, no special machinery is required for the processing of bioplastics, with just some changes in the processing parameters depending on their properties.

Bio-based and Biodegradable 25.8 %

Othersa (1.4 %) Bio-based PA (4.9 %)

6.8 % PTT

12.3 % Bio-based PE

bBiodegradable cellulose

ester

11.4 % PLA

11.3 % Starch blends PHA (2.1 %) Regenerated Cellulose (1.7 %) Othersb (0.3 %) 10.8 % PBAT, PBS, PCL

37 % Bio-based PET

Bio-based and non Biodegradable 62.4 %

Fossil-based and Biodegradable 10.8 %

aContains durable

starch blends, Bio-PC, Bio-TPE, Bio-PUR (except thermosets)

Figure I.4. Global production of bioplastics and by market segment in 2013 (adapted from the (European-Bioplastics-Association).

1.1.1. Poly(lactic acid) PLA is a bio-based and biodegradable polymer with high commercial potential by its convenient properties, easy processing and relative low cost when compared to other bioplastics. PLA is compostable and biocompatible, but also processable with standard equipment, permitting a reduction of costs and consequently making it competitive against all other bioplastics and most of the petroleum-based commodities.

~ 11 ~

Introduction

Properties and applications PLA has a significant presence in the market; with a production capacity about 185,000 tonnes in 2013 (European-Bioplastics-Association, 2015a). In addition, its relative low price has increased the potential of PLA as an alternative material to some conventional polymers, such as PET or HDPE, because of PLA’s favourable properties, such as high transparency, excellent printability, and low-temperature sealability (Auras, Harte, Selke and Hernández, 2003; Gupta and Kumar, 2007). PLA has certain limitations such as low deformation at break; low heat resistance; high modulus; hydrophilic properties; brittle behaviour; and insufficient barrier to oxygen, CO2 and water vapour compared to other benchmark packaging polymers, such as polyolefins and PET. These factors limit the use of PLA in the packaging industry (Hughes, Thomas, Byun and Whiteside, 2012). In consequence most research focus on improving the mechanical and barrier properties of PLA by adding nanoclays, natural fibres or antioxidant (AO) additives for the development of PLA-based films (Gonçalves et al, 2013; FarmahiniFarahani, Xiao and Zhao, 2014; Rawi, Jayaraman and Bhattacharyya, 2014; Cumkur, Baouz and Yilmazer, 2015). Other researchers placed emphasis on the improvement of thermal stability (Hughes, Thomas, Byun and Whiteside, 2012; Araújo, Botelho, Oliveira and Machado, 2014; Iturrondobeitia, Okariz, Guraya, Zaldua and Ibarretxe, 2014; Kovacevic, Bischof and Fan, 2015). PLA is classified as GRAS (Generally Recognized as Safe) by the FDA (Food and Drug Administration, USA) for its intended use as polymer for manufacturing articles that will hold and/or package food. It has been also proposed for the formulation of transparent films, but the inherent brittleness and poor flexural properties of the polymer make necessary the

~ 12 ~

Introduction

addition of plasticizers to obtain the adequate flexibility for stretching films with no risks of rupture (Ljungberg and Wesslén, 2005; Burgos, Martino and Jiménez, 2013; Burgos, Tolaguera, Fiori and Jiménez, 2014) or the processing of blends based on PLA with different biodegradable and non-biodegradable polymers, such as PCL (Zhao and Zhao, 2016), PP (Jain et al, 2015) or cellulose acetate (Kunthadong et al, 2015). Commercial PLA can be obtained in different grades, from pure poly(Llactic acid) (PLLA) to pure poly(D-lactic acid) (PDLA), including their copolymers at variable ratios. It is known that the PLA properties vary to a large extent depending on the PLLA/PDLA ratio and their arrangement in the polymer chains (Södergård and Stolt, 2010). PLA with PLLA content higher than 90 % tends to be crystalline and it is commercially produced in different grades, with a Tg value around 60-65 °C, crystallinity degree in the range of 15-74 %, melting temperature around 173-178 °C and tensile modulus of 2.7-16 GPa (Södergård and Stolt, 2002). These properties are adequate for the production of a transparent, tough and brittle polymer, with mechanical resistance similar to PET and PS, and application in rigid containers for food packaging. It was reported that Tg, Tm and crystallinity of PLA decrease by decreasing the PLLA/PLDA ratio (Jiang et al, 2010). Commercial PLA has distinctive properties over other bioplastics, such as good appearance; transparency; relatively high melting temperature; high tensile properties; good biocompatibility; and low toxicity that have helped to broaden its applications window. Indeed, it has been used in a variety of applications, in addition to food packaging, in the pharmaceutical and biomedical fields (Albertsson, Varma, Lochab, FinneWistrand and Kumar, 2010). In tissue engineering, PLA has demonstrated to be biocompatible and degradable into non-toxic components with an

~ 13 ~

Introduction

in-vivo controllable degradation rate having a long history of use in degradable surgical sutures (Lopes, Jardini and Filho, 2014; Salerno, Fernández-Gutiérrez, Gutiérrez, San Román del Barrio and Domingo, 2015) 2015).

Production Lactic acid (2-hydroxy hydroxy propionic acid) (LA) is the single monomer of PLA and it is produced either by fermentation of dextrose (the common commercial name for D-glucose) and other sugars extracted from plants, such as corn sugarcane, sugar beet and potatoes, or by chemical synthesis. LA has a chiral carbon atom and exists in two optically active stereoisomers, namely L- and D- enantiomers ((Figure I.5) (Gupta and Kumar, 2007; Jamshidian, Tehrany, Imran, Jacquot and Desobry, 2010) 2010).

D-Lactic Acid (D-LA)

L-Lactic Lactic Acid (L-LA)

Figure I.5. Chemical structures of LA.

PLA has variable molar mass, ranging from 1000 1000-2000 Da in the case of oligomers to more than 100 kDa in the case of the high molar mass polymer, which is mostly used in processing and development of products for the packaging industry (Jamshidian, Tehrany, Imran, Jacquot and Desobry, 2010).. Oligomers have been proposed for their use as PLA additives with plasticizer performance (Burgos, Martino and Jiménez, 2013; Burgos, Tolaguera, Fiori and Jiménez, 2014; Fiori and Ara, 2009). Several polymerization routes to obtain PLA A from L L-LA and D-LA have been described (Figure I.6) (Södergård and Stolt, 2010) 2010):

~ 14 ~

Introduction

i.

Polycondensation reactions. a.

Direct condensation.

b. Direct condensation in azeotropic solutions. c. Solid-state condensation. ii.

Chain extension.

iii.

Polymerization

through

lactide

formation

(Ring-opening

polymerization, ROP).

Low molar mass PLA Direct Condensation

Chain coupling agent

Polycondensation reactions

Mw: 1000-5000 High molar mass PLA Azeotropic Solution

D-LA

ROP Mw: 100-300 kDa

L-LA

Solid State

Melt State

L-Lactide / meso-Lactide / D-Lactide Oligo(lactic acid)

Polycondensation

Oligo(lactic acid)

Depolymerization

Figure I.6. Production routes of PLA (adapted from (Södergård and Stolt, 2002; Gupta and Kumar, 2007; Avérous, 2008).

Direct condensation is based on the polymerization of LA in the presence of catalysts at low pressures and high temperatures. The most common catalysts are antimony oxides and stannous organometallic complexes (Lopes, Jardini and Filho, 2014). PLA obtained by direct condensation is a low molar mass polymer, by the difficulty in removing water from the

~ 15 ~

Introduction

highly viscous reaction mixture, resulting in oligomers with no relevant applications in food packaging (Gupta and Kumar, 2007; Södergård and Stolt, 2010), but some potential as bio-based additives (Burgos, Tolaguera, Fiori and Jiménez, 2014). PLA can be also obtained by direct condensation in azeotropic solutions, permitting the production of high molar mass polymers. This technique allows working below the melting point of the polymer, preventing depolymerization (Gupta and Kumar, 2007). The azeotropic solutions used in PLA polymerization are mixtures of two or more liquids in such a ratio that its composition cannot be changed by simple distillation and they help in removing water as a by-product (Jamshidian, Tehrany, Imran, Jacquot and Desobry, 2010) resulting in highly pure PLA with molar mass up to 300 kDa. ROP is the most applied polymerization technique for PLA due to the possibility of an accurate control of the chemistry of the process resulting in polymers with high molar mass and low dispersion, which broadens their application fields (Albertsson, Varma, Lochab, Finne-Wistrand and Kumar, 2010). ROP is based on the synthesis of lactide, a cyclic dimer of lactic acid with three different forms: L-lactide, D-lactide, and mesolactide, and further polymerization of this reaction intermediate. PLLA, PDLA and poly(DL-lactic acid) (PDLLA) are synthesized from L-lactide, D-lactide and DL-lactide, respectively. A wide range of physical and mechanical properties and degradation rates can be achieved by controlling the ROP process and the molar mass and stereochemistry of the monomers (Gupta and Kumar, 2007). In addition, other parameters, such as racemization, lactide purity or the residual monomer content should be controlled to ensure the quality of the PLA obtained by ROP. Small amounts of impurities, such as residual monomers or oligomers

~ 16 ~

Introduction

formed during the process, nutrients, cell debris, and enantiomeric impurities of LA, could drastically change the PLA properties; in particular, crystallinity and degradation rate (Inkinen, Hakkarainen, Albertsson and Sodergard, 2011).

1.2. Active packaging The traditional concept for food packaging materials as mere containers with no interaction with the packaged food to avoid contamination (i.e. passive packaging) has been recently changed by the introduction of new paradigms where interactions have been permitted, promoting two new concepts: active and intelligent packaging. In fact, research in new developments in food packaging materials is aimed by the increase in the consumer’s preferences for fresh, natural, healthy and easy-to-prepare food, minimally processed and convenient from the nutritional point of view. Consequently, the food sector has experienced important changes aimed to meet these demands while adapting to the global market, which implies an increase in time of food transport and distribution (Singh, Wani and Saengerlaub, 2011; Sanches-Silva et al, 2014). The developments associated to the active packaging concept for food packaging producers and consumers should take into account the process of mass transfer and interactions between food and packaging materials (Figure I.7). These interactions could be divided into different individual processes, which could appear all together or be partially restricted in each particular case. These processes include migration of the material components (monomers or additives) to foodstuff, sorption and desorption of volatile compounds (flavours and aromas), changes in the food moisture content, permeability to gases and the possible degradation

~ 17 ~

Introduction

of food by external conditions (Silva-Weiss, Ihl, Sobral, Gómez-Guillén and Bifani, 2013). All these physico-chemical processes should be carefully studied to design and develop new packaging systems that leave behind the conventional passive container.

Figure I.7. Interaction processes between food packaging materials and the environment.

Active and intelligent packaging systems were firstly defined by the Framework

Regulation

(EC)

No

1935/2004

on

food

(Regulation_(EC)/No-1935/2004): i.

“Active food contact materials and articles” are defined as materials and articles that are intended to extend the shelf-life or to maintain or improve the condition of packaged food. They are designed to deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food.

ii.

“Intelligent food contact materials and articles” are defined as materials and articles that monitor the condition of packaged food or the environment surrounding the food.

~ 18 ~

Introduction

Active packaging includes the use of additives, which could be defined as “freshness enhancers”, able to diffuse and interact with food by increasing the packaging’s functionalities,, such as resistance to oxidation and microbiological spoilage or retention of natural aromas and flavours as well as food quality and safety (López-Gómez Gómez eet al, 2009). Figure I.8 summarizes the main causes of food spoilage and the strategies followed to limit or deactivate them.

Figure I.8. Mechanisms that can cause a loss of quality in food and active packaging applications.

The chemical nature of the additives used for such purpose is diverse, but all them should play their role efficiently by scavenging or absorbing oxygen, CO2, ethylene, moisture and/or odour and flavour taints; releasing oxygen, CO2, water vapour, ethanol, sorbates, AOs and/or other

~ 19 ~

Introduction

preservatives

and

antimicrobials

(AMs);

and/or

by

maintaining

temperature to avoid the food overheating during transport and distribution. New developments in active packaging materials, methods and effects on food have been under study in the last few years. These studies are based on chemical, physical or biological actions to modify and control the interactions between materials, food and the packaging headspace to achieve the desired outcome (Gómez-Estaca, López-deDicastillo, Hernández-Muñoz, Catalá and Gavara, 2014; Mellinas et al, 2015; Valdes et al, 2015). 1.2.1. Antimicrobial and antioxidant active packaging Antimicrobial active packaging is commonly designed to reduce the risk from pathogen attacks to food and to extend shelf-life by limiting spoilage effects caused by microorganisms. A wide range of agents with AM characteristics has been proposed, e.g. organic acids, bacteriocins, spice extracts, thiosulphates, enzymes, proteins, isothiocyanates, antibiotics, fungicides, chelating agents, parabens and metals (Sung et al, 2013). All them could be incorporated into or coated onto food packaging materials to get the desired effect (Singh, Wani and Saengerlaub, 2011). However, some important features should be considered to design an efficient AM packaging system. i.

They should be able to get the controlled release of the AM agent from the polymer film in the adequate time to maximize efficiency. The fast release of the AM agent from the packaging material to food is quite common and this is an important drawback of these systems, since the effect of the active compound is limited in time.

~ 20 ~

Introduction

ii.

The use of harmless substances is a requirement in food packaging and not all the proposed AM agents are currently included in the current legislation as chemicals intended to be in direct contact with food.

Antioxidant active packaging focuses on the improvement of the resistance to lipids oxidation retarding the natural processes that can lead to organoleptic deterioration and reduction of shelf-life of food products. The use of active packaging materials with AO properties is relevant in many types of food, but particularly for dried products and oxygensensitive food (Gómez-Estaca, López-de-Dicastillo, Hernández-Muñoz, Catalá and Gavara, 2014). Moreover, some authors reported that the addition of natural AOs to polymer matrices can protect the polymer from degradation during processing, with the double effect of protection for the material and food through controlled release mechanisms (Peltzer, Navarro, López and Jiménez, 2010; Wu, Qin, et al, 2014). Both types of active food packaging systems can be divided into two main groups. This classification is based on the incorporation of the active additive to the packaging material and the interaction between the agent and foodstuff (Bastarrachea, Dhawan and Sablani, 2011). Most of them are based on the use of thin films of polymer where the active agent is embedded. i.

Films that allow the release of the active additive by following a particular kinetic scheme. The AM and/or AO agent can be incorporated to the material either within the matrix or onto the material surface. The strategy for incorporation is relevant to control the release of active compounds from the material to the food surface. Different approaches have been proposed:

~ 21 ~

Introduction

a.

Packaging systems incorporating the active additive in a single layer, permitting a gradual release into food.

b. Packaging systems with an inner layer which can be useful to control the release rate of the AM and/or AO compounds from the outer layer. c. Packaging systems with a coating layer containing the active additive. ii.

Films that do not release the active additive but show a direct contact with food inhibiting the microbial growth or lipid oxidation on the food surface.

Figure I.9 shows the scheme of these different interaction mechanisms between active agents, packaging material and food. All these strategies for the incorporation of the active agents to the packaging material and further interaction with food have shown their potential, but some drawbacks related to the full control of the release kinetics of the AM and/or AO compounds to food have been described (Anbinder, Peruzzo, Martino and Amalvy, 2015; Fuciños et al, 2015). Recent studies have proposed the use of other strategies for the incorporation of active compounds to the packaging material permitting a more efficient release to food. These new techniques are encapsulation, a process by which small particles of core materials are packed within the wall material to form capsules to protect bioactive compounds from adverse environment and permitting the controlled release at targeted sites (Marques,

2010;

Ezhilarasi,

Karthik,

Chhanwal

and

Anandharamakrishnan, 2013; Dias et al, 2014; Noronha, de Carvalho, Lino and Barreto, 2014; Wen et al, 2016), grafting, which is one of the most promising methods to functionalize polymers (Schreiber, Bozell, Hayes and Zivanovic, 2013) and reinforcement with nanofillers with intrinsic

~ 22 ~

Introduction

AM properties, in particular metallic nanoparticles (Llorens, Lloret, Picouet, Trbojevich and Fernandez, 2012; Fortunati, Peltzer, Armentano, Jiménez and Kenny, 2013; Dias et al, 2014; Fortunati et al, 2014; Shankar, Teng, Li and Rhim, 2015). (a)

(b)

Release

Food

Food

Packaging Coating (c)

Food

Packaging

Outer Layer

Release

Release

Inner Layer (d)

Outer Layer

Food

Not release Direct contact

Packaging

Packaging

Figure I.9. Release of active substance in different applications of active packaging systems: films that allow the release of the active additive (a), (b) and (c); films that do not release the active additive (d); (adapted from (Bastarrachea, Dhawan and Sablani, 2011).

The introduction of bioplastics has permitted the design of systems where active agents are incorporated into biopolymer matrices, giving a surplus to these formulations by combining activity and sustainability. The proposal of new bioplastics, such as PLA or edible films and coatings, to substitute conventional plastics in active formulations has been recently reviewed (Rhim, Park and Ha, 2013; Mellinas et al, 2015) and many formulations have been proposed. Some of them are discussed below.

~ 23 ~

Introduction

Manzanarez-López et al reported a study based on PLA with 2.58 wt% of α-tocopherol as active additive with AO action for food packaging (Manzanarez-López, Soto-Valdez, Auras and Peralta, 2011). The main optical and thermal properties were evaluated as well as the kinetics of diffusion of the active agent from the PLA matrix to ethanol and vegetable oil, as food simulants. Their results showed a slower diffusion of α-tocopherol to soybean oil than to ethanol with 5.1 % and 12.9 % of release respectively after 60 days. Authors also studied the influence of temperature in the release kinetics by testing their systems at temperatures between 20 and 40 °C. The release of α-tocopherol from PLA films to soybean oil was enough to delay the oxidation at 20 and 30 °C, compared with the oil put in contact with pure PLA films with no active agent in their composition. Jamshidian et al used the solvent casting processing to obtain films based on PLA with natural AOs, including α-tocopherol, and synthetic phenolic AOs, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate or tert-butylhydroquinone (TBHQ) (Jamshidian, Tehrany and Desobry, 2013). They studied the release of all these AOs to different food simulants and calculated the kinetic coefficients. It was concluded that antioxidant active packaging was adequate for foodstuff protection and α-tocopherol can be used as a natural additive. Wu et al evaluated the AM activity of films based on PLA combined with PCL and thymol as active agent (Wu, Qin, et al, 2014). Results showed that the addition of thymol to the biopolymer matrix had a plasticizing effect by the decrease in the PLA Tg values and crystallinity, but not affecting the thermal stability of films. The AM activity was also evaluated and they reported that films with thymol showed inhibition against two foodborne bacteria: Escherichia coli and Listeria monocytogenes.

~ 24 ~

Introduction

Polysaccharides, lipids, proteins or their blends can be used as edible biopolymer matrices in active food packaging applications (Rhim, Park and Ha, 2013). Indeed, chitosan was proposed as edible matrix on bioactive coatings containing organic acids and nanoemulsions with carvacrol, mandarin, bergamot and lemon essential oils (EOs) (CruzRomero, Murphy, Morris, Cummins and Kerry, 2013). Gamma irradiation and modified atmosphere packaging (MAP) were proposed to increase efficiency of the active films in food (Severino et al, 2015). These authors confirmed the strong AM activity of carvacrol nanoemulsions against two Gram-negative pathogenic bacteria (Escherichia coli and Salmonella typhimurium). Authors stated that the bioactive coating with carvacrol onto the chitosan matrix resulted satisfactory in the total inhibition of these bacteria after 11 days of storage, highlighting the strong bactericidal effect of this coating. The effects of the addition of BHT and green tea extracts (GTE) on the physical, barrier, mechanical, thermal and AO properties of potato starch films were reported (u Nisa et al, 2015). The AO properties of these bioactive composites were evaluated by using the spectrophotometric method with formation of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) complex, permitting the determination of the radical scavenging ability of both (BHT and GTE starch films) after their contact with the fatty food simulant (ethanol 95 %, v/v). The formation of methamyoglobin was monitored while the lipid oxidation was evaluated by using the thiobarbituric acid reactive substances (TBARS) method. GTE and BHT films were individually applied to fresh beef samples stored at 4 °C and room temperature (RT) for 10 days. It was concluded that the addition of BHT and GTE extracts resulted in the decrease in the concentration of methamyoglobin and TBARS values.

~ 25 ~

Introduction

2. Natural additives The selection of the most adequate natural compounds to be used as AO or AM additives in active packaging formulations depends primarily on their activity against oxygen or the targeted microorganisms and their compatibility with the packaged food, while the continued release during storage and distribution is necessary to extend food shelf-life and quality (Manzanarez-López, Soto-Valdez, Auras and Peralta, 2011). But many other factors should be considered in the design of active packaging systems, such as their specific activity, resistance of microorganisms to the additives action, release kinetics and mechanisms, storage and distribution conditions, physical and mechanical properties of the packaging materials and organoleptic characteristics of food (Gómez-Estaca, López-deDicastillo, Hernández-Muñoz, Catalá and Gavara, 2014). All these factors should be carefully considered in agreement with the requirements stated in legal regulations (Dainelli, Gontard, Spyropoulos, van den Beuken and Tobback, 2008). All these considerations have led to the search for natural compounds to be used in active packaging formulations in substitution of the synthetic additives. Many studies have been performed to propose the use of compounds obtained from natural sources with AM and/or AO character (Srinivasan, 2012; Silva-Weiss, Ihl, Sobral, Gómez-Guillén and Bifani, 2013; Sung et al, 2013; Gómez-Estaca, López-de-Dicastillo, HernándezMuñoz, Catalá and Gavara, 2014; Gyawali and Ibrahim, 2014; Valdés, Mellinas, Ramos, Garrigós and Jiménez, 2014; Valdes et al, 2015). Natural additives can be obtained from different sources, including plants, animals, bacteria, algae, fungi and by-products generated during the fruits and vegetables processing. Figure I.10 summarizes the most relevant additives and other components with AM/AO activity obtained from

~ 26 ~

Introduction

different natural sources proposed for their use in food packaging applications.

Animal origin

Plant origin

•Casein and whey

•Plant-derived compounds

•Lysozyme

•EOs

•Lactoferrin

•Plant extracts

•Chitosan

•Phenolic compounds, quinones,

•Lipids

saponins, flavonoids, tannins, coumarins, terpenoids, and alkaloids •Plant by –products •Fruit pomace, seeds, peels, pulps, unused flesh and husks

Figure I.10. Compounds with potential activity from natural sources used in food packaging.

EOs are active agents obtained from plants, since they are fully-renewable additives, easy to extract and highly efficient in their AM and/or AO character. For example, the AO activity and AM effect against eight bacterial and nine fungal strains was evaluated in the EO of Mosla chinensis Maxim and its methanol extract (Cao et al, 2009). Results showed that this EO, whose main components are carvacrol (57 %), p-cymene (14 %), thymol acetate (13 %), thymol (7 %) and c-terpinene (2 %), exhibited great potential against microorganisms, in particular against two Grampositive bacteria common in many food products, Staphylococcus aureus and Listeria monocytogenes. Moreover, high AO activity was also reported for this EO.

~ 27 ~

Introduction

In general terms, EOs are rich in monoterpenes, sesquiterpenes, esters, aldehydes, ketones, acids, flavonoids and polyphenols (Ćavar Zeljković and Maksimović, 2015). All these chemicals have demonstrated their AO/AM character. Figure I.11. Chemical structures of some natural additives incorporated in active food packaging with AO/AM character (CAS numbers are indicated in parentheses).

Figure I.11 summarizes names and molecular composition of some of the main bioactive compounds that can be obtained from plants, in particular EOs and extracts. These compounds have been recently incorporated into or coated onto packaging films and their performance as active additives has been assessed. Li et al added different natural additives with AO properties, obtained from green tea, grape seeds, ginger and gingko leaf, into gelatin films with good results in the inhibition of the oxidation in selected food (Li, Miao, Wu, Chen and Zhang, 2014). Sánchez-Aldana et al used lime EO as AM agent on edible films with high activity against foodborne pathogenic bacteria (Escherichia coli O157:H7, Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes) determined by the agar-disc diffusion method (Sánchez Aldana, AndradeOchoa, Aguilar, Contreras-Esquivel and Nevárez-Moorillón, 2015). PCL with α-tocopherol (30, 50 and 70 wt%) incorporated by nanoencapsulation was proposed for the production of biodegradable and AO films based on methylcellulose (MC) (Noronha, de Carvalho, Lino and Barreto, 2014). Films were obtained by solvent casting and their mechanical and optical properties were determined together with their AO performance and release kinetics. The incorporation of α-tocopherol to PCL films produced a modification of their mechanical properties, decreasing their tensile strength around 60 % and the elastic modulus around 70 % when the nanocapsules percentage was high (70 wt%). These

~ 28 ~

Introduction

films showed high AO character by the incorporation of nanocapsules to permit the controlled release of α-tocopherol herol to food simulants. Carvacrol, (499-75-2)

Geraniol, (106-24 24-1)

Sorbic acid, (110-44-1)

Thymol, (89-83-8)

Linalool, (78-70--6)

Gallic acid, (149-91-7)

Tocopherol, (1406-66-2, 2, mixed of tocopherols)

α: (CH3; R1); (CH3; R2) β: (CH3; R1); (H; R2) γ: (H; R1); (CH3; R2) δ: (H; R1); (H; R2) Benzoic acid, (65-85-0)

Citric acid, (77-92 92-9)

(R)-(+)-Limonene, (5989-27-5)

Quercetin, (117-39-5)

(−)-Catechin, (18829 (18829-70-4)

3-Hydroxytyrosol, (10597-60-1)

Figure I.11. Chemical structures of some natural additives incorporated in active food packaging with AO/AM character (CAS numbers are indicated in parentheses).

2.1. Antimicrobial activity of essential oils The bacterial susceptibility to EOs and their extracts increases with the reduction in the pH of food products, since at low pHs the hydrophobic character of the oil increases, resulting in an easier dissolution in the cell

~ 29 ~

Introduction

membranes of the target bacteria (Burt, 2004). In addition, most common bacteria, in particular pathogens, have lower proliferation rate at low pHs. The mechanism of action of EOs against bacteria is not clear yet, since each compound present in the EO composition exhibits a unique mechanism of action that is specific to a particular range of food and microorganisms (Bastarrachea, Dhawan and Sablani, 2011). Different mechanisms have been identified: damage to the cell wall, interaction with and disruption of the cytoplasmic membrane, damage of membrane proteins, leakage of cellular components, coagulation of cytoplasm and depletion of the proton motive force. All these effects produce the microorganisms death by the modification of the structure and composition of the bacteria cells (Tajkarimi, Ibrahim and Cliver, 2010; Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2011b; Calo, Crandall, O'Bryan and Ricke, 2015). Two main types of bacterial cell wall structures have been studied, permitting their classification in Gram-positive and Gram-negative organisms (Figure I.12). Both types of cells have external cytoplasmic membranes but some details, such as the presence of a thin peptidoglycan layer in Gram-negative bacteria, make the difference (Aldred, Buck and Vall, 2009) and they should be more resistant to EO. These outer layers contain lipids, proteins and lipopolysaccharides in their composition, preventing the penetration of hydrophobic compounds, such as EOs (Feng et al, 2000; Maneerung, Tokura and Rujiravanit, 2008).

~ 30 ~

Introduction

Figure I.12. Structure of the bacterial cell wall (adapted from (Aldred, Buck and Vall, 2009).

It was reported that phenols, phenolic acids, quinones, saponins, flavonoids, tannins, coumarins, terpenoids and alkaloids present in EOs or plant extracts are responsible of their AM activity (Kuorwel, Cran, Sonneveld, eveld, Miltz and Bigger, 2011b; Sung et al, 2013). However, the total AM activity of EO cannot be attributed entirely to the mixture of their main components, since these complex matrices produce synergies between major and minor compounds to incr increase the AM action (Sánchez-González, Vargas, González-Martínez, Martínez, Chiralt and Cháfer, 2011).. Different authors have reported the effectiveness of EOs to inhibit different pathogenic and spoilage microorganisms, including Gram Grampositive bacteria, such as Staphylococcus aureus aureus, Listeria monocytogenes and Bacillus cereus; Gram-negative negative bacteria, such as Escherichia coli, Salmonella enteritidis,, Salmonella choleraesuis, Yersinia enterocoli enterocolitica and Pseudomonas aeruginosa; yeasts, such as Saccharomyces cerevisiae, Candida albicans, Debaryomyces hansenii; and molds, such as Alternaria alternate, Aspergillus niger, Botrytis cinerae, Aspergillus flavus, Penicllium roqueforti (Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2011b).

~ 31 ~

Introduction

2.2. Antioxidant activity of essential oils EOs and their main compounds are some of the most important additives to avoid food degradation by lipid oxidation due to their high reactivity with peroxyl radicals. The mechanism of action of these natural AOs in lipid oxidation reactions is focused on phenols and other compounds with hydroxyl groups presents in the EOs composition. Hydrogen atoms from phenol hydroxyl groups could react with the peroxyl radicals produced in the early stages of the oxidation mechanisms to yield stable phenoxyl radicals and, consequently, resulting in the termination of the lipid peroxidation chain reactions (Mastelic et al, 2008; Amorati, Foti and Valgimigli, 2013). However, the AO activity of these phenolic compounds depends on the electronic and steric effects of their ring substituents and on the strength of hydrogen-bonding interactions between the phenol and the solvent in the EO (Mastelic et al, 2008). A large variety of testing methods have been proposed to evaluate the AO activity of natural additives, either as pure compounds or plant extracts. Some of them are methods based on studies of the inhibition of the autoxidation reactions, which are commonly followed by monitoring the kinetics of oxygen consumption and the hydroperoxides formation. The analytical determination of secondary oxidation products (e.g. carbonyl compounds) has been also used and it is the basic reaction of the majority of the current testing methods even though they do not involve substrate autoxidation. These methods can be direct, such as ORAC (oxygen-radical antioxidant capacity) or TOSC (total oxidant scavenging capacity). But the most common are the indirect methods, which are based on the reduction of persistent radicals (e.g. DPPH and TEAC (Trolox-Equivalent Antioxidant Capacity) method), or inorganic oxidizing species (e.g. FRAP (Ferric Reducing Antioxidant Power) and Folin-Ciocalteu method)

~ 32 ~

Introduction

(Sánchez-Moreno, 2002; Amorati and Valgimigli, 2015). The DPPH method is based on the idea that the AO effect is proportional to the disappearance of the DPPH• free radical in test samples. Figure I.13 shows the mechanism by which the DPPH• free radical accepts hydrogen atoms from an AO (Moon and Shibamoto, 2009).

Figure I.13. Reaction between the DPPH• radical and AO to form the DPPH complex.

Barbosa-Pereira et al based their studies in the evaluation of the AO effectiveness of different commercial products containing natural additives incorporated into LDPE matrices by their effect on the delay of lipid oxidation in salmon muscles (Barbosa-Pereira et al, 2013). The AO activity of these films was tested by using the DPPH method and the effect in salmon muscle was evaluated by using the TBARS method. Results demonstrated that the natural products used in this trial had noticeable AO effectiveness, showing the best results the mixture of natural tocopherols with good possibilities to replace synthetic AOs in food packaging materials.

2.3. Carvacrol and Thymol Carvacrol (5-isopropyl-2-methylphenol) and thymol (2-isopropyl-5methylphenol) are two phenolic monoterpenes widely studied as natural additives in active packaging. Both compounds are isomers obtained from

~ 33 ~

Introduction

EOs obtained from many aromatic plants of the Labiatae family, including Origanum, Satureja, Thymbra, Thymus and Corydothymus species (Nostro and Papalia, 2012).. Their structures and main physico physico-chemical properties are shown in Table I.3. Table I.3. Structures and physico-chemical chemical properties of carvacrol and thymol. Carvacrol / 5-Isopropyl Isopropyl-2-methylphenol CAS number

499-75-22

Molecular weight

150.22 (g mol-1)

Boiling point

236-237 °C

Tm

3-4 °C

δ

0.976 g mL-1 at 20 °C

Physical state at RT

Liquid (oily)

Thymol / 2-Isopropyl Isopropyl-5-methylphenol CAS number

89-83-8

Molecular weight

150.22 (g mol-1)

Boiling point

232 °C

Tm

48-51 °C

δ

0.965 g mL-1 at 25 °C

Physical state at RT

Powder (White)

Both additives have been reported to present a wide variety of biological activities with potential interest in food applications, including antifungal, phytotoxic, insecticidal, AO, antitumor, antimutagenic, antiparasitic and AM (Ahn, Lee, Lee and Kim, 1998; Chizzola, Michitsch and Franz, 2008; Kordali et al, 2008; Xu, Zhou, Ji, Pei andd Xu, 2008; Nerioa, Olivero OliveroVerbel and Stashenko, 2010; Babili et al,, 2011) 2011). In this context, the effect of thymol and carvacrol against different microorganisms, such as Gram Grampositive bacteria, Gram-negative negative bacteria, food spoilage or pathogenic

~ 34 ~

Introduction

fungi and yeasts has been studied (Lambert, Skandamis, Coote and Nychas, 2001; Burt, 2004; Xu, Zhou, Ji, Pei and Xu, 2008; Gutierrez, Barry-Ryan and Bourke, 2009; Hazzit, Baaliouamer, Veríssimo, Faleiro and Miguel, 2009; Arana-Sánchez et al, 2010; Li et al, 2010; Babili et al, 2011; Du, Avena-Bustillos, Hua and McHugh, 2011; Nostro and Papalia, 2012; Friedman, 2014). Carvacrol and thymol can be used in food packaging since both additives have been approved as a safe food additive, in the U.S.A. due to their GRAS status and in Europe due to their classification as flavours (Commission_Regulation/(EU)/No-10/2011).

They

have

been

traditionally used as flavouring agents in foodstuff such as sweets, beverages and chewing gum (Commission_Decision/2002/113/EC; Nostro and Papalia, 2012). The presence of the hydroxyl group in the carvacrol and thymol structures enhance their AM and AO activities (Gyawali and Ibrahim, 2014). In both cases, the hydroxyl group acts as a proton exchanger, promoting the electrons delocalization and reducing the pH gradient through the cytoplasmic membrane. This interaction with the microorganisms cell membranes causes the collapse of the proton motive force, disrupting membrane structures and ultimately leading to the cells death (Ultee, Slump, Steging and Smid, 2000). More recently, Gyawali et al reported that thymol and carvacrol may show a different AM behaviour against Grampositive and Gram-negative bacteria due to the location of their hydroxyl groups, the meta position in thymol and the ortho position in carvacrol (Gyawali and Ibrahim, 2014).

~ 35 ~

Introduction

2.3.1. Use in packaging materials Active food packaging systems based on carvacrol and thymol have been tested in dry and fresh products, such as meat, cheese, fruits or vegetables. Table I.4 summarizes the most relevant studies in active packaging with carvacrol and/or thymol. These studies have evaluated different active materials in their thermal, mechanical and optical properties. Also, the release kinetics and mechanisms of some additives from the polymer matrix to food simulants or in direct contact with real food, as well as lipid oxidation or inhibition of foodborne bacteria have been investigated. All these studies reported that the addition of carvacrol and thymol to polymer matrices can produce some modification of their physicochemical properties. For example, edible films based on bovine gelatin with carvacrol showed a clear decrease in their TS, swelling and water uptake, increasing the εB, water solubility and WVP compared to the neat films with no additives (Kavoosi, Dadfar, Mohammadi Purfard and Mehrabi, 2013). These important changes in properties were related to the hydrophilic character of gelatin and chemical interactions with the hydrophobic carvacrol. Indeed, the hydrophobic domains of the gelatin structure may interact with carvacrol enhancing the interfacial interaction between the polymer matrix and additives, saturating the gelatin network with carvacrol molecules while water could not diffuse to the gelatin network, causing the decrease in swelling and water uptake. In addition, carvacrol showed some plasticizer effect when it was added to edible matrices resulting in changes in tensile properties, while some increase in the ductility of the polymer blend was also observed. Another important feature related to the use of thymol and carvacrol in food packaging systems is their high stability and control of their release to food over time (Kurek, Guinault, Voilley, Galić and Debeaufort, 2014).

~ 36 ~

Introduction

In fact, the release rate is a key parameter to allow a good and sustained microbial inhibition and AO activity of thymol and carvacrol. Recent works have reported the use of alternative techniques for their incorporation into polymers by using micro- or nano- encapsulation in cyclodextrins with the aim to improve and control their release rate (Tao, Hill, Peng and Gomes, 2014; Higueras, López-Carballo, Gavara and Hernández-Muñoz, 2015; Santos, Kamimura, Hill and Gomes, 2015).

~ 37 ~

Introduction

Table I.4. Carvacrol and thymol in active food packaging. Polymer base

Additives Amount

Potential studies

Effect of the additive

References

Inhibition of Escherichia coli and Listeria monocytogenes.

(Wu, Qin, et al, 2014)

AM Packaging PLA/PCL

Thymol 0, 3, 6, 9, and 12 wt%

PLA/poly(trimethylene carbonate), PTMC

Thymol 0, 3, 6, 9 and 12 wt%

Starch

Thymol and carvacrol 1, 3 and 5 wt%

TPS SPI/PLA

Thymol and carvacrol 3 and 4 wt% Thymol 2.5, 5, 10, 15, 20, 25 and 50 wt%

AM activity by observing the bacterial growth and counting the colony-forming units (CFU). Mechanical characterization, water vapour permeability (WVP), and AM activity. AM activity by using the agar disc diffusion method and inoculation on the surfaces of Cheddar cheese. Release of additives into isooctane as a fattyfood simulant. AM activity by using the agar disc diffusion method.

Chitosan

Carvacrol 0.01-3 % (w/v)

AM efficiency by the vapour phase.

Chitosan/cyclodextrin films

Carvacrol 2.3 g/g of dry film

AM activity of carvacrol-CS films on chicken breast fillets.

Chitosan/cyclodextrin films

Carvacrol 216.3, 133.3 and 56.8 % (g carvacrol/g dry film)

AM activity of films by using a microatmosphere method.

~ 38 ~

Inhibition of Escherichia coli, Staphylococcus aureus, Listeria, Bacillus subtilis, and Salmonella. Inhibition of Staphylococcus aureus in vitro or inoculated on the surfaces of Cheddar cheese. Controlled release of both additives. Inhibition of Staphylococcus aureus, Escherichia coli, Aspergillus spp. and Saccharomyces cerevisiae. Inhibition of Bacillus subtilis, Listeria innocua, Escherichia coli and Salmonella enteritidis. Inhibition of the total aerobic bacteria, and Pseudomonas spp., Enterobacteria, Lactic acid bacteria, yeasts and fungi. Inhibition of Staphylococcus aureus and Escherichia coli.

(Wu, Yuan, et al, 2014) (Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2011a) (Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2013) (González and Alvarez Igarzabal, 2013) (Kurek, Moundanga, Favier, Galić and Debeaufort, 2013) (Higueras, López-Carballo, Hernández-Muñoz, Catalá and Gavara, 2014) (Higueras, López-Carballo, Gavara and Hernández-Muñoz, 2015)

Introduction

Table I.4. (Cont.) Calcium caseinate Sodium caseinate

Carvacrol 10 wt%

Chitosan

Carvacrol 1 wt% (nanoemulsion)

Pectin-based apple, carrot, and hibiscus edible films

Carvacrol 0.5, 1.5 and 3 wt%

AM activity by using the agar disc diffusion method. AM effect of the combined treatment of coating, MAP and gamma-irradiation to green beans during storage. AM activity by the inoculation of ham samples with Listeria. monocytogenes.

Inhibition of Staphylococcus aureus and Escherichia coli.

(Arrieta, Peltzer, Garrigós and Jiménez, 2013; Arrieta, Peltzer, et al, 2014)

Inhibition of Escherichia coli and Salmonella typhimurium.

(Severino et al, 2015)

Inhibition of Listeria monocytogenes on contaminated ham samples.

(Ravishankar et al, 2012)

Controlled release of carvacrol.

(Peltzer, Wagner and Jiménez, 2009)

Inhibition of lipid oxidation and positive effect on the colour stability of beef patties during storage.

(Park et al, 2012)

Controlled release of thymol from LLDPE impregnated with this additive

(Torres, Romero, Macan, Guarda and Galotto, 2014)

Inhibition of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

(Kavoosi, Dadfar, Mohammadi Purfard and Mehrabi, 2013)

AO Packaging HDPE

Carvacrol 1 and 2 wt%

Corn-zein-laminated/LDPE

Thymol and carvacrol 1.5, 3 and 5 % (v/v)

Release of additives into water and olive oil as a food simulant. Minimum effective concentrations, release kinetics in the gas and liquid phases and study of the lipid oxidation in fresh ground beef packaging. AO and AM packaging

LLDPE

Thymol 1.48, 2.17, 3.81 %

Bovine gelatin Films

Carvacrol 1, 2, 3, 4 and 5 wt%

Characterization of the mass transfer during the migration process of thymol from LLDPE films AM activity by using the agar disc diffusion method and decolourization method with ABTS to AO activity.

~ 39 ~

Introduction

Table I.4. (Cont.)

Bovine gelatin Films

Thymol 1, 2, 3, 4 and 8 wt%

Chitosan

Carvacrol 0.5, 1.0 and 1.5 % (v/v)

Strawberry puree edible films

Carvacrol 0.75 wt%

AM activity by using the agar disc diffusion method and decolourization method with ABTS to AO activity. AM activity by using agar diffusion method and AO activity by using radical scavenging capacity using DPPH method TEAC. AO activity by using an adaptation of the DPPH method and identification of major fungal species in real food.

~ 40 ~

Inhibition of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. AO activity was increased with increasing the concentration of thymol in the films. Inhibition of Escherichia coli O157:H7 and Salmonella typhimurium. Increase of AO capacity. Inhibition of fungal species until 10 days in real food and improvement of fruit quality.

(Kavoosi, Dadfar and Purfard, 2013)

(López-Mata et al, 2013)

(Peretto et al, 2014)

Introduction

3. Nanotechnology in the food industry Nanotechnology has developed in the last decade into a multidisciplinary field of applied science and technology, representing a revolution in many concepts in materials science. The novel properties and functions provided by nanomaterials and the increasing possibilities offered by working at the nano-scale, between 1 and 100 nanometers, has resulted in advanced materials with a large number of potential applications, including food industry (Cushen, Kerry, Morris, Cruz-Romero and Cummins, 2012). In this general context, nanofillers have gained some space as additives in food packaging materials by their action in improving some of their key properties, including mechanical and barrier performance. There are many different terms to refer to nanofillers depending on their morphology, but the most accepted classification includes nanoparticles, nanofibrils, nanorods, nanocrystals and nanotubes (Zaman, Manshoor, Khalid and Araby, 2014). In particular, nanoparticles are defined as discrete entities with their three dimensions in the nano-scale (lower than 100 nm). Nanoparticles show larger surface area, aspect ratio and higher number of surface atoms than their microscale counterparts (de Azeredo, 2009). The research in the use of nanoparticles in food packaging materials has increased since their introduction has allowed creating, understanding, characterizing and using these compounds in material structures, devices and systems with novel and unseen properties and this is the main reason of their importance in food packaging applications (Cushen, Kerry, Morris, Cruz-Romero and Cummins, 2012). In this area, nanotechnologies have offered innovation and technological advances all over the production chain; from primary production at the farming level,

~ 41 ~

Introduction

due to advances in pesticides efficiency and delivery, to processing and properties of the final food product to improve taste, colour, flavour, texture

and

consistency

(Ezhilarasi,

Karthik,

Chhanwal

and

Anandharamakrishnan, 2013; Mihindukulasuriya and Lim, 2014). Other important features of the use of nanotechnologies in food industries include the increase in absorption and bioavailability of food and food ingredients (nutrients) by their nanoencapsulation. Different methods have been suggested to nanoencapsulate active principles into food packaging materials, such as spray-drying and electrospinning, showing promising results as novel delivery vehicles for supplementary food compounds working with aqueous solutions at RT (Ghorani and Tucker, 2015; Santos, Kamimura, Hill and Gomes, 2015; Wen et al, 2016). Figure I.14 shows some of the main nanofillers that have been reported for their incorporation into food contact materials to enhance their mechanical and barrier properties, to prevent their photodegradation and to preserve and extend the food shelf-life by their AM effect (Othman, 2014).

Inorganic origin

Organic origin

Natural biopolymers Chitosan Chitin Starch Cellulose

Carbon nanofillers Fullerenes Graphene Carbon nanotubes

Natural antimicrob. agents Nisin

Clays Mica Montmorillonite Sepiolite Laponite

Metals Silver Copper Gold Palladium Iron

Metal Oxides ZnO TiO2 MgO AgO CuO

Figure I.14. Nanofillers in food packaging applications.

~ 42 ~

Introduction

3.1. Nanoclays Many types of commercial nanofillers have been proposed for their incorporation into polymer matrices to form nanocomposites. The initial research in this field was focused on the use of layered silicates, also known as nanoclays. They typically have a stacked arrangement of silicate layers (nanoplatelets) with nanometric dimensions (de Azeredo, 2013; Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013). The most common crystalline arrangement in layered silicates is called the phyllosilicate structure, which is particularly remarkable in smectites which are based on 2:1 layers distribution made up of two tetrahedral coordinated silicon atoms forming an edge-shared octahedral sheet. These sheets show central holes where native metal atoms, such as aluminum or magnesium, could be found (Figure I.15) (Sinha Ray and Okamoto, 2003; Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013). The separation between layers is known as intergallery or interlayer spacing and it is due to the regular stacking of the layers, as observed in Figure I.15. The layers dimensions depend on the clay source and the preparation technique, but most of them show thicknesses around 1 nm and their length vary from tens of nanometres to more than one micron. Hence, phyllosilicates show very high aspect ratio (surface-to-volume) and surface area (Alexandre and Dubois, 2000).

Figure I.15. Crystalline structure of smectites, (2:1 layered silicate structure) (T, tetrahedral sheet; O, octahedral sheet; C, intercalated cations; d, interlayer distance) (Bordes, Pollet and Avérous, 2009).

~ 43 ~

Introduction

The substitution of the native metal atoms by other cations gives different functionalities and properties to these nanoclays. These substitutions could take place predominantly in the octahedral sheet; for example, Al3+ could be replaced by Mg2+ or Fe2+. These substitutions generate negative charges, which are counterbalanced by cations, such as Na+, K+, Li+ and Ca2+ located within the interlayer spacing, producing an increase of the clay hydrophilic character and charged surface, which is characterized by the cation exchange capacity (CEC), which is generally defined as milliequivalents of cations in 100 g of layered silicates (meq/100g). In general terms, CEC corresponds to the number of monovalent countercations within the interlayer spacing and it is a characteristic parameter in layered silicates (Sinha Ray and Okamoto, 2003). Smectites are the most common layered phyllosilicate due to their availability, low cost, significant enhancement in key properties of polymers and relatively simple processability (de Azeredo, 2009). Table I.5 shows the characteristics of the main smectites and their structural characteristics. Table I.5. Structural characteristics of common smectites (2:1 layered silicates) (adapted from (Bordes, Pollet and Avérous, 2009). Smectite group/Formula 2:1 Layered Silicates Saponites Ca

0.25

(Mg,Fe) ((Si,Al) O )(OH) nH O 3

4

10

2

2

Cation

Interlayer cations

CEC (meq/100g)

Aspect Ratio

Mg2+

Na+ Ca2+ Mg2+

86.6

50-60

Al3+

Na+ Ca2+ Mg2+

110

100-150

Mg2+

Na+ Ca2+ Mg2+

120

200-300

Montmorillonites (Na,Ca)0.33(Al,Mg)2(Si4O10)(OH)2nH2O Hectorites Na0.3(Mg,Li)3(Si4O10)(F,OH)2

~ 44 ~

Introduction

Montmorillonites (MMTs) are the most widely studied nanoclays since they show high swelling capacity in aqueous media favouring the dispersion of silicates into their individual layers, making them adequate for the formulation of nanocomposites with polymers. However, this high swelling capacity makes MMTs hydrophilic resulting in low compatibility with hydrophobic polymers (Raquez, Habibi, Murariu and Dubois, 2013). Therefore, the organo-modification of layered MMTs is a requirement to get stable nanocomposites. These modifications are usually performed via ion exchange reactions and have the main aim of matching the polymer polarity. Other techniques, such as organosilane grafting, use of monomers or block copolymers adsorption can be also used for such a purpose (Sreejarani and Suprakas, 2012). Ion exchange reactions in MMTs are based on the replacement of the original interlayer inorganic ions with organic cations, such as cationic surfactants. These compounds include primary, secondary, tertiary and quaternary alkyl-ammonium or alkyl-phosphonium cations with at least one long alkyl chain, called tallow chain (T) (Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013). Furthermore, the use of organomodified MMTs produce an increase of the interlayer spacing due to the large volume of the organo-modifying cations, favouring the dispersion of organo-modified montmorillonites (OMMTs) into their individual layers in the polymer matrix and thereby improving in a high degree the service properties of nanocomposites, in particular their mechanical and barrier performance. Table I.6 shows the most common commercial OMMTs besides their characteristic features. In addition, the organic substituent can provide specific functional groups able to react with the polymer matrix or in some cases initiate the polymerization process to improve the

~ 45 ~

Introduction

strength of the interface between the silicate and the polymer matrix (Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013). Interactions between clays and polymer matrices are illustrated in Figure I.16 and they can be classified into three main groups (Bordes, Pollet and Avérous, 2009): i.

Microcomposites, where clay particles are dispersed within the polymer matrix but their layers are still stacked, resulting in two phases since the polymer is not intercalated within the silicate layers due to the poor polymer-clay affinity. These materials can show phase separation.

ii.

Intercalated nanocomposites, where the polymer chains are partially intercalated between the silicate layers, but the system still remains well ordered in a stacked type of arrangement with some increase in the interlayer spacing.

iii.

Exfoliated nanocomposites, where the silicate layers are completely delaminated from each other and the clay platelets are well-dispersed between the polymer chains. In this case, the layered structure of clays is not observed since polymer and clay form a unique continuous phase.

Figure I.16. Polymer-clay structures according to the distribution of layered silicates into the polymer matrix (de Azeredo, 2009).

~ 46 ~

Introduction

Araújo et al studied the influence of the clay organic modifier on the thermal stability of PLA-based nanocomposites with different commercial OMMTs, Cloisite®30B (C30B), Cloisite®15A (C15A) and Dellite®43B (D43B) at different concentrations (3 and 5 wt%) (Araújo, Botelho, Oliveira and Machado, 2014). All nanocomposites were submitted to thermo-oxidative degradation at 140 °C for 120 hours by using an oven under air atmosphere. Authors reported that the better dispersion achieved with C30B could be associated to the strong interactions between the carbonyl functions of PLA chains and the hydroxyl functions of the modifier, which improve the dispersion of this nanoclay through the PLA matrix. They calculated the interlayer spacing values (d-spacing values) by using Bragg's law and results showed high increases in dspacing of nanocomposites with respect to the original matrix (1.60 nm for C30B and 1.65 nm for D43B).

~ 47 ~

Introduction

Table I.6. Commercial organo-modified montmorillonites (adapted from (Bordes, Pollet and Avérous, 2009; Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013). OMMTs / Designation

Organo-modifiying

CEC

Interlayer

WLignition

typea

(meq/100g)

spacing (Å)

(%)b

Used by…

none

-

11.7

7

125

19.3

39

(Shemesh, Goldman, et al, 2015)

125

31.5

43

(Araújo, Botelho, Oliveira and Machado, 2014; Shemesh,

Supplier: Southern Clay Products (USA) Cloisite® Na / CNA

(Dias et al, 2014; Shemesh, Goldman, et al, 2015)

Cloisite®10A / C10A

N+(Me)2(benzyl)

Cloisite®15A / C15A

N+(Me)2(T)2

Cloisite®20A / C20A

N+(Me)2(T)2

95

24.2

38

(Olivares-Maldonado et al, 2014; Shemesh, Goldman, et al, 2015)

Cloisite®25A / C25A

N+(Me)2(C8H17)(T)

95

18.6

34

(Olivares-Maldonado et al, 2014)

Cloisite®93A / C93A

NH+(Me)(T)2

90

23.6

40

(Olivares-Maldonado et al, 2014; Xia, Rubino and Auras, 2015)

Cloisite®30B / C30B

N+(Me)(EtOH)2(T)

90

18.5

30

(Fukushima, Tabuani and Abbate, 2011; Araújo, Botelho, Oliveira

(T)

Goldman, et al, 2015)

and Machado, 2014; Efrati et al, 2014) Supplier: Laviosa Chimica Mineraria (Italy) Dellite® 43B / D43B aTallow

N+(Me)2(benzyl)(T)

66

16.6

(T): ~ 65 % C18; ~ 30 % C16; ~5 % C14

bWLignition:

Weight Loose on ignition

~ 48 ~

(Scatto et al, 2013; Araújo, Botelho, Oliveira and Machado, 2014)

Introduction

3.2. Silver nanoparticles (Ag-NPs) Metallic nanofillers have found some space in the packaging technologies, in particular in active systems since they are able not only to enhance barrier and mechanical properties when they are incorporated into materials in direct contact with food, but also to improve the food preservation and shelf-life by their AM performance (Jokar, Abdul Rahman, Ibrahim, Abdullah and Tan, 2010; Erem, Ozcan, Erem and Skrifvars, 2013; Kanmani and Rhim, 2014b; Pagno et al, 2015). Copper, zinc, titanium, gold and silver NPs and some of their metallic oxides have been proposed as active additives to extend food shelf-life and to provide affordable and safe innovative strategies (Llorens, Lloret, Picouet, Trbojevich and Fernandez, 2012). Ag-NPs are those most widely used for the development of active packaging materials by their high surface-to-volume ratio which provides better contact with microorganisms, showing their efficiency in AM behaviour compared to ionic silver. In addition, Ag-NPs show unique properties in their electric, optical, catalytic, and thermal stability performance (Dallas, Sharma and Zboril, 2011). The AM activity of silver and silver salts has been well known for centuries in their application in curative and preventive health care (Dallas, Sharma and Zboril, 2011). Some examples include the use of silver nitrate in the treatment of venereal diseases, fistulae and abscesses or in the treatment of burn wounds (Klasen, 2000; Rai, Yadav and Gade, 2009). In the last decade, many researchers have reported the strong AM activity of silver, in particular when it is used as nanoparticles, against a wide variety of Gram-positive and Gram-negative bacteria, viruses and fungi (Kim et al, 2007; Rai, Yadav and Gade, 2009; Sharma, Yngard and Lin,

~ 49 ~

Introduction

2009). However, the AM mechanism of the Ag-NPs is a highly controversial subject, in particular when referred to materials in direct contact with food. These controversies are mainly due to their small dimensions, required to achieve a significant AM effect, the requirement of an oxidized surface and the subsequent feasible exchange of silver ions. For these reasons, the mechanism of the AM action of Ag-NPs is not well known yet. The proposals for mechanisms suggested by several authors are supported by the morphological and structural changes found in the bacterial cells and the possibilities of Ag-NPs to penetrate inside the bacterial structurel due to their attachment to the cell membrane (Reidy, Haase, Luch, Dawson and Lynch, 2013). Rhim et al proposed another mechanism to explain the activity of silver ions and Ag-NPs against bacteria (Rhim, Park and Ha, 2013). Silver ions interact with negatively charged groups in the enzymes and nucleic acids, causing direct damage to cell walls and membranes by structural changes and deformation and leading to disruption of metabolic processes followed by cells death. It has been reported that the increase in the surface area of Ag-NPs is associated with the high release rate of silver ions and consequently the electrostatic attraction between the negativecharged cell membranes and the positive-charged nanoparticles is improved causing direct damage to the cell membranes (Kim et al, 2007). The accumulation of Ag-NPs in the bacterial cytoplasmic membrane can also produce a significant increase in permeability with the result of AgNPs entering into the bacterial cells and altering the respiratory chain, cells division and finally leading to death (Kim et al, 2007; Rhim, Park and Ha, 2013). Sondi et al studied the surface morphology of Escherichia coli inoculated in agar plates supplemented with Ag-NPs from 10 to 100 μg cm−3 (Sondi

~ 50 ~

Introduction

and Salopek-Sondi, 2004). Scanning electron microscopy (SEM) images of these bacteria cell walls are shown in Figure I.17. Figure I.17A shows changes in the treated bacterial cells resulting in major damage due to the formation of “pits” in cell walls. The energy dispersive X-ray analysis (EDAX) of these samples (Figure I.17B) showed that Ag-NPs were incorporated into the membrane of the treated bacterial cells since the characteristic optical absorption peak of Ag at around 3 keV is observed due to surface plasmon resonance.

(A)

(B)

Figure I.17. (A) SEM micrographs of native Escherichia coli cells (a) and cells treated with 50 μg cm−3 of Ag-NPs in liquid medium for 4 hours (b); (B) EDAX spectra of native Escherichia coli (a) and Escherichia coli treated with 50 μg cm−3 of Ag-NPs in liquid medium for 4 hours (b).(Sondi and Salopek-Sondi, 2004).

Ag-NPs can be synthesized either by ex situ synthesis by chemical reduction or in situ with direct contact with bacteria cells (de Azeredo,

~ 51 ~

Introduction

2013). Both methods include the use of polymer matrices as carriers, biological

macromolecules,

mesoporous

inorganic

materials

and

hydrogels. Other environmentally-friendly approaches to obtain Ag-NPs have been proposed (Sharma, Yngard and Lin, 2009; Rajan, Chandran, Harper, Yun and Kalaichelvan, 2015). Extracts of Skimmia laureola have been used by Ahmed et al to synthesize Ag-NPs (Ahmed, Murtaza, Mehmood and Bhatti, 2015). Authors reported that the obtained spherical nanoparticles showed around 40 nm diameter and AM activity against Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli. When Ag-NPs are immobilized in polymer matrices, they can display their AM activity by the release of metal ions due to the high water sorption created by the hydrophilic character of some biopolymers. The moisture sensitivity and the associated plasticizing effect due to the water sorption induce the uncontrolled release of immobilized nanoparticles besides the oxidation of silver, releasing gradually silver ions (Llorens, Lloret, Picouet, Trbojevich and Fernandez, 2012). Echegoyen and Nerín studied the release of Ag-NPs incorporated into polyolefins in two food simulants: ethanol 50 % (v/v) and acetic acid 3 % (v/v) at two testing conditions: 40 °C for 10 days and 70 °C for 2 hours in three cycles (Echegoyen and Nerín, 2013). Results showed that the overall migration of silver under these conditions was far below the limits stated by the European legislation in all cases ensuring the possibilities of these formulations with Ag-NPs in food packaging applications.

~ 52 ~

Introduction

3.3. Nanocomposites in food packaging The use of nanocomposites is gaining some space in the food and beverage packaging market, although it is not yet widely introduced by the increase in costs of the final material and the strict legislative requirements regarding the use of materials in the nanoscale in food applications. Research is raising fast in this area and Table I.7 summarizes some examples of nanocomposites in food packaging applications. The use of nanocomposites in food packaging materials has resulted in improving some of their key properties, such as strength and flexibility, barrier to gases, moisture stability and higher resistance to heat and cold (Restuccia et al, 2010; Cushen, Kerry, Morris, Cruz-Romero and Cummins, 2012). For example, the addition of low amounts of metal nanoparticles or nanofillers to PLA matrices results in improvements in the intrinsic poor mechanical resistance, thermal, and gas barrier properties of this biopolymer. These are essential characteristics for packaging materials and have joined to key properties of PLA, including thermoplasticity, high transparency and biocompatibility, for its use as a valuable and sustainable packaging material (Araújo, Botelho, Oliveira and Machado, 2014). In general terms, the studies based on the use of PLA with nanofillers showed the clear increase in toughness and tensile strength of its nanocomposites after the addition of nanoclays and/or metal nanoparticles.

For

example,

tensile

properties

of

PLA-based

nanocomposites can be improved with the addition of C30B at different concentrations. The increase in their elastic modulus and tensile strength compared with the unfilled PLA was around 40 % and 50 % respectively. Jollands and Gupta reported that the elastic modulus was around 4200 MPa for unfilled PLA and 5900 MPa for PLA with 4 wt% of C30B, while

~ 53 ~

Introduction

tensile strength was 32 MPa and 59 MPa respectively (Jollands and Gupta, 2010). Fukushima et al. also reported that the highest thermo-mechanical and mechanical improvements in PLA matrices were obtained upon the addition of 10 wt% of nanoclay, and they are associated with the good dispersion level observed by using wide angle X-ray scattering (WAXS) and to the high clay content (Fukushima, Tabuani, Arena, Gennari and Camino, 2013).

~ 54 ~

Introduction

Table I.7. Representative examples of nanocomposites application in food packaging. Polymer matrix

Nanofiller

Amount

Processing

Quinoa starch

Au nanoparticles

2.5 and 5 % (v/v)

Solvent casting (82 °C)

Foodgrade agar

Ag-NPs

0, 0.2, 0.5, 1.0 and 2.0 wt%

Solvent casting (95 °C)

Gelatinbased

ZnO nanoparticles

N.R.

Solvent casting (80 °C)

LDPE

TiO2 nanoparticles

0.05, 0.08 and 0.11 g TiO2 in 100 mL ethyl methyl ketone

Manual Coating

PE

TiO2 nanoparticles

PLA

TiO2 nanoparticles

1, 3.5 and 8 wt%

LDPE

Ag-NPs

0.1, 0.3, 0.5, 3 and 5 wt%

PLA

Silver/MMT

1, 5 and 10 wt%

3 wt%

Melt extrusion (130 °C) Melt blending (180 °C) Melt blending (140 °C) Solvent casting (RT)

~ 55 ~

Effect of nanofiller Inhibition of 99 % against Escherichia coli and 98 % against Staphylococcus aureus. Improvement in mechanical and optical performance, maintaining the thermal and barrier properties. Increase in WVP and surface hydrophobic character. Strong AM activity against Listeria monocytogenes and Escherichia coli. Antibacterial activity against Gram-positive and Gram-negative bacteria. Strong activity against Listeria monocytogenes. Enhanced thermal stability. AM activity of the films exposed to fluorescent and UV radiation increased with the TiO2 nanoparticles concentration. Improved barrier properties. Excellent AM activity against Pseudomonas spp. and ethylene photodegradation. Improvement of E and crystallization temperature. AM activity increase under UVA irradiation.

References

(Pagno et al, 2015)

(Rhim, Wang and Hong, 2013) (Shankar, Teng, Li and Rhim, 2015) (Othman, Abd Salam, Zainal, Kadir Basha and Talib, 2014) (Bodaghi, Mostofi, Oromiehie, Ghanbarzadeh and Hagh, 2015) (Fonseca et al, 2015)

AM activity against Staphylococcus aureus and Escherichia coli.

(Jokar, Abdul Rahman, Ibrahim, Abdullah and Tan, 2010)

Migration levels of silver, within the legislation and high AM activity against Salmonella spp.

(Busolo, Fernandez, Ocio and Lagaron, 2010)

Introduction

Table I.7. (cont) Corn starch

Chitosan-MMT Laponite RD

5 wt%

Chitosan

C30B

5 wt%

PLA

C30B Fluoro-hectorite/SOMASIF MEE (SOMMEE)

5 and 10 wt%

PLA

Cellulose nanocrystals from Posidonia oceanica

1 and 3 wt%

Blending (RT) Solvent casting (60 °C) Melt blending (165 °C) Solvent casting (RT)

(Not reported, N.R.)

~ 56 ~

Reinforcing effect: Improvement of E and tensile strength. Reinforcing effect: Improvement of E and tensile strength. Reduction of oxygen transmission rate (OTR).

(Chung et al, 2010) (Rodríguez, Galotto, Guarda and Bruna, 2012)

Acceleration in the degradation of PLA in compost at 40 °C.

(Fukushima, Tabuani, Arena, Gennari and Camino, 2013)

Migration levels into two food simulants well below the European legislative limits.

(Fortunati et al, 2015)

Introduction

PLA-based nanocomposites with D43B showed higher thermal stability than those with C15A and C30B after thermo-oxidative degradation experiments due to the hydrophobic character caused by the aromatic ring in the D43B structure (Araújo, Botelho, Oliveira and Machado, 2014). PLA is a hydrophobic polymer due to the presence of methyl groups and the D43B can be easily dispersed into the polymer structure. Moreover, the ability of D43B to absorb moisture is lower than in the cases of C15A and C30B, retarding the PLA hydrolysis. In general terms, gas barrier properties can be greatly improved with the inclusion of particulate nanomaterials into polymer matrices. The mechanism for this increase in barrier to gases is based on the higher tortuosity of the path to be followed by gas molecules in the presence of nanoparticles. Carboxy(methylcellulose)

films

reinforced

with

MMT

improved

significantly their barrier properties to oxygen (around 50 %). This effect was due to the high degree of exfoliation/intercalation reached in these nanocomposites and the possible interactions between the polymer and the nanoclay (Quilaqueo Gutiérrez, Echeverría, Ihl, Bifani and Mauri, 2012). 3.3.1. Preparation and processing Processing techniques for nanocomposites should be optimized to obtain well-dispersed nanoparticles with high structural integrity and to minimize their adverse effects to the polymer matrix, such as their possible degradation at high temperatures. Nanocomposites are usually obtained by using three main techniques: (i) in-situ intercalation, where the layered silicates are swollen in a monomer solution before polymerization; (ii) solvent intercalation, consisting of

~ 57 ~

Introduction

swelling the layered silicates in a suitable solvent to promote the diffusion of the macromolecular chains in the clay galleries; and (iii) meltintercalation, where usual polymer processing in the molten state, such as extrusion, are used. Other techniques, such as electrospinning (Ghorani and Tucker, 2015) and electrospraying (Tapia-Hernández et al, 2015) have been recently proposed for the preparation of homogeneous nanobiocomposites. The use of supercritical conditions, such as those offered by supercritical CO2 as blowing agent to obtain PLA-based foams with C30B has been also reported (Keshtkar, Nofar, Park and Carreau, 2014). Yang et al used supercritical CO2 to pre-disperse commercial organic MMTs with further solvent mixing with PS to form nanocomposites with significant dispersion and interfacial enhancement (Yang, Manitiu, Kriegel and Kannan, 2014). X-Ray Diffraction (XRD) results showed that nanocomposites with C10A and C20A increased the interlayer spacing of these nanoclays. Nanocomposites with C10A showed diffraction values 2θ = 2.4° corresponding to an increase in the interlayer spacing from 1.05 to 2.68 nm and nanocomposites with C20A experienced an increase from 1.77 to 2.68 nm, suggesting that the polymer chains had been intercalated into the clay galleries. Obviously, the melt intercalation process is highly preferred for food packaging producers since there is no need of organic solvents and the production can be easily scaled-up to industry.

4. Active Nanocomposites Active nanocomposites are particularly useful in emerging technologies in food packaging due to their improved structural integrity and barrier properties by the addition of nanomaterials (either nanoclays or metal

~ 58 ~

Introduction

nanoparticles), and the increase in AM and/or AO properties in most cases by the action of active additives and/or the own nanofiller. Nevertheless, the selection of the most adequate AM and/or AO agent to be combined with nanofillers is often a complex task by the lack of compatibility of many active compounds with some polymer matrices or by the poor heat resistance of active agents, polymers or nanofillers hampering their stability during processing. The effect of active nanocomposites in direct contact with food depends on the specific activity of each active agent against spoilage microorganisms and/or oxidation processes, besides the nanofiller and the polymer as well as other additives, such as plasticizers (Rhim and Ng, 2007). In this sense, the polymer matrix plays the most important role in regulating the action of additives and nanofillers by controlling the particle release or the homogeneous distribution of nanofillers. Much research is currently ongoing in this area by evaluating the possibility to mix different types of nanofillers (nanoclays, nanocelluloses, Ag-NPs, etc.) with AM and/or AO additives in conventional plastics or bioplastics. EOs obtained from rosemary (Abdollahi, Rezaei and Farzi, 2012; Gorrasi, 2015), clove ,cumin, caraway, marjoram, cinnamon and coriander (Alboofetileh, Rezaei, Hosseini and Abdollahi, 2014) or Zataria multiflora Boiss (Shojaee-Aliabadi et al, 2014) have shown promising possibilities in active nanocomposites. The main compounds of many EOs, such as α-tocopherol (Dias et al, 2014) and hydroxytyrosol (Beltrán, Valente, Jiménez and Garrigós, 2014) as well as plant extracts, such as pomegranate rind powder extract (Qin et al, 2015) or alcoholic extracts of red propolis (Costa, Druzian, Machado, De Souza and Guimaraes, 2014), play an important role in novel active nanocomposites. Table I.8 shows the comparison between two active nanocomposites obtained by solvent

~ 59 ~

Introduction

casting

and

based

on

edible

films

with

different

nanofillers

(nanocrystalline cellulose (CNC) and MMT) with two different EOs, savory (S-EO) and Zataria multiflora Boiss (ZMB-EO). Results suggested that mechanical properties were largely influenced by the amount and kind of clay. However, the WVP values showed different behaviour depending on the active component used in each formulation. In the presence of ZMB-EO, WVP values clearly decreased and this result was related to the hydrophobic

nature

of

the

EO,

which

affects

the

hydrophilic/hydrophobic character of films and increases the tortuosity of the polymer internal structure. In addition, the presence of low amounts of the EO likely changes the hydrogen-bonding network within the polymer structure and allows better intercalation of κ-carragenan molecules into the silicate galleries in MMT nanocomposites. On the other hand, S-EO in active nanocomposites based on agar film solutions resulted in significant increases in the WVP due to the formation of cracks or fractures in the nanocomposite structure enhancing the diffusion of moisture molecules through the films and thereby increasing the WVP values. Nanocomposites with metal nanoparticles are gaining some space in active packaging, since they could play a double role, as nanofillers (increasing mechanical and barrier properties) and active agents with AM performance. Silver, gold and TiO2 nanoparticles (Busolo, Fernandez, Ocio and Lagaron, 2010; Bodaghi, Mostofi, Oromiehie, Ghanbarzadeh and Hagh, 2015; Mihaly Cozmuta et al, 2015; Pagno et al, 2015) have been proposed in active formulations and will be briefly discussed below. For example, gelatin-based AM films with Ag-NPs and C30B were produced and characterized (Kanmani and Rhim, 2014a). The AM activity was measured by the agar diffusion and the colony count methods.

~ 60 ~

Introduction

Results showed that nanocomposites with Ag-NPs and C30B exhibited different AM activity against Gram-positive bacteria. While the strong action of Ag-NPs is well known, in the case of C30B the AM effect is mainly due to the strong activity of the organic tallow used in the modification of the native MMT. The combination of C30B and Ag-NPs into gelatin films showed their synergic effect against Listeria monocytogenes. The TS of the AM nanocomposite with C30B and Ag-NPs (20.8 ± 3.1 MPa) and the nanocomposite with only C30B (19.5 ± 4.3 MPa) showed slight increase when compared to the unfilled film (15.5 ± 3.9 MPa). However, no increase was observed in the film with only Ag-NPs (15.3 ± 3.5 MPa). Similar increases in TS of the active nanocomposite films by the addition of nanoclays, such as MMT, have been frequently observed with other biopolymer matrices such as κ-carrageenan (Rhim and Wang, 2014). Lavorgna et al synthesized AM nanocomposites by loading chitosan with Ag-MMT nanoparticles by replacing Na+ ions in native MMT with Ag ions by exchange reactions (Lavorgna et al, 2014). The Ag-MMT nanocomposites were submitted to analysis with XRD and X-Ray Photoelectron Spectroscopy (XPS) to have a deep understanding of their chemical structure. The main diffraction peaks assigned to the MMT were modified in terms of shape and intensity and an additional peak appeared in Ag-NPs, corresponding to the (1 1 1) plane reflection of silver. In addition, the successful intercalation and the interaction between chitosan and Ag-NPs led to the enhancement of the thermal stability of active nanocomposites with clear improvement of their TS, mainly due to the better load transfer between matrix and fillers. AM tests were performed and results showed that a significant delay in microbial growth was observed after 24 hours with the active nanocomposites.

~ 61 ~

Introduction

Munteanu et al studied the antibacterial property of Ag-NPs and the AO activity of Vitamin E when they were combined within PLA nanofibers via electrospinning (Munteanu, Aytac, Pricope, Uyar and Vasile, 2014). Results showed strong AM effect against Escherichia coli, Listeria monocytogenes and Salmonella typhymurium, and the AO activity was determined as 94 % by using the DPPH method. Other studies showed that carvacrol and thymol can be also incorporated as active additives into polymer nanocomposites to enhance their AM and/or AO performance (Tunç and Duman, 2011; Efrati et al, 2014; Shemesh, Krepker, et al, 2015; Tawakkal, Cran and Bigger, 2016). Pérez et al reported that LDPE-based films with Nanomer® I44P and carvacrol at 5 and 10 wt% produced an increase in the interlayer spacing favoured by the addition of carvacrol. The addition of the nanoclay improved the crystallinity and reduced the permeability to oxygen. Similar results were obtained by (Shemesh, Goldman, et al, 2015). In this study, authors reported a new approach to use MMTs and OMMTs, as active carriers for carvacrol, aiming to minimise its loss throughout the polymer compounding. Different nanoclays were pre-treated with carvacrol, resulting in the intercalation of molecules between the clay galleries, enhancing the carvacrol thermal stability. The active nanocomposites exhibited excellent and prolonged AM activity against Escherichia coli compared with binary LDPE/carvacrol films.

~ 62 ~

Introduction

Table I.8. Comparison between two active nanocomposites based on edible films and S-EO and ZMB-EO. Active nanocomposite

Nanofiller/EO (%) 2.5/0 2.5/0.5

Agar film solution/CNC/S-EO 2.5/1 2.5/1.5 0/0 5/0 κ-carragenan/MMT/ZMB-EO

5/1 5/2 5/3

AM test against

εB (%)

E (MPa)

TS (MPa)

WVP (g s−1 m−1 Pa−1·10−10)

Listeria monocytogenes Staphylococcus aureus Bacillus cereus Escherichia coli

51.7 ± 2.3

55.8 ± 4.2

31.2 ±0.8

1.60 ± 0.01

46.2± 2.8

63.0 ± 3.8

28.3 ± 0.8

1.53 ± 0.13

49.4 ±3.1

57.0 ± 2.5

28.1 ± 1.6

1.82 ± 0.12

51.7 ± 4.9

46.5 ± 4.2

20.4 ± 1.7

2.34 ± 0.13

Staphylococcus aureus Bacillus cereus Escherichia coli Pseudomona Aeruginosa Salmonella typhimurium

36.5 ±1.0

N.R.

26.3 ± 2.9

2.31 ± 0.05

27.4 ±1.6

N.R.

32.9 ± 2.1

1.72 ± 0.09

38.6 ± 1.6

N.R.

18.1 ± 1.2

0.58 ± 0.04

40.6 ± 1.2

N.R.

14.8 ± 1.1

0.48 ± 0.02

44.4 ± 1.9

N.R.

13.2 ± 0.9

0.36 ± 0.04

~ 63 ~

References

(Atef, Rezaei and Behrooz, 2015)

(Shojaee-Aliabadi et al, 2014)

Introduction

4.1. End-of-life for active nanocomposites Polymer degradation could induce changes in polymer properties due to chemical, physical or biological reactions resulting in bond scission and subsequent chemical transformations. These structural modifications could induce changes in the material properties, such as mechanical, optical or electrical characteristics, erosion, discoloration, phase separation or delamination, chemical transformations and formation of new functional groups (Shah, Hasan, Hameed and Ahmed, 2008). Biodegradable polymers should be decomposed in Nature by the action of microorganisms, such as bacteria, fungi, and algae, in aerobic or anaerobic conditions but these processes could vary considerably depending on the environment where they take place, since the microorganisms responsible for the degradation differ from each other and they have their own optimal growth conditions (e.g. industrial composting plants, soil, fresh water, marine water). These microorganisms are able to produce changes in the chemical structure of biodegradable polymers by reducing the long chains into simple chemical substances like water and CO2 during aerobic biodegradation; besides minerals with formation of other intermediate products like biomass and humic materials or water, methane and CO2 during anaerobic biodegradation (UNE-EN_13432, 2000; Shah, Hasan, Hameed and Ahmed, 2008; Vaverková, Toman, Adamcová and Kotovicová, 2012; Araújo, Botelho, Oliveira and Machado, 2014). The polymer characteristics, mainly mobility, tacticity, crystallinity, molar mass, type of functional groups and substituents present in their structure, as well as the presence of plasticizers and other additives also play an important role in the degradation rate and mechanisms (Shah, Hasan, Hameed and Ahmed, 2008). This process can be evaluated under aerobic

~ 64 ~

Introduction

or anaerobic conditions by International standard methods (UNEEN_13432, 2000). Biodegradation is also associated with chemical deterioration occurring in two steps (Shah, Hasan, Hameed and Ahmed, 2008).: i.

Fragmentation of the long polymer chains into lower molar mass species by means of either abiotic reactions, such as oxidation, photodegradation or hydrolysis, or by microorganisms.

ii.

Bio-assimilation of the polymer fragments by microorganisms with further mineralization.

In the case of nanocomposites, they are also submitted to biodegradation processes after their shelf-life. Relevant results have been obtained in the last decade showing a remarkable improvement in biodegradability for nanocomposites prepared with organically modified layered silicates. Fukushima et al studied the biodegradation of amorphous PLA and the corresponding nanocomposites prepared with OMMT and modified kaolinite by using composting conditions at the laboratory scale at 32 °C (Fukushima, Giménez, Cabedo, Lagarón and Feijoo, 2012). Results showed that the PLA biodegradation rate was significantly enhanced in nanocomposites due to the presence of terminal hydroxylated edge groups in modified kaolinite which started the heterogeneous hydrolysis of the PLA matrix after absorbing water from the composting medium. However, in the early stages (initial 6 weeks in compost), the presence of OMMT retained PLA degradation, likely due to its higher dispersion level into the polymer matrix as compared to modified kaolinite, causing a high barrier effect of OMMTs layers towards microbial attack to PLA ester groups, as well as reducing the loss of oligomers which could catalyse PLA hydrolysis through chain-end hydroxyl groups.

~ 65 ~

Introduction

Disintegration under composting conditions at the laboratory scale for nanocomposite films based on PLA-PHB blends and CNC was also reported (Arrieta, Fortunati, et al, 2014). The addition of CNC to PLA increased the disintegration rate in composting conditions. Authors also reported that the disintegrability degree values could fit to the Boltzmann equation permitting the theoretical prediction of the half-life in disintegration processes for biopolymers.

4.2. Risk assessment and migration in active nanocomposites The high surface-to-volume ratio and reactivity of nanofillers provide nanocomposites with enhanced properties and different migration levels. These effects and the presence of materials in the nanoscale in formulations intended to be in direct contact with food raise some potential health and environmental risks to be studied before using these active nanocomposites at the industrial scale (Sanchez-Garcia, LopezRubio and Lagaron, 2010). The potential toxicity, mutagenicity and carcinogenicity of some nanofillers have been put under discussion. The main concerns over the risks associated with the use of nanocomposites in food packaging are based on the lack of sufficient knowledge about nanofillers and the significance of their interaction at the cellular and molecular level in the human body (Huang, Li and Zhou, 2015). The evaluation of potential risks should be based on considering the nanomaterials properties and their transfer rate through cell walls. No migration of nanofillers should be expected in normal cases, but poor characteristics of packaging materials and the subsequent ingestion of food previously in contact with nanocomposites can be considered as a potential exposure route (Huang,

~ 66 ~

Introduction

Li and Zhou, 2015). Consequently, the investigation of the possibility to apply nanomaterials in food packaging and clear assessment of the safety of these materials is necessary to permit the commercial distribution of active nanocomposites. The main studies should focus on migration analysis under controlled conditions to determine the real possibilities of nanomaterials to be considered a real hazard in food packaging. Migration is the result of the diffusion, dissolution and equilibrium processes involving the mass transfer of low molecular weight compounds initially present in the packaging material into food samples or food simulants (Manzanarez-López, Soto-Valdez, Auras and Peralta, 2011). Several factors of the migrant compound, such as the original concentration, particle size, molecular weight, solubility and diffusivity of the specific substance in the polymer, as well as the pH value, temperature, polymer structure and viscosity, mechanical stress, contact time, and food composition, are the main controlling parameters in migration studies (Song, Li, Lin, Wu and Chen, 2011). It has been stated that migration rates depend on mass transport parameters and the thermodynamic equilibrium between materials and food (Torres, Romero, Macan, Guarda and Galotto, 2014). Many factors are essential to estimate the magnitude of the migration process from packaging films into food or food simulants and to know the concentration change of migrating species with time. The key point in designing a specific migration model in food contact materials is the determination of two fundamental parameters, the diffusion and partition coefficients which are specific for each system. In most cases, the migration of a particular substance from polymer films is controlled by the molecular diffusion of the migrant through the polymer internal structure,

~ 67 ~

Introduction

which can be described by the Fick’s second law (Poças, Oliveira, Brandsch and Hogg, 2012; Huang, Li and Zhou, 2015)

= (I.1)

where Cp refers to the concentration of the migrant in the material at time t and position x, and D is the diffusion coefficient which measures the rate at which the diffusion process occurs. D could be either a constant or a concentration-dependent value and characterizes the migration kinetics, as the rate at which the transferred substances move through the system. Active nanocomposites can be used in packaging applications as twodimensional delivery systems based on the release of active additives to extend the shelf-life of food products by their action against microorganisms and oxidative degradation processes. Indeed, the main role of active packaging materials is the release of functional additives onto the food surfaces in a controlled and systematic process, depending on the consumer’s nutritional needs and tastes, including mineral, probiotics, vitamins, phytochemicals, oils and other active agents. Migration tests in food contact materials should cover all those requirements established by the EU Regulation No 10/2011 on plastic materials in contact with foodstuff. Although the best approach to test migration is to work with real food matrices, it is not often possible by the complex compositions of most foodstuffs resulting in non-reliable, tedious and time-consuming procedures. The current legislation marks the valid route to assess the mass transport processes by evaluating the overall and specific migration of targeted substances using food simulants (Commission_Regulation/(EU)/No-10/2011; Huang, Li and Zhou, 2015). Table I.9 shows the food simulants which are selected as model systems according to the current legislation.

~ 68 ~

Introduction

At the end of the contact period and depending on the selected food simulant, accurate analytical methods should be applied to determine the precise amount of migrant in contact with food under the testing conditions. It is necessary to identify and determine the target substance(s) in the food or food simulants to estimate the specific migration level. However, no standardized analytical methods have been proposed up to now to identify and determine nanoparticles and/or active additives in food simulants. Table I.9. Food simulants established by EU Regulation No 10/2011. Food Simulant

Abbreviation

Ethanol 10 % (v/v)

A

Acetic acid 3 % (w/v)

B

Ethanol 20 % (v/v)

C

Ethanol 50 % (v/v)

D1

Vegetable Oil

D2

Poly(2.6-diphenyl-p-phenylene oxide), also known as MPPO and TENAX®

E

Chromatography has been classically used for identification and quantification of migrated compounds in passive packaging materials, particularly for common additives, such as plasticizers or colourants. Recent studies have proposed the use of different chromatographic techniques to evaluate the migration of active agents, such as gas chromatography-flame ionization detector (GC-FID) (Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2013; Muriel-Galet, Cran, Bigger, Hernández-Muñoz and Gavara, 2015), gas chromatography–mass spectrometry (GC/MS) (Efrati et al, 2014) or high performance liquid chromatography-UV detection (HPLC-UV) (Muriel-Galet, Cran, Bigger, Hernández-Muñoz and Gavara, 2015).

~ 69 ~

Introduction

In most cases, a previous step to chromatographic analysis involves the preparation of an appropriate sample based on analytical procedures to achieve a concentration and/or isolation of analytes by using sample preparation techniques, such as solid phase extraction (SPE) (Ridgway, Lalljie and Smith, 2007; Viñas and Campillo, 2014). Inductively coupled plasma with different detectors, such as mass spectrometry (ICP-MS), atomic emission spectrometry (ICP-AES) and optical emission spectrometry (ICP-OES) can be also used in quantitative analysis of potentially migrating nanofillers. These techniques are highly selective, sensitive and accurate, making them the most efficient in determining trace metal ions, such as those present in nanofillers and nanocomposites intended for the use in food packaging. For example, Lavorgna et al quantified the concentration of silver released in aqueous solutions at RT from multifunctional active nanocomposites based on chitosan with silver-MMT by using ICP-MS (Lavorgna et al, 2014). Artiega et al also used this technique to evaluate the Ag-NPs migration from commercial food containers. Results demonstrated that the amount of silver migrated increased with storage time and temperature although, in general, silver showed a low tendency to migrate into food simulants (Artiaga, Ramos, Ramos, Cámara and Gómez-Gómez, 2015).

5. Legislation The legislative framework associated to food contact materials includes many regulations, not always applicable in all countries, which have been discussed and turned on by considering legal and scientific assumptions applicable to the formulation, processing and use of materials intended to

~ 70 ~

Introduction

be in direct contact with food. The European Union (EU) has made great efforts in unifying the legislation of different member countries. Traditional packaging systems intended to come in contact with food must comply with the legislation set up by the EU and extrapolated to the national level. But, the raising interest in production of active packaging systems has forced the EU and other administrations to set up the applicable legislation in food contact materials (Amenta et al, 2015). The current legislative framework is represented in Figure I.18, in particular for active packaging systems (surrounded by green lines). The Framework Regulation (EC) No 1935/2004 refers to all materials in contact with food, but it is mainly focused on active and intelligent materials, since it establishes the basic principles that should accomplish all

materials

intended

for

their

use

in

food

packaging

(Regulation_(EC)/No-1935/2004). The article 3 indicates: 1.

Materials and articles, including active and intelligent materials and articles, shall be manufactured in compliance with good manufacturing practice so that, under normal or foreseeable conditions of use, they do not transfer their constituents to food in quantities which could: a) endanger human health b) bring about an unacceptable change in the composition of the food c) bring about a deterioration in the organoleptic characteristics thereof

2.

The labelling, advertising and presentation of a material or article shall not mislead the consumers.

The introduction of active and intelligent food packaging systems, which are supposed to interact with food and/or the package headspace, represents the appearance of new challenges for the evaluation of their safety and their harmless character to human health. From the beginning of the research in active packaging for their possible commercial use it was clear that regulations limiting or even banning migration should be changed to avoid the incorrect use of packaging due to the insufficient

~ 71 ~

Introduction

labelling or non-efficient operation of the packaging materials (Dainelli, Gontard, Spyropoulos, van den Beuken and Tobback, 2008). The Framework Regulation (EC) No 1935/2004 on materials and articles intended to come into contact with food contains some general provisions on the safety of active and intelligent packaging and sets the framework for the European Food Safety Agency (EFSA) evaluation processes.

Directive 2005/31/EC Ceramic articles Regulation (UE) 10/2011 Plastics materials

Regulation (EC) 282/2008 Recycled plastic materials

Regulation (EC) 450/2009 Active and Intelligent materials Regulation (EC) 2023/2006 Good manufacturing practice

RD 847/2011 Positive list of permitted substances Regulation (EC) 1935/2004

Directive 2007/42/EC Regenerated cellulose film

General aspects Labelling Traceability

Safeguard measures Declaration of compliance

General requirements Active and intelligent

Evaluation and authorisation of substances

Figure I.18. European legislation in food contact materials and general aspects of the Framework Regulation (EC) No 1935/2004 on materials and articles intended to come into contact with food and legislation applied to active packaging (surrounded by green lines).

However, specific legislation devoted to active and intelligent materials was introduced in 2009. Regulation (EC) No 450/2009 was published to cover this particular situation in the case of materials designed to

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intentionally interact with food. This regulation established specific rules for active and intelligent materials and should be applied in harmonization with the general requirements established in the Framework Regulation (EC) No 1935/2004 (Commission_Regulation/(EC)/No-450/2009). This regulation also mentions that the substances responsible for the active and intelligent functions can be either directly incorporated into the packaging material or contained in separate containers (e.g. sachets or labels). It also describes the procedures for the authorization of active substances in the EU. The main requirement indicated in this regulation is based on the risk assessment that the EFSA should perform to all the active compounds to be proposed for their commercial use. In addition, a list of substances or group/combination of them to be used in active and intelligent packaging materials should be drawn up following the risk assessment of these substances by the EFSA (Valdés, Mellinas, Ramos, Garrigós and Jiménez, 2014). Therefore, only those substances included in the positive list of authorized substances drawn by the EFSA may be used as valid components of active and intelligent packaging materials and articles, with the exception of these substances already authorized in other EU legislations, such as food additives, flavourings, enzymes, etc (AINIA and EOI, 2015). The list of authorized substances is continuously growing after the positive evaluations by the EFSA, which allows the submission of new proposals for active substances, which must be accompanied by a risk assessment study (Restuccia et al, 2010). All passive parts of active and intelligent food packaging systems are also under the EU legislation (Figure I.18). The Regulation (EU) No 10/2011 and subsequent amendments and corrections (the last of them being introduced on February 2015), stated the specific measures to be taken into account on active packaging systems. This regulation provides the

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overall migration limits admissible for materials in direct contact with food (10 mg per 1 dm2 of food surface area or 60 mg per kg food) to ensure the safety of the final material or article. It also establishes the specific migration limits for substances incorporated inside the polymer matrix that can be released after the extended contact with food. These limits ensure that the material in contact with food does not pose a risk to human health. It is indicated that the amount of active substances released from packaging materials could exceed the overall migration requirements indicated in the EU or national legislations if these substances have been approved as harmless by the EFSA. The transfer of these active substances to food should not be included in the calculation of the overall migration limit (Valdés, Mellinas, Ramos, Garrigós and Jiménez, 2014). On the other hand, as previously discussed, the growing concern associated with nanotechnologies and the human health has forced the legislative bodies to set up new regulations regarding the safe use of nanomaterials in food packaging applications (Bumbudsanpharoke and Ko, 2015). The EU proposes the use of the European Food Information to Consumers Regulation (EU) No 1169/2011 as a guideline and reference for nanotechnology applied in food contact materials. These guidelines were published on the provision of pre-packed food information to consumers on general food labelling and nutrition labelling (EFSA, 2011). The main novelty of this regulation and the application to nanomaterials was that all food ingredients with a form of engineered nanomaterials must be indicated in the list of ingredients, warning consumers of their use (Bumbudsanpharoke and Ko, 2015).

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INTERNATIONAL AND NATIONAL REGULATIONS Commission Decision 2002/113/EC Regards the Register of Flavouring Substances Used in or on Foodstuffs. Commission Regulation (EC) No 450/2009. Active and Intelligent Materials and Articles Intended to Come into Contact with Food. Commission Regulation (EU) No 10/2011. Plastic Materials and Articles Intended to Come into Contact with Food. EFSA, (2011). Scientific Committee. Guidance on the Risk Assessment of the Application of Nanoscience and Nanotechnologies in the Food and Feed Chain. . EFSA Journal, 9, 2140-2176. Regulation (EC) No 1935/2004. Materials and Articles Intended to Come into Contact with Food. UNE-EN 13432:2000-Requirements for Packaging Recoverable through Composting and Biodegradation. Test Scheme and Evaluation Criteria for the Final Acceptance of Packaging. 2000.

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II.

Objectives

Objetives

The main objective of the present work is the development and characterization of innovative active systems based on the combination of different polymer matrices and additives, to extend the shelf-life of packaged foodstuff. For this purpose, two main research lines are proposed: (1) active films based on PP as polymer matrix widely used in food packaging applications in combination with carvacrol and/or thymol; and (2) active nanocomposites based on PLA as biopolymer matrix with the addition of thymol and two different nanofillers (Dellite®43B nanoclay and silver nanoparticles). This approach would lead to propose the following specific objectives: i.

Development and characterization (morphological, mechanical, thermal and oxygen barrier properties) of active films based on PP with carvacrol and/or thymol. The antimicrobial activity of the obtained films was evaluated against two typical foodborne bacteria: Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). The release of the active additives from PP films into different aqueous and fatty food simulants was also investigated, including kinetic diffusion study and evaluation of the antioxidant activity of the obtained extracts. Finally, the efficiency of the active films in increasing food shelf-life was evaluated by their application to two food samples (strawberries and sliced bread) stored at different conditions.

ii.

Development of active nanocomposites based on PLA with thymol. Two different formulations were proposed by the addition of nanomaterials: a) PLA/thymol bio-films with the addition of a commercial organo-modified montmorillonite (Dellite®43B, D43B). A full characterization (morphological, mechanical, thermal,

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Objectives

optical and oxygen barrier properties) of the obtained nanocomposites was performed. Their disintegration rate under composting conditions; a kinetic release study of thymol into aqueous food simulants; and their antioxidant and antimicrobial activity were also evaluated. b) PLA/thymol bio-systems with silver nanoparticles (Ag-NPs). Two different morphologies were proposed to evaluate the effect of processing on the nanocomposites properties: films and dog-bone bars. A full characterization (morphological, mechanical, thermal, optical, oxygen barrier properties and water vapour permeability) of all systems was carried out. Disintegration under composting conditions was also investigated. In addition, a kinetic release study of thymol and Ag-NPs into aqueous food simulants, as well as the antioxidant and antimicrobial activities of films were evaluated. Special attention was paid to the antimicrobial performance of films including both active additives, thymol and Ag-NPs.

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III.

Results and Discussion

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Results and Discussion

This section presents and discusses the main results obtained in this work following the research lines specified in the previous section. These results are divided in two chapters, corresponding to formulations based on PP and PLA, respectively: Chapter 1: this study is focused on the development of active films with antioxidant and antimicrobial performance with PP as a conventional polymer matrix and carvacrol and thymol as active natural additives. Films were processed by melt-blending/compression moulding and they were further characterized in their physico-chemical and mechanical properties, while their functionality for the intended use in active food packaging was also studied by different in-vitro tests and by reproduction of their real behaviour on food contact assays. Chapter 2: PLA was used as biopolymer matrix for the development of nanocomposites with antioxidant and antimicrobial performance with thymol as active additive and two different nanofillers to improve some of the PLA properties: Section 2.1: a commercial nanoclay, Dellite®43B, was added to PLA/thymol formulations to obtain films which were fully characterized

in

their

physico-chemical

properties.

Their

functionality for active food packaging was also studied by using different tests. The biodegradable character of the obtained films was also evaluated under composting conditions. Section 2.2: silver nanoparticles (Ag-NPs) were used as nanofillers with antimicrobial properties to obtain different PLA/thymol/Ag-NPs formulations. Films and dog-bone bars were processed by extrusion and all formulations were fully characterized in their pyhysico-chemical properties. Their

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Results and Discussion

functionality for active food packaging was also evaluated by using different tests. The biodegradable character of the obtained nanocomposites was also evaluated under composting conditions. Part of the experimental work of this doctoral thesis was carried out at international and well-recognised institutions in the fields of material science and food technology. This work was divided in two different stays under the supervision of: -

Prof. Jose Maria Kenny at the Università degli studi di Perugia, Terni (Italy), Dipartamento di Ingegneria Civile e Ambientale, Group of Scienza e Tecnologia dei Materiali (NovemberDecember 2010 and September-December 2012 for a total time of 6 months).

-

Prof. Joseph Kerry at the University College Cork, (Ireland), Department of Food and Nutritional Sciences, Food Packaging Group (October 2013-February 2014 for a total time of 5 months).

Some of the results presented in this PhD thesis have been already published or are now under review in different scientific books and journals with high impact factor in the fields of analytical and food chemistry (Table III.1 and Table III.2). Additionally, the obtained results were disseminated by participation in several international conferences (Table III.3). In particular, six oral communications were presented, three of them invited. Finally, Table III.4 and Table III.5 summarizes other papers or chapters published by the doctoral candidate not directly related with this Ph.D. work.

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Results and Discussion

Table III.1. Publications of results in peer-reviewed journals. Title

Authors

Book/Journal

Characterization and antimicrobial activity studies of polypropylene films with carvacrol and thymol for active packaging.

Marina Ramos; Alfonso Jiménez; Mercedes Peltzer; Maria C. Garrigós. Marina Ramos; Ana Beltrán; Mercedes Peltzer; Artur J.M. Valente; María C. Garrigós. Marina Ramos; Ana Beltrán; Arantzatzu Valdés; Mercedes Peltzer; Alfonso Jiménez; María C. Garrigós; Gennady Zaikov. Marina Ramos; Alfonso Jiménez; Mercedes Peltzer; María C. Garrigós. Marina Ramos; Elena Fortunati.; Mervedes Peltzer; Franco Dominici; Alfonso Jiménez; María C. Garrigós; Jose M. Kenny.

Journal of Food Engineering. 2012; 109, 513-519.

Release and antioxidant activity of carvacrol and thymol from polypropylene active packaging films.

Carvacrol and Thymol for Fresh Food Packaging Development of novel nano-biocomposite antioxidant films based on poly (lactic acid) and thymol for active packaging. Influence of thymol and silver nanoparticles on the degradation of poly(lactic acid) based nanocomposites: Thermal and morphological properties.

LWT-Food Science and Technology. 2014; 58, 470-477. Journal of Bioequivalence & Bioavailability. 2013; 5, 154-160. Food Chemistry. 2014; 162, 149-155. Polymer Degradation and Stability. 2014; 108, 158-165.

Table III.2. Publications of results in peer-reviewed books. Title

Authors

Book/Journal

Characterization of PP Films with Carvacrol and Thymol as Active Additives.

Marina Ramos; Mercedes Peltzer, María C. Garrigós.

Biodegradable Polymers and Sustainable Polymers (BIOPOL-2009): Nova Science Publishers. 2011; 105-116. ISBN: 978-161209-520-2. (Chapter 7)

Estudio de películas activas de PP con agentes antioxidantes y antimicrobianos de origen natural derivados del orégano.

Marina Ramos; Mercedes Peltzer; María C. Garrigós

EAE, Editorial Académica Española. 2012. ISBN: 9783-659-04935-4.

Carvacrol-based films: usage and potential in antimicrobial packaging.

Marina Ramos; Alfonso Jiménez; María C. Garrigós

Antimicrobial Food Packaging: Academic Press, Elsevier. 2016. In press. ISBN: 978-0-12-800723-5. (Chapter 26)

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Results and Discussion

Table III.3. Publications of results in international conferences. Title

Contribution

Conference

Novel nanocomposite films based on poly (lactic acid) and thymol for active packaging.

Invited Oral

Food Chemistry and Technology (FCT-2015), San Francisco (United States), November 2015.

Novel nanocomposites based on PLA, Ag-nanoparticles and thymol for active packaging.

Invited Oral

International Conference on Bio-friendly Polymers and Polymer Additives, BPPA14, Budapest (Hungary), May 2014.

Degradation of nano-biocomposites based on active poly(lactic acid): physical and thermal properties.

Oral

4th International Conference on Biodegradable and Biobased Polymers (BIOPOL), Rome (Italy), October 2013.

Degradation of nano-biocomposites based on active poly(lactic acid): Physical and thermal properties

Poster

4th International Conference on Biodegradable and Biobased Polymers (BIOPOL), Rome (Italy), October 2013.

Development and characterization of novel nano-biocomposite films based on poly(lactic acid) with thymol and silver nanoparticles as active additives.

Poster

3th International Symposium Frontiers in Polymer Science, Sitges (Spain), May 2013.

Antimicrobial and antioxidant activities of thymol released from novel nano-biocomposites films based on poly(lactic acid).

Poster

5th International Symposium on Food Packaging Scientific Developments supporting Safety and Innovation, Berlin (Germany), November 2012.

Characterization and antimicrobial activity studies of polypropylene films with carvacrol and thymol for active packaging.

Invited Oral

Polyolefin Additives 2012, Cologne (Germany). October 2012.

Release of carvacrol and thymol from polypropylene active films for bread and strawberries packaging based on HS-SPME-GC/MS analysis.

Poster

5th International symposium on Recent Advances in Food Analysis, Prague (Czech Republic), November 2011.

Novel nano-biocomposites with antioxidant activity based on poly(lactic acid) and thymol as active additive.

Oral

Polymers for Advanced Technologies Conference. Lodz (Poland), October 2011.

Development and characterization of novel nano-biocomposites based on poly (lactic acid) and thymol as active additive.

Poster

3rd International Conference on Biodegradable and/or Biobased Polymers (BIOPOL). Strasbourg, (France). August 2011.

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Results and Discussion

Table III.2.cont. Title

Contribution

Conference

Study of migration and antioxidant activity of carvacrol and thymol in active packaging.

Oral

6th International packaging congress. Istanbul (Turkey), September 2010.

Optimization and validation of a SPE-GC/MS method for the simultaneous determination of carvacrol and thymol in aqueous food simulants.

Poster

International Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers. Bruges (Belgium). January 2010.

Study of the antimicrobial activity of active PP films additivated with carvacrol and thymol.

Poster

4th International Symposium on Recent Advances in Food Analysis. Prague (Czech Republic). November 2009.

Characterization of PP films with carvacrol and thymol as active additives.

Poster

2nd International Conference on Biodegradable Polymers and Sustainable Composites (BIOPOL). Alicante (Spain). August 2009.

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Results and Discussion

Table III.4. Other publications in peer review journals. Title Active edible films: current state and future trends.

Use of herbs, spices and their bioactive compounds in active food packaging. New Trends in Beverage Packaging Systems: A Review.

Authors Ana C. Mellinas; Arantzatzu Valdés; Marina Ramos; Nuria Burgos; María C. Garrigós; Alfonso Jiménez. Arantzatzu Valdés; Ana C. Mellinas; Marina Ramos; Nuria Burgos; AlfonsoJiménez; María C. Garrigós. Marina Ramos; Arantzazu Valdés; Ana C. Mellinas; María C. Garrigós

Book/Journal Journal of Applied Polymer Science. 2015; 132. In Press. RSC Advances. 2015; 5, 40324-40335. Beverages. 2015; 1, 248-272.

Characterization and degradation characteristics of poly(ɛ-caprolactone)based composites reinforced with almond skin residues.

Arantzatzu Valdés; Marina Ramos; Ana Beltrán; María C. Garrigós.

Polymer Degradation and Stability. 2014; 108, 269-279.

Natural additives and agricultural wastes in biopolymer formulations for food packaging.

Arantzazu Valdés; Ana C. Mellinas; Marina Ramos; María C. Garrigós; Alfonso Jiménez.

Frontiers in Chemistry. 2014; 2, 1-10.

Classification of Almond Cultivars Using Oil Volatile Compounds Determination by HS-SPME-GC/MS.

Ana Beltrán; Marina Ramos; Nuria Grané; María L. Martín; María C. Garrigós.

Journal of The American Oil Chemists Society. 2011; 88, 329336.

Monitoring the oxidation of almond oils by HS-SPME-GC/MS and ATRFTIR. Application of volatile compounds determination to cultivar authenticity.

Ana Beltrán; Marina Ramos; Nuria Grané; María L. Martín; María C. Garrigos.

Food Chemistry. 2011; 126, 603-609.

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Results and Discussion

Table III.5. Other publications in peer review books. Title

Polymers extracted from bio-mass.

Multifunctional antimicrobial nanocomposites for food packaging applications.

Authors Arantzazu Valdés; Marina Ramos; Esther García; María C. Garrigós; Alfonso Jiménez Elena Fortunati; Debora Puglia; Ilaria Armentano; Arantzazu Valdés; Marina Ramos, Nerea Juárez, María C. Garrigós, José M. Kenny

Book/Journal Reference module in Food Science. Elsevier. In press, accepted. Available in 2016

Multi-Volume SET "Nanotechnology in Food Industry, Volume VI: Food Preservation. Elsevier. In press, accepted. Available in 2016

Ultrasonic-assisted derivatization of fatty acids from edible oils and determination by GC-MS.

Marina Ramos; Ana Beltran; Iván P. Roman; María L. Martin; Antonio Canals; Nuria Grane

Food Process Engineering Emerging Trends in Research and Their Applications. Series. Innovations in Agricultural and Biological Engineering Vol. 5. (Chapter 6) ISBN: 978-1-771-884020. Available in April 2016

Desarrollo de biopelículas activas para envasado de alimentos. Aplicación en materiales para envasado de alimentos.

Marina Ramos; Arancha Valdés; Ana Beltrán

EAE, Editorial Académica Española. 2012. ISBN: 978-3-659-04482-3.

Estudio de la estabilidad oxidativa de las almendras en base a diferentes técnicas y parámetros. Aplicación a la clasificación de variedades.

Ana Beltrán; Marina Ramos; Maria C. Garrigós

EAE, Editorial Académica Española. 2012. ISBN: 978-3-659-04863-0.

Characterization of PLA, PCL and Sodium Caseinate Active Bio-Films for Food Packaging Applications.

Marina Ramos; Marina P. Arrieta; Ana Beltrán; Maria C. Garrigós

Linoleic Acid Content and Antioxidant Properties of Different Tree Nuts: a Review.

Ana Beltrán; Marina Ramos; Arantzazu Valdés; María C. Garrigós

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Food Packaging: Procedures, Management and Trends: Nova Science Publishers. 2012; 63-78. ISBN: 978-1-62257-319-6. (Chapter 3) Linoleic Acids: Sources, Biochemical Properties and Health Effects: Nova Science Publishers. 2012; 83-96. ISBN: 978-1-62257-384-4. (Chapter 2)

1

Chapter 1

Antioxidant/antimicrobial polypropylene films with carvacrol and thymol for active food packaging

Results and Discussion. Chapter 1

Figure 1.1. General scheme of the experimental work presented in Chapter 1.

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Results and Discussion. Chapter 1

1. Introduction Food active packaging systems consisting on polymer matrices with the addition of compounds with antimicrobial and/or antioxidant properties are increasing their use to extend foodstuff shelf-life and while improving consumer’s safety (Vermeiren, Devlieghere, Van Beest, De Kruijf and Debevere, 1999; Fernández, 2000; Appendini and Hotchkiss, 2002; Del Nobile et al, 2009). The migration of active compounds may be achieved by direct contact between food and the packaging material or through gasphase diffusion from the inner packaging layers to food surface (Conte, Buonocore, Bevilacqua, Sinigaglia and Del Nobile, 2006; Coma, 2008; Gemili, Yemenicioǧlu and Altinkaya, 2009; Mastromatteo, Mastromatteo, Conte and Del Nobile, 2010). Food can be subjected to microbial contamination that is mainly caused by bacteria, yeasts and fungi. Many of these microorganisms can cause undesirable reactions and can deteriorate organoleptic and nutritional properties of food (Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2011b). The most common procedure to incorporate AM agents (most of them synthetic) into food is by direct addition permitting to diminish food spoilage by microorganisms. But this strategy has several disadvantages, such as the rising consumer’s concerns for foods with synthetic additives in their composition, as well as some mistaken procedures in the addition of these agents to the food bulk when spoilage occurs primarily on the surface. The undesirable modification of organoleptic properties is another drawback of the use of these strategies in processed food. AM packaging is increasing the attention from food industries to protect their products from microbial contamination (Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2013) due to the increasing consumer demands for minimally processed and preservative-free products (López, Sánchez,

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Batlle and Nerín, 2007a). Food packaging films allow the controlled release of AM additives into food in prolonged periods of time (including storage and distribution operations) and limit possible undesirable flavours caused by the direct addition of synthetic additives into food (Suppakul, Miltz, Sonneveld and Bigger, 2003; Ho Lee, Soon An, Cheol Lee, Jin Park and Sun Lee, 2004; Suppakul, Miltz, Sonneveld and Bigger, 2006; López, Sánchez, Batlle and Nerín, 2007a; Peltzer, Wagner and Jiménez, 2009). Active food packaging with AO abilities is also growing as an adequate alternative to common procedures oxidation. The addition of AOs directly to food samples in combination with vacuum or modified atmosphere has been proposed as a good possibility to preserve fat food from fast oxidative degradation (Lopez-de-Dicastillo et al, 2011). In these systems, the AO additives incorporated directly into polymer matrices can play a double role: (i) food protection by their release in controlled conditions and rates, avoiding oxidation of fats and pigments (Del Nobile et al, 2009); and (ii) to protect the polymer from oxidative degradation during processing. In fact, the addition of AOs to polyolefins is a common practice for food-grade film manufacturing (Tovar, Salafranca, Sanchez and Nerin, 2005; Siró et al, 2006). The new possibilities offered by the use of natural additives in food packaging have produced a clear increase in the number of studies based on natural active compounds wit AM/AO abilities, such as -tocopherol (Barbosa-Pereira et al, 2013), aromatic plant extracts (Dopico-García et al, 2011; Lopez-de-Dicastillo et al, 2011) and polyphenols extracted from natural oils (Peltzer, Wagner and Jiménez, 2009; Park et al, 2012). AM/AO additives derived from EOs are perceived by consumers as healthy compounds and they have been proposed in the last decade as potential

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alternatives to synthetic additives, such as BHT (Valentao et al, 2002). Many studies have focused on the AMs present in EOs extracted from plants or spices (basil, thyme, oregano, cinnamon, clove, rosemary) consisting on complex mixtures of different biological active compounds including terpenoids, phenolic acids, esters, aldehydes, ketones and alcohols (Dorman and Deans, 2000). Extracts derived from herbs and EOs contain many natural compounds such as thymol, linalool and carvacrol with a broad AM activity against different pathogenic and spoilage microorganisms, including Gram-negative (López, Sánchez, Batlle and Nerín, 2007b; Suppakul, Sonneveld, Bigger and Miltz, 2011a) and Gram-positive species (López, Sánchez, Batlle and Nerín, 2007a; Gutiérrez, Escudero, Batlle and Nerín, 2009); as well as against yeast (Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2011a) and moulds (Rodriguez-Lafuente, Nerin and Batlle, 2010). Therefore, there is a rising interest in the evaluation and application of these compounds for minimizing the surface contamination of food, in particular meats, fruits and vegetables, decreasing the microbial growth rate of microorganisms responsible for food degradation (Appendini and Hotchkiss, 2002; Lundbäck, Hedenqvist, Mattozzi and Gedde, 2006; Sanchez-Garcia, Ocio, Gimenez and Lagaron, 2008; Persico et al, 2009; Peltzer, Navarro, López and Jiménez, 2010; Suppakul, Sonneveld, Bigger and Miltz, 2011a) Carvacrol and thymol, which are major compounds present in thyme and oregano EOs (Al-Bandak and Oreopoulou, 2007), are isomeric phenolic monoterpenes that exhibit a significant antifungal and in vitro antibacterial activity against several strains (Halliwell, Aeschbach, Löliger and Aruoma, 1995). They also exhibit high AO activity (Tomaino et al, 2005) and are generally recognized as safe (possess “GRAS” status) (Persico et al, 2009) and as flavouring substances according to the European Commission

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Decision 2002/113/EC. Their AO activity can be easily evaluated by using diverse methods such as DPPH, a simple, rapid, sensitive, and reproducible procedure (Ozcelik, Lee and Min, 2003). The addition of both additives into polymer matrices might represent an advantage in food applications due to their possible synergistic effect against several microorganisms (Lambert, Skandamis, Coote and Nychas, 2001; Guarda, Rubilar, Miltz and Galotto, 2011). The use of high initial concentrations of these volatile additives was previously reported, since some loss during processing was expectable (Sanchez-Garcia, Ocio, Gimenez and Lagaron, 2008; Del Nobile et al, 2009; Persico et al, 2009; Mascheroni, Guillard, Gastaldi, Gontard and Chalier, 2011; Tunç and Duman, 2011). Active compounds have been usually added to packaging materials by the incorporation of their precursor EOs (Salafranca, Pezo and Nerín, 2009). Rodríguez et al studied the addition of EOs to wax coatings to develop AM active packaging materials able to preserve strawberries from microorganisms contamination by the release of additives from the coating to food surface (Rodríguez, Batlle and Nerín, 2007). Authors reported that there was no direct contact between EOs and food, so it was concluded that the natural volatile compounds (eugenol, carvacrol, thymol) present in the headspace packaging were the main responsible for the inhibition of the pathogens growth. In other study, carvacrol was added to chitosan-based films for active packaging and their antimicrobial efficiency against food spoilage microorganisms was demonstrated by using a headspace chromatographic technique (Kurek, Moundanga, Favier, Galić and Debeaufort, 2013). Gutiérrez et al used a cinnamonbased active material to increase more than 3 times the shelf-life of a complex bakery product with minimal changes in packaging and no

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additional manipulation steps (Gutiérrez, Sánchez, Batlle and Nerín, 2009). It is also known that changes in the food macroscopic properties can also induce biochemical reactions and chemical alterations in tissues, such as changes in the volatile profile (Chiralt et al, 2001) and development of undesirable chemicals (i.e. ethanol or acetaldehyde) associated with changes in the respiratory paths (Tovar, Garcı́a and Mata, 2001). In fact, flavour is one of the main factors influencing consumer’s food choice (Pozo-Bayón, Guichard and Cayot, 2006) and volatile aromatic compounds are important contributors to flavour and odour of fruits. As an example, the flavour of strawberries is comprised of a complex mixture of esters, aldehydes, alcohols, furans and sulphur compounds, being esters the main headspace volatiles. The amount of methyl esters increases with the plant maturation, while it does not change significantly for ethyl esters during the fruit growth stages (Rizzolo, Gerli, Prinzivalli, Buratti and Torreggiani, 2007). Bread is another example where more than 540 different compounds were described in its complex volatile fraction (Ruiz, Quilez, Mestres and Guasch, 2003), being alcohols, aldehydes, esters, ketones, acids, pyrazines and pyrrolines the major volatile components, while furans, hydrocarbons and lactones were also identified (Poinot et al, 2007). Solid phase microextraction (SPME) has become one of the preferred techniques in analysis of aromas and volatile compounds, offering solvent-free, rapid, low-cost and reliable analytical methods with easy preparation. SPME is also sensitive, selective and usually offers low detection limits (Ho, Wan Aida, Maskat and Osman, 2006). When used in the sample headspace, HS-SPME is a non-destructive and non-invasive method that has been used to evaluate volatile and semi-volatile

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compounds released from a great number of foods (Quílez, Ruiz and Romero, 2006; Poinot et al, 2007). The release rate of AOs from packaging materials can be evaluated by migration studies, using aqueous and fatty food simulants and conditions specified in the European food packaging regulations launched in 2011 and later amendments (Commission_Regulation/(EU)/No-10/2011; Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2013). Migration is the result of diffusion, dissolution and equilibrium processes involving the mass transfer of low molecular mass compounds initially present in the package into food samples or food simulants; and it is often described by Fick’s second law (Manzanarez-López, Soto-Valdez, Auras and Peralta, 2011). Chromatographic methods are commonly used for identification and quantification of migrated compounds (Salafranca, Pezo and Nerín, 2009), considering that concentration and/or isolation of analytes into suitable solvents should be performed prior to chromatographic analysis. Some sample preparation and purification techniques, such as SPE, have been proposed to improve detection and quantification in the analysis of migrated compounds from polymer matrices (Burman, Albertsson and Höglund, 2005; Ridgway, Lalljie and Smith, 2007). In spite of the increasing concern on the use of synthetic polymers in massive applications, such as food packaging, due to their poor biodegradability and high permanence in the environment after use, these materials show many advantages including low cost, good processability and excellent mechanical and physical properties. Polyolefin-based films have been proposed for the development of active packaging systems by the combination of the polymer good properties (mechanical, barrier, optical and thermal) and the AM/AO effect given by the additives (Suppakul, Miltz, Sonneveld and Bigger, 2006; López, Sánchez, Batlle and

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Nerín, 2007a; Peltzer, Wagner and Jiménez, 2007; Peltzer, Wagner and Jiménez, 2009). This study is focused on the development of AM/AO films based on PP with carvacrol and thymol at different initial concentrations (4, 6 and 8 wt% of both additives as well as an equimolar mixture) (Figure 1.1). Polymer and additives were processed by melt-blending followed by compression moulding to obtain films at the laboratory scale. A full physico-chemical characterization of these films was carried out by determination of their main thermal, structural, mechanical and functional properties. The release of these compounds from films into different food simulants was also studied; including a kinetics diffusion study and the evaluation of the antioxidant efficiency by the DPPH method. Fast and reliable analytical procedures were developed and validated for the analysis of the studied AOs in selected food simulants. For aqueous food simulants, SPE followed by GC/MS analysis was used. Fatty food simulants (isooctane and ethanol 95 % (v/v)) were directly analysed by GC/MS and HPLC-UV, respectively. The AM activity of films was also evaluated against two typical food-borne bacteria: Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). Finally, the effectiveness of the developed active films to increase the post-harvest shelf-life of fresh food was evaluated by studying the headspace volatile composition of two food samples (sliced bread and strawberries) by headspace solid phase microextraction and gas chromatography mass spectrometry (HS-SPME-GC/MS) at controlled conditions. Results were correlated with the AM activity by visual observation of the fungal growth in the studied food.

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2. Experimental 2.1. Materials and chemicals Polypropylene PP ECOLEN HZ10K (Hellenic Petroleum, Greece) was kindly supplied in pellets by Ashland Chemical Hispania (Barcelona, Spain). Melt flow index (MFI) was 3.2 g 10 min-1 determined according to ASTM D1238 standard (230 °C, 2.16 kg), and density 0.9 g cm-3. All reagents used in this work were analytical or chromatographic grade and were purchased from Panreac (Barcelona, Spain). Standards of carvacrol (≥ 98 %), thymol (99.5 %), and 2,2-diphenyl-1-picrylhydrazyl (DPPH, 95 %) were acquired from Sigma-Aldrich Inc. (St. Louis, MO, USA). Ultrapure water was obtained from a Millipore Milli-Q system (Millipore, Bedford, MA, USA).

2.2. Films preparation PP active films were obtained by melt-blending in a Haake Polylab QC mixer (ThermoFischer Scientific, Walham, USA) at 190 °C for 6 min at rotation speed of 50 rpm. Both additives were introduced in the mixer once the polymer was already in the melt state, in order to avoid unnecessary losses and to ensure the presence of the maximum amount of them remaining in the final material. Nine active formulations were obtained: PP containing 4, 6 and 8 wt% of thymol (PPT4, PPT6 and PPT8) or carvacrol (PPC4, PPC6 and PPC8); and PP with the combination of an equimolar mixture of both additives at 4, 6 and 8 wt% (PPTC4, PPTC6 and PPTC8) to study the possible synergies between both compounds. An additional sample without any active compound was also prepared and used as control (PP0).

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Results and Discussion. Chapter 1

Active films were obtained by compression-moulding at 190 °C in a hot press (Carver Inc, Model 3850, USA). The material was kept between plates at atmospheric pressure for 5 min until melting and then it was successively pressed under 2 MPa for 1 min, 3.5 MPa for 1 min and finally 5 MPa for 5 min, in order to liberate the trapped air bubbles. The average thickness of films was around 200 μm measured with a Digimatic Micrometer Series 293 MDC-Lite (Mitutoyo, Japan) at five random positions around the film. The final appearance of the films was completely transparent and homogenous (Figure 1.2).

Figure 1.2. Visual observation of neat PP and active films.

2.3. Films characterization The active films were characterized by using different techniques in order to study their thermal, mechanical and oxygen barrier properties. 2.3.1. Scanning electron microscopy (SEM)

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The films surfaces and cross sections were analysed by using a JEOL model JSM-840 (Jeol USA Inc., Peabody, MA, USA) microscope operated at 12 kV. Samples were coated with a gold layer prior to analysis to increase their electrical conductivity. Images were registered at 300x and 500x of magnification to study their homogeneity. 2.3.2. Mechanical properties Tensile tests were carried out by using a 3340 Series Single Column System Instron Instrument, LR30K model (Fareham Hants, UK) equipped with a 2 kN load cell. Tests were performed in rectangular probes (100 x 10 mm2), an initial grip separation of 60 mm and crosshead speed of 25 mm min-1. Average tensile strength, elongation at yield and elastic modulus were calculated from the resulting stress-strain curves according to the ASTM D882-09 Standard procedure (ASTM, 2009). Results were the average of five measurements (± standard deviation). 2.3.3. Thermal properties

Thermogravimetric analysis (TGA) tests were performed in a TGA/SDTA 851e Mettler Toledo thermal analyser (Schwarzenbach, Switzerland). Approximately 5 mg samples were weighed in alumina pans (70 µL) and were heated from 30 to 700 °C at a heating rate 10 °C min-1 under inert nitrogen atmosphere (flow rate 50 mL min-1).

Differential scanning calorimetry (DSC) tests were conducted by using a TA DSC Q-2000 instrument (New Castle, DE, USA) under inert nitrogen atmosphere. 3 mg of films were introduced in aluminium pans (40 µL) and were submitted to the following thermal program: heating from 0 to 180 °C at 10 °C min-1 (3 min hold), cooling to 0 °C at 10 °C min-1 (3 min hold) and heating to 180 °C at 10 °C min-1. The percentage

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Results and Discussion. Chapter 1

of crystallinity (χ %) for each material was calculated according to Equation 1.1: (%) = Δ

Δ

∙ 100 (1.1.)

where Hm (J g-1) is the melting enthalpy, W is the PP weight fraction in the sample, and Hmo is the theoretical melting enthalpy for 100 % crystalline PP, 138 J g-1 (Joseph et al, 2003). Oxidative induction parameters. The AO performance of carvacrol and thymol was studied by determining the oxidation induction parameters by DSC, i.e. oxidation onset temperature, OOT (°C) and oxidation induction time, OIT (min) (Pospı́šil et al, 2003; Archodoulaki, Lüftl and Seidler, 2006). OOT is defined as the minimum temperature where oxidation takes place in pure oxygen atmosphere. The OIT value is defined as the time to the onset of an exothermic oxidation peak in oxygen atmosphere and it was determined as the difference between the time at the intersection between the baseline and the tangent of the exothermic oxidation peak and the time for the gas switching in two different atmospheres: pure oxygen and air. All tests were performed in triplicate for each formulation. OOT (°C) is a relative measurement of the degree of thermo-oxidative stability of the material evaluated at a given heating rate in oxidative atmosphere (Peltzer and Jiménez, 2009). Samples were heated up at 10 °C min-1 under pure oxygen (50 mL min-1) from 30 °C to the temperature where the exothermic oxidation peak was observed. OOT was calculated as the temperature for the intersection between the baseline and the slope of the exothermic peak in each case. OIT tests were carried out by heating samples at 100 °C min-1 under nitrogen (50 mL min-1) to the set temperature (200 °C). After 5 min, the

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Results and Discussion. Chapter 1

atmosphere was switched to pure oxygen or air (50 mL min-1). The heat flow was then recorded in isothermal conditions up to the detection of the exothermic peak indicating the beginning of the oxidation reaction. 2.3.4. Oxygen transmission rate (OTR) OTR is defined as the amount of oxygen passing through a defined area of the parallel surface of a plastic film per time unit. An oxygen permeation analyser (8500 model Systech, Metrotec S.A, Spain) was used. Tests were carried out by introducing pure oxygen into the upper half of the diffusion chamber while pure nitrogen was injected into the lower half, where an oxygen sensor was located. Films were cut into 14 cm diameter circles for each formulation and they were clamped in the diffusion chamber at 25 °C before testing.

2.4. Migration study 2.4.1. Release tests The release of thymol and carvacrol from PP films containing the studied additives at 8 wt% was performed into five food simulants according to the European Standard EN 13130-2005 (UNE-EN_13130-1, 2005): distilled water (A), acetic acid 3 % (m/v) (B), and ethanol 10 % (v/v) (C) were used as aqueous food simulants; whereas ethanol 95 % (v/v) and isooctane were employed as fatty food simulants. Migration studies were conducted in triplicate at the experimental conditions indicated in the referred legislation (40 °C for 10 days) in an oven (J.P. Selecta, Barcelona, Spain), except for isooctane studies which were performed at 20 °C and 50 % RH for 2 days in a climatic chamber (Dycometal, Barcelona, Spain). Double-sided, total immersion migration

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tests were performed with 12 cm2 films and 20 mL of each simulant (areato-volume ratio of 6 dm2 L-1). A blank test for each simulant was also carried out. 2.4.2. Migration kinetics A kinetic study for the release of carvacrol and thymol to food simulants was performed by using acetic acid 3 % (m/v), ethanol 10 % (v/v), ethanol 95 % (v/v) and isooctane, at the same temperature conditions described in Section 2.4.1. Samples were taken in triplicate at 2, 6, 12, 24, 48 hours and 5, 10 and 15 days. The migration process is described by the diffusion kinetics of the additive through the film and it is expressed by the diffusion coefficient, D (m2 s-1) (Manzanarez-López, Soto-Valdez, Auras and Peralta, 2011). Considering the case of limited packaging, limited food, where migration occurring from a limited volume packaging film into a well-mixed limited volume of food, the diffusion coefficients of thymol and carvacrol can be determined by using a release kinetic model based in the Fick’s second law (Equation 1.2). In this case, the food simulant initially does not contain any migrating compound, and as migration occurs, their concentration increases from zero (CF,0) to its equilibrium value (CF,). Equation 1.2 is the most rigorous general model describing the migration controlled by Fickian diffusion in a packaging film (Crank, 1975; Chung, Papadakis and Yam, 2002): ∞ , ,∞

=1− =1

2 (1 + ) 1+ + 2 2

2

− 2

(1.2.)

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Results and Discussion. Chapter 1

where MF,t and MF, are the total amount of the diffusing substance (thymol and carvacrol) released by the film at time t and after infinite time, respectively; LP is the film thickness; qn are the non-zero positive roots of tanqn=-αqn; and α is the partition coefficient expressed as indicated in Equation 1.3.

=

(

∙

)

(1.3.)

where VF and VP are the volumes of the simulant and the polymer, respectively; and KFP is the partition coefficient of the active compound between the simulant and the polymer. A simplified migration model was proposed by Chung et al derived from Equation 1.2 and useful for linear regression analysis (Chung, Papadakis and Yam, 2002):

1

−

1

0.5 , ,

0.5

=−

∙

∙

0.5

+

1 0.5

(1.4.)

where MP,0 is the initial amount of migrant in the packaging film (for a complete migration MP,0= MF,). Thus, the diffusion coefficient can be directly calculated from the fitting of Equation 1.4 to experimental migration data.

2.5. Analysis of released active additives into food simulants The amount of released active additives into aqueous food simulants was analysed by GC/MS with a previous extraction and concentration step by SPE on an octadecyl cartridge (C18, 500 mg, 6 mL) (Teknokroma,

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Barcelona, Spain). A Büchi V-700 vacuum system (Flawil, Switzerland) and a vacuum manifold from Teknokroma (Barcelona, Spain) were used for SPE sample processing. Cartridges were previously conditioned with 4 mL methanol and 4 mL distilled water at 5 mL min-1. Then, the extract was loaded and the elution of thymol and carvacrol was carried out with 4 mL dichloromethane (1 mL min-1). Extracts obtained from isooctane and ethanol 95 % (v/v) were directly analysed by GC/MS and HPLC-UV, respectively. Stock (4000 mg kg-1) and working solutions of each active additive were prepared in the appropriate solvent (dichloromethane, isooctane or ethanol 95 % (v/v) depending on the food simulant and the chromatographic technique) and stored in a freezer. Carvacrol and thymol quantification was performed in triplicate using external calibration. 2.5.1. GC/MS analysis A Perkin Elmer TurboMass Gold GC/MS (Boston, MA, USA) operating in electronic impact ionisation mode (70 eV) with a SPB-5 capillary column (30 m × 0.25 mm × 0.25 m; Supelco, Bellefonte, PA, USA) was used. The column temperature was programmed from 60 °C (1 min) to 120 °C (1 min) at 10 °C min-1 and to 150 °C at 2 °C min-1 (2 min). Helium was used as carrier gas at 1 mL min-1. Ion source and GC/MS transfer line temperatures were 250 and 270 °C, respectively. The injector temperature was 270 °C and 1 µL of extracts were injected in all cases (split mode 1:100). Identification of thymol and carvacrol were performed in full scan mode (m/z 30-550) by using both, the NIST mass spectral library and retention times of standard compounds. Quantification of additives was performed by using selected ion monitoring (SIM) mode focused on m/z 91, 135 and

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150, typical of phenols. Retention times obtained for thymol and carvacrol were 10.7 and 11.0 min, respectively. 2.5.2. HPLC-UV analysis. A Shimadzu LC-20A liquid chromatograph equipped with UV detector and a LiChrospher 100 RP18 column (250 mm × 5 mm × 5 μm, Agilent Technologies) was used. The mobile phase consisted of acetonitrile: distilled water, 40:60 (v:v) at 1 mL min-1. 20 μL of sample were injected. Detection of carvacrol and thymol was performed at 274 nm with retention times of 18.9 and 21.0 min, respectively.

2.5.3. Determination of antioxidant activity The antioxidant activity of thymol and carvacrol released into food simulants was analysed in terms of radical scavenging ability by using the stable radical DPPH method (Byun, Kim and Whiteside, 2010) with some modifications. An aliquot of 100 μL of each simulant extract was mixed with 3.9 mL of a methanolic solution of DPPH (23 mg L-1) in a capped cuvette. The mixture was shaken quickly at room temperature and the absorbance of solutions was measured immediately at 517 nm in 1-min intervals until the absorbance value was completely stable (200 min), by using a Biomate-3 Ultraviolet-visible (UV-Vis) spectrophotometer (Thermospectronic, USA). All analyses were performed in duplicate. The ability to scavenge the stable radical DPPH was calculated as percentage of inhibition (I %) by Equation 1.5.

(%) =

∙ 100

(1.5.)

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Results and Discussion. Chapter 1

where AControl and ASample are the absorbances of the control at t = 0 min (using methanol as the blank solution) and of the tested sample at t = 200 min, respectively.

2.6. Antibacterial activity The evaluation of the antibacterial activity of PP films containing carvacrol, thymol and the equimolar mixture of both additives was carried out by using two test microorganisms: Escherichia coli (Gram-negative, ATCC (American Type Culture Collection) 25922) and Staphylococcus aureus (Gram-positive, ATCC 6538P). The PP0 sample was also tested as control. Overnight cultures of Escherichia coli and Staphylococcus aureus were grown in Tryptic Soy Broth at 35 °C for 24 h. The strains selection represented typical spoilage organism groups commonly occurring in food products. Antibacterial activity tests were carried out by using the agar disk diffusion method. Disks cut from films were placed on Petri dishes containing Mueller-Hinton agar (MHA) supplied by INSULAB S.L., (Valencia, Spain), previously spread with 0.1 mL of each inoculum. The concentration of bacterial cultures in the inoculum was 106 colonyforming unit (CFU) per mL, corresponding to the concentration that could be found in contaminated food, and standardized in the McFarland scale 0.5, as it has been already proposed (Suppakul, Sonneveld, Bigger and Miltz, 2011b). The Petri dishes were then incubated at 37 °C for 24 h and the antibacterial activity of each material was evaluated by observing the growth inhibition zone and measuring the diameter (mm) by a ruler. The bacterial growth under the film disks (area of contact with the agar

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surface) was also observed in Petri dishes. Tests were carried out in duplicate for each formulation.

2.7. Study of the effectiveness of the active films to preserve perishable food: shelf-life study

Food samples. Strawberries and sliced bread were purchased from a local market. Strawberries were selected for this study due to their rapid postharvest deterioration, which constitutes a problem on their commercial distribution. Sliced bread was selected due to the increasing consumer demand for fresh bread with long shelf-life. Damaged, non-uniform, unriped or overriped strawberries were removed and the selected fruits were stored for at least 2 h at 3 °C to ensure their thermal equilibrium before testing.

Food packaging. The effectiveness of the developed active films (with each additive at 8 wt% in different samples) was evaluated by putting them in contact with the above-referred food, which were appropriately cut to be placed on the base of disposable PP Petri dishes (inside dimensions: 88 mm diameter x 12 mm high). An additional test was carried out with uncut strawberries which were placed into polyethylene suitable food containers (250 mL, 4 cm high x 13 cm opening diameter) as shown in Figure 1.3. Active films were cut with the appropriate dimensions to match the top of the lid of the used containers in order to release the antimicrobial studied agents (carvacrol and thymol) into the packaging headspace. The final containers were then sealed with "Parafilm" to avoid losses of volatile compounds and were incubated at 25 °C and 50 % RH in a climatic

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chamber for 15 days. Food samples stored with the control film (without active compounds) were also studied for comparison.

PP0

PPT8

PPC8

Figure 1.3. Experimental assembly used for headspace analysis of whole strawberries by HS-SPME.

Shelf-life study. The observation of the occurrence of fungal growth on the studied samples was also performed. In addition, HS-SPME-GC/MS analysis was carried out for food samples (whole strawberries and sliced bread) in direct contact with the PP films. For this purpose, samples were extracted at selected times to determine the headspace composition. Containers were sealed and a polytetrafluoroethylene (PTFE)/silicone septum was placed on their top part to allow the insertion of the SPME fibre for volatiles extraction (Figure 1.3). Samples were then stored in a climatic chamber and tested at different temperatures and days of storage according to the conditions shown in Table 1.1. 25 °C and 4 °C were selected to simulate ambient and refrigerated storage conditions, respectively. Three replicates were performed for each food sample and day of study. HS-SPME analysis of volatile compounds for food samples was performed by following a method applied to bread samples (Poinot et al,

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2008) with slight modifications. Similar conditions to those proposed by Blanda et al were used in the study with strawberries (Blanda et al, 2009). Table 1.1. Storage and testing conditions in the headspace study of food by HS-SPMEGC/MS. Food sample

Temperature (°C)

Days of study

Sliced bread

25

0

2

5

10

15

-

Whole strawberries

25

0

2

4

7

10

-

Whole strawberries

4

0

2

4

7

10

15

Divinylbenzene/carboxen/polydimethylsiloxane

(DVB/CAR/PDMS)

fibres (50/30 µm, StableFlex, 1 cm long) mounted to an SPME manual holder assembly from Supelco (Bellefonte, PA, USA) (Figure 1.3) was used. Fibres were previously conditioned by following the manufacturer’s recommendations. The needle of the SPME device was inserted into the container through the septum and fibres were exposed to the food sample headspace for 30 min at room temperature. Fibres were then retracted into the needle assembly point, removed from the container, transferred to the injection port of the GC unit and immediately desorbed. Analysis of volatiles produced in the headspace of bread and strawberries packed samples was performed by using a Perkin Elmer TurboMass Gold GC/MS (Boston, MA, USA) equipped with a split/splitless injector and a quadrupole mass spectrometer operating in electronic impact (EI) ionisation mode (70 eV). A SPB-5 capillary column (30 m x 0.25 mm x 0.25 m; Supelco, Bellefonte, PA, USA) was used. The column temperature was programmed from 40 °C (hold 10 min) to 120 °C (hold 1 min) at 5 °C min-1, to 140 °C at 2 °C min-1 (hold 0 min) and to 230 °C at 5 °C min-1 (hold 8 min). Helium was used as carrier gas at 1 mL min-1 flow rate. Ion source and GC/MS transfer line temperatures were 250 and 270

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Results and Discussion. Chapter 1

°C, respectively, while the injector temperature was 270 °C and time for fibre desorption was fixed at 5 min in the splitless mode (1.5 min splitlessperiod). After every run, the SPME fibre was conditioned for 30 min at 270 °C in the injector of the gas chromatograph followed by a blank analysis to avoid the fibre carryover. Identification of volatile compounds in strawberries and sliced bread headspace was performed in full scan mode (m/z 30-550). Carvacrol and thymol were identified by using both, the NIST mass spectral library and gas chromatographic retention times of standard compounds. The rest of volatiles were tentatively identified by their GC/MS spectra. In this sense, the compounds having  90 % similarity with spectra in the NIST library were not taken into consideration. Chromatographic responses of detected volatile compounds (peak area counts) were monitored for comparative measurements of each compound in the studied samples.

2.8. Statistical analysis One way analysis of variance (ANOVA) was applied on experimental data with the aid of the statistical program “Statgraphics Centurion program v.16.1.18 (StatPoint, Inc., Warrenton, USA)” and significant differences among sample data were recorded at a confidence level of 95 % (p < 0.05) according to Tukey´s post hoc test.

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3. Results and discussion 3.1. Films characterization 3.1.1. Scanning electron microscopy (SEM) Homogeneous surface morphologies were observed for all materials with no apparent effect of additives on the PP morphology. However, certain porosity on the surface of those materials with additives at each concentration (4, 6 and 8 wt%) was observed. As an example, Figure 1.4 shows the SEM micrographs obtained for PP0 and samples with additives (8 wt%) the other tested formulations showed similar surfaces morphologies.

Figure 1.4. SEM micrographs (x500) of the edge surfaces for PP0 and samples with 8 wt% of the studied additives.

This behaviour could be due to the presence of a certain amount of carvacrol and thymol in the materials surface and the eventual partial evaporation of these additives from the polymer matrix during processing,

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Results and Discussion. Chapter 1

leading to a potential loss of some molecules resulting in microholes and some porosity. Figure 1.5 shows the SEM microgprahs of cross section of materials. It could be observed that the matrix remained homogeneous with no porosity. Therefore, it could be concluded that the possible diffusion of active additives through the polymer matrix is only a surfa surface phenomenon.

Figure 1.5. Cross section micrographs (x300) for PP0 and samples with 8 wt% of the studied additives.

3.1.2. Mechanical properties. Tensile tests were performed in order to study the effect of thymol and carvacrol on the polymer mechanical properties, by the evaluation of different parameters, such as the elastic modulus (E), elongation at yield

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(εy) and tensile strength (TS), in all materials. Results are shown in Table 1.2. Table 1.2. Mechanical properties of samples according to ASTM D882-09. Sample

E (MPa)

TS (MPa)

εy (%)

PP0

851 ± 37a

30 ± 1a

19 ± 1a

PPC4

601 ± 25bc

27 ± 2 a

23 ± 2b

PPC6

597 ± 42bc

27 ± 1 a

23 ± 1 b

PPC8

543 ± 44b

27 ± 3 a

24 ± 2 b

PPT4

593 ± 25bc

28 ± 2 a

23 ± 2 b

PPT6

680 ± 96c

28 ± 1 a

24 ± 1 b

PPT8

585 ± 40bc

28 ± 2 a

25 ±1 b

PPTC4

681 ± 30c

28 ± 2 a

23 ± 2 b

PPTC6

646 ± 34c

28 ± 1 a

22 ± 2 b

PPTC8

677 ± 53c

27 ± 3 a

22 ± 1 ab

Results are represented as mean ± standard deviation, n=5. Different superscripts within the same column indicate statistically significant different values (p < 0.05).

The addition of carvacrol and thymol to PP resulted in a slight modification of tensile properties. A significant decrease (p < 0.05) in elastic modulus was observed for the materials with additives when compared with PP0, being this effect more pronounced for PPC8 and PPT8 films. A certain increase in elongation at yield for these samples was also observed (p < 0.05). This behaviour could be explained by some plasticizing effect caused by the addition of both additives to the polymer matrix resulting in the increase in ductile properties, which would also result in changes in the materials crystallinity. This behaviour has been also reported for LDPE-based samples with carvacrol (Persico et al, 2009).

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Results and Discussion. Chapter 1

3.1.3. Thermogravimetric Analysis (TGA). The effect of carvacrol and thymol on the thermal stability of PP films was studied by TGA under nitrogen atmosphere. Figure 1.6 shows the TGA curves obtained for PP0 and PPC samples. As it is well reported for PP thermal degradation, a single degradation step was observed for PP0 sample (Navarro, Torre, Kenny and Jiménez, 2003). However, samples with carvacrol showed a first degradation step at low temperatures (about 115 °C) and a second step corresponding to the thermal degradation of the polymer matrix. The TGA patterns of other formulations were quite similar in all cases. In this way, the first degradation step observed in active films was associated to the degradation of carvacrol and/or thymol. 100 PPC4 PPC6 PPC8 PP0

Weight (%)

80

60 105 100

40

95 20

90 20

120

220

320

420

0 20

120

220

320 420 Temperature (ºC)

520

620

720

Figure 1.6. TGA curves obtained for PP0 and formulations with carvacrol under nitrogen.

Therefore, it was possible to determine the remaining amount of additive in the polymer matrix after processing. The remaining concentrations were approximately 1, 2 and 3.5 wt% for formulations where the initial amounts were 4, 6 and 8 wt%, respectively. In conclusion, TGA results

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Results and Discussion. Chapter 1

gave an indirect confirmation of the presence of both compounds in the polymer matrix after processing and consequently their ability to act as active agents in these materials as it has been reported by other authors (Persico et al, 2009). Table 1.3 summarizes the initial degradation temperature, Ti, determined at 5 % of weight loss, and the temperature for the maximum rate, Tmax, for the main degradation step, ascribed to the PP thermal degradation. As can be observed, no appreciable differences were observed for Ti and Tmax values in all samples. These results show that the addition of carvacrol and thymol to the PP matrix does not significantly affect the thermal degradation profile in inert atmosphere. However, it is expectable that a certain amount of carvacrol and thymol would be lost during processing, since these materials are submitted to temperatures above the volatilization point of the additives. Therefore, the processing parameters, in particular temperature and time, should be optimized to avoid excessive evaporation and therefore the loss of these additives incorporated to PP permitting the permanence of significant amounts of additives in PP matrices (Dobkowski, 2006). 3.1.4. Differential Scanning Calorimetry (DSC).

Determination of thermal parameters in inert atmosphere. Thermal properties of samples were also studied by DSC, where four parameters were

determined:

crystallization

temperature,

Tc

(°C);

melting

temperature, Tm (°C); crystallization enthalpy, ∆Hc (J g-1); and melting enthalpy, ∆Hm (J g-1). Results are summarized in Table 1.3.

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Results and Discussion. Chapter 1

Table 1.3. TGA and DSC parameters obtained for all samples. Sample

Ti (C)

Tmax (C)

Tc (°C)

Tm (°C)

ΔHc (J g-1)

ΔHm (J g-1)

 (%)

PP0

411

461

119

161

95.2

99.1

72

PPC4

417

462

119

161

91.4

49.6

37

PPC6

414

461

117

160

93.6

52.2

40

PPC8

407

462

118

161

89.2

48.1

38

PPT4

417

462

119

161

92.2

52.0

39

PPT6

406

461

117

160

86.5

47.3

36

PPT8

408

462

115

159

88.9

49.5

39

PPTC4

412

462

118

160

93.9

53.5

40

PPTC6

398

461

117

160

89.6

49.0

38

PPTC8

404

463

114

162

83.0

52.6

41

Melting and crystallization temperatures and crystallization enthalpy did not show important differences for all materials. Nevertheless, it should be highlighted that the melting enthalpy of the PP0 sample was clearly higher than those obtained for the active materials. This observation could indicate some decrease in PP crystallinity caused by the presence of additives. In this sense, crystallinity,  (%), of samples was calculated according to Equation 1.1. Results are also shown in Table 1.3, where a higher value for  (%) was obtained for PP0. From these results, it could be concluded that the PP crystallinity decreases significantly with the addition of thymol and carvacrol, confirming the observed changes in the mechanical properties where a decrease in the elastic modulus was noticed. This decrease in crystallinity could be due to the interactions between the polymer matrix and additive molecules in the PP macromolecular network, in particular by the formation of hydrogen bonds between the hydroxyl groups in the additive and the crosslinking

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Results and Discussion. Chapter 1

points in the polymer macrochains with increase in the disorder of the PP structure and consequent decrease in crystallinity. A similar effect was reported for PP with the addition of other antioxidants (Alin and Hakkarainen, 2010).

Evaluation of oxidation induction parameters (OIT and OOT). The evaluation of the antioxidant performance of carvacrol and thymol in PP matrices is relevant in this study, since these additives are not only supposed to play the role of active additives for food, but also to protect the polymer against oxidative degradation during processing and further use. The determination of OOT and OIT parameters is considered a reliable, simple and fast method for the evaluation of the AOs efficiency (Pomerantsev and Rodionova, 2005). Both parameters correspond to relative measurements of the stability against oxidation of materials at high temperatures. Table 1.4 shows the OOT and OIT results for all the tested materials. The onset degradation temperatures for all active materials were significantly higher (at least in 25 °C) than the OOT obtained for the PP0 sample (p < 0.05). Therefore, it is remarkable that some antioxidant effect in the material caused by the presence of the active additives in films was observed. In particular, the formulations with thymol showed significantly higher values than their counterparts with carvacrol (p < 0.05). Similar results were previously reported for PP matrices with α-tocopherol and hydroxytyrosol, two other natural antioxidants (Peltzer and Jiménez, 2009).

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Results and Discussion. Chapter 1

Table 1.4. Oxidation induction parameters, oxygen transmission rate obtained for all formulations. OOT (C)a

PP0

195 ± 1a

0.9 ± 0.3a

1.3 ± 0.4g

82.2

PPC4

219 ± 3b

5.9 ± 0.5b

13.1 ± 1.6f

104.7

PPC6

225 ±

1bc

0.2bc

2.0ef

146.9

PPC8

224 ± 1bcd

8.5 ± 1.0bc

20.7 ± 2.8de

159.6

PPT4

234 ± 2ef

11.4 ± 1.8cd

28.9 ± 2.2c

114.3

PPT6

233 ±

3ef

2.8de

1.2bc

142.5

PPT8

235 ± 1f

15.4 ± 1.7e

38.8 ± 0.6a

155.5

PPTC4

223 ± 2bc

8.4 ± 1.2bc

24.1 ± 1.6d

112.2

PPTC6

226 ±

2cd

1.1bc

2.3cd

126.7

PPTC8

230 ± 3de

35.5 ± 1.7ab

158.0

8.2 ±

13.2 ±

8.7 ±

10.7 ± 1.2cd

OIT (min) aira

OTR·e (cm3 mm m-2 day)b

Sample

OIT (min) O2a

17.8 ±

33.7 ±

26.4 ±

Different superscripts within the same column indicate statistically significant different values (p < 0.05). aMean ± standard deviation (n = 3). be: Thickness, mm.

According to the ASTM D3895-07 Standard, the results obtained for OIT are dependent on the type of atmosphere used for the analysis (ASTM, 2007). For this reason, the evaluation of OIT in this work was carried out in two different atmospheres at 200 °C. This temperature was fixed since these tests require values slightly higher than those obtained for OOT of the pure polymer, and OOT for PP0 was 195 ± 1 °C. Air was selected since it would represent a situation according to the real processing or shelf-life conditions for these materials, while pure oxygen was also used since it would represent the most aggressive conditions for the oxidative degradation. Table 1.4 shows the results obtained for OIT in both

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Results and Discussion. Chapter 1

atmospheres. In the case of pure oxygen, PPT6 and PPT8 films showed higher efficiency as AOs in such aggressive conditions when compared to carvacrol (p < 0.05). This behaviour was also reported by other authors who demonstrated that the AO efficiency of thymol was higher than that of carvacrol in sunflower oil samples (Yanishlieva, Marinova, Gordon and Raneva, 1999). In the case of air atmosphere, as expected, OIT values were higher than those obtained in pure oxygen, since the experiment under air is less aggressive to materials (Riga, Collins and Mlachak, 1998), but results showed a similar trend since thymol showed higher antioxidant performance than carvacrol. In all cases, the increase in OOT and OIT values for PP with additives showed certain AO effect after processing to protect PP from oxidative degradation. These results are an additional confirmation that certain amounts of thymol and carvacrol still remained in all formulations after processing and they would be able to be released from the material to foodstuff as active additives, since it will be discussed in further sections of this chapter. Finally, an increase in both parameters was also observed when the additive concentration increased. So, it can be concluded that the best results for AO performance in these formulations would be obtained in those samples with higher amount of additives, in particular thymol. 3.1.5. Oxygen Transmission Rate (OTR) Barrier properties to oxygen were studied by the determination of oxygen transmission rate per film thickness (e), OTR∙e. Results are shown in Table 1.4. As can be observed, PP0 showed lower OTR∙e values than all active films, obtaining the maximum values for formulations with 8 wt% of carvacrol or thymol. This increase in oxygen transmission for the active

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Results and Discussion. Chapter 1

films could be due to the modification of the polymer matrix structure by the presence of additives, reducing consequently the resistance of films to oxygen diffusion through them (Sothornvit and Krochta, 2000). This behaviour could be due to two related causes. On one hand, the increase in free volume in the polymer structure caused by the chemical interaction between polymer chains and additive molecules and, on the other hand, the decrease in the material crystallinity already observed by DSC and the presence of certain porosity in the films surface observed by SEM (Amstrong, 2002).

3.2. Migration study 3.2.1. Validation of the developed methods The analytical methods developed in this study for the evaluation of the migration of carvacrol and thymol into food simulants were validated by assessing

the

main

analytical

characteristics:

linearity,

precision

(repeatability), detection (LOD) and quantification (LOQ) limits and accuracy (recovery test). Linear ranges were calculated with five calibration points, each of them in triplicate (0.15-2.10 mg kg-1 in dichloromethane for the SPE-GC/MS method and aqueous food simulants; 0.15-4.00 mg kg-1 for isooctane and ethanol 95 % (v/v) for direct GC/MS and HPLC-UV analyses, respectively). The calculated calibration curves gave an acceptable level of linearity for thymol and carvacrol with determination coefficients (R2) ranging between 0.9963-0.9989, as shown in Table 1.5. Repeatability was evaluated by analysing three replicates of standard solutions processed in the same day. All methods showed similar results for relative standard deviation (RSD), lower than 10 %. LOD and LOQ values were

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Results and Discussion. Chapter 1

determined by using regression parameters from the calibration curves (3 Sy/x/a and 10 Sy/x/a, respectively; where Sy/x is the standard deviation of the residues and a is the slope of the curves). As can be seen in Table 1.5, the lowest values for LOD and LOQ were obtained for thymol by using the HPLC-UV method. On the other hand, carvacrol showed lower values for these parameters considering the SPE-GC/MS method. In general terms, the LODs and LOQs values obtained for these active additives ranged between 0.16-0.22 mg kg-1 and between 0.50-0.74 mg kg1,

respectively.

Recovery tests for the SPE-GC/MS method were accomplished in triplicate to evaluate accuracy, by spiking aqueous food simulants with known amounts of each additive at three concentration levels (0.03, 0.27 and 2.60 mg kg-1). A working solution containing thymol and carvacrol (4000 mg kg-1) in methanol was used. Satisfactory results were obtained for mean recoveries at all the tested levels (Table 1.6), ranging from 86.7108.2 % with RSD values between 2.1-11.0 %. In conclusion, the results obtained in the validation of the analytical methods developed in this study can be considered acceptable for the determination of the migration of carvacrol and thymol in aqueous and fatty food simulants.

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Results and Discussion. Chapter 1

Table 1.5. Main analytical parameters obtained for the studied active additives using the optimized methods. Parameter Analyte

Carvacrol

Thymol

Method Slope ± SD

Intercept ± SD

Linearity (R2)a

LOD (mg/kg)b

LOQ (mg/kg)c

SPE-GC/MS

18416 ± 1907

-3881 ± 2372

0.9968

0.16

0.54

Direct HPLC-UV

13510 ± 333

1195 ± 82

0.9963

0.20

0.66

Direct GC/MS

-2241 ±721

15103 ± 320

0.9982

0.22

0.73

SPE-GC/MS

19792 ± 1120

-3868 ± 2377

0.9972

0.15

0.50

Direct HPLC-UV

13936 ± 177

1091 ± 43

0.9989

0.10

0.34

Direct GC/MS

-1852 ± 701

14568 ± 316

0.9981

0.22

0.74

a

Number of calibration points = 5. Linear range: 0.15 – 2.10 (SPE-GC/MS); 0.15 – 4.00 (Direct HPLC-UV and GC/MS). b Calculated for 3 Sy/x. c Calculated for 10 S y/x SD: Standard deviation The results are represented as mean ± standard deviation, n=3.

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Results and Discussion. Chapter 1

Table 1.6. Mean recoveries (%) and RSD values (%) in parentheses obtained for each active additive in aqueous simulants by SPE-GC/MS. Rresults are represented as mean ± standard deviation, n=3. Spiking level (mg) Analyte

Carvacrol

Thymol

Simulant 0.03

0.27

2.60

Distilled water

98.1 (5.4)

94.7 (9.7)

108.2 (2.1)

Ethanol 10 % (v/v)

95.2 (4.3)

100.3 (3.3)

89.8 (2.4)

Acetic acid 3% (m/v)

99.4 (6.5)

88.0 (3.2)

97.2 (10.9)

Distilled water

96.8 (5.8)

101.0 (3.6)

106.1 (2.4)

Ethanol 10 % (v/v)

94.1 (3.8)

99.1 (3.4)

88.4 (2.5)

Acetic acid 3% (m/v)

94.8 (4.9)

86.7 (3.2)

95.8 (11.0)

3.2.2. Release of active additives into food simulants Both active additives were readily released into aqueous and fatty food simulants from all PP films (Table 1.7). Similar behaviour was observed for thymol and carvacrol migration under the same experimental conditions. However, thymol showed a trend to higher migration than carvacrol in distilled water. The amount of active additives released into fatty food simulants from PP-based films with 8 wt% thymol (PPT8) and carvacrol (PPC8) was significantly higher than those obtained in the aqueous simulants (p < 0.05). In particular, the highest migration levels were obtained into isooctane at 20 °C for 2 days compared with the rest of simulants tested (40 °C and 10 days). This behaviour might result from the higher affinity of PP to non-polar liquids, such as isooctane, than to the highly polar solvents, such as ethanol 95 % (v/v) or other polar food simulants. Therefore the extraction of additives becomes an issue in isooctane, showing a diffusion behaviour rather than migration, ultimately leading to high migration values (Torres, Romero, Macan, Guarda and Galotto, 2014).

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Results and Discussion. Chapter 1

The higher migration observed for thymol and carvacrol into fatty food simulants could be also attributed to two factors: the higher solubility of the migrated active additives into these solvents and the phenomenon of swelling of the polymer matrix when the films come into contact with fatty food simulants (Suppakul, Sonneveld, Bigger and Miltz, 2011a). Tehrany et al indicated that polarity can be a predominant controlling factor in migration kinetics and, consequently, a highly polar simulant should have a great effect on sorption of additives into polymer matrices (Tehrany, Mouawad and Desobry, 2007). In this sense, partitioning depends on the polarity and solubility of the migrant in the food simulant. In our case, the higher release of carvacrol and thymol into ethanol 95 % (v/v) rather than ethanol 10 % (v/v) showed the influence of the simulant polarity and the solubility of thymol and carvacrol in the migration phenomenon. It can be also assumed that certain amount of simulants will penetrate into the matrix, enhancing the mobility of the target active additives inside the polymer chains, which could promote faster diffusion and in consequence higher migration. This behaviour has been also suggested in previous studies of the migration of some active compounds with antioxidant properties from polyolefins into fatty food simulants (Haider and Karlsson, 2000; Tovar, Salafranca, Sanchez and Nerin, 2005; Peltzer, Wagner and Jiménez, 2009; Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2013).

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Results and Discussion. Chapter 1

Table 1.7. Release of thymol and carvacrol (mg (kg-1 simulant)) obtained from PP films into aqueous and fatty food simulants under conditions in agreement with European Standard EN 13130-2005. Simulant Analyte

Film Watera

Ethanol (10 %, v/v)a

Acetic acid (3 %, m/v) a

Ethanol (95 %, v/v) a

Isooctaneb

PPC8

288 ± 20a

718 ± 54b

647 ± 47b

880 ± 27c

921 ± 157c

PPTC8

157 ± 19a

285 ± 26b

474 ± 44c

347 ± 21d

633 ± 34e

PPT8

433 ± 46a

656 ± 30b

689 ± 61b

829 ± 19c

1085 ± 112d

PPTC8

162 ± 18a

362 ± 53b

547 ± 51c

367 ± 21b

616 ± 49c

Carvacrol

Thymol Migration conditions: a 40 °C, 10 days; b 20 °C, 2 days The results are represented as mean ± standard deviation, n=3 Different superscripts within the same row indicate statistically significant different values (p < 0.05).

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Regarding aqueous food simulants, some migration of thymol and carvacrol was also observed although the solubility of both compounds in aqueous solutions is low; with migration values increasing by using acetic acid 3 % (m/v) and ethanol 10 % (v/v). The migration of these additives into the tested simulants might be due mainly to two factors: the hydrophilic character of these additives described in literature (Peltzer, Wagner and Jiménez, 2009); and the small size of their molecules, permitting faster diffusion since the diffusion rate is governed by the mobility of the additives molecules which is determined by their size and geometry (Haider and Karlsson, 2000; Reynier, Dole, Humbel and Feigenbaum, 2001). This behaviour was also observed by Mastromatteo et al who reported that the release of thymol from a swelling homogeneous polymeric network could be viewed as the result of the diffusion from the outer water solution into the polymer matrix, the macromolecular matrix relaxation and the diffusion of the active compound from the swollen polymeric network into the outer water solution (Mastromatteo, Barbuzzi, Conte and Del Nobile, 2009). The difference in polarity between the polar migrated substances and the non-polar polymer should be also taken into account. 3.2.3. Antioxidant activity of migration extracts. The AO activity of carvacrol and thymol has been already reported in previous studies, (Yanishlieva, Marinova, Gordon and Raneva, 1999) although the mechanism of such activity is not fully understood yet. The antioxidant activity of these compounds depends not only on their structure but also on many other factors, such as their concentration, temperature, light, simulant type and physical parameters inherent to the particular food to be put in contact with these compounds (e.g. pH).

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Figure 1.7. Radical scavenging activity measured by the DPPH method, expressed as percentage of inhibition for migration extracts (isooctane: 20 °C, 2 days; rest of simulants: 40 °C, 10 days) (mean ± standard deviation, n=3)). Different letters represent significant difference at p < 0.05.

The AO performance of the obtained extracts w was evaluated by the DPPH radical test and results are shown in Figure 1.7. Results for the DPPH inhibition in samples submitted to contact with acetic acid 3 % (m/v) were not introduced in Figure 1.7,, since no particular inhibition was observed in all cases. This result could be possibly due to the low pH conditions during the test, influencing in the same way the DPPH complex mechanisms for formation. It is kno known that the absorbance of DPPH decreases by light exposure, high oxygen content, low pHs, and solvent type (Ozcelik, Lee and Min, 2003).. Regarding pH, it has been reported that the increase of hydrogen ion concentration leads to the decrease in the rate of the chromogen radical scavenging reaction, whereas

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Results and Discussion. Chapter 1

under basic conditions proton dissociation in polyphenols would enhance the reducing capacity of these compounds (Pyrzynska and Pekal, 2013). On the other hand, all the other extracts showed a noticeable AO activity, showing a significant inhibition of the DPPH radical under the test conditions. A higher AO activity was observed for thymol extr extracts, with the highest inhibition obtained into isooctane (42.2 ± 1.1 %). The ANOVA results showed that regardless of the variations introduced by the use of the different simulants, the AOactivity of the formulation with 8 wt% of thymol was significantly different from all the other (Figure 1.8).

Figure 1.8. Mean DPPH inhibition values (%) for P PPT8, PPC8 and PPTC8. Different letters represent significant differences at p < 0.05.

These results showed that the AO activity of thymol was higher to that of carvacrol, possibly due to the larger steric hindrance of the thymol phenolic group; as concluded by different authors when considering the mechanisms of action of these compounds in the DPPH test (Yanishlieva, Marinova, Gordon and Raneva, 1999; Wu, Luo and Wang, 2012) 2012). Other

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Results and Discussion. Chapter 1

compounds with sterically-hindered hydroxyl groups, such as BHT, have been also reported to possess high antioxidant activity (Mastelic et al, 2008). The inhibition values obtained in DPPH tests were correlated with the amount of thymol and carvacrol released from films (Table 1.7). As previously discussed, the highest amount of released additives was observed into fatty food simulants and no significant differences between isooctane and ethanol 95 % (v/v) were observed for different formulations at p < 0.05 significance level (Figure 1.7). These results suggest that a considerable amount of both additives remain in the polymer matrix after processing and consequently could act as active agents in these PP-based formulations. In this sense, the PP films obtained in this study could be used as AO films for food packaging applications in order to extend the shelf-life of food products, retarding oxidation processes. Finally, the study of the combined activity of carvacrol and thymol introduced in the matrix at 4 wt% of each compound (PPTC8) showed some additive effect between them since similar results were obtained for samples with 8 wt% of each compound (PPC8 and PPT8) separately. This effect was more evident into fatty food simulants (Figure 1.7). 3.2.4. Release kinetics of thymol and carvacrol from active films Information about diffusion coefficients of additives through packaging materials is very useful to evaluate their performance in active systems. The critical point in antimicrobial and antioxidant performance of these materials is the release kinetics of the active additives through the polymer bulk and to food surfaces. A deeper knowledge of the migration mechanism of the target AOs from PP films is necessary to obtain

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information about the real possibilities ies of carvacrol and thymol in active PP-based materials. Therefore, some experiments were designed and carried out by using four different food simulants and getting contact between the PP-based formulations containing thymol and carvacrol at 8 wt% and them during 15 days. Figure 1.9 and Figure 1.10Figure 1.10 show the release of thymol and carvacrol from PPT8 and PPC8 films with time, respectively. A similar behaviour was observed for bothh compounds, being rapidly released from films into food simulants, with the expected increase in their release with time and reaching a steady state after approximately 120 h.

Figure 1.9. Release of thymol from PPT8 into different food simulants over 15 days. (A) Ethanol 10 % (v/v), 40 °C; (B) acetic acid, 40 °C; (C) Ethanol 95 % (v/v), 40 °C; and (D) isooctane, 20 °C. Solid lines were obtained by fitting Equation 1.4 to experimental data.

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Results and Discussion. Chapter 1

Figure 1.10. Release of carvacrol from PPC8 into different food simulants over 15 days. (A) Ethanol 10 % (v/v), 40 °C; (B) acetic acid, 40 °C; (C) Ethanol 95 % (v/v), 40 °C; and (D) isooctane, 20 °C. Solid lines were obtained by fitting Equation 1.4 to experimental data.

Table 1.8. Diffusion coefficients (D×10−14, m2 s-1) calculated from Equation 1.4 for the release of carvacrol and thymol from PP films into different food simulants (mean ± standard deviation, n=3). Simulant Analyte (Film)

Parameter

Ethanol 10 % (v/v)a

Acetic acid 3 % (m/v)a

Ethanol 95 % (v/v)a

Isooctaneb

αap

1.38  0.07

0.96  0.04

1.27  0.07

1.4  0.1

D

1.20  0.05

1.7  0.1

1.99  0.07

9.4  0.6

αap

1.56  0.1

1.44  0.05

1.56  0.08

2.4  0.2

D

1.75  0.08

2.51  0.02

1.01  0.03

5.9  0.1

Carvacrol (PPC8)

Thymol (PPT8) Migration temperature: a 40 °C; b 20 °C

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Results and Discussion. Chapter 1

In a first approach, a significantly higher amount of migrated analytes occurred into isooctane (see α values in Table 1.8). Furthermore, for this simulant, at t > 120 h, the equilibrium was not well defined and in the case of PPT8 in isooctane the release values obtained at the end of the experiment were close to 100 %. This result can be explained by the “swelling-controlled” model already proposed by other authors (Suppakul, Sonneveld, Bigger and Miltz, 2011a). According to this model, simulants would firstly penetrate into the polymer matrix to dissolve the active agents, thereby enabling their subsequent release. Indeed, it could be expected that the simulant uptake would cause polymer swelling. The migration of thymol and carvacrol is thus expected to increase with an increase in the simulant penetration into the PP-based film, reaching a plateau when the matrix is saturated with the simulant. However, many interactions take place during the migration of species from polymers into liquids. Moreover, it has been pointed out that a time-dependent relaxation process could occur as the result of the swelling that takes place during the diffusion of the liquid through the polymer. Consequently, release rates change continuously and the accurate mathematical analysis of the migration is difficult. The rapid diffusion of simulant molecules through the polymer bulk facilitates further penetration by the plasticization of the polymer matrix caused by the presence of additives, until a plateau is reached. As pointed out before, for isooctane an increase of migration after reaching the equilibrium was observed for both active additives at 360 h. This could be due to a combination of temperature and longer times in which the PP-based films were penetrated by isooctane producing the increase on the released amount of additives. It can be also assumed that the sorption of isooctane by the PP matrix and the consequent creation of void spaces could favour

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the migration of the phenolic compounds (Manzanarez-López, SotoValdez, Auras and Peralta, 2011). The experimental release data shown in Figure 1.9 and Figure 1.10 were further analysed in terms of a diffusion model in agreement with Equation 1.4. However, the use of this equation to compute diffusion coefficients needs the previous knowledge of the partition values. According to the previous discussion, it can be assumed that the amount of thymol or carvacrol released from the matrix can be estimated as being constant after 120 h, and once Equation 1.4 can only be applied to MF,t/MP,0 < 0.6; the use of this equation do not interfere with the assumed effect of the swelling-process in the mass transport by diffusion. Under these circumstances, the release of thymol and carvacrol from PPT8 and PPC8 to different simulants can be modelled by using Equation 1.6:

, ,

,∞

=

,

1−

− ′

(1.6.)

where k’ is a constant related to the release rate constant. By fitting Equation 1.6 to the experimental data (see solid lines in Figure 1.9 and Figure 1.10), the values of MF,∞/MP,0 can be obtained and, finally apparent partition coefficients (αap) can be calculated through Equation 1.7:

,∞ ,

=

1+ (1.7.)

It should be stressed that the Equation 1.6 has been selected once considered the border and limit conditions of the experiments reported in this work, since it describes a first order kinetic process (Reis, Guilherme,

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Results and Discussion. Chapter 1

Rubira and Muniz, 2007). In general terms, the fitting of the determination coefficients with experimental data are higher than 0.96 in all cases but isooctane-containing systems where determination coefficients were 0.819 (PPC8) and 0.713 (PPT8). The results obtained for thymol and carvacrol release are shown in Figure 1.11A and Figure 1.11B, respectively. As can be seen, the linearity of the plot

 1 1 M F ,t         M F , 0 

0 .5

versus t 0 .5 was very good for both active additives and

all simulants tested, resulting in determination coefficient values (R2) ranging from 0.961-0.995 for thymol and 0.983-0.992 for carvacrol, suggesting that the experimental release data are well described by the proposed diffusion model for short-range times. The analysis of the diffusion coefficients (D) (Table 1.8) shows that the diffusion process for thymol and carvacrol in different simulants are independent on theactive additives, with D values ranging from 1×10−14 to 2×10−14 m2 s-1. This behaviour was expected if considering that carvacrol and thymol are isomers having similar molecular weights, chemical structure and polarity (Licciardello, Muratore, Mercea, Tosa and Nerin, 2013). The exception occurs for the diffusion of these compounds into isooctane. In fact, D values for thymol and carvacrol are 4 and 6 times higher for this simulant than the average values for the other ones. This is, however, in line with the previous discussion and with results reported in Section 3.2. as well. It is also worth noticing that the magnitude of D values found for these films are one order of magnitude lower than those obtained for similar AOs and films (Suppakul, Sonneveld, Bigger and Miltz, 2011a), suggesting that these films can provide a long-term release, or higher retention inside films, of AOs making them very useful for active packaging systems.

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Results and Discussion. Chapter 1

Figure 1.11. Plots of

 1 1 M F ,t         M F , 0 

0.5



1 versus t 0 .5 for the migration of thymol  0.5

(A) and carvacrol (B) from PPT8 and PPC8 films into different food simulants. Isooctane (◊), 20 °C; acetic acid (o), 40 °C; ethanol 10 % (v/v) (□),), 40 °C; and ethanol 95 %(v/v) (∆), 40 °C.

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Results and Discussion. Chapter 1

3.3. Antibacterial properties Figure 1.12 shows the results of the antibacterial tests for the active films with 8 wt% of additives against Staphylococcus aureus and Escherichia coli by using the agar disk diffusion method. This method is very simple and it is based on the measurement of the clear zone caused by growth inhibition produced by a film disk containing the antimicrobial agent when putting in direct contact with a bacterial culture (Singh, Singh, Bhunia and Singh, 2003; Weerakkody, Caffin, Turner and Dykes, 2010). In this sense, when the PP-based films with the active agents were placed on top of the culture media, it is expected that both additives will diffuse from the polymer matrix into the agar in a radial manner, producing a clear zone of growth inhibition around the active film.

(a) S. aureus

PP0

PPC8

PPT8

PPTC8

PPC8

PPT8

PPTC8

(b) E. coli PP0

Figure 1.12. Antimicrobial activity of PP films with 8 wt% active additives: (a) Staphylococcus aureus; (b) Escherichia coli.

As can be seen in Figure 1.12a, films containing 8 wt% of thymol were the most effective against Staphylococcus aureus showing the largest inhibition

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Results and Discussion. Chapter 1

zone. This behaviour was also observed for the rest of materials with 8 wt% of additives (carvacrol and the equimolar mixture of both compounds), but with smaller inhibition zone (Table 1.9). These results demonstrate the antimicrobial action of both additives at high concentrations (8 wt%). The study of the combined activity by carvacrol and thymol in the same film (PPTC8) showed that some additive effect between them took place considering the results obtained for samples with 4 wt% of each compound (PPC4 and PPT4) separately, where an insufficient inhibition with no growth under the film was observed, as it was already reported by other authors (Guarda, Rubilar, Miltz and Galotto, 2011). Finally, samples with additives at initial concentrations 6 wt% were also not enough to achieve an adequate inhibition under these conditions. Table 1.9. Inhibition zone against Staphylococcus aureus obtained for all formulations.

Sample Inhibition zone diameter (cm) against Staphylococcus aureus

PP0 n.d.

PPC4

PPC6

PPC8

n.d

n.d

2.75 ± 0.07

PPT4

PPT6

PPT8

n.d

n.d

3.70 ± 0.14

PPTC4

PPTC6

PPTC8

n.d

n.d

3.25 ± 0.07

n.d.: No inhibition zone detected. Mean ± standard deviation (n = 2).

On the other hand, lower inhibition was observed when using these materials for Escherichia coli (Figure 1.12b). There was just some effect only under the film, but the inhibition zone was not observed. However, it is still possible to attribute certain antibacterial properties of these films

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Results and Discussion. Chapter 1

against Escherichia coli, since this inhibition is categorized as “sufficient” as it is described in the SNV 195920-1992 Standard (Pollini, Russo, Licciulli, Sannino and Maffezzoli, 2009). The low antibacterial activity against Escherichia coli could be due to the higher resistance of Gram-negative microorganisms to polyphenols making necessary either the use of higher concentrations of carvacrol and thymol in these films or the use of these active agents in the vapour phase, which has been reported to be more efficient against Escherichia coli (Becerril, Gómez-Lus, Goñi, López and Nerín, 2007). Other studies have described the antimicrobial effect of thymol and carvacrol against Escherichia coli (Xu, Zhou, Ji, Pei and Xu, 2008), attributing this effect to their ability to permeate and depolarize the cytoplasmic membrane. These authors observed areas with coagulated material in the outer wall of cells caused by the precipitation of some proteins. However, other authors reported higher effectiveness of oregano essential oil in Gram-positive bacteria (López, Sánchez, Batlle and Nerín, 2005). Therefore it can be concluded that experimental conditions for such tests are important to get high or low resistance of specific microorganisms against these compounds (López, Sánchez, Batlle and Nerín, 2007a; Weerakkody, Caffin, Turner and Dykes, 2010). As a general conclusion, the addition of carvacrol and thymol to PP films demonstrated some antimicrobial activity in bacterial strains potentially present in food, in particular against Gram-positive bacteria.

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Results and Discussion. Chapter 1

3.4. Study of the effectiveness of the active films to preserve perishable food 3.4.1. Observation of fungal growth This study was conducted to evaluate the antimicrobial activity of the developed films and their ability to act in active packaging formulations to increase the shelf-life in fresh food. These tests were based on the visual observation of the inhibition of the fungal growth in food samples by the action of the highly volatile active additives, carvacrol and thymol. In this sense, some previous studies by other authors showed the effectiveness of these compounds against different fungal strains of particular interest in the food industry (López, Sánchez, Batlle and Nerín, 2007a).

Figure 1.13. Study of the effectiveness of PP0 and active films containing 8 wt% of thymol (PPT8) to preserve cut bread and strawberries by observation of fungal growth.

~ 164 ~

Results and Discussion. Chapter 1

Figure 1.13 shows the appearance of sliced strawberries and bread samples at the beginning of the experiment (day 0) and after the observation of microbial growth for some days. Regarding strawberries, satisfactory results were obtained for samples in contact with the active films, since no fungal growth was observed until six days of storage. In the case of strawberries in contact with the pure PP film (PP0), a rapid growth of microorganisms was observed at the third day of treatment. On the other hand, the presence of microorganisms was observed in bread samples in contact with the PP0 film after 13 days of storage, in contrast to bread with the active films where no evidence of microbial contamination after 45 days of storage was observed.

Figure 1.14. Evaluation of the effectiveness of PP0 and active film containing 8 wt% of thymol (PPT8) to preserve uncut strawberries by observation of fungal growth.

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Results and Discussion. Chapter 1

However, it was noticed that cut strawberries lost their organoleptic properties after few days of storage, even before the visual evidence of fungal growth. For this reason, this study was also conducted for uncut strawberries (Figure 1.14). For the PP0 film microbial growth was observed after 6 days of storage. However, strawberries in contact with the PPT8 film remained unaltered after 13 days. At this storage time strawberries presented a physical deterioration due to the storage conditions, but it is important to highlight that microbial growth was not observed until the end of the study (15 days). Similar studies were performed with uncut strawberries by other authors, getting satisfactory results for samples in contact with films treated with EOs, such as cinnamon, oregano and thyme (Rodríguez, Batlle and Nerín, 2007). Regarding thyme and oregano EOs, their antimicrobial activity is attributed to the high amount of carvacrol and thymol in their composition (Hazzit, Baaliouamer, Faleiro and Miguel, 2006). Other studies conducted in different fruits and vegetables also demonstrated the effectiveness of the constituents of different EOs (eugenol, thymol, menthol or eucalyptol) to improve the organoleptic quality of food as well as to reduce the microbial growth, in particular when using a modified atmosphere for packaging (Mastromatteo, Conte and Del Nobile, 2010). In conclusion, results obtained from food samples in contact with PPbased films containing carvacrol and thymol evidenced the effectiveness of these compounds to improve the shelf-life of perishable food, such as strawberries and bread. Accordingly, these results also suggest the potential to use these films in active packaging systems to replace the direct addition of preservatives in food formulations.

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Results and Discussion. Chapter 1

3.4.2. Headspace analysis by HS-SPME-GC/MS Figure 1.15 shows the levels of carvacrol, in terms of peak area counts, reached in the headspace of the containers with bread slices after 0, 2, 5, 10 and 15 days of storage at room temperature described in the previous section. As can be seen, an increase in the amount of carvacrol released from the PP-based films was observed with time for the bread samples. A high release of carvacrol was observed after 2 days with slower release rate after 5, 10 and 15 days of storage. This mechanism of controlled release could lead to the improvement in shelf-life of the stored samples retarding the post-harvest deterioration. A similar behaviour was also observed for strawberries. Regarding the thymol release, a similar trend was shown for both test food samples. Bread stored at room temperature with PPC films 1E+10 9E+09

Bread stored at room temperature with PPC8 films

Abundance

8E+09

Days of study

Peak area (x106)

7E+09

0

644

6E+09

2

1086

5

1443

10

1616

15

2178

5E+09 4E+09 3E+09

Day 0 Day 2 Day 5 Day 10 Day 15

2E+09 1E+09 0 20

21

22

23

24

25

26

27

28

Time (min) Figure 1.15. Release of carvacrol in the headspace of bread slices after 0, 2, 5, 10 and 15

days of storage at room temperature.

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Results and Discussion. Chapter 1

Equilibrium modified atmosphere packaging (EMAP) is the most common packaging technology used to reduce the high respiration rate of strawberries. It is known that a suitable atmosphere composition can reduce the respiration rate of fruits and fungal growth with minimal alteration of organoleptic properties (Rizzolo, Gerli, Prinzivalli, Buratti and Torreggiani, 2007). In this sense, the identification of the main compounds present in the headspace of the packaged strawberries was carried out (Table 1.10) with PP0 films after 4 days of storage at room and refrigerated conditions. One of the most important processes occurring during fruit ripening is the increase in volatiles contributing to fruit aroma and flavour, so the determination of volatiles is a very adequate tool to predict the decomposition degree of packaged fruits. In this case, the major volatiles identified for strawberries stored at room temperature include methyl-isopentanoate, 2-methyl-butylacetate, methyl-hexanoate and hexyl-acetate. Methyl-butanoate and methyl-hexanoate were also found in the headspace composition of refrigerated strawberries at 4 °C. These results are in accordance with those obtained by other authors when studying volatile compounds in the same food samples (Rizzolo, Gerli, Prinzivalli, Buratti and Torreggiani, 2007; Blanda et al, 2009). The addition of thymol and carvacrol to PP films modified significantly the atmosphere inside packages during the storage of food samples due to their release from films. This fact could be related to the inhibition of the volatile compounds identified under these conditions (Table 1.10) that were not detected in samples in contact with PPT8 and PPC8 films after 4 days of storage.

~ 168 ~

Results and Discussion. Chapter 1

Table 1.10. Identified compounds present in the headspace of food samples packed with PP0 films after 4 days. Bread stored at room temperature Time Compound (min) 1.4 5.7 6.6

Ethanol 2,4-dimethyl heptane 2,4-dimethyl heptene

7.8

Isononane

13.0

4-methyloctane

Strawberries stored at room temperature Time Compound (min) Methyl 3.2 isopentanoate 2-methyl 5.8 butylacetate Methyl 7.4 hexanoate 10.6

Hexylacetate

Strawberries refrigerated at 4 °C Time (min)

Compound

1.9

Hexane

3.1

Methylbutanoate

3.9

Toluene

6.8

Isopropyl butyrate

12.2

Methyl hexanoate

On the other hand, ethanol was the main volatile compound found in the headspace of bread in contact with the PP0 film and stored at room temperature for 4 days. Ethanol results from the fermentation and/or lipid oxidation in bread as it has been reported by other authors (Poinot et al, 2007). In this sense, commercial bread samples in contact with PPT8 or PPC8 films after 4 days of storage were characterized by significantly lower amounts of ethanol, suggesting a reduction on oxidation reactions by the presence of thymol and carvacrol. The improvement on the oxidative stability of bread could be attributed to the release of carvacrol and thymol resulting in the increase of shelf-life. From these results it can be concluded that the release of both additives from active films to the headspace of the studied packaged foodstuff increased with the storage time, as expected. The volatiles profile obtained by HS-SPME-GC/MS was found to be different for samples in contact with PP0 and those with PPT8 and PPC8, due to the modification of the food headspace composition by the presence of these additives. Therefore, the release of thymol and carvacrol from the active PP films has shown to be effective in maintaining the quality of strawberries and bread submitted to different storage conditions. Finally, it can be

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Results and Discussion. Chapter 1

concluded that PP films with carvacrol and thymol could be a promising alternative to increase the foodstuff shelf-life.

4. Conclusions Carvacrol and thymol have shown their potential as active additives in PPbased films for food packaging with the double effect of their controlled release to foodstuff and their possibility to protect food from oxidative and microbiological degradation processes. Characterization of the active PP-based films was carried out by using different analytical techniques in order to evaluate the effect of carvacrol and thymol in the polymer matrix and their stabilization performance during processing. SEM micrographs showed certain porosity for films with the highest additives concentrations (8 wt%). Some decrease in the elastic modulus was observed for the active formulations compared with neat PP. The presence of additives did not affect the thermal stability of PP, but resulted in decreasing crystallinity and oxygen barrier properties. The presence of thymol and carvacrol also increased the stabilization against thermo-oxidative degradation, with higher oxidation induction parameters; suggesting that the polymer is well stabilized and a certain amount of these compounds remained in the polymer matrix after processing at high temperatures. The release study of carvacrol and thymol from PP films into aqueous and fatty food simulants was also accomplished. Analytical methods for the determination of the target compounds in the studied food simulants were successfully developed and validated. The release of additives from films was dependent on the food simulant and the amount incorporated into the polymer matrix. In particular, high migration levels were obtained for

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Results and Discussion. Chapter 1

both additives into isooctane, showing higher migration for thymol. The antioxidant activity of migration extracts was confirmed by the DPPH method, showing thymol a higher antioxidant capacity especially into isooctane with a 42.2 % of inhibition. The results obtained for the migration kinetics study showed that carvacrol and thymol incorporated into PP films at 8 wt% were readily released into different food simulants, but some quantities still remaining in the polymer matrix after 15 days. The release kinetics of both additives from PP films showed a Fickian behaviour with diffusion coefficients ranging from 1-2×10−14 m2 s-1; except for the diffusion into isooctane where values 4-6 times higher were obtained. Finally, thymol showed higher inhibition against bacterial strains present in food than carvacrol, leading to higher antimicrobial activity, in particular against Gram-positive bacteria. The obtained results provided evidences that exposure to carvacrol and thymol is an effective way to enlarge the quality of strawberries and bread samples during distribution and sale. As a general conclusion of this chapter, it could be stated that the addition of AO/AM additives, such as carvacrol and thymol to PP matrices in food packaging applications shows high potential to improve quality and safety aspects. In particular, the high efficiencies of their release from PP active films points out the great potential of these systems in AO/AM packaging of different food products to extend their shelf-life while avoiding the direct addition of additives to food.

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Results and Discussion. Chapter 1

5. References Al-Bandak, G., Oreopoulou, V. (2007). Antioxidant Properties and Composition of Majorana Syriaca Extracts. European Journal of Lipid Science and Technology, 109, 247-255. Alin, J., Hakkarainen, M. (2010). Type of Polypropylene Material Significantly Influences the Migration of Antioxidants from Polymer Packaging to Food Simulants During Microwave Heating. Journal of Applied Polymer Science, 118, 1084-1093. Amstrong, R.B. (2002). Effects of Polymer Structure on Gas Barrier of Ethylene Vinyl Alcohol (EVOH) and Considerations for Package Development. Tappi 2002 Place Conference, 285-311. Appendini, P., Hotchkiss, J.H. (2002). Review of Antimicrobial Food Packaging. Innovative Food Science & Emerging Technologies, 3, 113-126. Archodoulaki, V.M., Lüftl, S., Seidler, S. (2006). Oxidation Induction Time Studies on the Thermal Degradation Behaviour of Polyoxymethylene. Polymer Testing, 25, 83-90. ASTM D 3895 - 07. Standard Test Method for Oxidative-Induction Time of Polyolefins by Differential Scanning Calorimetry. 2007 ASTM D 882 - 09. Standard Test Method for Tensile Properties of Thin Plastic Sheeting. 2009 Barbosa-Pereira, L., Cruz, J.M., Sendón, R., et al (2013). Development of Antioxidant Active Films Containing Tocopherols to Extend The shelf Life of Fish. Food Control, 31, 236-243. Becerril, R., Gómez-Lus, R., Goñi, P., López, P., Nerín, C. (2007). Combination of Analytical and Microbiological Techniques to Study the Antimicrobial Activity of a New Active Food Packaging Containing Cinnamon or Oregano against E. Coli and S. Aureus. Analytical and Bioanalytical Chemistry, 388, 1003-1011.

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Results and Discussion. Chapter 1

Blanda, G., Cerretani, L., Cardinali, A., et al (2009). Osmotic Dehydrofreezing of Strawberries: Polyphenolic Content, Volatile Profile and Consumer Acceptance. LWT - Food Science and Technology, 42, 30-36. Burman, L., Albertsson, A.C., Höglund, A. (2005). Solid-Phase Microextraction for

Qualitative

and

Quantitative

Determination

of

Migrated

Degradation Products of Antioxidants in an Organic Aqueous Solution. Journal of Chromatography A, 1080, 107-116. Byun, Y., Kim, Y.T., Whiteside, S. (2010). Characterization of an Antioxidant Polylactic Acid (PLA) Film Prepared with α-Tocopherol, BHT and Polyethylene Glycol Using Film Cast Extruder. Journal of Food Engineering, 100, 239-244. Coma, V. (2008). Bioactive Packaging Technologies for Extended Shelf Life of Meat-Based Products. Meat Science, 78, 90-103. Commission Regulation (EU) No 10/2011. Plastic Materials and Articles Intended to Come into Contact with Food. Conte, A., Buonocore, G.G., Bevilacqua, A., Sinigaglia, M., Del Nobile, M.A. (2006). Immobilization of Lysozyme on Polyvinylalcohol Films for Active Packaging Applications. Journal of Food Protection, 69, 866-870. Crank, J., (1975). The Mathematics of Diffusion, Place: Oxford University Press. Oxford Chiralt, A., Martínez-Navarrete, N., Martínez-Monzó, J., et al (2001). Changes in Mechanical Properties Throughout Osmotic Processes: Cryoprotectant Effect. Journal of Food Engineering, 49, 129-135. Chung, D., Papadakis, S.E., Yam, K.L. (2002). Simple Models for Assessing Migration from Food-Packaging Films. Food Additives and Contaminants, 19, 611-617. Del Nobile, M.A., Conte, A., Buonocore, G.G., et al (2009). Active Packaging by Extrusion Processing of Recyclable and Biodegradable Polymers. Journal of Food Engineering, 93, 1-6. Dobkowski, Z. (2006). Thermal Analysis Techniques for Characterization of Polymer Materials. Polymer Degradation and Stability, 91, 488-493.

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Dopico-García, M.S., Castro-López, M.M., López-Vilariño, J.M., et al (2011). Natural Extracts as Potential Source of Antioxidants to Stabilize Polyolefins. Journal of Applied Polymer Science, 119, 3553-3559. Dorman, H.J.D., Deans, S.G. (2000). Antimicrobial Agents from Plants: Antibacterial Activity of Plant Volatile Oils. Journal of Applied Microbiology, 88, 308-316. Fernández, M. (2000). Revisión: Envasado Activo De Los Alimentos / Review: Active Food Packaging Food Science and Technology International, 6, 97-108. Gemili, S., Yemenicioǧlu, A., Altinkaya, S.A. (2009). Development of Cellulose Acetate Based Antimicrobial Food Packaging Materials for Controlled Release of Lysozyme. Journal of Food Engineering, 90, 453-462. Guarda, A., Rubilar, J.F., Miltz, J., Galotto, M.J. (2011). The Antimicrobial Activity of Microencapsulated Thymol and Carvacrol. International Journal of Food Microbiology, 146, 144-150. Gutiérrez, L., Escudero, A., Batlle, R.N., Nerín, C. (2009). Effect of Mixed Antimicrobial Agents and Flavors in Active Packaging Films. Journal of Agricultural and Food Chemistry, 57, 8564-8571. Gutiérrez, L., Sánchez, C., Batlle, R., Nerín, C. (2009). New Antimicrobial Active Package for Bakery Products. Trends in Food Science & Technology, 20, 9299. Haider, N., Karlsson, S. (2000). Kinetics of Migration of Antioxidants from Polyolefins in Natural Environments as a Basis for Bioconversion Studies. Biomacromolecules, 1, 481-487. Halliwell, B., Aeschbach, R., Löliger, J., Aruoma, O.I. (1995). The Characterization of Antioxidants. Food and Chemical Toxicology, 33, 601617. Hazzit, M., Baaliouamer, A., Faleiro, M.L., Miguel, M.G. (2006). Composition of the Essential Oils of Thymus and Origanum Species from Algeria and Their Antioxidant and Antimicrobial Activities. Journal of Agricultural and Food Chemistry, 54, 6314-6321.

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Ho, C.W., Wan Aida, W.M., Maskat, M.Y., Osman, H. (2006). Optimization of Headspace

Solid

Phase

Microextraction

(HS-SPME)

for

Gas

Chromatography Mass Spectrometry (GC/MS) Analysis of Aroma Compound in Palm Sugar (Arenga Pinnata). Journal of Food Composition and Analysis, 19, 822-830. Ho Lee, C., Soon An, D., Cheol Lee, S., Jin Park, H., Sun Lee, D. (2004). A Coating for Use as an Antimicrobial and Antioxidative Packaging Material Incorporating Nisin and α-Tocopherol. Journal of Food Engineering, 62, 323-329. Joseph, P.V., Joseph, K., Thomas, S., et al (2003). The Thermal and Crystallisation Studies of Short Sisal Fibre Reinforced Polypropylene Composites. Composites Part A: Applied Science and Manufacturing, 34, 253-266. Kuorwel, K.K., Cran, M.J., Sonneveld, K., Miltz, J., Bigger, S.W. (2011a). Antimicrobial Activity of Natural Agents against Saccharomyces Cerevisiae. Packaging Technology and Science, 24, 299-307. Kuorwel, K.K., Cran, M.J., Sonneveld, K., Miltz, J., Bigger, S.W. (2011b). Essential Oils and Their Principal Constituents as Antimicrobial Agents for Synthetic Packaging Films. Journal of Food Science, 76, R164-R177. Kuorwel, K.K., Cran, M.J., Sonneveld, K., Miltz, J., Bigger, S.W. (2013). Migration of Antimicrobial Agents from Starch-Based Films into a Food Simulant. LWT - Food Science and Technology, 50, 432-438. Kurek, M., Moundanga, S., Favier, C., Galić, K., Debeaufort, F. (2013). Antimicrobial Efficiency of Carvacrol Vapour Related to Mass Partition Coefficient When Incorporated in Chitosan Based Films Aimed for Active Packaging. Food Control, 32, 168-175. Lambert, R.J.W., Skandamis, P.N., Coote, P.J., Nychas, G.J.E. (2001). A Study of the Minimum Inhibitory Concentration and Mode of Action of Oregano Essential Oil, Thymol and Carvacrol. Journal of Applied Microbiology, 91, 453-462. Licciardello, F., Muratore, G., Mercea, P., Tosa, V., Nerin, C. (2013). Diffusional Behaviour of Essential Oil Components in Active Packaging

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Results and Discussion. Chapter 1

Polypropylene

Films

by

Multiple

Headspace

Solid

Phase

Microextraction–Gas Chromatography. Packaging Technology and Science, 26, 173-185. Lopez-De-Dicastillo, C., Nerin, C., Alfaro, P., et al (2011). Development of New Antioxidant Active Packaging Films Based on Ethylene Vinyl Alcohol Copolymer (EVOH) and Green Tea Extract. Journal of Agricultural and Food Chemistry, 59, 7832-7840. López, P., Sánchez, C., Batlle, R., Nerín, C. (2005). Solid- and Vapor-Phase Antimicrobial Activities of Six Essential Oils: Susceptibility of Selected Foodborne Bacterial and Fungal Strains. Journal of Agricultural and Food Chemistry, 53, 6939-6946. López, P., Sánchez, C., Batlle, R., Nerín, C. (2007a). Development of Flexible Antimicrobial Films Using Essential Oils as Active Agents. Journal of Agricultural and Food Chemistry, 55, 8814-8824. López, P., Sánchez, C., Batlle, R., Nerín, C. (2007b). Vapor-Phase Activities of Cinnamon, Thyme, and Oregano Essential Oils and Key Constituents against Foodborne Microorganisms. Journal of Agricultural and Food Chemistry, 55, 4348-4356. Lundbäck, M., Hedenqvist, M.S., Mattozzi, A., Gedde, U.W. (2006). Migration of Phenolic Antioxidants from Linear and Branched Polyethylene. Polymer Degradation and Stability, 91, 1571-1580. Manzanarez-López, F., Soto-Valdez, H., Auras, R., Peralta, E. (2011). Release of α-Tocopherol from Poly(Lactic Acid) Films, and Its Effect on the Oxidative Stability of Soybean Oil. Journal of Food Engineering, 104, 508517. Mascheroni, E., Guillard, V., Gastaldi, E., Gontard, N., Chalier, P. (2011). AntiMicrobial Effectiveness of Relative Humidity-Controlled Carvacrol Release from Wheat Gluten/Montmorillonite Coated Papers. Food Control, 22, 1582-1591. Mastelic, J., Jerkovic, I., Blazevic, I., et al (2008). Comparative Study on the Antioxidant and Biological Activities of Carvacrol, Thymol, and

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Eugenol Derivatives. Journal of Agricultural and Food Chemistry, 56, 39893996. Mastromatteo, M., Barbuzzi, G., Conte, A., Del Nobile, M.A. (2009). Controlled Release of Thymol from Zein Based Film. Innovative Food Science & Emerging Technologies, 10, 222-227. Mastromatteo, M., Conte, A., Del Nobile, M. (2010). Combined Use of Modified Atmosphere Packaging and Natural Compounds for Food Preservation. Food Engineering Reviews, 2, 28-38. Mastromatteo, M., Mastromatteo, M., Conte, A., Del Nobile, M.A. (2010). Advances in Controlled Release Devices for Food Packaging Applications. Trends in Food Science & Technology, 21, 591-598. Navarro, R., Torre, L., Kenny, J.M., Jiménez, A. (2003). Thermal Degradation of Recycled Polypropylene Toughened with Elastomers. Polymer Degradation and Stability, 82, 279-290. Ozcelik, B., Lee, J.H., Min, D.B. (2003). Effects of Light, Oxygen, and pH on the Absorbance of 2,2-Diphenyl-1-Picrylhydrazyl. Journal of Food Science, 68, 487-490. Park, H.Y., Kim, S.J., Kim, K.M., et al (2012). Development of Antioxidant Packaging Material by Applying Corn-Zein to LLDPE Film in Combination with Phenolic Compounds. Journal of Food Science, 77, E273-E279. Peltzer, M., Jiménez, A. (2009). Determination of Oxidation Parameters by DSC for Polypropylene Stabilized with Hydroxytyrosol (3,4-Dihydroxyphenylethanol). Journal of Thermal Analysis and Calorimetry, 96, 243-248. Peltzer, M., Navarro, R., López, J., Jiménez, A. (2010). Evaluation of the Melt Stabilization

Performance

of

Hydroxytyrosol

(3,4-Dihydroxy-

phenylethanol) in Polypropylene. Polymer Degradation and Stability, 95, 1636-1641. Peltzer, M., Wagner, J., Jiménez, A. (2007). Thermal Characterization of UHMWPE Stabilized with Natural Antioxidants. Journal of Thermal Analysis and Calorimetry, 87, 493-497.

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Peltzer, M., Wagner, J., Jiménez, A. (2009). Migration Study of Carvacrol as a Natural Antioxidant in High-Density Polyethylene for Active Packaging. Food Additives & Contaminants: Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment, 26, 938 - 946. Persico, P., Ambrogi, V., Carfagna, C., et al (2009). Nanocomposite Polymer Films Containing Carvacrol for Antimicrobial Active Packaging. Polymer Engineering & Science, 49, 1447-1455. Poinot, P., Arvisenet, G., Grua-Priol, J., et al (2008). Influence of Formulation and Process on the Aromatic Profile and Physical Characteristics of Bread. Journal of Cereal Science, 48, 686-697. Poinot, P., Grua-Priol, J., Arvisenet, G., et al (2007). Optimisation of HS-SPME to Study Representativeness of Partially Baked Bread Odorant Extracts. Food Research International, 40, 1170-1184. Pollini, M., Russo, M., Licciulli, A., Sannino, A., Maffezzoli, A. (2009). Characterization of Antibacterial Silver Coated Yarns. Journal of Materials Science: Materials in Medicine, 20, 2361-2366. Pomerantsev, A.L., Rodionova, O.Y. (2005). Hard and Soft Methods for Prediction of Antioxidants' Activity Based on the DSC Measurements. Chemometrics and Intelligent Laboratory Systems, 79, 73-83. Pospı́Šil, J., Horák, Z., Pilař, J., et al (2003). Influence of Testing Conditions on the Performance and Durability of Polymer Stabilisers in Thermal Oxidation. Polymer Degradation and Stability, 82, 145-162. Pozo-Bayón, M.A., Guichard, E., Cayot, N. (2006). Flavor Control in Baked Cereal Products. Food Reviews International, 22, 335-379. Pyrzynska,

K.,

Pekal,

A.

(2013).

Application

of

Free

Radical

Diphenylpicrylhydrazyl (DPPH) to Estimate the Antioxidant Capacity of Food Samples. Analytical Methods, 5, 4288-4295. Quílez, J., Ruiz, J.A., Romero, M.P. (2006). Relationships between Sensory Flavor Evaluation and Volatile and Nonvolatile Compounds in Commercial Wheat Bread Type Baguette. Journal of Food Science, 71, S423-S427.

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Reis, A.V., Guilherme, M.R., Rubira, A.F., Muniz, E.C. (2007). Mathematical Model for the Prediction of the Overall Profile of in Vitro Solute Release from Polymer Networks. Journal of Colloid and Interface Science, 310, 128-135. Reynier, A., Dole, P., Humbel, S., Feigenbaum, A. (2001). Diffusion Coefficients of Additives in Polymers. I. Correlation with Geometric Parameters. Journal of Applied Polymer Science, 82, 2422-2433. Ridgway, K., Lalljie, S.P.D., Smith, R.M. (2007). Sample Preparation Techniques for the Determination of Trace Residues and Contaminants in Foods. Journal of Chromatography A, 1153, 36-53. Riga, A., Collins, R., Mlachak, G. (1998). Oxidative Behavior of Polymers by Thermogravimetric Analysis, Differential Thermal Analysis and Pressure Differential Scanning Calorimetry. Thermochimica Acta, 324, 135-149. Rizzolo, A., Gerli, F., Prinzivalli, C., Buratti, S., Torreggiani, D. (2007). Headspace Volatile Compounds During Osmotic Dehydration of Strawberries (cv Camarosa): Influence of Osmotic Solution Composition and Processing Time. LWT - Food Science and Technology, 40, 529-535. Rodriguez-Lafuente, A., Nerin, C., Batlle, R. (2010). Active Paraffin-Based Paper Packaging for Extending the Shelf Life of Cherry Tomatoes. Journal of Agricultural and Food Chemistry, 58, 6780-6786. Rodríguez, A., Batlle, R., Nerín, C. (2007). The Use of Natural Essential Oils as Antimicrobial Solutions in Paper Packaging. Part II. Progress in Organic Coatings, 60, 33-38. Ruiz, J.A., Quilez, J., Mestres, M., Guasch, J. (2003). Solid-Phase Microextraction Method for Headspace Analysis of Volatile Compounds in Bread Crumb. Cereal Chemistry Journal, 80, 255-259. Salafranca, J., Pezo, D., Nerín, C. (2009). Assessment of Specific Migration to Aqueous Simulants of a New Active Food Packaging Containing Essential Oils by Means of an Automatic Multiple Dynamic Hollow Fibre Liquid Phase Microextraction System. Journal of Chromatography A, 1216, 3731-3739.

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Sanchez-Garcia, M.D., Ocio, M.J., Gimenez, E., Lagaron, J.M. (2008). Novel Polycaprolactone Nanocomposites Containing Thymol of Interest in Antimicrobial Film and Coating Applications. Journal of Plastic Film and Sheeting, 24 (3-4), 239-251 Singh, A., Singh, R.K., Bhunia, A.K., Singh, N. (2003). Efficacy of Plant Essential Oils as Antimicrobial Agents against Listeria Monocytogenes in Hotdogs. Lebensmittel-Wissenschaft und-Technologie, 36, 787-794. Siró, I., Fenyvesi, É., Szente, L., et al (2006). Release of Alpha-Tocopherol from Antioxidative Low-Density Polyethylene Film into Fatty Food Simulant: Influence of Complexation in Beta-Cyclodextrin. Food Additives and Contaminants, 23, 845-853. Sothornvit, R., Krochta, J.M. (2000). Oxygen Permeability and Mechanical Properties of Films from Hydrolyzed Whey Protein. Journal of Agricultural and Food Chemistry, 48, 3913-3916. Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W. (2003). Active Packaging Technologies with an Emphasis on Antimicrobial Packaging and Its Applications. Journal of Food Science, 68, 408-420. Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W. (2006). Characterization of Antimicrobial Films Containing Basil Extracts. Packaging Technology and Science, 19, 259-268. Suppakul, P., Sonneveld, K., Bigger, S.W., Miltz, J. (2011a). Diffusion of Linalool and Methylchavicol from Polyethylene-Based Antimicrobial Packaging Films. LWT - Food Science and Technology, 44, 1888-1893. Suppakul, P., Sonneveld, K., Bigger, S.W., Miltz, J. (2011b). Loss of AM Additives from Antimicrobial Films During Storage. Journal of Food Engineering, 105, 270-276. Tehrany, E.A., Mouawad, C., Desobry, S. (2007). Determination of Partition Coefficient of Migrants in Food Simulants by the PRV Method. Food Chemistry, 105, 1571-1577.

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Tomaino, A., Cimino, F., Zimbalatti, V., et al (2005). Influence of Heating on Antioxidant Activity and the Chemical Composition of Some Spice Essential Oils. Food Chemistry, 89, 549-554. Torres, A., Romero, J., Macan, A., Guarda, A., Galotto, M.J. (2014). Near Critical and Supercritical Impregnation and Kinetic Release of Thymol in Lldpe Films Used for Food Packaging. The Journal of Supercritical Fluids, 85, 4148. Tovar, B.Z., Garcı́A, H.S., Mata, M. (2001). Physiology of Pre-Cut Mango. I. ACC and ACC Oxidase Activity of Slices Subjected to Osmotic Dehydration. Food Research International, 34, 207-215. Tovar, L., Salafranca, J., Sanchez, C., Nerin, C. (2005). Migration Studies to Assess the Safety in Use of a New Antioxidant Active Packaging. Journal of Agricultural and Food Chemistry, 53, 5270-5275. Tunç, S., Duman, O. (2011). Preparation of Active Antimicrobial Methyl Cellulose/Carvacrol/Montmorillonite

Nanocomposite

Films

and

Investigation of Carvacrol Release. LWT - Food Science and Technology, 44, 465-472. UNE-EN 13130-1:2005. Materiales Y Artículos En Contacto Con Alimentos. Sustancias Plásticas Sometidas a Limitaciones. Parte 1: Guía de métodos de ensayo para la migración específica de sustancias procedentes de materiales plásticos a los alimentos y simulantes de alimentos, determinación de sustancias en los materiales plásticos y selección de las condiciones de exposición a los simulantes de alimentos. 2005 Valentao, P., Fernandes, E., Carvalho, F., et al (2002). Antioxidative Properties of Cardoon (Cynara Cardunculus L.) Infusion against Superoxide Radical, Hydroxyl Radical, and Hypochlorous Acid. Journal of Agricultural and Food Chemistry, 50, 4989-4993. Vermeiren, L., Devlieghere, F., Van Beest, M., De Kruijf, N., Debevere, J. (1999). Developments in the Active Packaging of Foods. Trends in Food Science & Technology, 10, 77-86.

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Weerakkody, N.S., Caffin, N., Turner, M.S., Dykes, G.A. (2010). In Vitro Antimicrobial Activity of Less-Utilized Spice and Herb Extracts against Selected Food-Borne Bacteria. Food Control, 21, 1408-1414. Wu, Y., Luo, Y., Wang, Q. (2012). Antioxidant and Antimicrobial Properties of Essential Oils Encapsulated in Zein Nanoparticles Prepared by Liquid– Liquid Dispersion Method. LWT - Food Science and Technology, 48, 283290. Xu, J., Zhou, F., Ji, B.P., Pei, R.S., Xu, N. (2008). The Antibacterial Mechanism of Carvacrol and Thymol against Escherichia Coli. Letters in Applied Microbiology, 47, 174-179. Yanishlieva, N.V., Marinova, E.M., Gordon, M.H., Raneva, V.G. (1999). Antioxidant Activity and Mechanism of Action of Thymol and Carvacrol in Two Lipid Systems. Food Chemistry, 64, 59-66.

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2

Chapter 2

Active nanocomposites based on PLA with thymol and nanomaterials for food packaging applications

Results and Discussion. Chapter 2

The main aim of this chapter is the presentation of the results obtained for the formulation, processing and characterization of PLA-based active nanocomposites with intended application in the manufacture of films for food packaging. PLA was selected as the polymer matrix by its adequate combination of mechanical and optical properties for the formulation of transparent films while preserving the biodegradable character of nanocomposites. Thymol was used as active additive while two different nano-reinforcements were selected, a commercial organo-modified montmorillonite (MMT), Dellite®43B, in Section 2.1, and silver nanoparticles (Ag-NPs) in Section 2.2. Both nanomaterials were selected by their commercial availability, good compatibility with the polymer matrix and possibility of positive modifications in the mechanical, barrier and antimicrobial properties of PLA.

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1. Introduction Research in biopolymers and their possible use in massive applications, such as food packaging, has gained a lot of attention in the last few years from technological and ecological points of view (Mellinas et al, 2015). In fact, the raising trend for the industrial use of environmentally-friendly materials, such as PLA, represents an interesting alternative to polymers derived from petroleum due to its renewable origin, biodegradability and biocompatibility (Alix et al, 2013; Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013). PLA is one of the most important commercially available bio-based and biodegradable thermoplastic polyesters (Inkinen, Hakkarainen, Albertsson and Sodergard, 2011) by its adequate optical and mechanical properties, possibilities of modification with additives without hampering its biodegradation/biocompatibility abilities. In addition, PLA is classified as GRAS for food packaging applications, fulfilling the requirements to be in direct contact with aqueous, acidic and fatty foods (Peelman et al, 2013). PLA is a highly transparent and rigid material with a relatively low crystallization rate, making it a promising candidate for the fabrication of biaxial oriented films, thermoformed containers and stretch-blown bottles (Inkinen, Hakkarainen, Albertsson and Sodergard, 2011). However, some properties of PLA are inadequate for food packaging applications, such as poor thermal stability and low glass transition temperature, gas barrier properties, ductility and toughness (Hwang et al, 2012). In the last few years, some work has been reported to improve some of these PLA properties. One of the most paved paths to overcome these drawbacks is by the reinforcement with nanomaterials, mostly layered silicates (Fukushima, Tabuani and Camino, 2009; Gamez-Perez et al, 2011; Lagaron and Lopez-Rubio, 2011; Picard, Espuche and Fulchiron, 2011).

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In this sense, the incorporation of lamellar nanofillers with high aspect ratio, such as MMTs, has resulted in significant improvement in PLA mechanical, gas barrier, and optical properties (Rhim, Hong and Ha, 2009). Silver nanoparticles (Ag-NPs) have also emerged as candidates for PLA modification by their strong AM effect to a wide range of microorganisms, joined to their stability at high temperatures and low volatility (Echegoyen and Nerín, 2013). Due to their unspecific mechanism of action, silver ions are active not only against a broad number of bacteria, but also against yeast, fungi and viruses (Sharma, Yngard and Lin, 2009). However, some concerns about the safety and environmental effects of products containing Ag-NPs in direct contact with food have raised recently (Reidy, Haase, Luch, Dawson and Lynch, 2013; Addo Ntim, Thomas, Begley and Noonan, 2015). According to the Council Directive 94/36/EC (1994), silver is accepted as food additive with the code E174 if used as “external coating of confectionary, decoration of chocolates, liqueurs”. Nevertheless, in food contact materials Ag-NPs are not yet allowed, although the presence of certain silver zeolites is already authorized in plastic food containers and rubber seals (Artiaga, Ramos, Ramos, Cámara and Gómez-Gómez, 2015). Nanoand thin-film technologies based on novel systems associating metal nanoparticles to biopolymer matrices open a broad range of new applications, such as active biofilms for food packaging. The AM effect of Ag-NPs loaded in selected polymer matrices against foodborne bacteria has been reported. For instance, Kanmani et al developed active nanocomposite films by blending aqueous solutions of gelatin with different concentrations of Ag-NPs (Kanmani and Rhim, 2014a). Nanocomposite films based on LDPE containing Ag-NPs were also

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Results and Discussion. Chapter 2

formulated,

showing

some

improvement

in

the

resistance

to

microbiological degradation of packed juice (Emamifar, Kadivar, Shahedi and Soleimanian-Zad, 2011). Shameli et al evaluated the AM performance of PLA/Ag-NPs nanocomposites against Escherichia coli and Staphylococcus aureus by the disk diffusion method with high success (Shameli et al, 2010). Fortunati et al reported that the antibacterial activity of Ag-NPs depends on the bacterial strain and on differences in the cell wall structure between Gram-negative and Gram-positive bacteria (Fortunati, Rinaldi, et al, 2014). Recently, food packaging companies are focusing on the improvement of quality and extending the food shelf-life while maintaining their natural properties. The increasing demand for natural additives has resulted in the development of new active materials based on polymer or biopolymer matrices with natural additives, such as plant extracts or essential oils, which are categorized as GRAS by the FDA as well as the current European Legislation (Commission Regulation (EU) No 10/2011. Plastic materials and articles intended to come into contact with food) (Guarda, Rubilar, Miltz and Galotto, 2011; Valdés, Mellinas, Ramos, Garrigós and Jiménez, 2014). The addition of natural additives with AM and/or AO properties into a polymer matrix allows their gradual release during storage and distribution, extending food shelf-life by decreasing lipid auto-oxidation and the spoilage by microorganisms, which are recognized as major causes of deterioration affecting both sensory and nutritional quality (Manzanarez-López, Soto-Valdez, Auras and Peralta, 2011). In this sense, thymol is one of the most promising natural additives to be used in active formulations since it has been reported to be effective as AO and AM (AlBandak and Oreopoulou, 2007; Amorati, Foti and Valgimigli, 2013; Gyawali and Ibrahim, 2014). In fact, the presence of the hydroxyl

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Results and Discussion. Chapter 2

functional group in the thymol structure plays an important role in its AM and AO activity. The hydroxyl group promotes the delocalization of electrons, acting as proton exchangers to reduce the gradient across the cytoplasmic membrane of bacterial cells and causing the collapse of the proton motive force and depletion of the ATP pool, leading to cell death (Gyawali and Ibrahim, 2014). The AO properties of thymol are due to the ability to donate H-atoms from the phenol hydroxyl groups, which could react with peroxyl radicals to produce stabilized phenoxyl radicals and terminating the lipid peroxidation chain reactions (Mastelic et al, 2008; Viuda-Martos, Navajas, Zapata, Fernández-López and Pérez-Álvarez, 2010). Several methods have been proposed to determine the high AO and AM activity of thymol as pure compound or extracted from plants (Sánchez-Moreno, 2002; Amorati and Valgimigli, 2015; Perricone, Arace, Corbo, Sinigaglia and Bevilacqua, 2015). The use of natural additives in combination with nanofillers to develop novel active nanocomposites has been recently proposed (Kanmani and Rhim, 2014a; Mihindukulasuriya and Lim, 2014; Qin et al, 2015; Shemesh et al, 2015; Tornuk, Hancer, Sagdic and Yetim, 2015). In these active systems, additives with AM and/or AO performance, such as thymol, are embedded into a matrix acting against bacteria and/or moulds extending food shelf-life while improving quality (Sung et al, 2013). The use of nanomaterials in these formulations could improve some key properties, such as flexibility, gas barrier and temperature/moisture stability (Priolo, Holder, Gamboa and Grunlan, 2011; Araújo, Botelho, Oliveira and Machado, 2014; Mihindukulasuriya and Lim, 2014). In the case of addition of AG-NPs, all these features could be complemented by the increase in AM properties given by the additive or synergic effect of both

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Results and Discussion. Chapter 2

components, active principle and nanoparticles (Fonseca et al, 2015; Pagno et al, 2015). In summary, the development of different nanocomposites based on PLA with nanoclays (Fukushima, Tabuani and Camino, 2009; Gamez-Perez et al, 2011; Picard, Espuche and Fulchiron, 2011; Souza, Morales, MarinMorales and Mei, 2013; Rawi, Jayaraman and Bhattacharyya, 2014; Fortunati et al, 2015) or active systems (Byun, Kim and Whiteside, 2010; López-Rubio and Lagaron, 2010; Hwang et al, 2012; Wu, Yuan, et al, 2014) has been extensively reported in the last few years. However, few works have reported the combination of thymol and nanofillers in biopolymer matrices resulting in nanocomposites with AO and AM properties for use in food packaging. Indeed, the formulation of this new generation active nanocomposites represents a promising alternative to enhance mechanical and gas barrier properties and extend foodstuff shelf-life by the increase in the resistance to oxidative and microbiological degradation. This study focuses on the development of AO/AM active films based on PLA with a natural additive (thymol), reinforced with a commercial organically modified montmorillonite [Dellite®43B (D43B)] (Section 2.1) and AgNPs (Section 2.2).

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3

Section 2.1.

Active nanocomposites based on PLA with thymol and layered montmorillonite nanoclay for food packaging applications

Results and Discussion. Chapter 2

Summary A full characterization of PLA-thymol-D43B ternary formulations was carried out including the determination of thermal, structural, mechanical and functional properties. After the incorporation of the active additive and the nanofiller, the presence of thymol in the nanocomposites was determined by HPLC-UV analysis and the release of thymol into aqueous food simulant was determined. The AO activity was evaluated by using the DPPH method and the antibacterial activity against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) was also studied. Finally, the post-use disposal of these active nanocomposite films was evaluated in a laboratory-scale composting condition test. The scheme in the next page shows the graphical flow of tests performed whose results will be discussed in this section.

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Results and Discussion. Chapter 2

Figure 2.1. General scheme of the experimental work presented in Section 2.1.

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Results and Discussion. Chapter 2

2. Experimental 2.1. Materials and chemicals Commercial PLA-4060D (Tg = 58 °C, 11-13 wt% D-isomer) was supplied in pellets by Natureworks Co., (Minnetonka, MN, USA). Thymol (99.5 %), 2,2-Diphenyl-1-picrylhydrazyl (DPPH, 95 %),methanol and ethanol (HPLC grade) were supplied by Sigma-Aldrich (Madrid, Spain). The commercial nanoclay was Dellite®43B (D43B) (Laviosa Chimica Mineraria S.p.A. Livorno, Italy), a dimethyl-benzyldihydrogenated tallow ammonium modified montmorillonite with a cation exchange capacity (CEC) 95 meq/100 g clay, a bulk density of 0.40 g cm−3 and a typical particle size distribution between 7-9 µm.

2.2. Films preparation The different nanocomposites were obtained by melt-blending in a Haake Polylab QC mixer (ThermoFischer Scientific, Walham, MA, USA) with a mixing time of 20 min at 160 °C. Two different rotor speeds were used: 150 rpm in the loading and mixing steps (15 min) and 100 rpm for the last 5 min, when thymol was added. This final addition of thymol was designed to limit degradation and to ensure the presence of the active additive in the final blends. Prior to the mixing step, PLA and the nanoclay were dried for 24 h at 80 and 100 °C, respectively. Thymol was used as received. Five different formulations (three binary and two ternary) were obtained by combining thymol at one concentration level (8 wt %) and D43B at two different loadings (2.5 and 5 wt %) in PLA matrices, as described in Table 2.1. An additional sample without any additive was also prepared and used as control (neat PLA).

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Films were obtained by compression-moulding at 180 °C in a hot-plate press (Carver Inc 3850, Wabash, IN, USA). Blends were kept at atmospheric pressure for 5 min until melting and pressed at 2 MPa for 1 min, 3.5 MPa for 1 min and finally 5 MPa for 5 min to eliminate air bubbles trapped in the film structure. Transparent films were obtained with average thickness 190  15 μm measured with a Digimatic Micrometer Series 293 MDC-Lite (Mitutoyo, Japan) at five random positions. Table 2.1. PLA-based films formulated in this study. Samples

PLA (wt%)

D43B (wt%)

Thymol (wt%)

PLA

100

-

-

PLA/D43B2.5

97.5

2.5

-

PLA/D43B5

95

5

-

PLA/T

92

-

8

PLA/T/D43B2.5

89.5

2.5

8

PLA/T/D43B5

87

5

8

2.3. Thymol quantification The actual amount of thymol in films after processing was determined by solid-liquid extraction followed by HPLC-UV analysis. 0.05 ± 0.01 g of each film were extracted with 10 mL of methanol at 40 °C and 50 % RH for 24 h in a climate chamber (Dycometal CM-081, Barcelona, Spain). Thymol was determined with a Shimadzu LC-20A liquid chromatograph (Kyoto, Japan) equipped with a UV detector at 274 nm. The column used was a LiChrospher 100 RP 18 (250 mm x 5 mm x 5 μm, Agilent Technologies, USA). The mobile phase was composed of acetonitrile and water (40:60) at 1 mL min-1 flow rate. 20 μL of the extracted samples were

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injected and analyses were performed in triplicate. Quantification of the active additive was carried out by comparison of the chromatographic peak areas with standards in the same concentration range. Calibration curves were run at five concentration levels from 100 to 500 mg kg-1 using appropriately diluted standards of thymol in methanol.

2.4. Films characterization Films were characterized by the determination of their thermal, mechanical, morphological, optical and barrier to oxygen properties. 2.4.1. Thermal analysis TGA tests were performed with a TGA/SDTA 851 Mettler Toledo thermal analyser (Schwarzenbach, Switzerland). Approximately 5 mg samples were heated from 30 to 700 °C at 10 °C min-1 under nitrogen atmosphere (flow rate 50 mL min-1). DSC tests were used to determine Tg of all materials with a TA DSC Q2000 instrument (New Castle, DE, USA) under nitrogen atmosphere (flow rate 50 mL min-1). Approximately 3 mg samples were heated from 30 to 200 °C at 10 °C min-1 (3 min hold), then cooled at 10 °C min-1 to -30 °C (3 min hold) and further heated to 200 °C at 10 °C min-1. 2.4.2. Structural analysis The nanocomposites structure was studied by XRD, including the nanoclay dispersion. XRD patterns were recorded at room temperature in the scattering angle (2θ) 2-30° (step size: 0.01°, scanning rate: 8 s step-1) using filtered Cu Kα radiation (λ: 1.54 Å). A Bruker D8-Advance model

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diffractometer (Madison, WI, USA) was used to determine the interlayer distance (d-spacing) and intercalation of the nanoclay. 2.4.3. Morphological analysis The nanocomposites morphology was studied by transmission electron microscopy (TEM) micrographs which were performed by using a JEOL JEM-2010 (Tokyo, Japan) with accelerating voltage 100 kV. Prior to analysis, films were ultra-microtomed to obtain slices of 100 nm thick (RMC, model MTXL). 2.4.4. Mechanical properties Tensile properties of all films were determined with a 3340 Series Single Column System Instron Instrument, LR30K model (Fareham Hants, UK) equipped with a 2 kN load cell. The main tensile parameters, such as elastic modulus and elongation at break, were calculated from stress-strain curves according to the ASTM D882-09 Standard procedure (ASTM, 2009). Prior to testing, all samples were conditioned for 48 h at 25 °C and 50 % RH. Tests were performed with 100 x 10 mm2 rectangular probes and initial grip separation of 60 mm. The specimens were stretched at 10 mm min-1 until breaking. Results were the average of five measurements (± standard deviation). 2.4.5. Oxygen transmission rate (OTR) OTR is defined as the quantity of oxygen circulating through a determined area of the parallel surface of a plastic film per time unit. An oxygen permeation analyser (8500 model Systech, Metrotec S.A, Spain) was used for OTR tests. Pure oxygen (99.9 %) was introduced into the upper half of the diffusion chamber while nitrogen was injected into the lower half,

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where an oxygen sensor was located. Films were cut into 14 cm diameter circles for each formulation and they were clamped in the diffusion chamber at 25 °C before testing. Tests were performed in triplicate and mean values were expressed as oxygen transmission rate per film thickness (OTR∙e). 2.4.6. Colour tests Colour modifications on PLA-based films caused by the addition of the active additive and the nanoclay were followed by using a Konica CM3600d COLORFLEX-DIFF2 colorimeter, HunterLab, (Reston, VA, USA). Colour values were expressed as L* (lightness), a* (red-green) and b* (yellow-blue) coordinates in the CIELab colour space. These parameters were determined at five different locations in the film surfaces and the average values were calculated. Total colour difference (∆E*) was calculated according to Equation 2.1.

∆

∗

1

= [(∆ ∗ )2 + (∆ ∗ )2 + (∆ ∗ )2 ]

2

(2.1)

where ∆L*, ∆a* and ∆b* are the coordinate differences between control (neat PLA) and samples.

2.5. Degradation in compost Disintegration tests under composting conditions were performed by following the European Standard ISO 20200. The test method determines, at the bench-scale, the degree of disintegration of plastic materials under simulated intensive aerobic composting conditions (UNEEN_20200, 2006). Materials can be considered disintegrable according to

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this standard when 90 % of the plastic sample weight is lost within 180 days of analysis. Samples for disintegration tests were cut in pieces (20 x 20 mm2). A solid synthetic bio-waste was prepared, with certain amount of sawdust, rabbit food, compost inoculums, starch, oil, sugar and urea, as shown in Table 2.2. Water was periodically added and compost was mixed by hand at certain time intervals to ensure the aerobic conditions in the process. Samples were buried at 5 cm depth in perforated boxes and incubated at 58 °C for 35 days. Different times were selected to recover samples from their burial and further tested: 0, 2, 4, 7, 10, 14, 21, 28 and 35 days. Table 2.2. Composition of synthetic bio-waste used to simulate the disintegrability in composting conditions. Composition

Quantity (g)

Sawdust

240

Rabbit food

180

Starch

60

Compost inoculum

60

Sugar

30

Oil

18

Urea

12

Deionised water

600

TOTAL

1200 g

Samples were immediately washed after recovery with special care to remove traces of compost extracted from the container and further dried at 37 °C for 24 h before gravimetrical analysis. The degree of disintegration was calculated in percentage by normalizing the sample weight at different stages of incubation to the initial weight by using Equation 2.2.

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(%) =

−

∙ 100

(2.2)

where mi is the initial dry plastic mass and mt is the dry plastic material after the test. Fourier transform infrared spectroscopy (FTIR) and DSC analysis of all materials after testing at different times were performed, while sample photographs were taken for visual study. DSC analysis of samples at different disintegration times was carried out under nitrogen from -25 to 200 °C at a heating rate of 10 °C min-1. FTIR spectra of the degraded samples were recorded by a Jasco FT-IR 615 spectrometer, in attenuated total reflection (ATR) mode, in the 400–4000 cm−1 range.

2.6. Applicability of films for food packaging applications 2.6.1. Release study The release of thymol from nanocomposite films was performed into ethanol 10 % (v/v) as food simulant according to the European Standard EN 13130-2005 (UNE-EN_13130-1, 2005) and the Commission Regulation

(EU)

n°

10/2011

(Commission_Regulation/(EU)/No-

10/2011). Double-sided, total immersion migration tests were performed with films (12 cm2) and 20 mL of food simulant (area-to-volume ratio around 6 dm2 L-1) in triplicate at 40 °C for 10 days in an oven (J.P. Selecta, Barcelona, Spain). A kinetic study of the release of thymol from the film to the food simulant during a suitable period of time (15 days) was performed. Samples were taken after 2, 6, 12, 24, 48 hours and 5, 10 and 15 days, in triplicate. A blank test was also carried out. The obtained extracts were recovered after

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the removal of samples and stored at -4 °C before the chromatographic analysis. HPLC-UV was used to determine the amount of thymol released from films at different incubation times with an Agilent 1260 Infinity-HPLC Diode Array Detector (DAD) (Agilent, Santa Clara, CA) and Agilent eclipse plus C18 (100 mm x 4.6 mm x 3.5 μm) column. The mobile phase was composed of acetonitrile/water (40:60) at 1 mL min-1 flow rate. 20 μL of the extracts were injected and detected at λ = 274 nm. Analyses were performed in triplicate. Calibration standards were run at different concentrations between 12.5 and 780 mg kg-1 from a stock solution (1000 mg kg-1) using appropriately diluted standards of thymol in ethanol 10 % (v/v). This method was validated by the calculation of the main analytical parameters affecting the determination of thymol in the studied food simulant. LOD and LOQ values were determined by using regression parameters from the calibration curve (3 Sy/x/a and 10 Sy/x/a, respectively; where Sy/x is the standard deviation of the residues and a the slope of the calibration curve) being 0.29 mgThymol kg-1 and 0.96 mgThymol kg-1, respectively. A good linearity was obtained which was determined by the calculation of the determination coefficient, R2 (0.9994). 2.6.2. Antioxidant activity of released thymol The AO activity of thymol released from the food simulant was evaluated by using the spectrophotometric method based on the formation of the stable radical DPPH (Scherer and Godoy, 2009; Byun, Kim and Whiteside, 2010). 500 μL of extracts were mixed with 2 mL of a methanolic solution of DPPH (0.06 mM) in a capped cuvette. The mixture was shaken vigorously at room temperature and the absorbance of the solution was registered at 517 nm with a Biomate-3 UV-Vis

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spectrophotometer (Thermospectronic, Mobile, AL, USA). DPPH radical absorbs at 517 nm but, upon reduction, its absorption at this particular wavelength decreases. The decay in absorbance was measured at 1 min intervals until it reached the steady state

to

complete the reaction (200

min). All analyses were performed in triplicate. The scavenging ability of the stable radical DPPH was calculated as percentage of inhibition (I %) with the Equation 2.3:

(%) =

−

∙ 100

(2.3)

where AControl is the absorbance of the blank sample at t = 0 min and ASample is the absorbance of the tested sample at t = 200 min. 2.6.3. Antibacterial activity Escherichia coli RB (E. coli RB) and Staphylococcus aureus 8325-4 (S. aureus 8325-4) were used in this study. E. coli RB was an isolate strain provided by the “Zooprofilattico Institute of Pavia” (Italy), whereas S. aureus 8325-4 was a gift from Mr. Timothy J. Foster (Department of Microbiology, Dublin, Ireland). E. coli RB and S. aureus 8325-4 were grown overnight under aerobic conditions at 37 °C using a shaker incubator (New Brunswick Scientific Co., Edison, NJ, USA) in Luria Bertani Broth (LB) and Brian Heart Infusion (BHI) (Difco Laboratories Inc., Detroit, MI, USA), respectively. The final density of these cultures was established at 1 x 1010 cells mL-1, determined by comparison of the OD600 of samples with a standard curve relating OD600 to cell number. The evaluation of the antibacterial activity of neat PLA and PLA-based active nanocomposites was performed in 100 µL of an overnight diluted cell suspension (1 x 104) of E. coli RB and S. aureus 8325-4. Bacterial strains

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were added to each sample, seeded at the bottom of a 96-well tissue culture plate and incubated at three different temperatures: 4 °C, 24 °C and 37 °C for 3 h and 24 h, respectively. Furthermore, 96-well flat-bottom sterile polystyrene culture plates used as controls were incubated under the same conditions. At the end of each incubation period, bacterial suspensions were serially diluted and plated on the LB (E. coli RB) or BHI (S. aureus 8325-4) agar plates. They were then incubated for 24/48 h at 37 °C. Cell survival was expressed as percentage of CFU of bacterial growth on PLA active nanocomposite films compared to those obtained for the neat PLA film.

2.7. Statistical analysis Statistical analysis of results was performed with SPSS commercial software (Version 15.0, Chicago, IL). A one-way analysis of variance (ANOVA) was carried out. Differences between means were assessed on the basis of confidence intervals using the Tukey test at a p < 0.05 significance level.

3. Results and discussion 3.1. Determination of thymol in films One of the most important issues in the development of active materials when volatile additives are involved is to ensure that a significant amount of these chemicals remain in the polymer matrix after processing. In this case, the amount of thymol determined by HPLC-UV which is presented in formulations after processing is reported in Table 2.3.

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Table 2.3. Quantification of thymol (HPLC-UV) and thermal parameters (TGA, DSC) obtained for all nanocomposite films and neat PLA. Sample

Extracted thymol (wt%)

Weight loss (wt%) (1st step)

Tini (°C)

Tmax (°C)

Tg (°C)

PLA

n.d.

n.d.

335

369

57

PLA/D43B2.5

n.d.

n.d.

334

363

57

PLA/D43B5

n.d.

n.d.

340

369

57

0.01a

6.6

331

366

43

PLA/T/D43B2.5

5.99 ± 0.03b

6.3

336

366

41

PLA/T/D43B5

5.78 ± 0.02c

7.1

339

369

44

PLA/T

5.57 ±

n.d. Not detected Tg: determined by DSC from the first heating scan at 10 °C min-1. Weight loss (wt%, 1st degradation step), Tini and Tmax (2nd degradation step): determined by TGA at 10 °C min-1 in N2 atmosphere. Results are represented as mean ± standard deviation, n=3. Different superscripts within the same column indicate statistically significant different values (p < 0.05)

Results showed that in all cases approximately 70 % of the initial thymol remained in the polymer structure after processing. Losses of thymol during processing are caused by evaporation or degradation due to high temperatures. Nevertheless, these losses (around 30 %) are in the same range than those reported for other common antioxidants in PLA-based formulations, such as BHT (Ortiz-Vazquez, Shin, Soto-Valdez and Auras, 2011). These losses can be due to several factors, such as poor mixing in the extruder, evaporation, thermal degradation and the own AO action of these additives to protect the polymer during processing. But, it is remarkable that the volatility of active additives is desirable in food packaging applications to promote their migration from the polymer surface to food (Wessling, Nielsen and Giacin, 2001). Therefore, thymol can be considered a good active additive in food packaging materials since a large amount remains after processing and may be released from the polymer matrix to improve foodstuff shelf-life. It should be also

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highlighted that the amount of thymol after processing was slightly higher in nanocomposite films containing D43B indicating that the nanoclay can retard thymol evaporation during processing (p < 0.05).

3.2. Films characterization 3.2.1. Thermal analysis Figure 2.2 shows the weight loss and derivative thermogravimetric analysis (DTG) curves obtained for neat PLA and all nanocomposite films. The main degradation peak for PLA was observed in all samples around 365370 °C. A first degradation step around 120 ºC was observed in those materials containing thymol and it was attributed to the evaporation and/or loss of thymol from the polymer matrix, as already discussed in Chapter 1 and in agreement with Tawakkal et al. (Tawakkal, Cran and Bigger, 2014). These authors used TGA to study the retention of thymol in PLA-based films after processing and suggested that the evaporation and/or volatilization of thymol from the polymer matrix started at low temperatures, remaining active for a broad temperature range. This result is another indication of the presence of thymol after processing. The amount of thymol (weight loss, wt%, 1st degradation step) was calculated from the TGA curves and results are shown in Table 2.3. The obtained results were quite similar to those values obtained from the determination of thymol by HPLC-UV already discussed in Section 3.1. The Tini, or onset temperature determined at 5 % of weight loss, and Tmax (temperature for the maximum degradation rate) of PLA are also shown in Table 2.3. No noticeable differences were observed in all materials regardless of their formulation. Therefore, it could be concluded that the

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addition of thymol and D43B did not affect the thermal degradation profile of the nanocomposite films.

(a)

PLA

100 Weight Loss (wt%)

PLA/D43B2.5 PLA/D43B5 PLA/T PLA/T/D43B2.5

50

PLA/T/D43B5

0 0

100

200

(b) 0

100

200

300 400 500 Temperature (ºC) Temperature (ºC) 300 400 500

600

600

700

700

DTG (mg s-1)

0,00 PLA PLA/D43B2.5 PLA/D43B5 PLA/T PLA/T/D43B2.5 PLA/T/D43B5

-0,02

-0,04

-0,06

Figure 2.2. Weight loss (wt%) (a) and DTG (b) curves obtained for PLA-based films.

Some authors have considered several molecular mechanisms to explain the PLA thermal degradation. The primary cause of this process is a nonradical, 'backbiting' which is refered to the formation of cyclic compounds through intramolecular reactions between the carboxylic end group of the PLA chain and the ester bond of the chain. This reaction can produce lactide, oligomers of lactic acid, acetaldehyde, carbon monoxide and water, depending upon the size of the cyclic transition state (Barrere et al, 2014){

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Results and Discussion. Chapter 2

Other authors have proposed radical reactions for the PLA degradation mechanism, which start with either alkyl-oxygen or acyl-oxygen homolysis leading to the formation of several types of oxygen- and carbon-centred macro-radicals and carbon monoxide (Fukushima, Abbate, Tabuani, Gennari and Camino, 2009). DSC was used to determine the Tg values in all PLA-based films (Table 2.3). It is known that this parameter is dependent upon the polymer structural arrangement and corresponds to the torsion oscillation of the carbon backbone giving a clear indication of the toughness and ductility of the polymer (Hughes, Thomas, Byun and Whiteside, 2012). Tg results of these materials showed that the addition of D43B to PLA did not produce important variations in the polymer structure, as reported by other authors for other PLA-based nanocomposites (Lewitus, McCarthy, Ophir and Kenig, 2006; Scatto et al, 2013). However, the effect of thymol on the PLA macromolecular structure (and consequently on Tg) was more important. In fact, the presence of thymol induced a decrease in more than 10 °C in Tg values, regardless of the presence of D43B (Figure 2.3),. A similar behaviour was reported by other authors in PLA formulations with thymol (Tawakkal, Cran and Bigger, 2014) and other active additives (Byun, Kim & Whiteside, 2010; Hwang et al., 2012). This decrease in Tg values could be explained by some plasticizing effect caused by the addition of thymol resulting in an increase in the molecular mobility of the macromolecular chains of the polymer matrix and the ductility of the final blend of PLA with thymol, with some reduction in the polymer toughness as will be discussed for the tensile properties of all materials. No other significant peaks were observed in the DSC curves (Figure 2.3), and it could be concluded that the addition of thymol and D43B to the polymer matrix did not change the inherent amorphous structure of PLA.

~ 210 ~

Results and Discussion. Chapter 2

Heat flow (W g-1)

(a)

Heat flow (W g-1)

(b)

Figure 2.3. DSC thermograms for PLA-based based films for the first heating (a) and the second heating scan (b).

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Results and Discussion. Chapter 2

3.2.2. Structural analysis XRD is a very useful technique to determine d-spacing and gives an accurate estimation of the layer separation in silicate nanocomposites. The XRD pattern of neat PLA is characterized by a broad band approximately at 2θ = 15°, confirming its amorphous structure (Fukushima, Abbate, Tabuani, Gennari and Camino, 2009). No significant differences were found in the XRD patterns of all materials at this angle range, suggesting that the polymer structure and crystallinity were not influenced by the presence of D43B and/or thymol and corroborating the DSC results. The most significant differences in XRD patterns of these materials were detected in the low angle range (2-10°) (Figure 2.4). D43B is characterized by a single diffraction peak at 2θ = 4.6° corresponding to the (001) plane of the silicate layers, accounting for a 19.2 Å interlayer distance. A shift of the clay diffraction peak to lower angles would mean a higher distance between layers and consequently suggests the good interaction of D43B with the polymer matrix (Scatto et al, 2013). In fact, the results obtained for XRD patterns of PLA nanocomposites with D43B showed a diffraction peak around 2.6º, corresponding to an interlayer distance of 35.6 Å. In addition, a significant decrease in this peak intensity was observed with the addition of the nanofiller, accounting for the formation of a more disordered structure. This result also suggests the formation of an intercalated nanocomposite structure, as indicated by other authors (Picard, Espuche and Fulchiron, 2011). The broad diffraction peak observed at 2θ around 5.2° (d-spacing equal to 17.0 Å) in the nanocomposite XRD patterns could be attributed to the fraction characterized by different alkylammonium chain arrangements in the interlayer space corresponding to the (002) basal plane (Persico et al, 2009). These observations were coincident to those by Araujo et al who

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reported the XRD analysis of PLA macromolecules concluding that they could diffuse and insert between the clay mineral layers (Araújo, Botelho, Oliveira and Machado, 2014). 12000 D43B PLA PLA/T PLA/D43B2.5 PLA/D43B5 PLA/T/D43B2.5 PLA/T/D43B5

10000

Intensity (counts)

8000 6000 4000 2000 0 2

2,5

3

3,5

4

4,5

5

5,5

6

6,5

7

7,5

8

8,5

9

9,5 10

2θ (°)

Figure 2.4. WAXS patterns of D43B, neat PLA and nanocomposite films.

The lowest peak intensity for the nanocomposites studied by XRD was obtained for PLA/T/D43B2.5. This result could be attributed to the presence of thymol favouring the nanoclay exfoliation making more effective the interaction between the silicate layers and the polymer macromolecules, as already reported in other plasticized nanocomposites studied in our research group (Martino, Ruseckaite, Jiménez and Averous, 2010) It could be concluded that thymol could promote the swelling of the nanoclay stacks, as also reported by other authors (Persico et al, 2009). However, the intensity of peaks for formulations with 5 wt% of D43B were higher. This behaviour could be due to the unfavourable effect in the polymer-clay interactions by swelling at high loadings. In conclusion, XRD results suggest the effective intercalation of PLA macromolecules into the D43B galleries achieved by mixing PLA with 2.5 wt% of D43B and 8 wt% of thymol.

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3.2.3. Morphological analysis The dispersion of the nanoclay in the PLA matrix was also evaluated by TEM at different magnifications (Figure Figure 2. 2.5). A high degree of clay delamination was observed in all cases. In fact, m micrographs obtained for PLA/T/D43B2.5 nanocomposite showed the high degree of exfoliation of the clay layers into the PLA matrix.. Single dispersed clay layers (dark regions in Figure 2.5) were randomly distributed through the PLA matrix (clear areas) and some regions with complete exfoliation of nanoclay layers were recognised.. These results obtained by TEM analyses also suggested the good dispersion of D43B and thymol through the PLA matrix, already asserted by the XRD patterns, since no important aggregates were observed.

Figure 2.5. TEM images of PLA/T/D43B2.5 active nanocomposite film.

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Results and Discussion. Chapter 2

3.2.4. Mechanical properties It is known that Tg for amorphous PLA is in the range of 50-60 °C. Below that temperature, PLA shows high tensile strength and is quite brittle, resulting in difficulties for the manufacture of flexible films. However, the addition of plasticizers leads to the enhancement in the ductile properties of the polymer matrix by increasing the plastic elongation and reducing brittleness (Byun, Kim and Whiteside, 2010). Conversely, the addition of nanoclays to polymer matrices results in improvement in toughness, particularly in exfoliated nanocomposites. In this study, the results obtained for the tensile properties of all materials are shown in Table 2.4. It was observed that elastic modulus (E, MPa) and elongation at break (εB, %) of PLA suffered some modification by the action of thymol and D43B. In fact, the addition of thymol in binary PLA films resulted in a significant decrease (around 15 %) in the E value (p < 0.05). This change in the polymer toughness could be explained, once again, by some plasticizing effect caused by thymol to PLA matrices, already discussed previously by the observed decrease in Tg values. Similar results were reported in PLA and LDPE formulations with active compounds, such as resveratrol, carvacrol or -tocopherol (Persico et al, 2009; Hwang et al, 2012; Tawakkal, Cran and Bigger, 2014). As expected, the addition of D43B to PLA in binary systems without thymol increased significantly the E values and decreased significantly εB (p < 0.05). This effect was related to the reinforcement provided by silicate layers to the PLA structure and the high aspect ratio, surface area and good dispersion of the nanoclay layers throughout the polymer matrix with strong interactions (Quilaqueo Gutiérrez, Echeverría, Ihl, Bifani and Mauri, 2012; Scatto et al, 2013).

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Table 2.4. Tensile properties (ASTM D882-09), oxygen transmission rate and CIELab colour parameters obtained for PLA-based formulations. Sample

E (MPa)a

εB (%)a

OTR·e b

L*

a*

b*

ΔE*c

PLA

2575 ± 76a

3.5 ± 0.1a

22.1 ± 1.5a

30.3

-0.11

-0.20

-

PLA/D43B2.5

2739 ± 151a

2.1 ± 0.4d

20.1 ± 2.0 a

30.7

0.02

-0.01

0.5

PLA/D43B5

2715 ± 95a

1.5 ± 0.2c

17.1 ± 2.3 a

32.0

-0.24

-0.81

1.9

PLA/T

2167 ± 196b

4.3 ± 0.1b

23.0 ± 0.2 a

33.3

-0.49

-1.10

3.2

135b

0.1d

0.1 a

32.0

-0.22

-1.14

2.0

22.7 ± 1.3 a

34.4

-0.58

-1.48

4.4

PLA/T/D43B2.5 PLA/T/D43B5

2246 ±

2140 ± 116b

2.4 ±

2.4 ± 0.2d

20.1 ±

an

= 5, mean ± standard deviation. (cm3 mm m-2 day); n =3, mean ± standard deviation (e: thickness, mm) c PLA film was used as control Different superscripts within the same column indicate statistically significant different values (p < 0.05) b OTR·e

The combination of thymol and D43B with PLA in ternary nanocomposites results in the effective combination of all their components, modifying the tensile properties of PLA-based films. These ternary combinations showed E and εB values significantly different to those of neat PLA and binary nanocomposites (p < 0.05). It is possible to assert that thymol has an important influence on the mechanical performance of these nanocomposites. In fact, the addition of 8 wt% of thymol results in a clear decrease of E and a significant improvement in εB (p < 0.05). 3.2.5. Oxygen transmission rate Permeability to oxygen of polymer films is an important parameter in the selection of materials for food packaging applications. Therefore, the improvement in barrier properties in PLA films is an important issue and should be studied. Table 2.4 summarizes the OTR·e results obtained for all the studied formulations. It is recognized that the incorporation of

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Results and Discussion. Chapter 2

nanofillers to polymer matrices can lead to significant enhancement in their barrier properties. However, the barrier to oxygen of PLA-based films with 2.5 and 5 wt% of D43B did not show significant differences between neat PLA and all the active nanocomposite films (p > 0.05) (Table 2.4). These results can be attributed to the relatively low amount of D43B added to PLA being insufficient to achieve an effective intercalation into the PLA matrix that could produce a tortuous pathway for oxygen molecules to permeate through the film (Martino, Ruseckaite, Jiménez and Averous, 2010; Quilaqueo Gutiérrez, Echeverría, Ihl, Bifani and Mauri, 2012; Reddy, Vivekanandhan, Misra, Bhatia and Mohanty, 2013). Regarding thymol, its addition did not modify the properties of PLA-based films as other authors reported by attributing the decrease in the oxygen permeability of PLA nanocomposites to the increase in the mobility produced by the addition of plasticizers (Jamshidian et al, 2012). A similar trend was obtained for PLA-based ternary nanocomposites, where results showed that the oxygen barrier of neat PLA was not modified with no significant improvement or decrease with the addition of thymol and the nanofiller (p > 0.05) as in conventional polymers used in food packaging like PS, PET or HDPE (Auras, Harte, Selke and Hernández, 2003). 3.2.6. Optical properties Colour and transparency are important factors for materials intended to be used in food packaging since they have great influence in their consumer acceptance and commercial success. Figure 2.6 shows the visual aspect of all studied PLA-based films, which showed high transparency and no visual discontinuities, suggesting that no agglomerations were present in the nanocomposite structure. Moreover, the uniform

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Results and Discussion. Chapter 2

distribution of the colour observed with the naked eye throughout the films (Figure 2.6)) also suggests that additives were uniformly distributed within the polymer matrix. However, some differences in CIELab coordinat coordinates (L*, a*, b*) and E* between neat PLA and nanocomposite films were observed ((Table 2.4). These differences could be attributed to the intrinsic colour of the added additives (white for thymol and yellowish for D43B). Neat PLA showed the lowest L* value, indicating that brightness ss increased with the addition of thymol and D43B. A yellowish-reddish reddish tone was obtained for PLA/T formulation, while PLA/T/D43B5 ternary nanocomposite showed the higher value for ∆E*, as expected, due to the high concentrations of the additives (5 wt% D43B B and 8 wt% thymol). Similar colour differences were reported when using other active additives, such as -tocopherol and resveratrol, into PLA matrices, where the presence of these compounds contributed to increase the films colour (Byun, Kim and Whiteside, 2010) 2010).

Figure 2.6. Visual observation of neat PLA and nanocomposite films.

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Results and Discussion. Chapter 2

3.3. Disintegrability under composting conditions The disintegrability of PLA under composting conditions has been studied by some authors who mentioned that PLA suffers hydrolysis reactions induced by the diffusion of water into the polymer structure, producing a reduction in the molecular weight by random non-enzymatic chain scissions of the ester groups and resulting in the formation of oligomers and lactic acid (Luzi et al, 2015). Furthermore, these oligomers, when buried under composting conditions can be decomposed by microorganisms, including fungi and bacteria resulting in simple molecules, mainly water, carbon dioxide and biomass monomers. In fact, once started the water diffusion through the PLA matrix, the molecular weight decreases up to 10.000-20.000 Da and microorganisms start metabolizing these macromolecules into organic matter, and simple molecules. In this work, disintegrability under composting conditions of PLA and PLA-based active nanocomposites were studied. Figure 2.7 shows the visual observation of films submitted to the disintegrability test and Table 2.5 summarizes the values of weight loss obtained at different times. It was observed that, after 2 days, the disintegration rate of PLA-based materials increased significantly (p < 0.05) for binary and ternary systems. In fact, after 4 days samples changed their appearance (Figure 2.7) with a general whitening effect, loss of transparency and evident deformation and size reduction. These results were indicative of the beginning of the hydrolytic degradation process caused by simultaneous changes in the refractive index due to water absorption, with formation of low molecular weight by-products and increase in the PLA crystallinity (Fukushima, Tabuani, Arena, Gennari and Camino, 2013).

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Results and Discussion. Chapter 2

However, the faster appearance of visual signs of degradation observed in nanocomposites when compared to neat PLA could be due to the presence of hydroxyl groups from thymol and the organic modifier of D43B (Fukushima, Abbate, Tabuani, Gennari and Camino, 2009; Fukushima, Tabuani, Arena, Gennari and Camino, 2013; Fortunati, Luzi, et al, 2014). Hydroxyl groups can contribute to the heterogeneous hydrolysis of PLA by absorbing water from the medium resulting in a noticeable formation of labile bonds in the PLA structure with the consequent significantly higher disintegrability rate (p < 0.05) (Sinha Ray, Yamada, Okamoto and Ueda, 2003; Proikakis, Mamouzelos, Tarantili and Andreopoulos, 2006).

Figure 2.7. Visual observations of PLA-based films at different times under composting conditions at 58 °C.

Binary and ternary systems suffered physical breakage (Figure 2.7) and the weight loss considerably increased (Table 2.5) after 7 days, showing

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Results and Discussion. Chapter 2

significant differences in the disintegrability values regarding neat PLA (p < 0.05). Results at longer testing times showed that the physical degradation progressed with burial time, resulting in the complete disintegration of the initial sample after 35 days where the degree of disintegration exceeded 90 % covering the ISO 20200 requirements. FTIR analysis of neat PLA and PLA-based nanocomposite films obtained during the disintegrability tests provided the PLA characteristic bands: 1750 cm−1 (C=O), 1440 cm−1 (CH(CH3)), and 1267 cm−1 (C-O-C) as well as three peaks at 1123, 1082 and 1055 cm−1 related to the C-C-O groups. Figure 2.8 shows the FTIR spectra of neat PLA, PLA/T and PLA/T/D43B5 after 0, 7 and 21 days of the study. A general reduction in the intensity of the three peaks related to the C-C-O groups was detected after 7 days, resulting in their disappearance at 21 days in composting conditions for all the studied formulations. Similar results were reported by Fortunati et al, who proposed that the decrease in intensity of the peaks corresponding to C-C-O groups was related to the scission of the PLA interchain bonds caused by the hydrolysis during the disintegration tests (Fortunati, Luzi, et al, 2014). Some decrease in the intensity for the band corresponding to the C-O-C stretching vibration at 1267 cm−1 was also observed in all samples. Both modifications can be due to the depletion of the lactic acid and oligomer molecules caused by microorganisms, leaving highly reactive carboxylate ions end groups (Khabbaz, Karlsson and Albertsson, 2000). FTIR results are in agreement with the disintegration weight loss above discussed, where a progressive disintegration occurred with the increase in testing time.

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Results and Discussion. Chapter 2

Table 2.5. Disintegrability values (%) of PLA and nanocomposite films at different times under composting conditions at 58 °C. Sample

2 Days

4 Days

7 Days

10 Days

14 Days

21 Days

28 Days

35 Days

PLA

0.30 ± 0.10a

0.38 ± 0.08a

42.2 ± 3.8a

56.3 ± 4.6a

72.4 ± 2.8a

73.2 ± 6.7a

77.3 ± 1.4a

95.7 ± 0.7a

PLA/T

5.08 ± 0.44c

3.5 ± 0.2b,c

51.3 ± 0.2b

72.0 ± 4.3b

65.6 ± 3.3a

79.5 ± 2.4a

81.9 ± 1.6a

98.0 ± 0.5a

PLA/T/D43B2.5

8.4 ± 0.9d

5.2 ± 1.2c,d

57.9 ± 2.3b

76.0 ± 1.3b

68.9 ± 3.9a

82.4 ± 3.4a

82.2 ± 0.8a

95.5 ± 0.9a

PLA/T/D43B5

7.2 ± 0.3d

6.0 ± 0.2d

49.8 ± 0.9b

77.3 ± 4.9b

65.5 ± 4.5a

81.8 ± 2.8a

79.7 ± 6.8a

97.8 ± 0.5a

PLA/D43B2.5

2.4 ± 0.7b

2.3 ± 0.2a,b

53.0 ± 2.2b

67.8 ± 1.5b

64.9 ± 6.4a

82.2 ± 4.3a

76.2 ± 1.6a

97.2 ± 1.2a

PLA/D43B5

1.19 ± 0.03a,b

1.98 ± 0.12a,b

54.5 ± 1.8b

69.9 ± 4.3b

64.0 ± 3.8a

78.2 ± 1.6a

77.5 ± 4.9a

96.5 ± 1.7a

(mean ± standard deviation, n = 3) Different superscripts within the same column indicate statistically significant different values (p < 0.05)

~ 222 ~

(C=O)

(CH(CH3)

Transmitance (a.u.)

Results and Discussion. Chapter 2

(C-O-C) (C-C-O)

Day 21 1800

Day 0

1600 1400 Wavenumber (cm-1)

1200 PLA

1000

800

1200 PLA/T

1000

800

1200

1000

800

Transmitance (a.u.)

2000

Day 7

Day 21 1800

Day 0

1600 1400 Wavenumber (cm-1)

Transmitance (a.u.)

2000

Day 7

Day 21 2000

1800

Day 7 1600

Day 0 1400

Wavenumber

(cm-1)

PLA/T/D43B5

Figure 2.8. FTIR spectra of PLA, PLA/T and PLA/T/D43B5 before (0 days) and after different incubation times (7 and 21 days) in composting conditions.

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Results and Discussion. Chapter 2

Figure 2.9 shows the DSC thermograms obtained from the first heating scan for all PLA-based films at different composting times. The endothermic peak observed immediately after Tg at day 0 corresponds to the enthalpic relaxation process for all the tested materials. This effect was related to the ageing process of PLA and it was previously observed by other authors (Hughes, Thomas, Byun and Whiteside, 2012; Burgos, Martino and Jiménez, 2013). However, the initially amorphous PLA-based materials developed multiple endothermic peaks just after 7 days of testing. In fact, the enthalpic relaxation peak gradually disappeared due to the hydrolytic reactions at the beginning of the disintegration process. Yang et al related this behaviour with the moisture absorption under composting conditions, since water could serve as a plasticizer agent in PLA matrices (Yang, Fortunati, Dominici, Kenny and Puglia, 2015). The gradual disintegration suffered when increasing the testing time allows the observation of new melting peaks related to the formation of crystalline structures with different perfection degrees in the PLA matrix (Figure 2.9). These results are correlated with visual changes, since hydrolysis promotes crystallization in the polymer matrix, resulting in important changes in the disintegrability behaviour. Similar results were reported by other authors, who suggested that the appearance of multiple melting peaks could be related to the formation of different crystal structures due to the polymer chains scission produced during degradation (Fukushima, Abbate, Tabuani, Gennari and Camino, 2009; Fortunati, Armentano, Iannoni, et al, 2012; Gorrasi and Pantani, 2013; Yang, Fortunati, Dominici, Kenny and Puglia, 2015). Figure 2.10 shows the DSC thermograms recorded during the second heating scan for samples submitted to the disintegration test. It was observed that after 2 days, all PLA-based films showed an important decrease in Tg.

~ 224 ~

Results and Discussion. Chapter 2

Previous reported work in our research group showed that this decrease was due to the increase in the mobility of the polymer chains as a consequence of the hydrolytic process (Burgos, Martino and Jiménez, 2013) and the formation of lactic acid oligomers and low molecular weight by-products with a plasticizing effect in the polymer structure and the consequent changes in their visual appearance.

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Results and Discussion. Chapter 2

Figure 2.9. DSC curves (1st heating scan) of PLA-based films after different composting times.

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Results and Discussion. Chapter 2

Figure 2.10. DSC curves (2nd heating scan) of PLA-based films after different composting times.

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Results and Discussion. Chapter 2

3.4. Release study Active nanocomposites used in food packaging should release the desired chemicals at suitable rates to result in a noticeable enhancement of foods shelf-life and quality during storage. The incorporation of active additives, such as thymol, to polymer matrices should permit their gradual release to food, minimizing surface contamination, but obviously satisfying the requirements stated in the current food packaging legislation. The use of nanofillers in active packaging systems has revealed some ability in retarding the release of active additives from polymer matrices, improving the action of the additives and extending foodstuff shelf-life (Campos-Requena, Rivas, Pérez, Garrido-Miranda and Pereira, 2015). In this work, migration tests were carried out to evaluate the effect of the nanoclay in controlling the release kinetics of thymol from the PLA matrix to ethanol 10 % (v/v) (Figure 2.11). It was observed that the addition of D43B resulted in the delay in the thymol release due to the larger tortuosity effect imposed by the dispersed nanoclay, as reported by Sanchez-Garcia et al (Sanchez-Garcia, Ocio, Gimenez and Lagaron, 2008). The formulation with the highest amount of D43B (PLA/T/D43B5) showed lower migration rates, retaining higher amounts of thymol in the polymer structure. The final amounts of thymol migrated in ethanol 10 % (v/v) at 40 ºC after 10 days were 285.0 ± 3.3, 275.5 ± 13.8 and 235.3 ± 19.4 mgthymol kg-1Food simulant

for PLA/T, PLA/T/D43B2.5 and PLA/T/D43B5, respectively.

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Results and Discussion. Chapter 2

mgThymol kg-1Food Simulant

400 350 300 250 200 PLA/T PLA/T/D43B2.5 PLA/T/D43B5

150 100 50 0 0

50

100

150

200 Time (h)

250

300

350

400

Figure 2.11. Thymol release profiles of PLA/T, PLA/T/D43B2.5 and PLA/T/D43B5 active nanocomposite films.

The release mechanism of thymol in PLA nanocomposites was evaluated by using kinetic studies with results obtained for the release of thymol at different times for 15 days. Figure 2.12 shows the normalized plots for the mass of thymol released to the food simulant, MF,t, by the mass of thymol released at time t→∞, MF,∞, vs time t.

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Results and Discussion. Chapter 2

1,2

MFt/MF∞

1 0,8 0,6 0,4 0,2 PLA/T

0 0

60

120

180 Time (hours)

240

300

360

1

MFt/MF∞

0,8 0,6 0,4 0,2 PLA/T/D43B2.5

0 0

60

120

180 Time (hours)

240

300

360

1

MFt/MF∞

0,8 0,6 0,4 0,2 PLA/T/D43B5

0 0

60

120

180 Time (hours)

240

300

360

Figure 2.12. Normalized migration of thymol from different polymer matrices: PLA/T, PLA/T/D43B2.5, and PLA/T/D43B5.

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Results and Discussion. Chapter 2

The quantitative assessment of MF,∞ allows the further quantitative analysis of the diffusion process. For such purpose, Equation (2.4) was fitted to experimental data, where MP,0 is the initial amount of thymol inside the polymeric matrix, previously calculated in TGA tests, MF,∞, and k’ is a constant (see Table 2.6).

, ,0

⎛ =⎜ ⎝

,∞

⎞ ,0 ⎟

∙ (1 −

− ′

)

⎠

(2.4)

Apparent partition coefficients (αap) can be calculated through Equation (2.5) from the values obtained for MF,∞/MP,0 by fitting Equation. (2.4)

,∞ ,0

= 1 (1 + )

(2.5)

where α is defined as

=

,

(2.6)

where VP and VF are, respectively, the volumes of polymer sample (P) and food simulant (F), and KP,F is the partition coefficient of thymol related to the relative solubility of thymol at the equilibrium between PLA and the food simulant (Silva, Cruz Freire, Sendón, Franz and Paseiro Losada, 2009). The corresponding data for α and KP,F (see Table 2.6) have been computed taking into account that VF was 20 cm3 of ethanol 10 %(v/v) and the area of PLA-based films used in these tests was 12 cm2. From this analysis two different conclusions can be obtained: i.

By increasing the amount of D34B in the PLA matrix the cumulative amount of thymol released to ethanol, 10 % (v/v) decreased from 38 % (without D34B) to 35 and 31 % for 2.5 and 5 wt % of D34B, respectively.

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Results and Discussion. Chapter 2

The analysis of the partition coefficients (α and KP,F) showed that the

ii.

assessment of diffusion of thymol is clearly influenced by them in the different tested times. Thus, assuming that the thymol migration is governed by the Fick’s 2nd law Equation (2.7)

= (2.7)

where D is the diffusion coefficient, c is the concentration of the released species and x is the space coordinate. For a plane sheet of thickness l, and the initial condition l/2 < x < l/2, considering constant the concentration of thymol released and with a boundary condition of a partition coefficient between both phases, the analytical solution of Equation (2.7) for one-dimensional diffusion of thymol in a limited volume solution is expressed as Equation (2.8)(Crank, 1975):

M F ,t M F ,

2 1      Dqn2t  exp  2  2 2 n 1 1     qn  l  

 1 

(2.8)

where qn are the non-zero positive roots of tanqn = -α qn and l is the polymeric matrix half-thickness. Taking into account these conditions, the diffusion coefficients (D, cm2 s1)

were obtained from a least-square fit of Equation (2.8) to experimental

data (solid lines in Figure 2.12). The D values (Table 2.6), calculated for each sample, were determined by minimizing the root mean square errors (RMSE) of the measured and

~ 232 ~

Results and Discussion. Chapter 2

estimated values between the calculated (yi) and observed (ŷi) values of MF,t/MF,∞, (Equation (2.9) and providing a reliable indication of their fit. 1

(

=

− ŷ )2

2

=1

(2.9)

It can be seen from the analysis of Figure 2.12, the RMSE values calculated by Equation (2.9) and taking into account the experimental error of the MF,t/MF,∞ ratio. A reasonable fit was obtained for the following systems: PLA (RMSE: 0.0773) and PLA/T/D34B2.5 (RMSE: 0.0698), but it was poor for the active nanocomposite film with the highest content of D43B (RMSE: 0.114), in particular for long-range times. Table 2.6. Characteristic parameters for the release of thymol from PLA-based films to ethanol 10 % (v/v). PLA/T

PLA/T/D43B2.5

PLA/T/D43B5

MP,0 (mg)

16.6  0.2

17.82  0.09

17.21  0.05

MF,∞ (mg)

6.25  0.22

6.31  0.27

5.29  0.29

l / cm

0.0167

0.0215

0.0180

α

1.65

1.82

2.25

KP,F

60.3

42.4

41.1

D (cm2 s-1)

3.36×10-11

4.86×10-11

2.25×10-11

RMSE

0.0773

0.0698

0.114

Equation (2.8) and (2.9)

Equation (2.10) and (2.9) D’ (cm2 s-1)

5.95×10-12

7.45×10-12

5.82×10-12

RMSE

0.00362

0.00306

0.00370

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Results and Discussion. Chapter 2

In fact, a deeper analysis of the fitting between experimental and calculated values showed that positive deviations of the fitting line for short times (i.e., MF,t/MF,∞,0.60 were observed in all cases. These results allow estimating the kinetics of thymol migration for short times. For such purpose, a simplified migration model derived from Equation (2.8) and useful for linear regression analysis was used (Equation (2.10)) (Chung, Papadakis and Yam, 2002).

 1 1 M F,t        MP ,0  

0.5



D'0.5

 l

t

0. 5



1  0.5

(2.10)

Diffusion coefficients for short times, (D’, cm2 s-1), were computed by using the linear fitting of Equation (10) to the experimental data (Figure 2.13). Results obtained showed a very good fit between computed and experimental

 1 1 M F, t         M F,0 

0.5

values as a function of

t 0 .5 , with

determination coefficients (R2) higher than 0.999, suggesting that the experimental release data are well described by the proposed diffusion model for short-range times. Even though the whole data range was not characterised by a Fickian diffusion process (probably due to the fitting controlled by the last points in the plot), the fitting to the first data (called short-range time) is poor. Indeed, with Equation (2.10), results lead to best fitting values. Therefore, the discrepancy in D values obtained (D and D') from Equation (2.8) and Equation (2.10) is enough to conclude that a non-Fickian migration model is observed in this system.

~ 234 ~

[(1/)-(MF,t/(MP,0))]

0.5

Results and Discussion. Chapter 2

0,56

0,54

0,52

0,50

0,48 0

100

200 0.5

300

400

-0.5

t (s )  1 1 M F ,t        M F ,0  Figure 2.13. Plots of 

0.5

versus t

0 .5

for the migration of thymol from:

PLA/T ( ), PLA/T/D43B2.5 ( ), and PLA/T/D43B5 ( ), into ethanol 10 % (v/v).

In fact, the inability of the model to predict the release kinetics in ethanol 10 % (v/v) according to a Fickian diffusion process could be due to the structural modifications of the PLA matrix caused by sorption of ethanol, which could act as a PLA plasticizer and could favour the opening of the structure creating void spaces and favouring the thymol release at long times (Mascheroni, Guillard, Nalin, Mora and Piergiovanni, 2010). Likewise, as the diffusion rate increased (D > D'), the intermolecular interaction between ethanol and PLA chains was enhanced at long times (Samsudin, Soto-Valdez and Auras, 2014). At the best of our knowledge, there are no reported values of the diffusion coefficient of thymol in PLA nanocomposite films. However, in the release study presented in Chapter 1 the value of the diffusion coefficient for thymol in PP-based films in ethanol 10 % (v/v) was higher (1.75 × 10−10 cm2 s−1 ) than those reported in Table 1.5. Torres et al

~ 235 ~

Results and Discussion. Chapter 2

reported the values of the diffusion coefficient of thymol in LLDPE films in ethanol 10 % (v/v), ranging from 7.5 × 10−8 to 1.8 ×10−8 cm2 s−1, which were higher than those obtained in this work. This differences could be explained by the lower density of LLDPE resulting in higher mass transport properties (Torres, Romero, Macan, Guarda and Galotto, 2014). When considering the thymol release profiles shown in Figure 2.11, the D values are consistent with these results. The presence of the nanoclay leads to the decrease in the thymol release at long-time. Beltran et al reported the same behaviour when studied PCL with hydroxytyrosol and C30B. These authors related the decrease in the hydroxytyrosol release rate with the interactions between this compound and C30B (Beltrán, Valente, Jiménez and Garrigós, 2014). In conclusion, these results suggest that it is possible to control the release of active additives with interest in the design of novel active nanocomposites through the incorporation of laminar nanoclays, since the increase observed in the interlayer distance and intercalation results in the decrease in the diffusion of thymol through the polymer matrix by the tortuous path imposed with the incorporation of nanoclays to PLA-based films (Campos-Requena, Rivas, Pérez, Garrido-Miranda and Pereira, 2015).

3.5. DPPH radical scavenging ability The AO activity of the extracts obtained during the quantification of thymol (Section 3.1) and migration tests (Section 3.4) was estimated by their scavenging activity against DPPH radicals. Table 2.7 shows the results expressed as percentage of inhibition corresponding to the amount

~ 236 ~

Results and Discussion. Chapter 2

of thymol at the beginning of the study (IP,0.), and after 10 days of the migration test (IF,10

days).

As expected, all extracts containing thymol

showed important AO activity. It was observed that the amount of thymol after 10 days of contact with the food simulant (MF,10 days) was lower than the amount retained into the polymeric matrix (MP,0). But the released amount of thymol was enough to permit considering this additive as an efficient AO in PLA-based nanocomposites, since the inhibition values were around 77 % after 10 days, quite close to IP,0. These results were in agreement with those obtained in PP-based films (Chapter 1) where thymol and carvacrol were used as active additives. Table 2.7. Radical scavenging activity of thymol measured by the DPPH method for PLA-based formulations. Samples

MP,0 (mg)

I (%)P,0

MF,10 days (mg)

I (%)F,10 days

PLA/T

16.6 ± 0.2a

71.1 ± 0.2a

5.6 ± 0.1a

77.8 ± 0.1a

PLA/T/D43B2.5

17.8 ± 0.1b

84.3 ± 0.3b

5.4 ± 0.3a

77.0 ± 0.4a

PLA/T/D43B5

17.2 ± 0.1c

83.5 ± 0.1b

4.6 ± 0.4b

77.8 ± 0.8a

MP,0: Initial amount of thymol in the polymer matrix (mg) MF,10 days: Amount of thymol released after 10 days (mg) (mean ± standard deviation, n = 3) Different superscripts within the same column indicate statistically significant different values (p < 0.05)

DPPH radical scavenging activities of migration extracts were also determined and results were consistent with the expected increase in the thymol release in all films (Figure 2.14), suggesting the continuous release of the active additive in these formulations. Park et al reported a similar behaviour for corn-zein-laminated LLDPE (Park et al, 2012). These authors concluded that some relation should exist between the release of

~ 237 ~

Results and Discussion. Chapter 2

the active compound from the swelled polymer network and the intrinsic antioxidant properties of the natural additives.

90

350

80

300 250

60 50

200

40

150

30

100

20

DPPH results in PLA/T extracts

10

mgthymol kg food simulant -1

Inhibition (%)

70

50

Release of thymol from PLA/T

0

0 0

50

100

150

200

250

300

350

400

Time (hours)

Figure 2.14. AO activity obtained from migration extracts of PLA/T (left axis) and migration of thymol from PLA/T films (right axis) by using DPPH method.

3.6. Antibacterial activity The antibacterial activity of all active nanocomposite films used in this study was evaluated by placing small pieces of films in contact with a certain amount of inoculums of both microorganisms (Escherichia coli and Staphylococcus aureus) and measuring the viability of each bacteria in a controlled medium. Figure 2.15 shows the viability of microorganism’s cells onto PLA-based films after 3 h and 24 h incubated at 4, 24 (room temperature) and 37 °C, respectively. Some cell viability for both bacterial strains was observed, but interesting features could be drawn with some decrease for the active formulation (PLA/T) when compared to the non-active counterparts

~ 238 ~

Results and Discussion. Chapter 2

(PLA/D43B2.5 and PLA/D43B5). It has been stated that the antibacterial activity of phenolic monoterpenes, including thymol, is related with their ability to react with phospholipids present in the cell membranes, causing some increase in the cell wall permeability and the consequent leakage of cytoplasm as well as their interaction with some enzymes located on the cell wall (Burt, 2004; Emiroglu, Yemis, Coskun and Candogan, 2010). Indeed, and as discussed in a previous chapter, thymol has the ability to be released from polymer matrices and then it can disrupt the lipid structure of the bacteria cell wall, leading to the destruction of cell membranes, cytoplasmic leakage and ultimately microorganisms death (Kavoosi, Dadfar and Purfard, 2013). Formulations with D43B also showed some antibacterial activity against Escherichia coli and Staphylococcus aureus strains, but some special features should be highlighted. De Azeredo et al reported that OMMT could produce the rupture of cell membranes resulting in inactivation of both, Gram-positive and Gram-negative bacteria, due to the presence of quaternary ammonium groups able to react with lipids and proteins in the microorganism cell wall (de Azeredo, 2013). Nevertheless, taking into account the migration results previously discussed, the very low migration rate for D43B and the controlled release of thymol from the active nanocomposite films, it could be assumed that the antibacterial action of these nanocomposites is controlled by two factors, as already stated by other authors (Nigmatullin, Gao and Konovalova, 2008), i.e. the solid surface of D43B and the controlled release of thymol already reported in these PLA-based active nanocomposites. Indeed, the highest percentages of viability in this study were obtained for the active ternary nanocomposites with thymol and D43B.

~ 239 ~

Results and Discussion. Chapter 2

Figure 2.15. Antibacterial activity of PLA-based films at different temperatures against E. coli RB and S. aureus 8325-A. Cells were incubated on PLA with thymol and D43B for 3 h and 24 h at 4, 24 and 37 °C respectively. Results are expressed on a PLA-basis and are represented as mean ± standard deviation, n=3

Regarding the incubation times, the percentage of bacterial survival was quite similar after 3 and 24 h (Figure 2.15) for both strains and all tested temperatures. In general terms, the antibacterial activity was dependent on the combination of thymol and D43B, showing the highest value for Staphylococcus aureus. These results are in agreement with other studies where the antibacterial performance in active nanocomposites was reported (Shemesh et al, 2015). In conclusion, these results demonstrate the important role of the nanoclay as active carrier for the highly volatile thymol inside the polymer matrix. Therefore, the active nanocomposite films based on PLA with the

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Results and Discussion. Chapter 2

addition of thymol and D43B may be used to inhibit growth of different microorganisms on active packaging formulations.

4. Conclusions Active nanocomposite films based on PLA with thymol and D43B were processed and characterized. Different analytical techniques were used to evaluate the effect of the incorporation of D43B and thymol to the PLA matrix on the nanocomposites physico-chemical properties. The addition of thymol did not significantly affect the thermal stability of PLA, but some decrease in the elastic modulus was observed due to the slight plasticizing effect induced by the active additive. The incorporation of D43B and thymol did not result in a clear enhancement of oxygen barrier properties, but tensile behaviour was improved due to the intercalation and partial exfoliation of nanoparticles through the polymer matrix, as observed by XRD and TEM. Some differences in films colour were observed by the addition of thymol and D43B, being larger for films with the highest concentration of the nanoclay. Nevertheless, the intrinsic transparency of PLA was not affected by the addition of both components. It was observed that most of the thymol initially added to the PLA matrices (around 70-75 %) remained in the nanocomposites after processing, ensuring their posterior applicability to active systems. Results of the disintegrability tests under composting conditions showed that the incorporation of 8 wt% of thymol to PLA-based formulations could favour the disintegration of the polymer matrix, due to the presence of the reactive hydroxyl group in the thymol structure, while the presence of D43B did not show any influence in the disintegration performance.

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The combination of both additives induced higher degradation rates, suggesting their advantages in industrial applications where biodegradation could be an issue, such as food packaging. The applicability of these active nanocomposite films in food packaging was evaluated by studying the release of thymol into an aqueous simulant and their antioxidant and antimicrobial activity. The amount of thymol released into the aqueous food simulant was measured by HPLC-UV and a kinetic model was proposed, suggesting that the release of thymol is influenced by the presence of D43B and the PLA matrix. This continuous release favoured the antioxidant activity of these films determined by using the spectrophotometric DPPH method, resulting in a high percentage of inhibition. Finally, the addition of D43B has some effect in the improvement of the antibacterial activity of thymol-based films, showing higher inhibition against Staphylococcus aureus and Escherichia coli. In summary, the combination of thymol and D43B introduced into a commercial PLA matrix, in particular the combination of 8 wt% of thymol and 2.5 wt% of D43B, showed high potential to develop new biobased active films with application in fresh food packaging. The improvement in the functional properties of PLA-based films due to the addition of the active additive and the nanoclay also increased their antimicrobial and antioxidant properties, demonstrating the activity and high potential for packaging applications.

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4

Section 2.2.

Active nanocomposites based on PLA with thymol and silver nanoparticles for food packaging applications

Results and Discussion. Chapter 2

Summary The present work aims to develop biodegradable active nanocomposites with AM and AO properties based on PLA with thymol and Ag-NPs as active additives. In order to achieve this objective, firstly, injection moulded dog-bone bars were obtained and characterized to evaluate the thermal, morphological and mechanical properties as a preliminary approach to obtain active packaging systems for the food industry. Secondly, thin nanocomposite films (around 40 μm thick) were developed and characterized in order to evaluate their thermal, optical and barrier properties. The influence of thymol and Ag-NPs on the degradation of PLA-based active nanocomposites (films and dog-bone bars) in composting conditions was also studied. Migration tests were carried out to study the kinetic release of the nanocomposite films performance in food contact. Finally, the AO performance and AM activity of the developed films were evaluated by using the DPPH free radical scavenging method and against two typical foodborne bacteria (Escherichia coli and Staphylococcus aureus), respectively. These points are of great interest in order to prove the potential applicability of the developed systems as food active packaging solutions through controlled release formulations. The scheme in the next page shows the graphical flow of tests performed whose results will be discussed in this section.

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Results and Discussion. Chapter 2

Figure 2.16. General scheme of the experimental work presented in Section 2.2.

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5. Experimental 5.1. Materials Commercial poly(lactic acid) PLA-4060D (Tg = 58 °C, 11-13 wt% Disomer) was supplied in pellets by NatureWorks Co., (Minnetonka, MN, USA). Ethanol (EtOH, HPLC grade), 2,2-diphenyl-1-picrylhydrazyl (DPPH, 95 %) and thymol (99.5 %) were supplied by Sigma-Aldrich (Madrid, Spain). Commercial silver nanoparticles, P203, with a size distribution range between 20 and 80 nm, were purchased from Cima Nano-Tech (Saint Paul, MN, USA). Ag-NPs were treated at 700 °C for 1 h to condition the nanomaterial as reported elsewhere (Fortunati, Armentano, Iannoni and Kenny, 2010).

5.2. Active nanocomposites preparation PLA-based

nanocomposites

were

processed

in

a

twin-screw

microextruder (Dsm Explore 5&15 CC Micro Compounder, Heerlen, The Netherlands). PLA pellets were dried overnight at 45 °C before extrusion to prevent polymer hydrolysis during processing. A 170-180-190 °C temperature profile and a screw speed of 150 rpm were used. Different binary and ternary PLA-based formulations were obtained: binary systems, containing 6 (PLAT6) and 8 wt% (PLA/T8) of thymol or 1 wt% of AgNPs (PLA/Ag); and ternary systems, with 6 wt% of thymol and 1 wt% of Ag-NPs (PLA/Ag/T6), and 8 wt% of thymol and 1 wt% of Ag-NPs (PLA/Ag/T8). An additional sample without any additive was also prepared as control, as summarized in Table 2.8. A total mixing time of 6 min were used in binary systems. Thymol was added in the last 3 minutes of the extrusion process and the screw speed was then reduced to 100 rpm to limit losses by vaporization and thymol

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decomposition by high temperatures and shear. For ternary systems (PLA/Ag/T6, PLA/Ag/T8), a masterbatch of PLA and Ag-NPs was first processed in the extruder for 3 min and then it was combined with 6 or 8 wt% of thymol for 3 additional min. After mixing, two different morphologies were obtained. i.

Tensile dog-bone bars (ISO 527-2/5A) were prepared by means of a DSM Xplore 10-mL injection moulding machine. The injection pressure was set to 12.5 bars and the temperature was maintained at 200 °C.

ii.

Film forming process with a head force of 2500 N and a maximum temperature of 195 °C was performed to obtain films with mean thickness around 40 µm, which was determined with a 293 MDC-Lite Digimatic Micrometer (Mitutoyo, Japan) at five random positions. Table 2.8. PLA active nanocomposites formulated in this study.

Materials

PLA (wt%)

Ag-NPs (wt%)

Thymol (wt%)

Film thickness (µm)*

PLA

100

-

-

35 ± 4a

PLA/Ag

99

1

-

39 ± 4a

PLA/T6

94

-

6

40 ± 2a

PLA/T8

92

-

8

41 ± 5a

PLA/Ag/T6

93

1

6

42 ± 3a

PLA/Ag/T8

91

1

8

39 ± 6a

*Results are represented as mean ± standard deviation, n=3. Different superscripts within the same column indicate statistically significant different values (p < 0.05).

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5.3. Active nanocomposites characterization PLA-based

active

nanocomposites

were

characterized

by

the

determination of their thermal, mechanical, morphological, optical (colour and opacity) and barrier (oxygen transmission, and water vapour permeability) properties. 5.3.1. Thermal properties TGA tests were performed by using a TGA Seiko Exstar 6300 (USA). Samples (around 7 mg) were heated from 25 to 700 °C at 10 °C min-1 heating rate under nitrogen atmosphere (flow rate 50 mL min-1). Analyses were performed in triplicate. DSC tests were conducted, in triplicate, by using a DSC Mettler Toledo 822/e (Schwerzenbach, Switzerland) under nitrogen atmosphere (50 mL min-1). Samples (around 3 mg) were introduced in aluminium pans (40 µL) and were submitted to a thermal program: -25 to 250 °C at 10 °C min-1, with two heating and one cooling scans. Tg was determined from the second heating scan. 5.3.2. Field emission scanning electron microscopy (FESEM) The surface of neat PLA and PLA active nanocomposites and the cross section of PLA/Ag/T6 and PLA/Ag/T8 ternary composites in dog-bone bars were analysed by FESEM (Supra 25-Zeiss, Jena, Germany) to study their homogeneity and influence of thymol and Ag-NPs on the PLA morphology. Samples were coated with a gold layer prior to analysis in order to increase their electrical conductivity.

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5.3.3. Mechanical properties of injection moulded samples Tensile tests were used to evaluate the mechanical behaviour of neat PLA and the new active nanocomposites by using a digital Lloyd instrument LR 30K with a cross-head speed of 1 mm min-1 and a load cell of 30 kN. Dog-bone bars (2 mm thick) were prepared for testing by following the UNE ISO 527 Standard. Important parameters related with this study (εb, TS and E) were calculated from the resulting stress-strain curves according to the ASTM D882-09 Standard procedure (ASTM, 2009). Tests were carried out at room temperature and all values reported were the average of five measurements. 5.3.4. Optical properties of films The light transmission of PLA-based films was determined, in triplicate, by using a Perkin Elmer Lambda 35 UV-Vis spectrophotometer (Waltham, MA, USA). The measurements were carried out at 500 nm in the transmittance (%) mode to evaluate the transparency of the developed films in the visible region. Each film was cut in 2.5 x 2.5 cm2 strips. Modifications on colour caused by the addition of additives into the PLA matrix were determined with a Konica CM-3600d COLORFLEX-DIFF2 colorimeter (Reston, VA, USA) using the CIELab colour parameters: L* (lightness), a* (red-green coordinate) and b* (yellow-blue coordinate). Measurements were taken at five different locations around the film surface and average values were calculated. Total colour difference (∆E*) was calculated using Equation 2.11 using neat PLA as control.

∆

∗

1

= [(∆ ∗ )2 + (∆ ∗ )2 + (∆ ∗ )2 ]

2

(2.11)

where ∆L*= L*standard – L*sample, ∆a* = a*standard – a*sample and ∆b*= b*standard – b*sample

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5.3.5. Barrier properties of films An oxygen permeation analyser (8500 model Systech, Metrotec S.A, Spain) was used for OTR tests with pure oxygen (99.9 %). Film samples were cut into 14 cm diameter circles and they were clamped in the diffusion chamber at 25 °C before testing. Tests were performed in triplicate and average values were expressed as oxygen transmission rate per film thickness (OTR·e). WVP was determined gravimetrically in accordance with the test method indicated in the ASTM E 96M-05 Standard for water vapour transmission of materials. Films were cut in circles of 95 mm diameter and mounted on stainless steel permeation cells containing anhydrous calcium chloride, sealed with paraffin. The cells were placed in a climatic chamber (Dycometal, Barcelona, Spain) at 23.0 °C and 50% RH. The amount of water vapour transferred through films and absorbed by the desiccant was determined as the weight gain of the cell after 24 h. A minimum of seven determinations were taken to plot weight variation vs time resulting in a linear characteristic graph. Water vapour transmission (WVT) was calculated using Equation 2.12.

=

(

∙ ℎ−1 ∙

−2

)

(2.12)

where A is the film area exposed (0.005 m2) and G/t is the slope obtained from the weight gained in the permeation cell (G, grams) versus time (t, hours). The water vapour permeability (WVP) of film samples was determined, in triplicate, using Equation 2.13.

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Results and Discussion. Chapter 2

(

∙

∙

−1

∙

−1

∙

−2 )

=

∙ ∙

(

1

−

2)

(2.13)

where e is the film thickness, S is the saturation vapour pressure at 23 °C, and (R1-R2) is the difference in relative humidity between the exterior and interior of the permeation cell (0.5).

5.4. Quantification of thymol in PLA-based films after processing The actual amount of thymol present in PLA-based films after processing was determined by solid-liquid extraction followed by HPLC-UV analysis. 0.05 ± 0.01 g of each film were extracted with 10 mL of methanol at 40.0 °C and 50 % RH for 24 h in a climate chamber (Dycometal CM-081, Barcelona, Spain). Three replicates were carried out for each formulation. A Shimadzu LC-20A liquid chromatograph (Kyoto, Japan) with UV detector and a LiChrospher 100 RP18 column (250 mm × 5 mm × 5 μm, Agilent Technologies, USA) were used. An isocratic elution of 40:60 (v:v) acetonitrile:water at 25 °C and a flow rate 1 mL min-1 were applied. 20 μL of the extracted samples were injected and analyses were performed in triplicate at 274 nm. Standard solutions of thymol in methanol at concentrations between 100 and 500 mg kg-1 were used to elaborate the calibration curve for the thymol quantification.

5.5. Identification of thymol and Ag-NPs in PLA-based films FTIR analysis was performed by using a Jasco FTIR 615 spectrometer (Easton, MD, USA) equipped with a DTGS detector to confirm the presence of thymol into the PLA-based films. Spectra were recorded in the absorbance mode in the 4.000-400 cm−1 range, using 64 scans and 4

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Results and Discussion. Chapter 2

cm−1 resolution, and they were corrected against the background spectrum of air. Two spectra replicates were obtained for each sample. UV-Vis spectroscopy was used to detect the characteristic bands of AgNPs and thymol. A Perkin Elmer Instruments (Lambda 35) UV-Vis spectrophotometer (Waltham, MA, USA) operating in the 250-500 nm range was used. XRD patterns were recorded at room temperature in the scattering angle (2θ) from 2.5 to 80° (step size = 0.05º min-1) by using filtered Cu Kα radiation (λ = 1.54 Å). A Bruker D8-Advance diffractometer (Madison, WI, USA) was used, with voltage and current of X-ray tubes of 40 kV and 40 mA, respectively.

5.6. Disintegrability under composting conditions Disintegration tests under composting conditions were performed in dogbone bars and films by following the ISO-20200 standard method (UNEEN_20200, 2006). A commercial compost with certain amount of sawdust, rabbit food, starch, sugar, oil and urea was used. Testing samples (15 x 5 x 2 mm3 for injection moulded samples and 20 x 20 mm2 for films) were buried at 5 cm depth in perforated boxes and incubated at 58 °C. The aerobic conditions were guaranteed by mixing the compost softly and by the periodical addition of water according to the standard requirements. Different disintegration times were selected to recover samples from their burial and further tested: 0, 7, 14, 21, 28, 35 and 57 days for injection moulded samples; and 0, 1, 2, 4, 7 and 14 days for films. Samples were immediately washed with distilled water to remove traces of compost extracted from the container and further dried at 37 °C for 24 h before

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Results and Discussion. Chapter 2

gravimetrical analysis. The disintegrability value for each material at different times was obtained by normalizing the sample weight with the value obtained at the initial time. Photographs of recovered samples were also taken for visual evaluation. For injection moulded samples, the evolution of thermal properties upon disintegrability tests was also studied by DSC from -25 to 250 °C, at 10 °C min-1. Morphological changes in the surface of the recovered samples after testing at 0 and 14 days were studied by FESEM. FTIR spectra (Jasco FT-IR 615, USA) were also recorded in the 400-4000 cm-1 range, in ATR mode.

5.7. Release tests from PLA-based films The release of thymol and Ag-NPs from PLA-based nanocomposite films was studied in ethanol 10 % (v/v) as food simulant, in agreement with the European Standard EN 13130-2005 (UNE-EN_13130-1, 2005) and the Commission Regulation (EU) No 10/2011 on plastic materials and articles intended

to

come

into

contact

(Commission_Regulation/(EU)/No-10/2011).

with

Double-sided,

food total

immersion migration tests were performed, in triplicate, with 12 cm2 films and 20 mL of the simulant (area-to-volume ratio of 6 dm2 L-1) at 40 °C in an oven (J.P. Selecta, Barcelona, Spain) for 15 days. A blank test was also carried out. Extracts were taken at different times after film samples removal and they were stored at -4 °C before analysis. The amount of thymol and Ag-NPs released from the PLA-based films to the food simulant was determined by HPLC-UV and ICP/MS, respectively.

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Results and Discussion. Chapter 2

5.7.1. Silver release study The release of Ag-NPs from nanocomposite films into ethanol 10 % (v/v) at different times was directly determined by using an Agilent 7700x ICP/MS (Santa Clara, CA, USA) under conditions reported by Song et al with some modifications in the experimental parameters (Song, Li, Lin, Wu and Chen, 2011). A Scott-type spray chamber (Agilent Technologies) was used for sample introduction connected to a MicroMist small volume nebulizer. Sampling depth was 8.0 mm and argon was used as the carrier gas. ICP/MS operating conditions were: RF power, 1500 W; plasma gas flow rate, 15.0 L min-1; auxiliary gas flow rate, 0.9 L min-1; carrier gas flow rate, 1.0 L min-1; and make-up gas flow rate, 0.56 L min-1. Rhodium was used as internal standard and it was introduced by using a peristaltic pump in line with the sample solution. Suspensions were sonicated for 1 minute prior to analysis. Calibration standard working solutions were obtained by dilution of a stock solution (0.1 mg kg-1) of Ag-NPs in ethanol 10 % (v/v) to avoid matrix effects. Dilutions were prepared by accurately weighing the corresponding aliquot of the stock solution after 1 minute sonication (± 0.1 mg). LOD and LOQ values were calculated from the regression parameters obtained from the calibration curve (3 Sy/x/a and 10 Sy/x/a, respectively; where Sy/x is the standard deviation of the residues and a is the slope), resulting in 1.19 µg kg-1 and 3.98 µg kg-1, respectively. An acceptable level of linearity was obtained from the calculated calibration curve (R2 = 0.9972). 5.7.2. Thymol release study The amount of thymol released into ethanol 10 % (v/v) was determined in triplicate with an Agilent 1260 Infinity HPLC Diode Array Detector

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Results and Discussion. Chapter 2

(DAD) (Agilent, Santa Clara, CA) and Agilent eclipse plus C18 (100 mm x 4.6 mm x 3.5 μm) column. The mobile phase was acetonitrile/water (40:60) at 1 mL min-1 flow rate. 20 μL of the extracted samples were injected and detection was performed at λ = 274 nm. Analyses were performed in triplicate. Different standards between 5 and 500 mg kg-1 and working solutions of thymol were prepared in ethanol 10 % (v/v). LOD and LOQ values obtained from the calibration curve were 0.08 mg kg-1 and 0.26 mg kg-1, respectively. A high level of linearity was obtained from the calibration curve (R2 = 0.9999). A kinetic release study of thymol into ethanol 10 % (v/v) was performed for 15 days by taking samples at different times (2, 4, 6, 12, 24, 48 hours and 5, 10 and 15 days). Tests were performed in triplicate.

5.8. Determination of the antioxidant activity The AO activity of thymol released into ethanol 10 % (v/v) was analysed in terms of radical scavenging ability by using the DPPH method, as proposed by Byun et al with slight modifications (Byun, Kim and Whiteside, 2010). An aliquot of 100 μL of each extract was mixed with 3.9 mL of a methanolic solution of DPPH (23 mg L-1) in a capped cuvette. The mixture was shaken vigorously and it was kept in the dark at room temperature for 200 min. The absorbance of each solution was determined at 517 nm by using a Biomate-3 UV-Vis spectrophotometer (Thermo Scientific, USA). All analyses were performed in triplicate and the AO capacity was expressed as the ability to scavenge the stable radical DPPH by using Equation (2.14).

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Results and Discussion. Chapter 2

−

(%) =

∙ 100

(2.14)

where AControl and ASample are the absorbances of the blank control at t = 0 min and the tested sample at t = 200 min, respectively.

5.9. Antibacterial activity of PLA-based films The microorganisms used in this study were Escherichia coli RB (E. coli RB) and Staphylococcus aureus 8325-4 (S. aureus 8325-4). E. coli RB was an isolate strain provided by the “Zooprofilattico Institute of Pavia” (Italy) whereas S. aureus 8325-4 was a gift from Dr. Timothy J. Foster (Department of Microbiology, Dublin, Ireland). E. coli RB and S. aureus 8325-4 were routinely grown overnight in Luria Bertani Broth (LB) and Brian Heart Infusion (BHI) (Difco Laboratories Inc., Detroit, MI, USA), respectively, under aerobic conditions at 37 °C using a shaker incubator (New Brunswick Scientific Co., Edison, NJ, USA). These cultures were reduced at a final density of 1 × 1010 cells mL-1 as determined by comparing the OD600 of samples with a standard curve relating OD600 to cell number. The evaluation of the antibacterial activity of neat PLA and PLA active nanocomposite films was carried out in 100 µL of overnight diluted cell suspensions (1×104) of E. coli RB or S. aureus 8325-4 . These suspensions were added to each sample and seeded at the bottom of a 96-well tissue culture plate, and further incubated at three different temperatures: 4, 24 and 37 °C for 3 h and 24 h, respectively. Furthermore, 96-well flat-bottom sterile polystyrene culture plates (TCP) used as controls were incubated under the same conditions. At the end of each incubation time, the bacterial suspension was then serially diluted and plated on the LB (E. coli RB) or BHI (S. aureus 8325-4) agar plates, respectively. Plates were then

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Results and Discussion. Chapter 2

incubated for 24 h/48 h at 37 °C. Cell survival was expressed as percentage of CFU of bacterial growth on PLA active nanocomposite films compared to that obtained for the neat PLA film.

5.10. Statistical analysis Statistical analysis of results was performed with SPSS commercial software (Version 15.0, Chicago, IL). A one-way analysis of variance (ANOVA) was carried out. Differences between means were assessed on the basis of confidence intervals using the Tukey test at a p < 0.05 significance level. Two-group comparisons were performed by application of the Student’s t-test for the antibacterial activity, and results were expressed by mean ± SD (standard deviation), by using GraphPad Prism 4.0 software (San Diego, CA, USA). Two-tailed p values < 0.05 were considered statistically significant.

6. Results and discussion 6.1. Characterization of injection moulded samples 6.1.1. Thermal properties The effect of the addition of thymol and Ag-NPs in the thermal stability of PLA-based nanocomposites was studied by TGA under nitrogen atmosphere. The weight loss (TG) and derivative (DTG) curves of binary and ternary systems are reported in Figure 2.17a and Figure 2.17b, respectively. Two thermal parameters were obtained from this study (Table 2.9): initial degradation temperature (Tini), determined at 5 % weight loss, and maximum degradation temperature (Tmax). All materials

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Results and Discussion. Chapter 2

showed a main peak associated to the PLA thermal degradation between 330 and 360 °C, as previously reported (Martino, Jiménez, Ruseckaite and Avérous, 2010; Hwang et al, 2012; Burgos, Martino and Jiménez, 2013) 2013). A slight reduction in the Tmax value was observed by the addition of Ag Ag-NPs (p > 0.05). Regarding Tini values, a significant reduction (p < 0.05) was observed rved suggesting some loss in the PLA thermal stability by the addition of Ag-NPs. Thermograms hermograms corresponding to the ternary nanocomposites showed a first degradation step around 120 °C, which was related to the thymol degradation as it was shown for PP PP-based films in Chapter 1 and PLA-based based active nanocomposites with D43B (Section 2.1). In summary, TGA results showed that besides the slight reduction in the nanocomposites thermal stability by the addition of thymol and Ag Ag-NPs, these formulations could be processed cessed at the same temperatures (up to 200 °C) than neat PLA without risking thermal degradation. (a)

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Results and Discussion. Chapter 2

(b)

Figure 2.17. TG (a) and DTG (b) curves of neat PLA and nanocomposite injection moulded samples with Ag-NPs and thymol.

Table 2.9. Thermal parameters and tensile properties obtained for injection moulded samples (neat PLA and active nanocomposites). Samples

Tg* (°C)

Tini* (°C)

Tmax* (°C)

E** (MPa)

εb** (%)

TS** (MPa)

PLA

56 ± 3a

324 ± 12a

363 ± 6a

3181 ± 35ab

3.5 ± 0.2a

60.3 ± 8.0a

PLA/Ag

56 ± 1a

317 ± 8ab

357 ± 13a

3000 ± 172bc

3.6 ± 0.2a

59.7 ± 2.9a

PLA/T6

50 ± 2b

327 ± 15a

358 ± 7a

3289 ± 28a

2.7 ± 0.2a

52.1 ± 1.3a

PLA/T8

42 ± 1bc

316 ± 14a

353 ± 9a

2930 ± 76bc

2.6 ± 0.3b

36.4 ± 3.2b

PLA/Ag/T6

44 ± 1bc

284 ± 9bc

337 ± 12a

2823 ± 121cd

3.6 ± 0.3b

36.9 ± 3.0b

PLA/Ag/T8

41 ± 1c

288 ± 5c

336 ± 14a

2547 ± 244d

2.8 ± 0.2b

36.3 ± 2.8b

* (n=3;

mean ± standard deviation) mean ± standard deviation) Tg: determined by DSC from the first heating scan at 10 °C min-1. Tini and Tmax: determined by TGA at 10 °C min-1 in N2 atmosphere. Corresponding to the 2nd degradation step. Results are represented as mean ± standard deviation, n=3. Different superscripts within the same column indicate statistically significant different values (p < 0.05). **(n=5;

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Results and Discussion. Chapter 2

DSC thermograms for all the tested materials are shown in Figure 2.18. Since the PLA used in this study was mostly amorphous, a Tg value could be determined in all samples (Table 2.9). It is known that Tg is dependent upon the polymer structural arrangement and corresponds to the torsion oscillation of the carbon backbone (Hughes, Thomas, Byun and Whiteside, 2012). So, it was expected that the addition of thymol could lead to some reduction in Tg, as observed in Table 2.9. In fact, binary and ternary systems with thymol showed a significant decrease in more than 10 °C on Tg values (p < 0.05). This reduction was associated with the already reported plasticizing effect of thymol in polymer matrices, also observed when thymol was added to a PP matrix (Chapter 1), increasing the molecular mobility of the polymer structure. A similar behaviour was reported by other authors for the addition of similar AOs to PLA with a remarkable reduction on Tg values (Byun, Kim and Whiteside, 2010; Hwang et al, 2012; Arrieta, López, Ferrándiz and Peltzer, 2013). DSC results also showed that the addition of Ag-NPs had no relevant effect on the Tg values of PLA, in agreement with previous studies (Fortunati, Armentano, Zhou, Iannoni, et al, 2012). Conversely, parameters related to crystallization or melting of PLA nanocomposites were not observed due to the mentioned amorphous structure of the polymer used in this study, which remained after the addition of thymol and Ag-NPs to the polymer matrix.

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Results and Discussion. Chapter 2

Figure 2.18. DSC thermograms for PLA, PLA/Ag, PLA/T8 and PLA/Ag/T8 injection moulded samples; first heating and cooling scans (a) and second heating scan (b).

6.1.2. Morphological characterization Figure 2.19 shows the FESEM micrographs obtained for neat PLA and PLA nanocomposites surfaces after processing. Homogeneous surface morphologies were observed for all materials, with no apparent effect of the addition of thymol and Ag-NPs to the PLA matrix. FESEM micrographs were also taken to cross section of ternary systems to evaluate the incorporation of both additives to the polymer matrix (Figure 2.20). It was noticed that Ag-NPs were well dispersed with no apparent agglomerates, which could be probably related to the presence of thymol in these formulations (Fortunati, Armentano, Zhou, Puglia, et al, 2012).

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Results and Discussion. Chapter 2

Figure 2.19. FESEM micrographs of the surface of nanocomposite injection moulded.

Figure 2.20. Cross section micrographs of PLA/Ag/T6 and PLA/Ag/T8 injection moulded samples after processing.

6.1.3. Mechanical properties The he tensile properties of neat PLA and nanocomposites were evaluated and results are reported in Table 2.9. The addition of 1 wt% of Ag Ag-NPs to PLA had no significant effect (p > 0.05) on the elastic modulus, tensile strength and elongation at break values as previously reported by other

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Results and Discussion. Chapter 2

authors (Kanmani and Rhim, 2014a). Some reduction in tensile strength values was observed for the PLA-thymol binary systems, being more evident for those with the highest content (8 wt%, p < 0.05). This effect could be due to the increase in polymer chains mobility caused by the presence of thymol in these formulations (Fabra, Talens and Chiralt, 2008). These results are also related with those obtained for mechanical properties of PP-based films in Chapter 1 and active PLA-based films with D43B. The combined action of thymol and Ag-NPs on the PLA mechanical behaviour was also evaluated. A significant decrease in elastic modulus of the ternary formulations was observed compared to neat PLA resulting in more flexible and stretchable materials (p < 0.05). It could be suggested that the presence of Ag-NPs contributed to the thymol ability to increase the PLA chain mobility, which also promoted a more effective dispersion of silver nanoparticles. These combined effects could be related to the presence of Van der Waals interactions between the hydroxyl groups of thymol molecules and the partial positive charge on the surface of the AgNPs which affects the mechanical response of the ternary nanocomposites (Fabra, Talens and Chiralt, 2008; Shameli et al, 2010; Aguirre, Borneo and León, 2013).

6.2. Films characterization 6.2.1. Thermal properties The effect of the addition of thymol and Ag-NPs on the thermal properties of PLA-based nanocomposite films was evaluated by DSC. The obtained results are summarized in Table 2.10. Tg of PLA and all the active nanocomposite films was clearly observed, once again indicative of

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the amorphous character of the commercial PLA used in this study. No crystallization or melting phenomena were detected. The addition of AgNPs to PLA (PLA/Ag) did not reveal significant differences with respect to neat PLA in terms of Tg (p > 0.05), in agreement with Fortunati et al (Fortunati, Rinaldi, et al, 2014) and with previous results obtained in this work for dog-bone bars. However, as expected from the results already discussed for injection moulded samples, thymol-containing binary and ternary nanocomposite films showed a significant decrease (p < 0.05) in Tg values with differences higher than 10 °C with respect to the glass transition temperature of neat PLA. This reduction in Tg is related to the higher mobility of the polymer macromolecules caused by the increase of the free volume in the matrix, promoting the torsion oscillation of the carbon backbone due to the plasticizing effect induced by the addition of thymol. It is well known that the addition of low molecular weight compounds decreases PLA toughness by reducing the glass transition temperature and increasing the mobility of macromolecules (Gonçalves et al, 2013). A similar shift to lower temperatures in Tg was observed by the incorporation of thymol to PP matrix producing a plasticization effect, as discussed in Chapter 1. A significant decrease in Tg was also reported by other authors for PLA-based films with thymol (Tawakkal, Cran and Bigger, 2014; Wu, Qin, et al, 2014). A similar behaviour was reported for α-tocopherol, resveratrol, BHT and tert-butylhydroquinone added to PLA (Byun, Kim and Whiteside, 2010; Hwang et al, 2012; Gonçalves et al, 2013). In all cases, an effective plasticizing effect was observed, resulting in the decrease in Tg. The thermal stability of neat PLA and the active nanocomposite films was studied by TGA under nitrogen atmosphere. A main degradation peak associated to the PLA thermal degradation between 330-360 °C was

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observed for all samples. A first degradation step starting at 120 °C was also detected (data not shown), which could be related to the thymol degradation. This result confirms, once again, the permanence of a detectable amount of thymol in the PLA-based nanocomposites after processing. The main TGA parameters obtained, i.e. Tini determined at 5 % weight loss, and Tmax for the main peak associated to the PLA thermal degradation, are shown in Table 2.10. As can be observed, the separate addition of thymol and Ag-NPs into PLA did not significantly affect the thermal behaviour of the polymer matrix in terms of Tmax and Tini (p > 0.05). However, the significant reduction observed in these parameters caused by the combined addition of Ag-NPs and thymol in ternary systems suggests some decrease in the PLA thermal stability (p < 0.05) for the active nanocomposite ternary films. This phenomenon was related with some degradation of these materials during processing.

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Table 2.10. Characterization of neat PLA and active nanocomposite films. Samples

Thymol extracted (wt%)

Tg (°C)

Tini (°C)

Tmax (°C)

WVP*10-14 (Kg m s-1 m-2 Pa-1)

Reduction in WVP (%)

OTR·e (cm3 mm m-2 day-1)

PLA

n.d.

56.3 ± 2.2a

320 ± 4a

363 ± 2a

1.84 ± 0.12a

-

19.9 ± 2.1a

PLA/Ag

n.d.

53.7 ± 0.8a

316 ± 4a

354 ± 5a

1.77 ± 0.01a

4

26.2 ± 8.4a

PLA/T6

4.38 ± 0.04a

43.3 ± 0.2b

321 ± 3a

351 ± 3a

1.33 ± 0.11b

27

18.5 ± 1.6a

PLA/T8

5.79 ± 0.07b

43.5 ± 1.0b

312 ± 2a

354 ± 3a

1.10 ± 0.09c

40

20.7 ± 1.8a

PLA/Ag/T6

4.41 ± 0.04c

42.6 ± 0.8b

281 ± 3b

332 ± 5b

1.12 ± 0.05b,c

39

18.3 ± 1.1a

PLA/Ag/T8

6.09 ± 0.09d

43.0 ± 0.4b

284 ± 5b

334 ± 6b

1.17 ± 0.09b,c

36

18.3 ± 1.9a

n.d.: not detected Tg: determined by DSC from the second heating scan at 10 °C min-1. Tini and Tmax: determined by TGA at 10 °C min-1 in N2 atmosphere. Corresponding to the 2nd degradation step. Results are represented as mean ± standard deviation, n=3. Different superscripts within the same column indicate statistically significant different values (p < 0.05).

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6.2.2. Morphology The surface morphology and microstructure of PLA and active nanocomposite films were studied by FESEM in order to evaluate the incorporation of thymol and Ag-NPs into the polymer matrix. As in the case of injection moulded probes, all films showed smooth and homogeneous surface structure with good structural integrity without pores or cavities (Figure 2.21). FESEM images also confirmed the uniform distribution of the Ag-NPs all through the PLA matrix since no particle agglomeration was detected. Similar morphologies were observed by other authors for PLA and other polymer matrices blended with AgNPs or thymol (Kumar and Münstedt, 2005; Rhim, Wang and Hong, 2013; Fortunati, Rinaldi, et al, 2014; Kanmani and Rhim, 2014b). Rhim et al reported smooth surfaces with evenly dispersed silver particles on the PLA film surface (Rhim, 2013). These results demonstrate a positive combination between PLA and thymol to obtain homogeneous surfaces when processing films.

Figure 2.21. FESEM surface images of PLA and active nanocomposite films.

6.2.3. Optical properties All the PLA-based films were visually homogeneous and transparent regardless of their composition (Figure 2.22). The colour distribution observed in all films suggests that additives were uniformly distributed

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during processing. However, active nanocomposite films containing Ag AgNPs showed some darkening in the initially clear surface as well as some decrease in transparency, which is an important physical property in food packaging films (Jamshidian, Tehrany, Imran, Jacqu Jacquot and Desobry, 2010). The incorporation of additives to the PLA matrix can lead to substantial modifications and transparency losses, and these variations may represent an important drawback for consumer acceptance (Raquez, Habibi, Murariu and Dubois, 2013). Rihm et al suggested that surface plasmon phenomena of silver nanoparticles and phenolic compounds, such as thymol, may modify PLA colour during processing and storage leading to darkening in films when the Ag-NPs NPs concentration is high (Rhim, Wang and Hong, 2013).

Figure 2.22. Visual observation of neat PLA and binary and ternary nanocomposite films.

Results for colour olour and transmittance at 500 nm of all films are shown in Table 2.11.. The surface colour of PLA binary and ternary films was significantly influenced (p < 0.05) depending on the addition of thymol andAg-NPs. While some decrease (pp < 0.05) in film lightness (L-value) was observed in PLA films containing Ag Ag-NPs, it slightly increased in those with thymol (p < 0.05) when compared to the neat PLA film. In

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addition, a* and b* parameters were modified by the presence of both additives (Table 2.11). In particular, Ag-NPs-based binary and ternary systems resulted in a significantly increase (p < 0.05) in a* and b* towards positive values, indicating a trend in redness and yellowness, respectively, of the active nanocomposite films. Consequently, ∆E* of those films with Ag-NPs increased significantly (p < 0.05). This behaviour can be explained by the development of brown colour in nanocomposites by the plasmonic effect of Ag-NPs (Rhim and Wang, 2014). Binary systems containing thymol showed similar colour parameters than neat PLA. Table 2.11. Optical properties of neat PLA and active nanocomposite films. Colour parameters

Transparency

Samples

L*

a*

b*

ΔE*

T500nm (%)

PLA

47.46 ± 0.09a

-0.19 ± 0.03a

-0.12 ± 0.02a,c

-

94.77 ± 0.01a

PLA/Ag

46.67 ± 0.29b

1.53 ± 0.03b

8.04 ± 0.07b

8.37 ± 0.09a

91.31 ± 0.01b

PLA/T6

48.25 ± 0.20c

-0.15 ± 0.02a

-0.22 ± 0.04c

0.89 ± 0.16b

93.53 ± 0.03c

PLA/T8

48.33 ± 0.31c

-0.28 ± 0.02c

-0.06 ± 0.02a

0.97 ± 0.34b

94.41 ± 0.03d

PLA/Ag/T6

45.47 ± 0.27d

1.21 ± 0.03d

8.83 ± 0.08d

9.25 ± 0.10c

90.21 ± 0.01e

PLA/Ag/T8

46.38 ± 0.22b

1.04 ± 0.02e

9.57 ± 0.06e

9.81 ± 0.06d

90.80 ± 0.02f

Results are represented as mean ± standard deviation, n=3. Different superscripts within the same column indicate statistically significant different values (p < 0.05).

PLA is a transparent polymer with a transmittance close to 95% in the visible region (Table 2.11), as already reported (Jamshidian, Tehrany, Imran, Jacquot and Desobry, 2010). The evaluation of the light transmission in the visible region (500 nm) revealed that all binary and ternary films with thymol and Ag-NPs were, in general, highly transparent, showing transmittance values higher than 90 % in all cases. A slight decrease (p < 0.05) in transmittance was observed in binary systems

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containing thymol, which might be due to the colourless transparent appearance of this additive. On the other hand, the inclusion of Ag-NPs into PLA films also produced some significant reduction (p < 0.05) in transparency, which was related to the prevention of light transmission by the nanoparticles homogeneously dispersed through the polymer matrix (Kanmani and Rhim, 2014a). Results suggest that the amount of both additives, thymol and Ag-NPs, used in these formulations did not affect dramatically the colour and transparency of PLA films. Therefore, their incorporation into the PLA matrix could be suitable for food packaging applications without compromising to an unacceptable degree its optical properties. 6.2.4. Barrier properties Results of the effect of the addition of thymol and Ag-NPs on the barrier properties (OTR and WVP) of PLA-based films are shown in Table 2.10. Biofilms with low oxygen permeability are desirable for food preservation as oxygen can accelerate lipids oxidation and facilitate the growth of aerobic microorganisms, thereby shortening the food shelf-life (Pagno et al, 2015). The OTR·e values obtained in this study showed that the oxygen barrier offered by neat PLA was not significantly modified (p > 0.05) in the presence of additives. The study of the water vapour barrier properties of biofilms is important to assess their possible performance as food packaging materials since one of their main functions is to decrease the moisture transfer between food and the surrounding environment keeping food quality and increasing shelf-life (Siracusa, Rocculi, Romani and Rosa, 2008). Water vapour barrier in biofilms could be considered as the balance between the hydrophobic/hydrophilic characteristics of all their components. In our

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case, WVP of PLA films was not significantly affected (p > 0.05) by the incorporation of Ag-NPs (PLA/Ag). This behaviour may be due to the spherical shape of silver particles and their homogeneous dispersion in the polymer matrix which may not develop efficiently the tortuous pathway necessary to retard water vapour diffusion (Rhim and Wang, 2014). It has been stated that high water vapour permeability of films intended for food packaging could restrict considerably their use (SánchezGonzález, Vargas, González-Martínez, Chiralt and Cháfer, 2011). In this case, the addition of thymol to PLA-based films resulted in a significant decrease (p < 0.05) in WVP values for binary and ternary systems, up to 40 % compared to those values obtained for the neat PLA film. These results could be explained by the repulsion to water molecules caused by the addition of hydrophobic components, such as thymol, at high concentrations (López-Mata et al, 2013). Therefore, these thymolcontaining nanocomposites allowed an important improvement in barrier properties to water vapour, which is a remarkable feature in food packaging applications, especially at high RH storage conditions. Similar results were found by other authors under the same conditions (23 °C, 45% RH), reporting a WVP value of 1.99 x 10-14 kg m m-2 s-1 Pa-1 for neat PLA, and a 25 % reduction in WVP for PLA films loaded with 4 wt% αtocopherol (Gonçalves et al, 2013). Meanwhile, the addition of 2 wt% marigold flower extract containing astaxanthin resulted in the decrease in 21 % in WVP of PLA, which was attributed to the hydrophobic nature of this extract (Samsudin, Soto-Valdez and Auras, 2014).

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6.3. Quantification of thymol in PLA-based films after processing The quantification of the actual amount of thymol present in the processed active films is an important issue since the high volatility of this additive could result in losses during extrusion and further film manufacturing at high temperatures. The final amount of thymol detected in binary and ternary formulations after processing is shown in Table 2.10. Results are indicative that the losses of thymol were lower than 30 %, regardless of the initial amount used in the formulations (6 or 8 wt %). These results can be considered acceptable and were reduced to the minimum by the optimization of processing conditions as previously discussed, allowing thymol to be inside the extruder for the minimum time to achieve a good dispersion into the polymer matrix. Therefore, it can be expected that the remaining amount of thymol in these PLA-based films after processing is high enough to obtain acceptable properties in terms of antibacterial activity, as it will be further discussed. Other authors have also reported some losses of volatile additives during processing. In this sense, losses of catechin and epicatechin during processing incorporated to PLA matrices were reported in the range of 20-35 %, where processing temperature, residence time of PLA into the extruder and the additives concentration mainly influenced their permanence in the final blends (Iñiguez-Franco et al, 2012). Moreover, losses of astaxanthin from marigold flower extract added to PLA up to 80 % were reported (Samsudin, Soto-Valdez and Auras, 2014). PLA/Ag/T8 nanocomposites showed a significantly higher amount of thymol (p < 0.05) remaining after processing (around 76 %) (Table 2.10). This result may indicate that the loss of thymol could be influenced by the presence of Ag-NPs, which could play a role in retarding the diffusion of thymol molecules through the polymer structure. A similar effect was

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observed by Efrati et al for active films based on LDPE with thymol and different contents of MMTs. These authors reported a decrease in the loss of thymol during processing due to the increase of MMT content (Efrati et al, 2014).

6.4. Identification of thymol and Ag-NPs in PLA-based films FTIR and UV-Vis absorption spectra of PLA and PLA active nanocomposite films are shown in Figure 2.23a and Figure 2.23b, respectively. FTIR results confirmed the presence of a significant amount of thymol remaining in the nanocomposite films after processing, since the flexion vibration of the methylene group (CH2) at 806 cm-1 (see zoomed area in Figure 2.23a) was observed for all formulations containing thymol, a clear indication of the presence of the additive in the processed formulations. The presence of thymol in binary and ternary systems after processing was also confirmed by UV-Vis spectrophotometry (Figure 2.23b). Thymol shows maximum absorption in this region (λmax) at 274 nm, corresponding to the characteristic band of the π-π* transition, also due to the auxochrome phenolic hydroxyl group in its structure (Rukmani and Sundrarajan, 2011). Furthermore, those formulations with Ag-NPs showed a low-intensity but characteristic band at the visible region around 400 nm, which some authors correlated with the surface plasmon resonance

(SPR)

transition

peak

(Vigneshwaran,

Nachane,

Balasubramanya and Varadarajan, 2006; Shameli et al, 2010; Kanmani and Rhim, 2014a).

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Results and Discussion. Chapter 2

(b)

Figure 2.23. FTIR (a) and UV-Vis Vis (b) spectra of PLA and active nanocomposite films.

X-ray ray diffraction patterns of active nanocomposite films with Ag Ag-NPs were clearly indicative of the presence of these nanoparticles embedded into to the polymer matrix in the films ((Figure 2.24). While the XRD diffractogram of the mostly amorphous PLA only shows the characteristic broad band around 2θ = 20º; the clear and sharp peak around 38.2º

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Results and Discussion. Chapter 2

observed in thos formulations with Ag-NPs can be attributed to the 111 crystallographic plane of face-centred cubic (fcc) silver crystals (Shameli et al, 2010; Rhim, Wang and Hong, 2013).

Figure 2.24. WAXS patterns of PLA and active nanocomposite films.

6.5. Disintegrability under composting conditions Biodegradability tests are necessary to estimate the environmental impact of plastic materials after their shelf-life and it is particularly relevant in short-term applications, such as food packaging. The disintegration study under composting conditions of PLA-based active nanocomposites (injection moulded samples and films) was carried out to evaluate the effect of the addition of Ag-NPs and thymol on the degradation properties of the PLA matrix.

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6.5.1. Disintegrability study for injection on moulded samples The visual evaluation of the injection moulded samples at different degradation times is shown in Figure 2.25..

Figure 2.25. PLA and PLA nanocomposites processed by injection moulding at different times under composting conditions at 58 °C.

All samples showed considerable ble modifications in colour and a general loss of transparency after 7 days. These surface modifications were indicative of the beginning of the polymer hydrolytic degradation process, which was related to the moisture absorption. Fukushima et al related the increase in the materials opacity to various simultaneous phenomena, such as the formation of low molar-mass mass degradation by by-products during hydrolysis and/or the changes in crystallinity in the polymer matrix (Fukushima, Tabuani, Arena, Gennari and Camino, 2013) 2013). Indeed, a general increase in the polymer and nanocomposite nanocomposites crystallinity took place at a higher rate in their amorphous zones (Paul et al, 2005), resulting in reduction of transparency and a general modification in colour of the injected materials, as expected due to the high amorphous character of

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the PLA used in this work, as already stated by other authors (Gorrasi and Pantani, 2013).. Further results at longer testing times showed that the physical degradation of these samples progressed with burial time resulting in a complete loss of the initial morphology and general rupture after 35 days. Figure 2.26aa reports the disintegrability percentage (weight loss) as a function of the testing ting time for all the injection moulded materials. Before 14 days, no important differences were observed between all samples. However, after 14 days, those formulations containing thymol increased their weight loss rate and in consequence the disintegrabi disintegrability ratio to values higher than 30 %; while neat PLA and the PLA/Ag nanocomposite showed slower degradation rate with values (20.8 ± 0.6) % and (24.4 ± 4.0) % after 21 days, respectively. These differences in the disintegrability rate between nanocomposites es with and without thymol in their formulations increased after 35 days (Figure Figure 2. 2.26b).

Figure 2.26. Disintegrability (%) of PLA and PLA nanocomposite processed by injection moulding at different times in compost at 58 °C. The line at 90 % represents the goal of disintegrability tests as required by the ISO 20200 Standard.

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Results and Discussion. Chapter 2

PLA/Ag/T8 showed the highest disintegration rate followed by PLA/Ag/T6 highlighting the high influence of thymol in the diffusion process of water molecules through the polymer structure, promoting hydrolysis due to the increase in chain mobility induced by the plasticizing effect already discussed. This behaviour was improved by the homogeneous dispersion of thymol into the PLA matrix, as it was observed in FESEM micrographs. In addition, the hydroxyl groups in thymol can contribute to the heterogeneous hydrolysis of the PLA matrix after absorbing water from the composting medium, resulting in a noticeable increase in disintegrability values for PLA nanocomposites with thymol after 14 days. In the initial stages of the composting test, some interactions between the thymol hydroxyl groups and water molecules with formation of hydrogen bonds could retain the hydrolysis process compensating the higher water diffusion rate in samples with thymol. This effect is no longer observable after 14 days. A similar behaviour was reported by Sinha Ray et al, who suggested that 14 days could be considered the critical time to start the heterogeneous hydrolysis process (Sinha Ray, Yamada, Okamoto and Ueda, 2003), as observed in this study. The presence of hydroxyl groups in the thymol molecules, finely dispersed in the PLA matrix, are responsible of the formation of labile bonds in the polymer structure and consequently the hydrolysis reaction is faster by formation of low molar mass chains (Sinha Ray, Yamada, Okamoto and Ueda, 2003; Proikakis, Mamouzelos, Tarantili and Andreopoulos, 2006). This effect could be even reinforced by synergies between Ag-NPs and thymol, since Ag atoms could catalyse the disintegration process (Fortunati, Armentano, Iannoni, et al, 2012).

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Finally, after 57 testing days, it was observed that all PLA nanocomposites appeared totally disintegrated satisfying the ISO Standard requirements for a biodegradable material (UNE-EN_20200, 2006). The evolution of thermal, chemical and morphological properties upon disintegrability tests was also studied for injection moulded samples.

Structural analysis PLA/Ag/T8 nanocomposites submitted to composting conditions up to 21 days were analysed by FTIR and the most relevant spectra are reported in Figure 2.27. The typical stretching band of the carbonyl group (-C=O) at 1750 cm-1, attributed to lactide, and the -C-O- bond stretching band in the PLA -CH-O- group at 1180 cm-1 were identified (Shameli et al, 2010). As previously discussed, the hydrolytic degradation in PLA takes place during the initial phases of the composting treatment, where the high molar mass PLA chains are hydrolysed to form low molar mass oligomers with plenty of available and highly reactive hydroxyl and carboxylic acid groups (Fukushima, Abbate, Tabuani, Gennari and Camino, 2009). FTIR spectra after 21 days showed a considerable decrease in the intensity of the peak related to the carbonyl group (-C=O) at 1750 cm-1 and the simultaneous appearance of a typical IR absorption corresponding to carbonyl groups in carboxylic acids formed by the hydrolytic scission of the ester groups (Fukushima, Feijoo and Yang, 2012). In addition, the band at 1180 cm-1 corresponding to the -C-O- stretching practically disappeared after 7 treatment days as already reported by other authors (Fortunati, Armentano, Iannoni, et al, 2012). However, these results did not reveal important differences between binary and ternary formulations regardless of the thymol concentration.

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Results and Discussion. Chapter 2

Figure 2.27. FTIR spectra of PLA/Ag/T8 at different times under composting conditions.

Morphological analysis FESEM micrographs of nanocomposite surfaces after 14 days of degradation test are shown in Figure 2.28.. Important differences in sample surfaces submitted tted to composting were obtained. Before the beginning of this test (day 0) all materials showed smooth and neat surfaces, but after 7 days, fractures appeared, as expected from the observation of the important changes in the visual study. The formation of fractures and surface holes for all samples was clearly indicative of the beginning of the hydrolytic degradation process (Fukushima, Tabuani, Arena, Gennari and Camino, 2013).. After 14 days, those formulations with thymol showed important fractures up to 2 µm in width (Figure XXX). In general terms, a higher amount of thymol ol resulted in more degraded materials submitted

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to composting conditions, especially in ternary nanocomposites. In fact, binary and ternary formulations containing 8 wt% of thymol (PLA/T8 and PLA/Ag/T8), showed highly irregular surfaces with holes and fractures ctures all around their perimeter. This result could be related to the higher hydrolysis rates with formation and release of low molecular weight compounds, such as simple alcohols and/or CO2. This transformation could be also related to the action of microorganisms, oorganisms, which are able to convert these low molecular weight structures into CO2 and water (Fukushima, Abbate, Tabuani, Gennari and Camin Camino, 2009).

Figure 2.28. FESEM micrographs of the surface of nanocomposite injection moulded samples before (0 days) and after 14 days of disintegration in compost at 58 °C (500x) and after 14 days with higher zoom (10.00 kx).

Thermal analysis Figure 2.29 shows the DSC thermograms obtained during the first heating scan for all samples submitted to the disintegration test as a function of the composting time. It was observed that all active nan nanocomposites were amorphous before the disintegration test, as expected from the intrinsic characteristics of the PLA used in this study. Endothermic peaks corresponding to the enthalpic relaxation process were observed in all

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materials just after Tg. These peaks are indicative of the PLA ageing before the beginning of the test, as it was reported in other studies on PLA structure (Hughes, Thomas, Byun and Whiteside, 2012; Burgos, Martino and Jiménez, 2013). However, these samples developed multiple endothermic peaks just after 7 days. These peaks are related to the formation of crystalline structures with different perfection degrees in the PLA matrix during degradation, which was promoted by the hydrolysis process. Similar results were already reported by other authors, who suggested that the appearance of multiple melting peaks could be related to the formation of different crystal structures due to the PLA chain scission produced during the degradation process (Fukushima, Abbate, Tabuani, Gennari and Camino, 2009; Fortunati, Armentano, Iannoni, et al, 2012; Gorrasi and Pantani, 2013). DSC thermograms recorded during the second heating scan did not reveal any crystallization and melting peaks, as it was expected. However, it was observed that Tg values decreased with the testing time, upon 21 days of study (Figure 2.30). This behaviour could be associated with the increase in the mobility of the polymer chains as a consequence of hydrolysis and formation of low molar mass oligomers, producing some plasticizing effect (Sinha Ray, Yamada, Okamoto and Ueda, 2003; Proikakis, Mamouzelos, Tarantili and Andreopoulos, 2006; Burgos, Martino and Jiménez, 2013; Gorrasi and Pantani, 2013). Active nanocomposites with thymol showed a clear decrease in Tg between 7 and 14 testing days, suggesting that the formation of lactic acid oligomers and the addition of thymol would increase the above-referred plasticizing effect.

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Figure 2.29. DSC thermograms obtained for nanocomposites processed by injection moulding at different times under composting conditions at 58 °C (first heating scan (10 °C min-1)).

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Results and Discussion. Chapter 2

60

Tg (ºC) (second heating scan)

55 50 45 40 35 30 25 20 0

3

PLA

PLA/Ag

6 PLA/T6

9 12 Time (days) PLA/T8

15 PLA/Ag/T6

18

21

PLA/Ag/T8

Figure 2.30. Tg values for nanocomposite submitted to injection moulding at 0 and 21 days of disintegration under composting conditions at 58 °C (second heating scan).

6.5.2. Disintegrability study for films The disintegrability of PLA and PLA active nanocomposite films in composting conditions was studied to evaluate the degradation in natural environment of these films. The visual evaluation of all samples at different degradation times showed considerable changes, with a clear whitening, transparency loss and evident deformation and size reduction after 2 days (Figure 2.31). These results were indicative of the beginning of the hydrolytic degradation as it was shown previously in the study with injection moulded samples. Other authors also related the hydrolytic degradation process in PLA nanocomposites and the increase in their opacity to various simultaneous phenomena, such as the formation of low molar-mass degradation by-products during hydrolysis due to the water absorption and increase in the PLA crystallinity (Fukushima, Tabuani,

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Results and Discussion. Chapter 2

Arena, Gennari and Camino, 2013).. After 4 days, all the tested materials became brittle and just small pieces of films were recovere recovered. The faster degradation of these active nanocomposite films when compared to the previously discussed injected probes can be explained by the lower thickness of films, which showed considerable modifications in colour and a general loss of transparency after fter 7 days under composting conditions.

Figure 2.31. Visual appearance of neat PLA and active nanocomposite films at different testing days under composting conditions at 58 °C.

Figure 2.32 shows the evolution of disintegrability values (%) of films vs time (days). A progressive degradation of samples with the burial time was observed, which was visually corroborated by the clear whitening and transparency loss and evident deformation observed in samples ((Figure

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Results and Discussion. Chapter 2

2.31). A similar behaviour was reported by Fortunati et al, indicating that the PLA hydrolysis begins in the amorphous region of the polymer structure producing an overall increase in the polymer crystallinity (Fortunati, Rinaldi, et al, 2014). Furthermore, the results obtained before the beginning of the burial test suggest the influence of thymol on PLA degradation, since significantly higher disintegration values were obtained for PLA/T6, PLA/T8, PLA/Ag/T6 and PLA/Ag/T8 compared to PLA or PLA/Ag samples (p < 0.05). As previously discussed, the thymol hydroxyl groups can contribute to the PLA hydrolysis after absorbing water from the composting medium, resulting in a noticeable increase in disintegrability values for thymol-containing PLA nanocomposites. After 4 days of treatment, no significant differences (p > 0.05) were observed for all samples regardless of their composition (Figure 2.32), showing similar weight loss and disintegrability ratio. It should be also highlighted that the testing temperature (58 °C) was higher than the glass transition temperature of the nanocomposites, previously reported in the 40-45 ºC range, resulting in some induction of the crystallization process into the amorphous zones in the polymer matrix and chain mobility, accelerating the hydrolytic degradation process. This behaviour could also be related to the low thickness of the tested samples (Fortunati, Armentano, Iannoni, et al, 2012). It was observed that after 14 days of composting test all films reached the degradation values higher than 90 % (as indicated in the ISO 20200 standard for a biodegradable material). These results suggest that these active nanocomposite films could be used as biodegradable materials requiring short disintegration times.

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Results and Discussion. Chapter 2

a a a a a a

100 90

Disintegration (%)

PLA 80

PLA/Ag

70

PLA/T6

a a a a a a

PLA/T8 60 50

PLA/Ag/T6 PLA/Ag/T8

a a a a a a

40 30 a a a a a b b b b b

20 10

a a b b d d c c

0 1

2

4 Time (days)

7

14

Figure 2.32. Disintegrability (%) of neat PLA and nanocomposite films at different times under composting conditions at 58 °C (mean ± SD, n = 3). The line at 90 % represents the goal of disintegrability test as required by the ISO 20200 standard. Different superscripts over different samples at the same time indicate statistically significant different values (p < 0.05).

6.6. Release tests from PLA-based films The controlled release of active compounds depends on many parameters, some of them intrinsic to the system components, such as additives mobility, which is determined by the particle size, molecular weight and geometry of the diffusing compounds (Huang, Li and Zhou, 2015), as well as the solubility and diffusivity of additives in the matrix (Peltzer, Wagner and Jiménez, 2009), pH value, temperature, polymer structure and

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viscosity, mechanical stress, contact time, and food composition (Huang, Li and Zhou, 2015). Migration tests to determine the amount of thymol and Ag-NPs released from PLA-based films with time were carried out at different temperatures in ethanol 10% (v/v) at 40 °C for 10 days by following the current legislation (Commission_Regulation/(EU)/No-10/2011). 6.6.1. Silver release The total amount of silver released from PLA nanocomposite films was directly determined by ICP-MS from migration solutions. Results obtained for 2, 4, 6, 12, 24, 48 and 120 hours were lower than the LOQ of the instrument (3.98 µg kg-1), indicating a low Ag release at low times. Table 2.12 shows the results obtained for the total release of from binary (PLA/Ag) and ternary systems (PLA/Ag/T6 and PLA/Ag/T8) after 10 days at 40 °C and calculated as the quantity of silver per kilogram of food simulant. Results were well below the legislation limits for silver (0.05 mg Ag kg-1 food) (EFSA, 2005) although there is not a specific legislation for Ag-NPs. Echegoyen et al described the release of Ag-NPs as a superposition of two simultaneous processes: a surface release and the oxidative dissolution of silver into the ethanol medium. Therefore, the silver species present in ethanol solutions and detected in our tests after 10 days of contact probably correspond to Ag+ ions liberated by the oxidation of the Ag-NPs (Song, Li, Lin, Wu and Chen, 2011; Echegoyen and Nerín, 2013). This result is important bto have an estimation of the eventual migration of Ag-NPs in these nanocomposites, but more work with other analytical techniques is required for the detection and characterization of nanoparticles migrated into food simulants and real foodstuff and to evaluate the toxicological effects of Ag-NPs.

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Table 2.12. Thymol and Ag-NPs migration (ethanol 10 % (v/v) after 10 days at 40 °C) and DPPH scavenging activity (%) of PLA-based films. Thymol and Ag-NPs migrated at 10 days Samples

DPPH Scavenging activity (%)

mgThy (kgsimulant)-1

µgAg-NPs (kgsimulant)-1

PLA

n.d.

n.d.

--

PLA/Ag

n.d.

5.9 ± 0.7a

--

PLA/T6

13.4 ±

1.1a

n.d.

36.9 ± 2.2a

PLA/T8

18.2 ± 2.5b

n.d.

48.0 ± 0.1b

PLA/Ag/T6

27.2 ± 0.7c

7.1 ± 1.8a

44.3 ±1.1c

PLA/Ag/T8

34.0 ± 1.7d

8.6 ± 0.3a

51.8 ± 0.3d

Results are represented as the means ± standard deviation, n=3. Different superscripts within the same column indicate statistically significant different values (p < 0.05).

6.6.2. Thymol release Table 2.12 also shows the amount of thymol released after 10 days of contact between films and ethanol 10 % (v/v) at 40 °C. As expected, thymol release increased at high contents, showing significant differences between all formulations (p < 0.05). The highest migration levels were obtained for the ternary systems; in particular PLA/Ag/T8 (34.0 ± 1.7 mg (kgsimulant)-1). This result could give an indication of some protection of AgNPs to limit thymol losses during processing permitting the preservation of the active component and limiting losses at the extrusion temperatures. This effect was also observed by Efrati et al when blending thymol with different clays (Efrati et al, 2014), concluding that the increase in the clay content permitted the significant reductionin thymol losses during processing. In this context, the release kinetics of thymol in ethanol 10 % (v/v) was studied for 15 days (Figure 2.33). As it can be seen, the incorporation of Ag-NPs into the polymer matrix resulted in the increase of the total amount of migrated thymol. Although migration tests were carried out for

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15 days, it can be observed that the migration steady state was not clearly reached for the whole testing time (Figure 2.33). Therefore, the estimation of the maximum concentration of thymol able to migrate in ethanol 10 % (v/v) at t→∞, C∞, was performed by using the Weibull approach (Equation 2.15) (see solid lines in Figure 2.33), =

∞ [1

− exp(−

′

) ] (2.15)

where Ct is the cumulative concentration (ppm) of thymol (mass of thymol per kilogram of food simulant) released at time t, and k’ and d are constant values. Initially, based on empirical observations, Equation (2.15) can be used for a first assessment of the diffusion mechanism, since d and k’ are closely related to the mechanism and rate release constant, respectively (Papadopoulou, Kosmidis, Vlachou and Macheras, 2006; Costa, Valente, Miguel and Queiroz, 2011). It can be seen that the fit of C∞ values increased with the incorporation of Ag-NPs to the PLA matrix according to previous results, which related some protection of Ag-NPs to thymol losses (Table 2.13).

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Results and Discussion. Chapter 2

40

(b)

(a)

Ct (ppm)

30

20

10

0 0

100

200

300

400

0

time (hours)

100

200

300

400

time (hours)

Figure 2.33. Release kinetics of thymol from binary systems (black dots) and ternary systems (white dots) at 6 wt% (a) and 8 wt% (b), at 40 °C. Solid lines were obtained by fitting the Equation (2.15) to the experimental data points.

The reliability of the fitting of Equation (2.15) to experimental data was confirmed by comparing the estimated C∞ values with the maximum concentration of thymol available to migrate into the food simulant. The computed C∞ values corresponded to around 19  2 % and 22  2 % of the initial amount of thymol loaded into PLA matrices (for PLA/T6 and PLA/T8, respectively) and 41  6 % and 31  3 % of the initial amount of thymol into PLA/Ag/T6 and PLA/Ag/T8, respectively; showing that the estimated values were well below the total amount of thymol present in PLA matrices, but the release could be potentially higher in active nanocomposites than in binary PLA/thymol combinations.

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Table 2.13. Fitting parameters of Equations (2.14), (2.15) and (2.16) to experimental migration data of thymol loaded in binary and ternary systems into ethanol 10% (v/v) at 40 °C. PLA/T6

PLA/Ag/T6

PLA/T8

PLA/Ag/T8

Equation (2.15). Weibull approach

C∞ (ppm)

18.7 ± 1.7

42.7 ± 6.4

23.3 ± 1.6

40.6 ± 3.0

k’ (10−3 h−1)

5.5 ±1.3

4.7 ± 2.0

10.4 ± 2.2

8.2 ± 1.9

d

0.76 ±0.05

0.65 ± 0.06

0.77 ± 0.07

0.78 ± 0.06

R2

0.9963

0.9965

0.9924

0.9969

Equations (2.16) and (2.17). Power law equation

n

0.69 ± 0.03

0.60 ± 0.03

0.63 ± 0.02

0.65 ± 0.04

MDT * (h)

104

137

68

84

R2

0.9910

0.9869

0.9934

0.9853

*

Mean dissolution time (MDT): calculated from Eq. (2.16) taking into account short-range time migration (Ct/C∞ 0.05. For calculation of the p values, PLA versus PLA-based nanocomposite films results were compared at 3 and 24 h for S. aureus 8325-4 and E. coli RB.

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A slight reduction in the bacterial growth was observed for binary systems containing thymol against Escherichia coli showing no significant differences (p > 0.05) after 3 and 24 h of incubation. Moreover, these films significantly inhibited the growth of Sthaphylococcus aureus after 3 h. These results are in agreement with those obtained in a previous study where the concentration of thymol added into PP films was not enough to inhibit the growth of Escherichia coli. The AM activity of thymol has been proposed as consisting on binding to membrane proteins by means of hydrogen bonding, thereby changing the permeability characteristics of the membrane. Therefore, the AM activity of thymol is strongly dependent on the physico-chemical characteristics and composition of the bacterial membranes (Trombetta et al, 2005). The mechanism of action is based on the disturbance of the cytoplasmic bacterial membrane, disrupting the proton motive force (PMF), electron flow, active transport and coagulation of cell contents (Burt, 2004). In fact, most studies investigating the action of EOs and their components against food spoilage organisms and foodborne pathogens agree that, generally, all these compounds (including thymol) are more active against Grampositive than against Gram-negative bacteria, such as Escherichia coli, since they possess an outer layer surrounding the cell wall, primarily composed of lipids, proteins and lipopolysaccharides and forming a hydrophilic permeability barrier providing protection against the diffusion of hydrophobic compounds through them. In contrast, the cell wall of Gram-positive bacteria, such as Sthaphylococcus aureus, does not contain lipopolysaccharides and, consequently, thymol can be more susceptible to inhibit its growth (Feng et al, 2000; Maneerung, Tokura and Rujiravanit, 2008).

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Results and Discussion. Chapter 2

Active nanocomposite films containing Ag-NPs showed relevant antibacterial activity against both tested bacteria obtaining significant differences (p < 0.001) for each incubation time at 3 and 24 h compared to the PLA control film. The antibacterial activity of ternary nanocomposites was higher with Sthaphylococcus aureus than with Escherichia coli regardless of the incubation time and temperatures, confirming similar results reported by other authors (Kvítek et al, 2008; Jokar, Abdul Rahman, Ibrahim, Abdullah and Tan, 2012). Erem et al evaluated the antibacterial activity of PLA fibres with Ag-NPs against Sthaphylococcus aureus and Klebsiella pneumonia (Gram-negative) bacteria, concluding that Ag-NPs were more effective against Sthaphylococcus aureus (Erem, Ozcan, Erem and Skrifvars, 2013). These results may be also attributed to the structure and mode of antibacterial action of Ag-NPs as well as to differences in the cell wall structure of Gram-positive and Gram-negative cells (Jokar, Abdul Rahman, Ibrahim, Abdullah and Tan, 2012; Reidy, Haase, Luch, Dawson and Lynch, 2013). However, the mechanism of action of Ag-NPs has not been well established and several possibilities have been proposed to explain the AM activity of Ag-NPs. Some authors have focused on cell membrane disruption due to the interaction of Ag-NPs with phosphorous and sulphur containing compounds of proteins, preventing DNA replication. Other studies focused on the binding of the positively charged Ag-NPs with negatively charged bacterial cell membranes, disrupting cell walls and surface proteins. A third mechanism is related to the penetration of AgNPs into bacteria, which inactivates enzymes producing H2O2. All these possible mechanisms finally lead to the cells death (Kanmani and Rhim, 2014a). Furthermore, some studies have also shown that the toxicity of Ag-NPs vary significantly depending on their dimensions and shape, since

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Results and Discussion. Chapter 2

small nanoparticles have larger relative surface areas for the Ag+ release, with higher protein binding efficiencies and passing easily through pores in bacterial membranes (Duncan, 2011). The effect of temperature on the antibacterial activity efficiency of PLA and PLA-based nanocomposite films with thymol and Ag-NPs was also investigated. Results for the studied films showed their high activity against both tested bacteria obtaining significant differences (p < 0.001) for each incubation temperature at 3 and 24 h compared to the PLA control sample, and slight differences between both bacteria. The presence of thymol results in increasing the antibacterial effect of Ag-NPs due to the bacterial membranes damage caused by thymol. The presence of both active agents (thymol and Ag-NPs) may cross the cell membranes more efficiently, penetrating the interior of cells and interacting with intracellular critical sites for the antibacterial activity. This fact could be related with the higher amount of Ag and thymol released from the PLA matrix when both additives are incorporated, since it could be suggested that bacteria inactivation is most likely caused by the Ag+ ions release. Further studies need to be performed to elucidate this aspect since the amount of silver and thymol released depends on several factors as it has been previously mentioned in the migration results.

7. Conclusions Active nanocomposites based on PLA, thymol and silver nanoparticles were successfully processed by extrusion in the form of injection moulded samples and films and they were further characterized in terms of thermal, optical, mechanical, morphological, and barrier properties. Disintegrability under composting conditions was also studied. The applicability of films

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Results and Discussion. Chapter 2

to food packaging was also evaluated by evaluating their migration, antibacterial and antioxidant performance. The identification of Ag-NPs and thymol into the PLA matrix was successfully carried out. FESEM micrographs showed good distribution of both additives through the PLA matrix, with homogenous surfaces and highlighting the presence of silver nanoparticles successfully embedded into the polymer matrix. Moreover, the determination of thymol by HPLC-UV demonstrated that this additive remains after processing in a significant amount and could act as active additive. The combination of both additives influenced PLA thermal properties. DSC results showed that the addition of thymol resulted in a decrease in the Tg of PLA, favouring some plasticization of the polymer matrix. The presence of thymol and Ag-NPs into the PLA-based films also influenced the thermal stability of the ternary systems. An enhancement in the barrier properties to water vapour was also obtained by the incorporation of thymol, providing improved protection to packaged food. The degradation study of all active nanocomposites under composting conditions showed that the inherent biodegradable character of PLA was improved by the addition of thymol and Ag-NPs, getting a faster degradation rate and meeting the biodegradation legal requirements. Active nanocomposite films containing Ag-NPs and thymol, in particular PLA/Ag/T8, showed positive results concerning antibacterial and AO activity, demonstrating their effectiveness in the inhibition of the growth of foodborne bacteria and the radical scavenging inhibition by DPPH method. The amount of thymol and Ag-NPs released into aqueous food simulant suggested that the release of thymol is influenced by the presence of Ag-NPs in the PLA matrix.

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Results and Discussion. Chapter 2

In conclusion, PLA/Ag/T8 showed potential to be applied as bio-based active film with biodegradable character and AM and AO properties to extend the shelf -life of food products.

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Prepared by Melt Extrusion and Solvent Casting. European Polymer Journal, 71, 126-139.

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IV.

General Conclusions

General Conclusions

According to the proposed objectives and the results obtained in the present work, the following conclusions can be obtained: I.

PP-based active films containing 4, 6 and 8 wt% of thymol and/or

carvacrol

were

obtained

by

melt-blending

and

compression-moulding and further characterized by using different analytical techniques. The active films showed their homogeneity after processing despite, certain porosity observed by SEM at the highest additives concentration (8 wt%). Some decrease in the elastic modulus was observed for the active formulations compared with neat PP. The presence of additives did not affect the thermal stability of PP, but resulted in some decrease in crystallinity and oxygen barrier properties. The presence of thymol and carvacrol also increased the stabilization against thermo-oxidative degradation of PP-based films, with higher oxidation induction parameters when using 8 wt% of thymol and carvacrol; suggesting that the polymer is well stabilized and a certain amount of these compounds remained in the polymer matrix after processing. II.

The incorporation of 8 wt% of carvacrol and thymol as active additives into PP films for food packaging resulted in the possibilities of their controlled release with possible activity in protecting food from oxidative and microbiological degradation. Analytical methods for the determination of the target compounds in aqueous and fatty food simulants were successfully developed and validated. The release of the studied additives from films was dependent on the food simulant used in the tests and their amount effectively incorporated into the polymer matrix. Diffusion coefficients were calculated and a Fickian-type

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General Conclusions

model was developed for the release kinetics of thymol and carvacrol. The addition of thymol and carvacrol in PP-based films showed higher inhibition against Gram-positive bacteria, in particular for thymol. PP-based active films were also tested in direct contact with food, clearly increasing the quality and shelflife of strawberries and bread samples. III.

Active nanocomposite films based on PLA with a commercial organo-modified montmorillonite were prepared with 2.5 and 5 wt% of D43B and 8 wt% of thymol by melt-blending and compression-moulding. Around 70-75 % of thymol remained in the active nanocomposites after processing, ensuring their posterior applicability in active systems. These films showed an intercalated structure of the D43B nanoparticles through the polymer matrix, exhibiting a partial exfoliation in ternary nanocomposites, with thymol and D43B. Likewise, some decrease in toughness was observed due to some slight plasticizing effect induced by the presence of thymol, also observed by a clear decrease in the PLA glass transition temperature. Nevertheless, the addition of thymol did not significantly affect the thermal stability of PLA and oxygen barrier properties. Some differences in films colour were observed by the addition of thymol and D43B, being larger for films with the highest concentration of the nanoclay. Nevertheless, the intrinsic transparency of PLA was not affected by the addition of both components resulting in fully transparent active films.

IV.

The release of thymol from PLA matrices was determined by HPLC-UV at different times and a kinetic model was proposed, suggesting that the release of thymol was influenced by the

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General Conclusions

presence of D43B since diffusion coefficients were different for binary and ternary nanocomposites. The continuous release of thymol favoured the antioxidant activity of these films in contact with

food,

which

was

determined

by

using

the

spectrophotometric DPPH method, resulting in a high percentage of inhibition. Finally, the addition of D43B showed some effect in the improvement in the antibacterial activity of thymol-based films, with higher inhibition against Staphylococcus aureus than against Escherichia coli. V.

Ag-NPs (1 wt%) and thymol (6 and 8 wt%) were added into PLA matrices to obtain active nanocomposites and processed by extrusion, successfully obtaining injection moulded samples and films. FESEM micrographs showed the good distribution of both additives all through the PLA matrix, resulting in an improvement in water vapour barrier properties. Some plasticization of the polymer matrix could be related with the addition of thymol and the consequent decrease in the glass transition temperature. Likewise, the presence of thymol and Ag-NPs into the PLAbased films also had some influence on the thermal stability of the ternary systems, slightly decreasing the PLA behaviour at high temperatures.

VI.

Active nanocomposite films containing Ag-NPs and thymol showed positive results concerning antibacterial and antioxidant activities, demonstrating their effectiveness in the inhibition of the growth of foodborne bacteria and in the radical scavenging activity. The amount of thymol and Ag-NPs released into aqueous food simulants suggested that the release of thymol was influenced by the presence of Ag-NPs in the PLA matrix

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General Conclusions

resulting in some protection of the nanoparticles to the thymol release. VII.

The degradation study of all active nanocomposites under composting conditions showed that the inherent biodegradable character of PLA remained after the incorporation of the active additive and the nanoparticles. In fact, the incorporation of 8 wt% of thymol to PLA-based formulations increased the disintegration rate of the polymer matrix, due to the presence of the reactive free hydroxyl groups. The combination of thymol and Ag-NPs or thymol and D43B induced higher degradation rates, suggesting their advantages in industrial applications where biodegradation could be an issue, such as in food packaging.

VIII.

In summary, PLA-based films with 2.5 wt% of D43B and 8 wt% of thymol and PLA-based films with 1 wt% of Ag-NPs and 8 wt% of thymol showed their potential as bio-based active films with biodegradable character and antimicrobial/antioxidant performance to extend the shelf-life and quality of food products.

As a general conclusion, it could be stated that the addition of phenolic compounds, such as carvacrol and thymol, to conventional or bio-based and biodegradable polymers for food packaging showed high potential to improve quality and safety aspects of the packed food. It should be highlighted that these systems could be a valid and successful commercial alternative to the current antioxidant/antimicrobial formulations in food industry, most of them involving the direct addition of chemicals to food.

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