desarrollo de sistemas de envasado activo mediante la
TRANSCRIPT
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 UNIVERSIDAD 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 MAT2014-59242-
C2-2-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
~ 3 ~
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
Resumen
~ 4 ~
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
Resumen
~ 5 ~
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 (5-
isopropil-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
Resumen
~ 6 ~
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
Resumen
~ 7 ~
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.
Resumen
~ 8 ~
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 físico-
químicas 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.
Resumen
~ 9 ~
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.
Resumen
~ 10 ~
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.
Resumen
~ 11 ~
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-
Resumen
~ 12 ~
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-2-
picril-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
Resumen
~ 13 ~
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
Resumen
~ 14 ~
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
Resumen
~ 15 ~
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 Ag-
NPs (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
Resumen
~ 16 ~
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
Resumen
~ 17 ~
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
Resumen
~ 18 ~
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
Resumen
~ 19 ~
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
Resumen
~ 20 ~
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
Resumen
~ 21 ~
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
Resumen
~ 22 ~
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 Ag-
NPs. 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
Resumen
~ 23 ~
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.
~ I ~
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. Active packaging ............................................................................................... 17
1.2.1. Antimicrobial and antioxidant active packaging ................................... 20
2. Natural additives .......................................................................................................... 26
2.1. Antimicrobial activity of essential oils .......................................................... 29
2.2. Antioxidant activity of essential oils ............................................................. 32
2.3. Carvacrol and Thymol ..................................................................................... 33
2.3.1. Use in packaging materials ........................................................................ 36
3. Nanotechnology in the food industry ...................................................................... 41
3.1. Nanoclays ........................................................................................................... 43
3.2. Silver nanoparticles (Ag-NPs) ........................................................................ 49
3.3. Nanocomposites in food packaging ............................................................. 53
3.3.1. Preparation and processing ....................................................................... 57
4. 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
II. OBJECTIVES .................................................................................................................... 99
III. RESULTS AND DISCUSSION ...................................................................... 103
1 Chapter 1 ................................................................................................................................. 113
1. Introduction ............................................................................................................... 115
2. Experimental .............................................................................................................. 122
2.1. Materials and chemicals ................................................................................ 122
Table of content
~ II ~
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. Oxygen transmission rate (OTR) .......................................................... 126
2.4. 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. Statistical analysis ........................................................................................... 135
3. 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
Table of content
~ III ~
3.2. Migration study .............................................................................................. 145
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. Study of the effectiveness of the active films to preserve perishable food 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
2 Chapter 2 ................................................................................................................................ 183
1. Introduction ............................................................................................................... 187
3 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
Table of content
~ IV ~
2.7. Statistical analysis ........................................................................................... 206
3. 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
4. Conclusions ................................................................................................................ 241
4 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
Table of content
~ V ~
5.7.2. 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
6. 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. Disintegrability study for films .............................................................. 285
6.6. 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
IV. General Conclusions .............................................................................................. 317
~ VII ~
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
Figures and Tables
~ VIII ~
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
Figures and Tables
~ IX ~
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 5.0
5.0
0,
, 111
F
tF
MM versus 5.0t 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
Figures and Tables
~ X ~
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 versus 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
0.5
,
,0
1 1 F t
F
MM
5.0t
Figures and Tables
~ XI ~
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
Figures and Tables
~ XII ~
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
Figures and Tables
~ XIII ~
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 HS-
SPME-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
Figures and Tables
~ XIV ~
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
Figures and Tables
~ XV ~
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
Abbreviations and symbols
~ XVI ~
Abbreviations and symbols 2θ Scattering angle (2theta) ∆E* Total colour difference in CIELAB space
∆Hc Crystallization enthalpy ∆Hm Melting enthalpy εB Elongation at break εY Elongation at yield ρ Density χ (%) Percentage of crystallinity a* 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 Elastic Modulus E. coli RB 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
Abbreviations and Symbols
~ XVII ~
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
Abbreviations and symbols
~ XVIII ~
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
I. Introduction
Introduction
~ 3 ~
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).
Introduction
~ 4 ~
39.6%Packaging
20.3%Building and construction
8.5%Automative
5.6%
4.3%
21.7%Others
Agriculture
Consumer and household appliances,furniture, sport, health and safety,…
46.3 Million tonnes
Electrical and Electronics
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
Introduction
~ 5 ~
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).
Introduction
~ 6 ~
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
Introduction
~ 7 ~
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
Introduction
~ 8 ~
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
Biotechnology and chemistry
Reuse
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/non-
renewable origin and biodegradable/non-biodegradable character (Figure
I.3):
Introduction
~ 9 ~
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 sugar-
rich 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 non-
biodegradable 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(ε-
caprolactone) (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.
Introduction
~ 10 ~
Bioplastic
e.g. Bio-based PE, Bio-based PET, Bio-
based PP
Bioplastic
e.g. PLA, PHA, Starch, Cellulose,
Gelatines
Conventional plastics
e.g. PE, PP, PET
Bioplastic
e.g. PBAT, PCL
Non-biodegradable Biodegradable
Fossil-based
Bio-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-Bioplastics-
Association, 2015b). This continuous growth in the bioplastics production
for food packaging is also possible by the similarity in machinery and
Introduction
~ 11 ~
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.
11.4 %PLA
11.3 %Starch blends
10.8 %PBAT, PBS, PCL
37 %Bio-based PET
12.3 %Bio-based PE
6.8 %PTT
Regenerated Cellulose (1.7 %)
Othersb (0.3 %)
Bio-based PA (4.9 %)Othersa (1.4 %)
PHA (2.1 %)
Bio-based and Biodegradable 25.8 %bBiodegradable cellulose ester
aContains durable starch blends, Bio-PC, Bio-TPE, Bio-PUR (except thermosets)
Bio-based and non Biodegradable 62.4 % Fossil-based and Biodegradable 10.8 %
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.
Introduction
~ 12 ~
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; Farmahini-
Farahani, 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
Introduction
~ 13 ~
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(L-
lactic 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, Finne-
Wistrand and Kumar, 2010). In tissue engineering, PLA has demonstrated
to be biocompatible and degradable into non-toxic components with an
Introduction
~ 14 ~
in-vivo controllable degradation rate having a long history of use in
degradable surgical sutures (Lopes, Jardini and Filho, 2014; Salerno,
Fernández-Gutiérrez, San Román del Barrio and Domingo, 2015)
Production Lactic acid (2-hydroxy propionic acid) (LA) is the single monomer of PLA
and it is produced either by fermentation of
commercial name for D-glucose) and other sugars extracted from plants,
such as corn sugarcane, sugar beet and potatoes,
LA has a chiral carbon atom and exists in two
stereoisomers, namely L- and D- enantiomers (
Kumar, 2007; Jamshidian, Tehrany, Imran, Jacquot and Desobry, 2010)
D-Lactic Acid (D-LA) L-Lactic Acid
Figure I.5. Chemical structures of LA.
PLA has variable molar mass, ranging from 1000
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
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 from L
been described (Figure I.6) (Södergård and Stolt, 2010)
controllable degradation rate having a long history of use in
(Lopes, Jardini and Filho, 2014; Salerno,
Gutiérrez, San Román del Barrio and Domingo, 2015).
hydroxy propionic acid) (LA) is the single monomer of PLA
and it is produced either by fermentation of dextrose (the common
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
enantiomers (Figure I.5) (Gupta and
Kumar, 2007; Jamshidian, Tehrany, Imran, Jacquot and Desobry, 2010).
Lactic Acid (L-LA)
Chemical structures of LA.
PLA has variable molar mass, ranging from 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
(Jamshidian, Tehrany, Imran, Jacquot and
. 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).
A from L-LA and D-LA have
(Södergård and Stolt, 2010):
Introduction
~ 15 ~
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).
D-LA L-LA
Direct Condensation
Azeotropic Solution
High molar mass PLA
Mw: 100-300 kDa
Melt State
Oligo(lactic acid)
Polycondensation Oligo(lactic acid)Depolymerization
L-Lactide / meso-Lactide / D-Lactide
ROP
Low molar mass PLA
Mw: 1000-5000
Chain coupling agent
Solid State
Polycondensation reactions
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
Introduction
~ 16 ~
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 meso-
lactide, 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
Introduction
~ 17 ~
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
Introduction
~ 18 ~
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.
~ 19 ~
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
well as food quality and safety (López-Gómez e
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
oxygen, CO2, ethylene, moisture and/or odour and flavour taints;
releasing oxygen, CO2, water vapour, ethanol, sorbates, AOs and/or other
Introduction
Active packaging includes the use of additives, which could be defined as
“freshness enhancers”, able to diffuse and interact with food by increasing
, such as resistance to oxidation and
of natural aromas and flavours as
Gómez et al, 2009). Figure I.8
summarizes the main causes of food spoilage and the strategies followed
Mechanisms that can cause a loss of quality in food and active packaging
The chemical nature of the additives used for such purpose is diverse, but
all them should play their role efficiently by scavenging or absorbing
, ethylene, moisture and/or odour and flavour taints;
, water vapour, ethanol, sorbates, AOs and/or other
Introduction
~ 20 ~
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-de-
Dicastillo, 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.
Introduction
~ 21 ~
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 oxygen-
sensitive 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:
Introduction
~ 22 ~
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
Introduction
~ 23 ~
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)
(c) (d)
Packaging
Inner Layer Outer LayerCoating Outer Layer
PackagingPackaging
Packaging
Food Food
FoodFood
Release Release
Release
Not release Direct contact
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.
Introduction
~ 24 ~
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.
Introduction
~ 25 ~
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) (Cruz-
Romero, 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.
Introduction
~ 26 ~
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-de-
Dicastillo, 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, Hernández-
Muñoz, 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
Introduction
~ 27 ~
different natural sources proposed for their use in food packaging
applications.
Animal origin Plant origin
•Casein and whey
•Lysozyme
•Lactoferrin
•Chitosan
•Lipids
•Plant-derived compounds
•EOs
•Plant extracts
•Phenolic compounds, quinones,
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 Gram-
positive bacteria common in many food products, Staphylococcus aureus and
Listeria monocytogenes. Moreover, high AO activity was also reported for this
EO.
Introduction
~ 28 ~
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, Andrade-
Ochoa, 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
~ 29 ~
films showed high AO character by the incorporation of nanocapsules to
permit the controlled release of α-tocopherol
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
Carvacrol, (499-75-2)
Geraniol, (106-24
Thymol, (89-83-8)
Linalool, (78-70-
Tocopherol, (1406-66-2, mixed of tocopherols)
α: (CH3; R1); (CH3; R2) β: (CH3; R1); (H; R2) γ: (H; R
Benzoic acid, (65-85-0)
Citric acid, (77-92
Quercetin, (117-39-5)
(−)-Catechin, (18829
Introduction
films showed high AO character by the incorporation of nanocapsules to
herol to food simulants.
Chemical structures of some natural additives incorporated in active food
packaging with AO/AM character (CAS numbers are indicated in parentheses).
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
24-1)
Sorbic acid, (110-44-1)
-6)
Gallic acid, (149-91-7)
2, mixed of tocopherols)
) γ: (H; R1); (CH3; R2) δ: (H; R1); (H; R2)
92-9)
(R)-(+)-Limonene, (5989-27-5)
(18829-70-4)
3-Hydroxytyrosol, (10597-60-1)
Introduction
~ 30 ~
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).
~ 31 ~
Figure I.12. Structure of the bacterial cell wall (adapted from
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
Sonneveld, Miltz and Bigger, 2011b; Sung
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
(Sánchez-González, Vargas, González-Martínez, Chiralt and Cháfer,
2011). Different authors have reported the effectiveness of EOs to inhibit
different pathogenic and spoilage microorganisms, including Gram
positive bacteria, such as Staphylococcus aureus
Bacillus cereus; Gram-negative bacteria, such as
enteritidis, Salmonella choleraesuis, Yersinia enterocoli
aeruginosa; yeasts, such as Saccharomyces
Debaryomyces hansenii; and molds, such as Alternaria alternate, Aspergillus niger,
Botrytis cinerae, Aspergillus flavus, Penicllium roqueforti
Sonneveld, Miltz and Bigger, 2011b).
Introduction
(adapted from (Aldred, Buck and Vall,
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,
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 increase the AM action
Martínez, Chiralt and Cháfer,
. Different authors have reported the effectiveness of EOs to inhibit
and spoilage microorganisms, including Gram-
Staphylococcus aureus, Listeria monocytogenes and
negative bacteria, such as Escherichia coli, Salmonella
, Salmonella choleraesuis, Yersinia enterocolitica and Pseudomonas
Saccharomyces cerevisiae, Candida albicans,
Alternaria alternate, Aspergillus niger,
Botrytis cinerae, Aspergillus flavus, Penicllium roqueforti (Kuorwel, Cran,
Introduction
~ 32 ~
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)
Introduction
~ 33 ~
(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-5-
methylphenol) are two phenolic monoterpenes widely studied as natural
additives in active packaging. Both compounds are isomers obtained from
Introduction
~ 34 ~
EOs obtained from many aromatic plants of the
Origanum, Satureja, Thymbra, Thymus and Corydothymus
Papalia, 2012). Their structures and main physico
shown in Table I.3.
Table I.3. Structures and physico-chemical properties of carvacrol and thymol.
Carvacrol / 5-Isopropyl
CAS number
Molecular weight
Boiling point
Tm
δ
Physical state at RT
Thymol / 2-Isopropyl
CAS number
Molecular weight
Boiling point
Tm
δ
Physical state at RT
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 and Xu, 2008; Nerioa, Olivero
Verbel and Stashenko, 2010; Babili et al, 2011)
of thymol and carvacrol against different microorganisms, such as Gram
positive bacteria, Gram-negative bacteria, food spoilage or pathogenic
EOs obtained from many aromatic plants of the Labiatae family, including
Corydothymus species (Nostro and
. Their structures and main physico-chemical properties are
chemical properties of carvacrol and thymol.
Isopropyl-2-methylphenol
499-75-22
150.22 (g mol-1)
236-237 °C
3-4 °C
0.976 g mL-1 at 20 °C
Liquid (oily)
Isopropyl-5-methylphenol
89-83-8
150.22 (g mol-1)
232 °C
48-51 °C
0.965 g mL-1 at 25 °C
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
(Ahn, Lee, Lee and Kim, 1998; Chizzola, Michitsch and Franz, 2008;
d Xu, 2008; Nerioa, Olivero-
, 2011). In this context, the effect
of thymol and carvacrol against different microorganisms, such as Gram-
negative bacteria, food spoilage or pathogenic
Introduction
~ 35 ~
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 Gram-
positive 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).
Introduction
~ 36 ~
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 physico-
chemical 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).
Introduction
~ 37 ~
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).
Introduction
~ 38 ~
Table I.4. Carvacrol and thymol in active food packaging.
Polymer base Additives Amount Potential studies Effect of the additive References
AM Packaging
PLA/PCL Thymol 0, 3, 6, 9, and 12 wt%
AM activity by observing the bacterial growth and counting the
colony-forming units (CFU).
Inhibition of Escherichia coli and Listeria monocytogenes. (Wu, Qin, et al, 2014)
PLA/poly(trimethylene carbonate), PTMC
Thymol 0, 3, 6, 9 and 12 wt%
Mechanical characterization, water vapour permeability (WVP), and
AM activity.
Inhibition of Escherichia coli, Staphylococcus aureus, Listeria, Bacillus
subtilis, and Salmonella. (Wu, Yuan, et al, 2014)
Starch Thymol and carvacrol 1, 3 and 5 wt%
AM activity by using the agar disc diffusion method and inoculation on
the surfaces of Cheddar cheese.
Inhibition of Staphylococcus aureus in vitro or inoculated on the surfaces
of Cheddar cheese.
(Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2011a)
TPS Thymol and carvacrol 3 and 4 wt%
Release of additives into isooctane as a fattyfood simulant.
Controlled release of both additives.
(Kuorwel, Cran, Sonneveld, Miltz and Bigger, 2013)
SPI/PLA Thymol
2.5, 5, 10, 15, 20, 25 and 50 wt%
AM activity by using the agar disc diffusion method.
Inhibition of Staphylococcus aureus, Escherichia coli, Aspergillus spp. and
Saccharomyces cerevisiae.
(González and Alvarez Igarzabal, 2013)
Chitosan Carvacrol 0.01-3 % (w/v) AM efficiency by the vapour phase.
Inhibition of Bacillus subtilis, Listeria innocua, Escherichia coli and
Salmonella enteritidis.
(Kurek, Moundanga, Favier, Galić and Debeaufort, 2013)
Chitosan/cyclodextrin films Carvacrol 2.3 g/g of dry film
AM activity of carvacrol-CS films on chicken breast fillets.
Inhibition of the total aerobic bacteria, and Pseudomonas spp.,
Enterobacteria, Lactic acid bacteria, yeasts and fungi.
(Higueras, López-Carballo, Hernández-Muñoz, Catalá and
Gavara, 2014)
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.
Inhibition of Staphylococcus aureus and Escherichia coli.
(Higueras, López-Carballo, Gavara and Hernández-Muñoz, 2015)
Introduction
~ 39 ~
Table I.4. (Cont.)
Calcium caseinate Sodium caseinate
Carvacrol 10 wt%
AM activity by using the agar disc diffusion method.
Inhibition of Staphylococcus aureus and Escherichia coli.
(Arrieta, Peltzer, Garrigós and Jiménez, 2013; Arrieta, Peltzer, et
al, 2014)
Chitosan Carvacrol 1 wt% (nanoemulsion)
AM effect of the combined treatment of coating, MAP and
gamma-irradiation to green beans during storage.
Inhibition of Escherichia coli and Salmonella typhimurium. (Severino et al, 2015)
Pectin-based apple, carrot, and hibiscus edible films
Carvacrol 0.5, 1.5 and 3 wt%
AM activity by the inoculation of ham samples with Listeria.
monocytogenes.
Inhibition of Listeria monocytogenes on contaminated ham samples. (Ravishankar et al, 2012)
AO Packaging
HDPE Carvacrol 1 and 2 wt%
Release of additives into water and olive oil as a food simulant. Controlled release of carvacrol. (Peltzer, Wagner and Jiménez,
2009)
Corn-zein-laminated/LDPE Thymol and carvacrol 1.5, 3 and 5 % (v/v)
Minimum effective concentrations, release kinetics in the gas and liquid
phases and study of the lipid oxidation in fresh ground beef
packaging.
Inhibition of lipid oxidation and positive effect on the colour
stability of beef patties during storage.
(Park et al, 2012)
AO and AM packaging
LLDPE Thymol 1.48, 2.17, 3.81 %
Characterization of the mass transfer during the migration
process of thymol from LLDPE films
Controlled release of thymol from LLDPE impregnated with this
additive
(Torres, Romero, Macan, Guarda and Galotto, 2014)
Bovine gelatin Films Carvacrol 1, 2, 3, 4 and 5 wt%
AM activity by using the agar disc diffusion method and
decolourization method with ABTS to AO activity.
Inhibition of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and
Pseudomonas aeruginosa.
(Kavoosi, Dadfar, Mohammadi Purfard and Mehrabi, 2013)
Introduction
~ 40 ~
Table I.4. (Cont.)
Bovine gelatin Films Thymol 1, 2, 3, 4 and 8 wt%
AM activity by using the agar disc diffusion method and
decolourization method with ABTS to AO activity.
Inhibition of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and
Pseudomonas aeruginosa. AO activity was increased with increasing the concentration of
thymol in the films.
(Kavoosi, Dadfar and Purfard, 2013)
Chitosan Carvacrol
0.5, 1.0 and 1.5 % (v/v)
AM activity by using agar diffusion method and AO activity by using radical scavenging capacity using
DPPH method TEAC.
Inhibition of Escherichia coli O157:H7 and Salmonella
typhimurium. Increase of AO capacity.
(López-Mata et al, 2013)
Strawberry puree edible films Carvacrol 0.75 wt%
AO activity by using an adaptation of the DPPH method and
identification of major fungal species in real food.
Inhibition of fungal species until 10 days in real food and
improvement of fruit quality. (Peretto et al, 2014)
Introduction
~ 41 ~
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,
Introduction
~ 42 ~
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).
Organic origin Inorganic origin
MetalsSilverCopperGoldPalladiumIron
Metal OxidesZnOTiO2MgOAgOCuO
ClaysMicaMontmorilloniteSepioliteLaponite
Natural biopolymersChitosanChitinStarchCellulose
Natural antimicrob. agents Nisin
Carbon nanofillersFullerenesGrapheneCarbon nanotubes
Figure I.14. Nanofillers in food packaging applications.
Introduction
~ 43 ~
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).
Introduction
~ 44 ~
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 Cation Interlayer
cations CEC
(meq/100g) Aspect Ratio
Saponites
Ca0.25
(Mg,Fe)3((Si,Al)
4O
10)(OH)
2nH
2O Mg2+ Na+
Ca2+ Mg2+
86.6 50-60
Montmorillonites
(Na,Ca)0.33
(Al,Mg)2(Si
4O
10)(OH)
2nH
2O Al3+ Na+
Ca2+ Mg2+
110 100-150
Hectorites
Na0.3
(Mg,Li)3(Si
4O
10)(F,OH)
2 Mg2+ Na+
Ca2+ Mg2+
120 200-300
Introduction
~ 45 ~
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 organo-
modified 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
Introduction
~ 46 ~
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).
Introduction
~ 47 ~
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 d-
spacing of nanocomposites with respect to the original matrix (1.60 nm
for C30B and 1.65 nm for D43B).
Introduction
~ 48 ~
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
typea
CEC
(meq/100g)
Interlayer
spacing (Å)
WLignition
(%)b
Used by…
Supplier: Southern Clay Products (USA)
Cloisite® Na / CNA none - 11.7 7 (Dias et al, 2014; Shemesh, Goldman, et al, 2015)
Cloisite®10A / C10A N+(Me)2(benzyl) (T) 125 19.3 39 (Shemesh, Goldman, et al, 2015)
Cloisite®15A / C15A N+(Me)2(T)2 125 31.5 43 (Araújo, Botelho, Oliveira and Machado, 2014; Shemesh,
Goldman, et al, 2015)
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
and Machado, 2014; Efrati et al, 2014)
Supplier: Laviosa Chimica Mineraria (Italy)
Dellite® 43B / D43B N+(Me)2(benzyl)(T) 66 16.6 (Scatto et al, 2013; Araújo, Botelho, Oliveira and Machado, 2014) aTallow (T): ~ 65 % C18; ~ 30 % C16; ~5 % C14 bWLignition: Weight Loose on ignition
Introduction
~ 49 ~
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,
Introduction
~ 50 ~
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 negative-
charged 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 Ag-
NPs 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
Introduction
~ 51 ~
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.
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,
(B)
(A)
Introduction
~ 52 ~
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.
Introduction
~ 53 ~
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
Introduction
~ 54 ~
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).
Introduction
~ 55 ~
Table I.7. Representative examples of nanocomposites application in food packaging. Polymer matrix Nanofiller Amount Processing Effect of nanofiller References
Quinoa starch Au nanoparticles 2.5 and 5 % (v/v)
Solvent casting (82 °C)
Inhibition of 99 % against Escherichia coli and 98 % against Staphylococcus aureus.
Improvement in mechanical and optical performance, maintaining the thermal and
barrier properties.
(Pagno et al, 2015)
Food-grade agar Ag-NPs 0, 0.2, 0.5, 1.0 and 2.0
wt%
Solvent casting (95 °C)
Increase in WVP and surface hydrophobic character.
Strong AM activity against Listeria monocytogenes and Escherichia coli.
(Rhim, Wang and Hong, 2013)
Gelatin-based ZnO nanoparticles N.R.
Solvent casting (80 °C)
Antibacterial activity against Gram-positive and Gram-negative bacteria. Strong activity
against Listeria monocytogenes. Enhanced thermal stability.
(Shankar, Teng, Li and Rhim, 2015)
LDPE TiO2 nanoparticles 0.05, 0.08 and 0.11 g TiO2 in 100 mL ethyl
methyl ketone
Manual Coating
AM activity of the films exposed to fluorescent and UV radiation increased with the TiO2
nanoparticles concentration.
(Othman, Abd Salam, Zainal, Kadir Basha and
Talib, 2014)
PE TiO2 nanoparticles 3 wt%
Melt extrusion (130 °C)
Improved barrier properties. Excellent AM activity against Pseudomonas spp.
and ethylene photodegradation.
(Bodaghi, Mostofi, Oromiehie, Ghanbarzadeh
and Hagh, 2015)
PLA TiO2 nanoparticles 1, 3.5 and 8 wt% Melt
blending (180 °C)
Improvement of E and crystallization temperature.
AM activity increase under UVA irradiation. (Fonseca et al, 2015)
LDPE Ag-NPs 0.1, 0.3, 0.5, 3 and 5 wt%
Melt blending (140 °C)
AM activity against Staphylococcus aureus and Escherichia coli.
(Jokar, Abdul Rahman, Ibrahim, Abdullah and Tan,
2010)
PLA Silver/MMT 1, 5 and 10 wt% Solvent casting (RT)
Migration levels of silver, within the legislation and high AM activity against Salmonella spp.
(Busolo, Fernandez, Ocio and Lagaron, 2010)
Introduction
~ 56 ~
Table I.7. (cont) Corn starch
Chitosan-MMT Laponite RD 5 wt% Blending
(RT) Reinforcing effect: Improvement of E and
tensile strength. (Chung et al, 2010)
Chitosan C30B 5 wt% Solvent casting (60 °C)
Reinforcing effect: Improvement of E and tensile strength.
Reduction of oxygen transmission rate (OTR).
(Rodríguez, Galotto, Guarda and Bruna, 2012)
PLA C30B
Fluoro-hectorite/SOMASIF MEE (SOMMEE)
5 and 10 wt% Melt
blending (165 °C)
Acceleration in the degradation of PLA in compost at 40 °C.
(Fukushima, Tabuani, Arena, Gennari and Camino,
2013)
PLA Cellulose nanocrystals from Posidonia oceanica 1 and 3 wt%
Solvent casting (RT)
Migration levels into two food simulants well below the European legislative limits. (Fortunati et al, 2015)
(Not reported, N.R.)
Introduction
~ 57 ~
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
Introduction
~ 58 ~
swelling the layered silicates in a suitable solvent to promote the diffusion
of the macromolecular chains in the clay galleries; and (iii) melt-
intercalation, 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 nano-
biocomposites. 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
Introduction
~ 59 ~
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
Introduction
~ 60 ~
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.
Introduction
~ 61 ~
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.
Introduction
~ 62 ~
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.
Introduction
~ 63 ~
Table I.8. Comparison between two active nanocomposites based on edible films and S-EO and ZMB-EO.
Active nanocomposite Nanofiller/EO (%) AM test against εB (%) E (MPa) TS (MPa) WVP
(g s−1 m−1 Pa−1·10−10) References
Agar film solution/CNC/S-EO
2.5/0 Listeria monocytogenes Staphylococcus
aureus Bacillus cereus Escherichia coli
51.7 ± 2.3 55.8 ± 4.2 31.2 ±0.8 1.60 ± 0.01
(Atef, Rezaei and Behrooz, 2015)
2.5/0.5 46.2± 2.8 63.0 ± 3.8 28.3 ± 0.8 1.53 ± 0.13
2.5/1 49.4 ±3.1 57.0 ± 2.5 28.1 ± 1.6 1.82 ± 0.12
2.5/1.5 51.7 ± 4.9 46.5 ± 4.2 20.4 ± 1.7 2.34 ± 0.13
κ-carragenan/MMT/ZMB-EO
0/0 Staphylococcus aureus
Bacillus cereus Escherichia coli Pseudomona Aeruginosa Salmonella
typhimurium
36.5 ±1.0 N.R. 26.3 ± 2.9 2.31 ± 0.05
(Shojaee-Aliabadi et al, 2014)
5/0 27.4 ±1.6 N.R. 32.9 ± 2.1 1.72 ± 0.09
5/1 38.6 ± 1.6 N.R. 18.1 ± 1.2 0.58 ± 0.04
5/2 40.6 ± 1.2 N.R. 14.8 ± 1.1 0.48 ± 0.02
5/3 44.4 ± 1.9 N.R. 13.2 ± 0.9 0.36 ± 0.04
Introduction
~ 64 ~
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
Introduction
~ 65 ~
or anaerobic conditions by International standard methods (UNE-
EN_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.
Introduction
~ 66 ~
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, Lopez-
Rubio 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,
Introduction
~ 67 ~
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,
Introduction
~ 68 ~
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 two-
dimensional 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.
흏푪풑흏풕
= 푫흏ퟐ푪풑흏풙ퟐ
Introduction
~ 69 ~
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).
Introduction
~ 70 ~
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
Introduction
~ 71 ~
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
Introduction
~ 72 ~
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.
Regulation (EC) 450/2009Active and Intelligent materials
Directive 2007/42/ECRegenerated cellulose film
Directive 2005/31/ECCeramic articles
Regulation (UE) 10/2011Plastics materials
Regulation (EC)1935/2004
Regulation (EC) 2023/2006Good manufacturing practice
Regulation (EC) 282/2008Recycled plastic materials
RD 847/2011Positive list of permitted substances
Labelling
Traceability Declaration of compliance
Safeguard measures General requirements
Active and intelligent
Evaluation and authorisation of substances
General aspects
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
Introduction
~ 73 ~
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
Introduction
~ 74 ~
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).
Introduction
~ 75 ~
6. References
Abdollahi, M., Rezaei, M., Farzi, G. (2012). A Novel Active Bionanocomposite
Film Incorporating Rosemary Essential Oil and Nanoclay into Chitosan.
Journal of Food Engineering, 111, 343-350.
Ahmed, M.J., Murtaza, G., Mehmood, A., Bhatti, T.M. (2015). Green Synthesis of
Silver Nanoparticles Using Leaves Extract of Skimmia Laureola:
Characterization and Antibacterial Activity. Materials Letters, 153, 10-13.
Ahn, Y.J., Lee, S.B., Lee, H.S., Kim, G.H. (1998). Insecticidal and Acaricidal
Activity of Carvacrol and β-Thujaplicine Derived from Thujopsis
Dolabrata Var. Hondai Sawdust. Journal of Chemical Ecology, 24, 81-90.
Albertsson, A.C., Varma, I.K., Lochab, B., Finne-Wistrand, A., Kumar, K. (2010).
Design and Synthesis of Different Types of Poly(Lactic Acid). In: Auras,
R., Lim, L.-T., Selke, S.E.M. & Tsuji, H. (eds.) Poly(Lactic Acid): Synthesis,
Structures, Properties, Processing, and Applications. John Wiley & Sons, Inc.,
Hoboken, NJ, USA, pp. 43-58.
Alboofetileh, M., Rezaei, M., Hosseini, H., Abdollahi, M. (2014). Antimicrobial
Activity of Alginate/Clay Nanocomposite Films Enriched with Essential
Oils against Three Common Foodborne Pathogens. Food Control, 36, 1-
7.
Aldred, E.M., Buck, C., Vall, K. (2009). Chapter 29 - Antimicrobials. In: Vall,
E.M.a.B.B. (ed.) Pharmacology. Churchill Livingstone, Edinburgh, pp.
217-221.
Alexandre, M., Dubois, P. (2000). Polymer-Layered Silicate Nanocomposites:
Preparation, Properties and Uses of a New Class of Materials. Materials
Science and Engineering R: Reports, 28, 1-63.
Amenta, V., Aschberger, K., Arena, M., et al (2015). Regulatory Aspects of
Nanotechnology in the Agri/Feed/Food Sector in EU and Non-EU
Countries. Regulatory Toxicology and Pharmacology, 73, 463-476.
Amorati, R., Foti, M.C., Valgimigli, L. (2013). Antioxidant Activity of Essential
Oils. Journal of Agricultural and Food Chemistry, 61, 10835-10847.
Introduction
~ 76 ~
Amorati, R., Valgimigli, L. (2015). Advantages and Limitations of Common
Testing Methods for Antioxidants. Free Radical Research, 49, 633-649.
Anbinder, P.S., Peruzzo, P.J., Martino, M.N., Amalvy, J.I. (2015). Effect of
Antioxidant Active Films on the Oxidation of Soybean Oil Monitored
by Fourier Transform Infrared Spectroscopy. Journal of Food Engineering,
151, 43-50.
Arana-Sánchez, A., Estarrón-Espinosa, M., Obledo-Vázquez, E.N., et al (2010).
Antimicrobial and Antioxidant Activities of Mexican Oregano Essential
Oils (Lippia Graveolens H. B. K.) with Different Composition When
Microencapsulated in β-Cyclodextrin. Letters in Applied Microbiology, 50,
585-590.
Araújo, A., Botelho, G., Oliveira, M., Machado, A.V. (2014). Influence of Clay
Organic Modifier on the Thermal-Stability of PLA Based
Nanocomposites. Applied Clay Science, 88–89, 144-150.
Arrieta, M.P., Fortunati, E., Dominici, F., et al (2014). PLA-PHB/Cellulose Based
Films: Mechanical, Barrier and Disintegration Properties. Polymer
Degradation and Stability, 107, 139-149.
Arrieta, M.P., Peltzer, M.A., Garrigós, M.C., Jiménez, A. (2013). Structure and
Mechanical Properties of Sodium and Calcium Caseinate Edible Active
Films with Carvacrol. Journal of Food Engineering, 114, 486-494.
Arrieta, M.P., Peltzer, M.A., López, J., et al (2014). Functional Properties of
Sodium and Calcium Caseinate Antimicrobial Active Films Containing
Carvacrol. Journal of Food Engineering, 121, 94-101.
Artiaga, G., Ramos, K., Ramos, L., Cámara, C., Gómez-Gómez, M. (2015).
Migration and Characterisation of Nanosilver from Food Containers by
AF4-ICP-MS. Food Chemistry, 166, 76-85.
Atef, M., Rezaei, M., Behrooz, R. (2015). Characterization of Physical,
Mechanical, and Antibacterial Properties of Agar-Cellulose
Bionanocomposite Films Incorporated with Savory Essential Oil. Food
Hydrocolloids, 45, 150-157.
Introduction
~ 77 ~
Auras, R.A., Harte, B., Selke, S., Hernández, R. (2003). Mechanical, Physical, and
Barrier Properties of Poly(Lactide) Films. Journal of Plastic Film and
Sheeting, 19, 123-135.
Avérous, L. (2008). Polylactic Acid: Synthesis, Properties and Applications. In:
Belgacem, M.N. & Gandini, A. (eds.) Monomers, Polymers and Composites
from Renewable Resources. Elsevier, Amsterdam, pp. 433-450.
Babili, F.E., Bouajila, J., Souchard, J.P., et al (2011). Oregano: Chemical Analysis
and Evaluation of Its Antimalarial, Antioxidant, and Cytotoxic
Activities. Journal of Food Science, 76, C512-C518.
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.
Bastarrachea, L., Dhawan, S., Sablani, S. (2011). Engineering Properties of
Polymeric-Based Antimicrobial Films for Food Packaging: A Review.
Food Engineering Reviews, 3, 79-93.
Beltrán, A., Valente, A.J.M., Jiménez, A., Garrigós, M.a.C. (2014).
Characterization of Poly(ɛ-Caprolactone)-Based Nanocomposites
Containing Hydroxytyrosol for Active Food Packaging. Journal of
Agricultural and Food Chemistry, 62, 2244-2252.
Bodaghi, H., Mostofi, Y., Oromiehie, A., Ghanbarzadeh, B., Hagh, Z.G. (2015).
Synthesis of Clay–TiO2 Nanocomposite Thin Films with Barrier and
Photocatalytic Properties for Food Packaging Application. Journal of
Applied Polymer Science, 132, In press. doi: 10.1002/app.41764.
Bordes, P., Pollet, E., Avérous, L. (2009). Nano-Biocomposites: Biodegradable
Polyester/Nanoclay Systems. Progress in Polymer Science, 34, 125-155.
Bumbudsanpharoke, N., Ko, S. (2015). Nano-Food Packaging: An Overview of
Market, Migration Research, and Safety Regulations. Journal of Food
Science, 80, R910-R923.
Burgos, N., Martino, V.P., Jiménez, A. (2013). Characterization and Ageing Study
of Poly(Lactic Acid) Films Plasticized with Oligomeric Lactic Acid.
Polymer Degradation and Stability, 98, 651-658.
Introduction
~ 78 ~
Burgos, N., Tolaguera, D., Fiori, S., Jiménez, A. (2014). Synthesis and
Characterization of Lactic Acid Oligomers: Evaluation of Performance
as Poly(Lactic Acid) Plasticizers. Journal of Polymers and the Environment,
22, 227-235.
Burt, S. (2004). Essential Oils: Their Antibacterial Properties and Potential
Applications in Foods-a Review. International Journal of Food Microbiology,
94, 223-253.
Busolo, M.A., Fernandez, P., Ocio, M.J., Lagaron, J.M. (2010). Novel Silver-Based
Nanoclay as an Antimicrobial in Polylactic Acid Food Packaging
Coatings. Food Additives and Contaminants - Part A Chemistry, Analysis,
Control, Exposure and Risk Assessment, 27, 1617-1626.
Calo, J.R., Crandall, P.G., O'bryan, C.A., Ricke, S.C. (2015). Essential Oils as
Antimicrobials in Food Systems – a Review. Food Control, 54, 111-119.
Cao, L., Si, J.Y., Liu, Y., et al (2009). Essential Oil Composition, Antimicrobial
and Antioxidant Properties of Mosla Chinensis Maxim. Food Chemistry,
115, 801-805.
Ćavar Zeljković, S., Maksimović, M. (2015). Chemical Composition and
Bioactivity of Essential Oil from Thymus Species in Balkan Peninsula.
Phytochemistry Reviews, 14, 335-352.
Costa, S.S., Druzian, J.I., Machado, B.a.S., De Souza, C.O., Guimaraes, A.G.
(2014). Bi-Functional Biobased Packing of the Cassava Starch, Glycerol,
Licuri Nanocellulose and Red Propolis. PLoS ONE, 9, DOI:
10.1371/journal.pone.0112554.
Cruz-Romero, M.C., Murphy, T., Morris, M., Cummins, E., Kerry, J.P. (2013).
Antimicrobial Activity of Chitosan, Organic Acids and Nano-Sized
Solubilisates for Potential Use in Smart Antimicrobially-Active
Packaging for Potential Food Applications. Food Control, 34, 393-397.
Cumkur, E.A., Baouz, T., Yilmazer, U. (2015). Poly(Lactic Acid)-Layered Silicate
Nanocomposites: The Effects of Modifier and Compatibilizer on the
Morphology and Mechanical Properties. Journal of Applied Polymer Science,
132, In press. doi: 10.1002/app.42553.
Introduction
~ 79 ~
Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M., Cummins, E. (2012).
Nanotechnologies in the Food Industry – Recent Developments, Risks
and Regulation. Trends in Food Science & Technology, 24, 30-46.
Chizzola, R., Michitsch, H., Franz, C. (2008). Antioxidative Properties of Thymus
Vulgaris Leaves: Comparison of Different Extracts and Essential Oil
Chemotypes. Journal of Agricultural and Food Chemistry, 56, 6897-6904.
Chung, Y.L., Ansari, S., Estevez, L., et al (2010). Preparation and Properties of
Biodegradable Starch–Clay Nanocomposites. Carbohydrate Polymers, 79,
391-396.
Dainelli, D., Gontard, N., Spyropoulos, D., Van Den Beuken, E.Z., Tobback, P.
(2008). Active and Intelligent Food Packaging: Legal Aspects and Safety
Concerns. Trends in Food Science & Technology, 19, S103–S112.
Dallas, P., Sharma, V.K., Zboril, R. (2011). Silver Polymeric Nanocomposites as
Advanced Antimicrobial Agents: Classification, Synthetic Paths,
Applications, and Perspectives. Advances in Colloid and Interface Science,
166, 119-135.
De Azeredo, H.M.C. (2009). Nanocomposites for Food Packaging Applications.
Food Research International, 42, 1240-1253.
De Azeredo, H.M.C. (2013). Antimicrobial Nanostructures in Food Packaging.
Trends in Food Science & Technology, 30, 56-69.
Dias, M.V., Machado Azevedo, V., Borges, S.V., et al (2014). Development of
Chitosan/Montmorillonite Nanocomposites with Encapsulated α-
Tocopherol. Food Chemistry, 165, 323-329.
Du, W.-X., Avena-Bustillos, R.J., Hua, S.S.T., Mchugh, T.H. (2011).
Antimicrobial Volatile Essential Oils in Edible Films for Food Safety. In:
Mendez-Vilas, A. (ed.) Science against Microbial Pathogens: Communicating
Current Research and Technological Advances. Formatex Research Center,
Badajoz, pp. 1124-1134.
Echegoyen, Y., Nerín, C. (2013). Nanoparticle Release from Nano-Silver
Antimicrobial Food Containers. Food and Chemical Toxicology, 62, 16-22.
Introduction
~ 80 ~
Efrati, R., Natan, M., Pelah, A., et al (2014). The Combined Effect of Additives
and Processing on the Thermal Stability and Controlled Release of
Essential Oils in Antimicrobial Films. Journal of Applied Polymer Science,
131, In press. doi: 10.1002/app.40564.
Erem, A.D., Ozcan, G., Erem, H., Skrifvars, M. (2013). Antimicrobial Activity of
Poly(L-Lactide Acid)/Silver Nanocomposite Fibers. Textile Research
Journal, 83, 2111-2117.
Ezhilarasi, P.N., Karthik, P., Chhanwal, N., Anandharamakrishnan, C. (2013).
Nanoencapsulation Techniques for Food Bioactive Components: A
Review. Food and Bioprocess Technology, 6, 628-647.
Fages, E., Pascual, J., Fenollar, O., Garcia Sanoguera, D., Balart Gimeno, R.
(2011). Study of Antibacterial Properties of Polypropylene (PP) Filled
with Surfactant-Coated Silver Nanoparticles (Ag Nps). Polymer
Engineering & Science, 51, 804-811.
Farmahini-Farahani, M., Xiao, H., Zhao, Y. (2014). Poly Lactic Acid
Nanocomposites Containing Modified Nanoclay with Synergistic Barrier
to Water Vapor for Coated Paper. Journal of Applied Polymer Science, 131,
In press. doi: 10.1002/app.40952.
Feng, Q.L., Wu, J., Chen, G.Q., et al (2000). A Mechanistic Study of the
Antibacterial Effect of Silver Ions on Escherichia Coli and Staphylococcus
Aureus. Journal of Biomedical Materials Research, 52, 662-668.
Fonseca, C., Ochoa, A., Ulloa, M.T., et al (2015). Poly(Lactic Acid)/TiO2
Nanocomposites as Alternative Biocidal and Antifungal Materials.
Materials Science and Engineering C, 57, 314-320.
Fortunati, E., Luzi, F., Puglia, D., et al (2015). Processing of PLA
Nanocomposites with Cellulose Nanocrystals Extracted from Posidonia
Oceanica Waste: Innovative Reuse of Coastal Plant. Industrial Crops and
Products, 67, 439-447.
Fortunati, E., Peltzer, M., Armentano, I., Jiménez, A., Kenny, J.M. (2013).
Combined Effects of Cellulose Nanocrystals and Silver Nanoparticles
Introduction
~ 81 ~
on the Barrier and Migration Properties of Pla Nano-Biocomposites.
Journal of Food Engineering, 118, 117-124.
Fortunati, E., Rinaldi, S., Peltzer, M., et al (2014). Nano-Biocomposite Films with
Modified Cellulose Nanocrystals and Synthesized Silver Nanoparticles.
Carbohydrate Polymers, 101, 1122-1133.
Friedman, M. (2014). Chemistry and Multibeneficial Bioactivities of Carvacrol (4-
Isopropyl-2-Methylphenol), a Component of Essential Oils Produced by
Aromatic Plants and Spices. Journal of Agricultural and Food Chemistry, 62,
7652-7670.
Fuciños, C., Míguez, M., Cerqueira, M., et al (2015). Functional Characterisation
and Antimicrobial Efficiency Assessment of Smart Nanohydrogels
Containing Natamycin Incorporated into Polysaccharide-Based Films.
Food and Bioprocess Technology, 8, 1430-1441.
Fukushima, K., Giménez, E., Cabedo, L., Lagarón, J.M., Feijoo, J.L. (2012). Biotic
Degradation of Poly(DL-Lactide) Based Nanocomposites. Polymer
Degradation and Stability, 97, 1278-1284.
Fukushima, K., Tabuani, D., Abbate, C. (2011). Chapter 5 Biopolymer-Based
Nanocomposites. In: Sharma, S.K. & Mudhoo, A. (eds.) A Handbook of
Applied Biopolymer Technology: Synthesis, Degradation and Applications. The
Royal Society of Chemistry, Cambridge, pp. 129-148.
Fukushima, K., Tabuani, D., Arena, M., Gennari, M., Camino, G. (2013). Effect
of Clay Type and Loading on Thermal, Mechanical Properties and
Biodegradation of Poly(Lactic Acid) Nanocomposites. Reactive and
Functional Polymers, 73, 540-549.
Ghorani, B., Tucker, N. (2015). Fundamentals of Electrospinning as a Novel
Delivery Vehicle for Bioactive Compounds in Food Nanotechnology.
Food Hydrocolloids, 51, 227-240.
Gocek, I., Adanur, S. (2012). Effect of Nanoclay Amount and Compatibilizer
Presence on Thermal, Morphological and Mechanical Behaviour of
Nanoclay Reinforced Composite Polypropylene Cast Film. Materials
Testing, 54, 318-329.
Introduction
~ 82 ~
Gómez-Estaca, J., López-De-Dicastillo, C., Hernández-Muñoz, P., Catalá, R.,
Gavara, R. (2014). Advances in Antioxidant Active Food Packaging.
Trends in Food Science & Technology, 35, 42-51.
Gonçalves, C.M.B., Tomé, L.C., Garcia, H., et al (2013). Effect of Natural and
Synthetic Antioxidants Incorporation on the Gas Permeation Properties
of Poly(Lactic Acid) Films. Journal of Food Engineering, 116, 562-571.
González, A., Alvarez Igarzabal, C.I. (2013). Soy Protein - Poly (Lactic Acid)
Bilayer Films as Biodegradable Material for Active Food Packaging. Food
Hydrocolloids, 33, 289-296.
Gorrasi, G. (2015). Dispersion of Halloysite Loaded with Natural Antimicrobials
into Pectins: Characterization and Controlled Release Analysis.
Carbohydrate Polymers, 127, 47-53.
Gupta, A.P., Kumar, V. (2007). New Emerging Trends in Synthetic
Biodegradable Polymers – Polylactide: A Critique. European Polymer
Journal, 43, 4053-4074.
Gutierrez, J., Barry-Ryan, C., Bourke, P. (2009). Antimicrobial Activity of Plant
Essential Oils Using Food Model Media: Efficacy, Synergistic Potential
and Interactions with Food Components. Food Microbiology, 26, 142-150.
Gyawali, R., Ibrahim, S.A. (2014). Natural Products as Antimicrobial Agents. Food
Control, 46, 412-429.
Hazzit, M., Baaliouamer, A., Veríssimo, A.R., Faleiro, M.L., Miguel, M.G. (2009).
Chemical Composition and Biological Activities of Algerian Thymus
Oils. Food Chemistry, 116, 714-721.
Higueras, L., López-Carballo, G., Gavara, R., Hernández-Muñoz, P. (2015).
Incorporation of Hydroxypropyl-β-Cyclodextrins into Chitosan Films
To tailor Loading Capacity for Active Aroma Compound Carvacrol.
Food Hydrocolloids, 43, 603-611.
Higueras, L., López-Carballo, G., Hernández-Muñoz, P., Catalá, R., Gavara, R.
(2014). Antimicrobial Packaging of Chicken Fillets Based on the Release
of Carvacrol from Chitosan/Cyclodextrin Films. International Journal of
Food Microbiology, 188, 53-59.
Introduction
~ 83 ~
Huang, J.Y., Li, X., Zhou, W. (2015). Safety Assessment of Nanocomposite for
Food Packaging Application. Trends in Food Science & Technology, 45, 187-
199.
Hughes, J., Thomas, R., Byun, Y., Whiteside, S. (2012). Improved Flexibility of
Thermally Stable Poly-Lactic Acid (PLA). Carbohydrate Polymers, 88, 165-
172.
Inkinen, S., Hakkarainen, M., Albertsson, A.C., Sodergard, A. (2011). From Lactic
Acid to Poly(Lactic Acid) (PLA): Characterization and Analysis of Pla
and Its Precursors. Biomacromolecules, 12, 523-532.
Iturrondobeitia, M., Okariz, A., Guraya, T., Zaldua, A.M., Ibarretxe, J. (2014).
Influence of the Processing Parameters and Composition on the
Thermal Stability of PLA/Nanoclay Bio-Nanocomposites. Journal of
Applied Polymer Science, 131, 9120-9127.
Jain, K., Madhu, G., Bhunia, H., et al (2015). Physico-Mechanical Characterization
and Biodegradability Behavior of Polypropylene/Poly(L-Lactide)
Polymer Blends. Journal of Polymer Engineering, 35, 407-415.
Jamshidian, M., Tehrany, E., Desobry, S. (2013). Antioxidants Release from
Solvent-Cast PLA Film: Investigation of Pla Antioxidant-Active
Packaging. Food and Bioprocess Technology, 6, 1450-1463.
Jamshidian, M., Tehrany, E.A., Imran, M., Jacquot, M., Desobry, S. (2010). Poly-
Lactic Acid: Production, Applications, Nanocomposites, and Release
Studies. Comprehensive Reviews in Food Science and Food Safety, 9, 552-571.
Jiang, X., Luo, Y., Tian, X., et al (2010). Chemical Structure of Poly(Lactic Acid).
In: Auras, R., Lim, L.T., Selke, S.E.M. & Tsuji, H. (eds.) Poly(Lactic Acid):
Synthesis, Structures, Properties, Processing, and Applications. John Wiley &
Sons, Inc., Hoboken, N.J., U.S.A., pp. 69-82.
Jokar, M., Abdul Rahman, R., Ibrahim, N.A., Abdullah, L.C., Tan, C.P. (2010).
Melt Production and Antimicrobial Efficiency of Low-Density
Polyethylene (LDPE)-Silver Nanocomposite Film. Food and Bioprocess
Technology, 1-10.
Introduction
~ 84 ~
Jollands, M., Gupta, R.K. (2010). Effect of Mixing Conditions on Mechanical
Properties of Polylactide/Montmorillonite Clay Nanocomposites. Journal
of Applied Polymer Science, 118, 1489-1493.
Kanmani, P., Rhim, J.W. (2014a). Physical, Mechanical and Antimicrobial
Properties of Gelatin Based Active Nanocomposite Films Containing
Agnps and Nanoclay. Food Hydrocolloids, 35, 644-652.
Kanmani, P., Rhim, J.W. (2014b). Physicochemical Properties of Gelatin/Silver
Nanoparticle Antimicrobial Composite Films. Food Chemistry, 148, 162-
169.
Kavoosi, G., Dadfar, S.M.M., Mohammadi Purfard, A., Mehrabi, R. (2013).
Antioxidant and Antibacterial Properties of Gelatin Films Incorporated
with Carvacrol. Journal of Food Safety, 33, 423-432.
Kavoosi, G., Dadfar, S.M.M., Purfard, A.M. (2013). Mechanical, Physical,
Antioxidant, and Antimicrobial Properties of Gelatin Films
Incorporated with Thymol for Potential Use as Nano Wound Dressing.
Journal of Food Science, 78, E244-E250.
Keshtkar, M., Nofar, M., Park, C.B., Carreau, P.J. (2014). Extruded PLA/Clay
Nanocomposite Foams Blown with Supercritical Co2. Polymer, 55, 4077-
4090.
Kim, J.S., Kuk, E., Yu, K.N., et al (2007). Antimicrobial Effects of Silver
Nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine, 3, 95-
101.
Klasen, H.J. (2000). A Historical Review of the Use of Silver in the Treatment of
Burns. II. Renewed Interest for Silver. Burns, 26, 131-138.
Kordali, S., Cakir, A., Ozer, H., et al (2008). Antifungal, Phytotoxic and
Insecticidal Properties of Essential Oil Isolated from Turkish Origanum
Acutidens and Its Three Components, Carvacrol, Thymol and ρ-
Cymene. Bioresource Technology, 99, 8788-8795.
Kovacevic, Z., Bischof, S., Fan, M. (2015). The Influence of Spartium Junceum L.
Fibres Modified with Montmorrilonite Nanoclay on the Thermal
Introduction
~ 85 ~
Properties of PLA Biocomposites. Composites Part B: Engineering, 78, 122-
130.
Kunthadong, P., Molloy, R., Worajittiphon, P., et al (2015). Biodegradable
Plasticized Blends of Poly(L-Lactide) and Cellulose Acetate Butyrate:
From Blend Preparation to Biodegradability in Real Composting
Conditions. Journal of Polymers and the Environment, 23, 107-113.
Kuorwel, K.K., Cran, M.J., Sonneveld, K., Miltz, J., Bigger, S.W. (2011a).
Antimicrobial Activity of Natural Agents Coated on Starch-Based Films
against Staphylococcus Aureus. Journal of Food Science, 76, M531-M537.
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., Guinault, A., Voilley, A., Galić, K., Debeaufort, F. (2014). Effect of
Relative Humidity on Carvacrol Release and Permeation Properties of
Chitosan Based Films and Coatings. Food Chemistry, 144, 9-17.
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.
Lavorgna, M., Attianese, I., Buonocore, G.G., et al (2014). MMT-Supported Ag
Nanoparticles for Chitosan Nanocomposites: Structural Properties and
Antibacterial Activity. Carbohydrate Polymers, 102, 385-392.
Introduction
~ 86 ~
Li, J.E., Nie, S.-P., Qiu, Z.H., et al (2010). Antimicrobial and Antioxidant
Activities of the Essential Oil from Herba Moslae. Journal of the Science of
Food and Agriculture, 90, 1347-1352.
Li, J.H., Miao, J., Wu, J.L., Chen, S.-F., Zhang, Q.-Q. (2014). Preparation and
Characterization of Active Gelatin-Based Films Incorporated with
Natural Antioxidants. Food Hydrocolloids, 37, 166-173.
Lim, L.T., Auras, R., Rubino, M. (2008). Processing Technologies for Poly(Lactic
Acid). Progress in Polymer Science, 33, 820-852.
Ljungberg, N., Wesslén, B. (2005). Preparation and Properties of Plasticized
Poly(Lactic Acid) Films. Biomacromolecules, 6, 1789-1796.
Lopes, M.S., Jardini, A.L., Filho, R.M. (2014). Synthesis and Characterizations of
Poly(Lactic Acid) for Biomedical Applications. Chemical Engineering
Transactions, 38, 331-336.
López-Gómez, A., Fernández, P., Palop, A., et al (2009). Food Safety
Engineering: An Emergent Perspective. Food Engineering Reviews, 1, 84-
104.
López-Mata, M., Ruiz-Cruz, S., Silva-Beltrán, N., et al (2013). Physicochemical,
Antimicrobial and Antioxidant Properties of Chitosan Films
Incorporated with Carvacrol. Molecules, 18, 13735-13753.
Llorens, A., Lloret, E., Picouet, P.A., Trbojevich, R., Fernandez, A. (2012).
Metallic-Based Micro and Nanocomposites in Food Contact Materials
and Active Food Packaging. Trends in Food Science & Technology, 24, 19-29.
Maneerung, T., Tokura, S., Rujiravanit, R. (2008). Impregnation of Silver
Nanoparticles into Bacterial Cellulose for Antimicrobial Wound
Dressing. Carbohydrate Polymers, 72, 43-51.
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, 508-
517.
Marques, H.M.C. (2010). A Review on Cyclodextrin Encapsulation of Essential
Oils and Volatiles. Flavour and Fragrance Journal, 25, 313-326.
Introduction
~ 87 ~
Mastelic, J., Jerkovic, I., Blazevic, I., et al (2008). Comparative Study on the
Antioxidant and Biological Activities of Carvacrol, Thymol, and
Eugenol Derivatives. Journal of Agricultural and Food Chemistry, 56, 3989-
3996.
Mellinas, C., Valdés, A., Ramos, M., et al (2015). Active Edible Films: Current
State and Future Trends. Journal of Applied Polymer Science, In press. doi:
10.1002/app.42631.
Mihaly Cozmuta, A., Peter, A., Mihaly Cozmuta, L., et al (2015). Active Packaging
System Based on Ag/Tio2 Nanocomposite Used for Extending the
Shelf Life of Bread. Chemical and Microbiological Investigations.
Packaging Technology and Science, 28, 271-284.
Mihindukulasuriya, S.D.F., Lim, L.T. (2014). Nanotechnology Development in
Food Packaging: A Review. Trends in Food Science & Technology, 40, 149-
167.
Moon, J.K., Shibamoto, T. (2009). Antioxidant Assays for Plant and Food
Components. Journal of Agricultural and Food Chemistry, 57, 1655-1666.
Munteanu, B., Aytac, Z., Pricope, G., Uyar, T., Vasile, C. (2014). Polylactic Acid
(PLA)/Silver-Np/Vitamin E Bionanocomposite Electrospun
Nanofibers with Antibacterial and Antioxidant Activity. Journal of
Nanoparticle Research, 16, 1-12.
Muriel-Galet, V., Cran, M.J., Bigger, S.W., Hernández-Muñoz, P., Gavara, R.
(2015). Antioxidant and Antimicrobial Properties of Ethylene Vinyl
Alcohol Copolymer Films Based on the Release of Oregano Essential
Oil and Green Tea Extract Components. Journal of Food Engineering, 149,
9-16.
Nerioa, L.S., Olivero-Verbel, J., Stashenko, E. (2010). Repellent Activity of
Essential Oils: A Review. Bioresource Technology, 101, 372–378.
Noronha, C.M., De Carvalho, S.M., Lino, R.C., Barreto, P.L.M. (2014).
Characterization of Antioxidant Methylcellulose Film Incorporated with
α-Tocopherol Nanocapsules. Food Chemistry, 159, 529-535.
Introduction
~ 88 ~
Nostro, A., Papalia, T. (2012). Antimicrobial Activity of Carvacrol: Current
Progress and Future Prospectives. Recent Patents on Anti-Infective Drug
Discovery, 7, 28-35.
Olivares-Maldonado, Y., Ramírez-Vargas, E., Sánchez-Valdés, S., et al (2014).
Effect of Organoclay Structure Characteristics on Properties of Ternary
PP-EP/EVA/Nanoclay Blend Systems. Polymer Composites, 35, 2241-
2250.
Othman, S.H. (2014). Bio-Nanocomposite Materials for Food Packaging
Applications: Types of Biopolymer and Nano-Sized Filler. Agriculture and
Agricultural Science Procedia, 2, 296-303.
Othman, S.H., Abd Salam, N.R., Zainal, N., Kadir Basha, R., Talib, R.A. (2014).
Antimicrobial Activity of TiO2 Nanoparticle-Coated Film for Potential
Food Packaging Applications. International Journal of Photoenergy, 2014, 6.
Pagno, C.H., Costa, T.M.H., De Menezes, E.W., et al (2015). Development of
Active Biofilms of Quinoa (Chenopodium Quinoa W.) Starch Containing
Gold Nanoparticles and Evaluation of Antimicrobial Activity. Food
Chemistry, 173, 755-762.
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., 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. (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.
Introduction
~ 89 ~
Peretto, G., Du, W.X., Avena-Bustillos, R.J., et al (2014). Increasing Strawberry
Shelf-Life with Carvacrol and Methyl Cinnamate Antimicrobial Vapors
Released from Edible Films. Postharvest Biology and Technology, 89, 11-18.
Poças, M.F., Oliveira, J.C., Brandsch, R., Hogg, T. (2012). Analysis of
Mathematical Models to Describe the Migration of Additives from
Packaging Plastics to Foods. Journal of Food Process Engineering, 35, 657-
676.
Qin, Y.-Y., Zhang, Z.H., Li, L., et al (2015). Physio-Mechanical Properties of an
Active Chitosan Film Incorporated with Montmorillonite and Natural
Antioxidants Extracted from Pomegranate Rind. Journal of Food Science
and Technology, 52, 1471-1479.
Quilaqueo Gutiérrez, M., Echeverría, I., Ihl, M., Bifani, V., Mauri, A.N. (2012).
Carboxymethylcellulose–Montmorillonite Nanocomposite Films
Activated with Murta (Ugni Molinae Turcz) Leaves Extract. Carbohydrate
Polymers, 87, 1495-1502.
Rai, M., Yadav, A., Gade, A. (2009). Silver Nanoparticles as a New Generation of
Antimicrobials. Biotechnology Advances, 27, 76-83.
Rajan, R., Chandran, K., Harper, S.L., Yun, S.I., Kalaichelvan, P.T. (2015). Plant
Extract Synthesized Silver Nanoparticles: An Ongoing Source of Novel
Biocompatible Materials. Industrial Crops and Products, 70, 356-373.
Raquez, J.M., Habibi, Y., Murariu, M., Dubois, P. (2013). Polylactide (PLA)-Based
Nanocomposites. Progress in Polymer Science, 38, 1504-1542.
Ravishankar, S., Jaroni, D., Zhu, L., et al (2012). Inactivation of Listeria
Monocytogenes on Ham and Bologna Using Pectin-Based Apple,
Carrot, and Hibiscus Edible Films Containing Carvacrol and
Cinnamaldehyde. Journal of Food Science, 77, M377-M382.
Rawi, N.F.M., Jayaraman, K., Bhattacharyya, D. (2014). Bamboo Fabric
Reinforced Polypropylene and Poly(Lactic Acid) for Packaging
Applications: Impact, Thermal, and Physical Properties. Polymer
Composites, 35, 1888-1899.
Introduction
~ 90 ~
Reddy, M.M., Vivekanandhan, S., Misra, M., Bhatia, S.K., Mohanty, A.K. (2013).
Biobased Plastics and Bionanocomposites: Current Status and Future
Opportunities. Progress in Polymer Science, 38, 1653-1689.
Reidy, B., Haase, A., Luch, A., Dawson, K., Lynch, I. (2013). Mechanisms of
Silver Nanoparticle Release, Transformation and Toxicity: A Critical
Review of Current Knowledge and Recommendations for Future
Studies and Applications. Materials, 6, 2295-2350.
Restuccia, D., Spizzirri, U.G., Parisi, O.I., et al (2010). New EU Regulation
Aspects and Global Market of Active and Intelligent Packaging for
Food Industry Applications. Food Control, 21, 1425–1435.
Rhim, J.W., Ng, P.K.W. (2007). Natural Biopolymer-Based Nanocomposite Films
for Packaging Applications. Critical Reviews in Food Science and Nutrition,
47, 411 - 433.
Rhim, J.W., Park, H.M., Ha, C.S. (2013). Bio-Nanocomposites for Food
Packaging Applications. Progress in Polymer Science, 38, 1629-1652.
Rhim, J.W., Wang, L.F. (2014). Preparation and Characterization of Carrageenan-
Based Nanocomposite Films Reinforced with Clay Mineral and Silver
Nanoparticles. Applied Clay Science, 97–98, 174-181.
Rhim, J.W., Wang, L.F., Hong, S.I. (2013). Preparation and Characterization of
Agar/Silver Nanoparticles Composite Films with Antimicrobial Activity.
Food Hydrocolloids, 33, 327-335.
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.
Rodríguez, F.J., Galotto, M.J., Guarda, A., Bruna, J.E. (2012). Modification of
Cellulose Acetate Films Using Nanofillers Based on Organoclays. Journal
of Food Engineering, 110, 262–268.
Salerno, A., Fernández-Gutiérrez, M., San Román Del Barrio, J., Domingo, C.
(2015). Bio-Safe Fabrication of PLA Scaffolds for Bone Tissue
Engineering by Combining Phase Separation, Porogen Leaching and
scCO2 Drying. The Journal of Supercritical Fluids, 97, 238-246.
Introduction
~ 91 ~
Sanches-Silva, A., Costa, D., Albuquerque, T.G., et al (2014). Trends in the Use of
Natural Antioxidants in Active Food Packaging: A Review. Food
Additives & Contaminants: Part A, 31, 374-395.
Sanchez-Garcia, M.D., Lopez-Rubio, A., Lagaron, J.M. (2010). Natural Micro and
Nanobiocomposites with Enhanced Barrier Properties and Novel
Functionalities for Food Biopackaging Applications. Trends in Food
Science & Technology, 21, 528-536.
Sánchez-González, L., Vargas, M., González-Martínez, C., Chiralt, A., Cháfer, M.
(2011). Use of Essential Oils in Bioactive Edible Coatings: A Review.
Food Engineering Reviews, 3, 1-16.
Sánchez-Moreno, C. (2002). Review: Methods Used to Evaluate the Free Radical
Scavenging Activity in Foods and Biological Systems. Food Science and
Technology International, 8, 121-137.
Sánchez Aldana, D., Andrade-Ochoa, S., Aguilar, C.N., Contreras-Esquivel, J.C.,
Nevárez-Moorillón, G.V. (2015). Antibacterial Activity of Pectic-Based
Edible Films Incorporated with Mexican Lime Essential Oil. Food
Control, 50, 907-912.
Santos, E.H., Kamimura, J.A., Hill, L.E., Gomes, C.L. (2015). Characterization of
Carvacrol β-Cyclodextrin Inclusion Complexes as Delivery Systems for
Antibacterial and Antioxidant Applications. LWT - Food Science and
Technology, 60, 583-592.
Scatto, M., Salmini, E., Castiello, S., et al (2013). Plasticized and Nanofilled
Poly(Lactic Acid)-Based Cast Films: Effect of Plasticizer and
Organoclay on Processability and Final Properties. Journal of Applied
Polymer Science, 127, 4947-4956.
Schreiber, S.B., Bozell, J.J., Hayes, D.G., Zivanovic, S. (2013). Introduction of
Primary Antioxidant Activity to Chitosan for Application as a
Multifunctional Food Packaging Material. Food Hydrocolloids, 33, 207-214.
Severino, R., Ferrari, G., Vu, K.D., et al (2015). Antimicrobial Effects of Modified
Chitosan Based Coating Containing Nanoemulsion of Essential Oils,
Modified Atmosphere Packaging and Gamma Irradiation against
Introduction
~ 92 ~
Escherichia Coli O157:H7 and Salmonella Typhimurium on Green Beans.
Food Control, 50, 215-222.
Shah, A.A., Hasan, F., Hameed, A., Ahmed, S. (2008). Biological Degradation of
Plastics: A Comprehensive Review. Biotechnology Advances, 26, 246-265.
Shankar, S., Teng, X., Li, G., Rhim, J.W. (2015). Preparation, Characterization,
and Antimicrobial Activity of Gelatin/ZnO Nanocomposite Films. Food
Hydrocolloids, 45, 264-271.
Sharma, V.K., Yngard, R.A., Lin, Y. (2009). Silver Nanoparticles: Green Synthesis
and Their Antimicrobial Activities. Advances in Colloid and Interface Science,
145, 83-96.
Shemesh, R., Goldman, D., Krepker, M., et al (2015). LDPE/Clay/Carvacrol
Nanocomposites with Prolonged Antimicrobial Activity. Journal of
Applied Polymer Science, 132, In press: doi: 10.1002/app.41261.
Shemesh, R., Krepker, M., Goldman, D., et al (2015). Antibacterial and Antifungal
LDPE Films for Active Packaging. Polymers for Advanced Technologies, 26,
110-116.
Shojaee-Aliabadi, S., Mohammadifar, M.A., Hosseini, H., et al (2014).
Characterization of Nanobiocomposite κ Carrageenan Film with Zataria
Multiflora Essential Oil and Nanoclay. International Journal of Biological
Macromolecules, 69, 282-289.
Silva-Weiss, A., Ihl, M., Sobral, P.J.A., Gómez-Guillén, M.C., Bifani, V. (2013).
Natural Additives in Bioactive Edible Films and Coatings: Functionality
and Applications in Foods. Food Engineering Reviews, 5, 200-216.
Singh, P., Wani, A.A., Saengerlaub, S. (2011). Active Packaging of Food Products:
Recent Trends. Nutrition and Food Science, 41, 249-260.
Sinha Ray, S., Okamoto, M. (2003). Polymer/Layered Silicate Nanocomposites: A
Review from Preparation to Processing. Progress in Polymer Science
(Oxford), 28, 1539-1641.
Siracusa, V., Rocculi, P., Romani, S., Rosa, M.D. (2008). Biodegradable Polymers
for Food Packaging: A Review. Trends in Food Science & Technology, 19,
634-643.
Introduction
~ 93 ~
Södergård, A., Stolt, M. (2002). Properties of Lactic Acid Based Polymers and
Their Correlation with Composition. Progress in Polymer Science, 27, 1123-
1163.
Södergård, A., Stolt, M. (2010). Industrial Production of High Molecular Weight
Poly(Lactic Acid). In: Auras, R., Lim, L.-T., Selke, S.E.M. & Tsuji, H.
(eds.) Poly(Lactic Acid): Synthesis, Structures, Properties, Processing, and
Applications. John Wiley & Sons, Inc., Hoboken, NJ, USA, pp. 27-41.
Sondi, I., Salopek-Sondi, B. (2004). Silver Nanoparticles as Antimicrobial Agent:
A Case Study on E. Coli as A model for Gram-Negative Bacteria. Journal
of Colloid and Interface Science, 275, 177-182.
Song, H., Li, B., Lin, Q.B., Wu, H.J., Chen, Y. (2011). Migration of Silver from
Nanosilver–Polyethylene Composite Packaging into Food Simulants.
Food Additives & Contaminants: Part A, 28, 1758-1762.
Sreejarani, K.P., Suprakas, S.R. (2012). Chapter 2: Chitosan-Based
Nanocomposites. In: Maya, J.J. & Sabu, T. (eds.) Natural Polymers. Volume
2 Nanocomposites. RSC Green Chemistry, Cambridge, U.K., pp. 33-68.
Srinivasan, K. (2012). Antioxidant Potential of Spices and Their Active
Constituents. Critical Reviews in Food Science and Nutrition, 54, 352-372.
Sung, S.Y., Sin, L.T., Tee, T.T., et al (2013). Antimicrobial Agents for Food
Packaging Applications. Trends in Food Science & Technology, 33, 110-123.
Tajkarimi, M.M., Ibrahim, S.A., Cliver, D.O. (2010). Antimicrobial Herb and
Spice Compounds in Food. Food Control, 21, 1199-1218.
Tao, F., Hill, L.E., Peng, Y., Gomes, C.L. (2014). Synthesis and Characterization
of β-Cyclodextrin Inclusion Complexes of Thymol and Thyme Oil for
Antimicrobial Delivery Applications. LWT - Food Science and Technology,
59, 247-255.
Tapia-Hernández, J.A., Torres-Chávez, P.I., Ramírez-Wong, B., et al (2015).
Micro- and Nanoparticles by Electrospray: Advances and Applications
in Foods. Journal of Agricultural and Food Chemistry, 63, 4699-4707.
Introduction
~ 94 ~
Tawakkal, I.S.M.A., Cran, M.J., Bigger, S.W. (2016). Interaction and
Quantification of Thymol in Active Pla-Based Materials Containing
Natural Fibers. Journal of Applied Polymer Science, 133, n/a-n/a.
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, 41-48.
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.
u Nisa, I., Ashwar, B.A., Shah, A., et al (2015). Development of Potato Starch
Based Active Packaging Films Loaded with Antioxidants and Its Effect
on Shelf Life of Beef. Journal of Food Science and Technology, DOI:
10.1007/s13197-015-1859-3.
Ultee, A., Slump, R.A., Steging, G., Smid, E.J. (2000). Antimicrobial Activity of
Carvacrol toward Bacillus Cereus on Rice. Journal of Food Protection, 63, 620-
624.
Valdes, A., Mellinas, A.C., Ramos, M., et al (2015). Use of Herbs, Spices and Their
Bioactive Compounds in Active Food Packaging. RSC Advances, 5,
40324-40335.
Valdés, A., Mellinas, A.C., Ramos, M., Garrigós, M.C., Jiménez, A. (2014).
Natural Additives and Agricultural Wastes in Biopolymer Formulations
for Food Packaging. Frontiers in Chemistry, 2, 1-10.
Vaverková, M., Toman, F., Adamcová, D., Kotovicová, J. (2012). Study of the
Biodegrability of Degradable/Biodegradable Plastic Material in a
Controlled Composting Environment. Ecological Chemistry and Engineering
S, 19, 347-358.
Viñas, P., Campillo, N. (2014). Chapter 7 - Gas Chromatography–Mass
Spectrometry Analysis of Polyphenols in Foods. In: Watson, R.R. (ed.)
Polyphenols in Plants. Academic Press, San Diego, pp. 103-157.
Introduction
~ 95 ~
Wen, P., Zhu, D.-H., Wu, H., et al (2016). Encapsulation of Cinnamon Essential
Oil in Electrospun Nanofibrous Film for Active Food Packaging. Food
Control, 59, 366-376.
Wu, Y., Qin, Y., Yuan, M., et al (2014). Characterization of an Antimicrobial
Poly(Lactic Acid) Film Prepared with Poly(ɛ-Caprolactone) and Thymol
for Active Packaging. Polymers for Advanced Technologies, 25, 948-954.
Wu, Y., Yuan, M.W., Yang, J.Y., et al (2014). Physical Properties and
Antimicrobial Activity of a Poly(Lactic Acid)/Poly(Trimethylene
Carbonate) Film Incorporated with Thymol. Advanced Materials Research,
884-885, 481-484.
Xia, Y., Rubino, M., Auras, R. (2015). Release of Surfactants from Organo-
Modified Montmorillonite into Solvents: Implications for Polymer
Nanocomposites. Applied Clay Science, 105–106, 107-112.
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.
Yang, F., Manitiu, M., Kriegel, R., Kannan, R.M. (2014). Structure, Permeability,
and Rheology of Supercritical CO2 Dispersed Polystyrene-Clay
Nanocomposites. Polymer, 55, 3915-3924.
Zaman, I., Manshoor, B., Khalid, A., Araby, S. (2014). From Clay to Graphene
for Polymer Nanocomposites—a Survey. Journal of Polymer Research, 21,
1-11.
Zhao, H., Zhao, G. (2016). Mechanical and Thermal Properties of Conventional
and Microcellular Injection Molded Poly (Lactic Acid)/Poly (ɛ-
Caprolactone) Blends. Journal of the Mechanical Behavior of Biomedical
Materials, 53, 59-67.
Introduction
~ 96 ~
WEB PAGES AINIA, EOI. 2015. Tendencias En La Tecnología De Envasado Activo. Informe De
Vigilancia Tecnológica. AINIA Centro Tecnológico and EOI Escuela De
Organización Industrial. http://www.eoi.es/savia/documento/eoi-
80444/tendencias-en-la-tecnologia-de-envasado-activo-informe-de-
vigilancia-tecnologica. Last access: September 2015.
European-Bioplastics-Association. 2014. European Bioplastics. Driving the Evolution of
Plastics. http://en.european-bioplastics.org/multimedia/. Last access:
September, 2015.
European-Bioplastics-Association. 2015a. Benefits of Biobased Rigid Packaging.
http://en.european-bioplastics.org/multimedia/. Last access:
September, 2015.
European-Bioplastics-Association. 2015b. Bioplastics Packaging - Combining
Performance with Sustainability.Pdf. http://en.european-
bioplastics.org/multimedia/. Last access: September, 2015.
Plastics-Europe. 2015. Plastics – the Facts 2014/2015. An Analysis of European
Plastics Production, Demand and Waste Data.Pdf.
http://www.plasticseurope.org/Document/plastics-the-facts-
20142015.aspx. Last access: September, 2015.
Introduction
~ 97 ~
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.
II. Objectives
Objetives
~ 101 ~
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,
Objectives
~ 102 ~
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.
~ 103 ~
III. Results and Discussion
Results and Discussion
~ 105 ~
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
Results and Discussion
~ 106 ~
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 (November-
December 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.
Results and Discussion
~ 107 ~
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.
Journal of Food Engineering. 2012;
109, 513-519.
Release and antioxidant activity of carvacrol and thymol from polypropylene active
packaging films.
Marina Ramos; Ana Beltrán; Mercedes Peltzer; Artur J.M. Valente; María
C. Garrigós.
LWT-Food Science and Technology. 2014;
58, 470-477.
Carvacrol and Thymol for Fresh Food Packaging
Marina Ramos; Ana Beltrán; Arantzatzu Valdés; Mercedes Peltzer; Alfonso
Jiménez; María C. Garrigós; Gennady Zaikov.
Journal of Bioequivalence &
Bioavailability. 2013; 5, 154-160.
Development of novel nano-biocomposite antioxidant films based on poly (lactic acid)
and thymol for active packaging.
Marina Ramos; Alfonso Jiménez; Mercedes Peltzer;
María C. Garrigós.
Food Chemistry. 2014; 162, 149-155.
Influence of thymol and silver nanoparticles on the degradation of
poly(lactic acid) based nanocomposites: Thermal and morphological properties.
Marina Ramos; Elena Fortunati.; Mervedes
Peltzer; Franco Dominici; Alfonso Jiménez; María C. Garrigós; Jose M. Kenny.
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: 978-1-
61209-520-2. (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: 978-
3-659-04935-4.
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)
Results and Discussion
~ 108 ~
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.
Results and Discussion
~ 109 ~
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.
Results and Discussion
~ 110 ~
Table III.4. Other publications in peer review journals.
Title Authors Book/Journal
Active edible films: current state and future trends.
Ana C. Mellinas; Arantzatzu Valdés; Marina Ramos; Nuria Burgos; María C. Garrigós;
Alfonso Jiménez.
Journal of Applied Polymer Science. 2015;
132. In Press.
Use of herbs, spices and their bioactive compounds in active food packaging.
Arantzatzu Valdés; Ana C. Mellinas; Marina Ramos; Nuria Burgos; AlfonsoJiménez; María
C. Garrigós.
RSC Advances. 2015; 5, 40324-40335.
New Trends in Beverage Packaging Systems: A Review.
Marina Ramos; Arantzazu Valdés; Ana C. Mellinas; María
C. Garrigós
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, 329-
336. Monitoring the oxidation of almond oils by HS-SPME-GC/MS and ATR-
FTIR. 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.
Results and Discussion
~ 111 ~
Table III.5. Other publications in peer review books.
Title Authors Book/Journal
Polymers extracted from bio-mass.
Arantzazu Valdés; Marina Ramos; Esther
García; María C. Garrigós; Alfonso
Jiménez
Reference module in Food Science. Elsevier. In press, accepted. Available
in 2016
Multifunctional antimicrobial nanocomposites for food packaging
applications.
Elena Fortunati; Debora Puglia; Ilaria
Armentano; Arantzazu Valdés;
Marina Ramos, Nerea Juárez, María C.
Garrigós, José M. Kenny
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
Food Packaging: Procedures, Management and Trends: Nova Science Publishers. 2012; 63-78.
ISBN: 978-1-62257-319-6. (Chapter 3)
Linoleic Acid Content and Antioxidant Properties of Different
Tree Nuts: a Review.
Ana Beltrán; Marina Ramos; Arantzazu Valdés; María C.
Garrigós
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
~ 114 ~
Figure 1.1. General scheme of the experimental work presented in Chapter 1.
Results and Discussion. Chapter 1
~ 115 ~
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 gas-
phase 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,
Results and Discussion. Chapter 1
~ 116 ~
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
Results and Discussion. Chapter 1
~ 117 ~
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
Results and Discussion. Chapter 1
~ 118 ~
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 cinnamon-
based active material to increase more than 3 times the shelf-life of a
complex bakery product with minimal changes in packaging and no
Results and Discussion. Chapter 1
~ 119 ~
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, Garcı a 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
Results and Discussion. Chapter 1
~ 120 ~
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
Results and Discussion. Chapter 1
~ 121 ~
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.
Results and Discussion. Chapter 1
~ 122 ~
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).
Results and Discussion. Chapter 1
~ 123 ~
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)
Results and Discussion. Chapter 1
~ 124 ~
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
Results and Discussion. Chapter 1
~ 125 ~
of crystallinity (χ %) for each material was calculated according to
Equation 1.1:
(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) (Pospıšil 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
휒(%) = Δ퐻푚푊Δ퐻푚표 ∙ 100
Results and Discussion. Chapter 1
~ 126 ~
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
Results and Discussion. Chapter 1
~ 127 ~
tests were performed with 12 cm2 films and 20 mL of each simulant (area-
to-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):
(1.2.)
푀퐹,푡
푀퐹,∞= 1 −
2훼(1 + 훼)1 + 훼 + 훼2푞푛2
푒푥푝−퐷푞푛2푡퐿푃2
∞
푛=1
Results and Discussion. Chapter 1
~ 128 ~
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):
(1.4.)
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,
훼 =(퐾퐹푃∙푉퐹)
푉푃
1휋−
1훼푀퐹,푡
푀푃,표
0.5
= −퐷0.5
훼 ∙ 퐿푃∙ 푡0.5 +
1휋0.5
Results and Discussion. Chapter 1
~ 129 ~
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
Results and Discussion. Chapter 1
~ 130 ~
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.
(1.5.)
퐼(%) = 퐴퐶표푛푡푟표푙 퐴푆푎푚푝푙푒퐴퐶표푛푡푟표푙 ∙ 100
Results and Discussion. Chapter 1
~ 131 ~
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 colony-
forming 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
Results and Discussion. Chapter 1
~ 132 ~
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 post-
harvest 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
Results and Discussion. Chapter 1
~ 133 ~
chamber for 15 days. Food samples stored with the control film (without
active compounds) were also studied for comparison.
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,
PP0 PPT8 PPC8
Results and Discussion. Chapter 1
~ 134 ~
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-SPME-
GC/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
Results and Discussion. Chapter 1
~ 135 ~
°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 splitless-
period). 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.
Results and Discussion. Chapter 1
~ 136 ~
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,
~ 137 ~
leading to a potential loss of some molecules resulting in microholes and
some porosity. Figure 1.5 shows the SEM microgprahs 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
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
Results and Discussion. Chapter 1
molecules resulting in microholes and
shows the SEM microgprahs of cross section of
It could be observed that the matrix remained homogeneous
it could be concluded that the possible
diffusion of active additives through the polymer matrix is only a surface
Cross section micrographs (x300) for PP0 and samples with 8 wt% of the
studied additives.
Tensile tests were performed in order to study the effect of thymol and
polymer mechanical properties, by the evaluation of
elastic modulus (E), elongation at yield
Results and Discussion. Chapter 1
~ 138 ~
(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).
Results and Discussion. Chapter 1
~ 139 ~
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.
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
0
20
40
60
80
100
20 120 220 320 420 520 620 720
Wei
ght (
%)
Temperature (ºC)
PPC4PPC6PPC8PP0
90
95
100
105
20 120 220 320 420
Results and Discussion. Chapter 1
~ 140 ~
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.
Results and Discussion. Chapter 1
~ 141 ~
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
Results and Discussion. Chapter 1
~ 142 ~
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).
Results and Discussion. Chapter 1
~ 143 ~
Table 1.4. Oxidation induction parameters, oxygen transmission rate obtained for all
formulations.
Sample OOT (C)a OIT (min) O2a
OIT (min) aira
OTR·e (cm3 mm m-2 day)b
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 8.2 ± 0.2bc 17.8 ± 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 13.2 ± 2.8de 33.7 ± 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 8.7 ± 1.1bc 26.4 ± 2.3cd 126.7
PPTC8 230 ± 3de 10.7 ± 1.2cd 35.5 ± 1.7ab 158.0
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
Results and Discussion. Chapter 1
~ 144 ~
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
Results and Discussion. Chapter 1
~ 145 ~
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
Results and Discussion. Chapter 1
~ 146 ~
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 kg-
1, 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 86.7-
108.2 % 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.
Results and Discussion. Chapter 1
~ 147 ~
Table 1.5. Main analytical parameters obtained for the studied active additives using the optimized methods.
Analyte Method Parameter
Slope ± SD Intercept ± SD Linearity (R2)a
LOD (mg/kg)b
LOQ (mg/kg)c
Carvacrol
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
Thymol
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 Sy/x
SD: Standard deviation The results are represented as mean ± standard deviation, n=3.
Results and Discussion. Chapter 1
~ 148 ~
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.
Analyte Simulant Spiking level (mg)
0.03 0.27 2.60
Carvacrol
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)
Thymol
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).
Results and Discussion. Chapter 1
~ 149 ~
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).
Results and Discussion. Chapter 1
~ 150 ~
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.
Analyte Film Simulant
Watera Ethanol (10 %, v/v)a Acetic acid (3 %, m/v) a Ethanol (95 %, v/v) a Isooctaneb
Carvacrol PPC8 288 ± 20a 718 ± 54b 647 ± 47b 880 ± 27c 921 ± 157c
PPTC8 157 ± 19a 285 ± 26b 474 ± 44c 347 ± 21d 633 ± 34e
Thymol PPT8 433 ± 46a 656 ± 30b 689 ± 61b 829 ± 19c 1085 ± 112d
PPTC8 162 ± 18a 362 ± 53b 547 ± 51c 367 ± 21b 616 ± 49c
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).
~ 151 ~
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).
Results and Discussion. Chapter 1
~ 152 ~
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
DPPH radical test and results are shown in
DPPH inhibition in samples submitted to contact with
(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
conditions during the test, influencing in the same way the DPPH
complex mechanisms for formation. It is kno
DPPH decreases by light exposure, high oxygen content,
solvent type (Ozcelik, Lee and Min, 2003).
reported that the increase of hydrogen ion concentration leads to the
decrease in the rate of the chromogen radical scavenging
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.
of the obtained extracts was evaluated by the
test and results are shown in Figure 1.7. Results for the
in samples submitted to contact with acetic acid 3 %
, since no particular inhibition was
possibly due to the low pH
conditions during the test, influencing in the same way the DPPH
It is known that the absorbance of
oxygen content, low pHs, and
. Regarding pH, it has been
reported that the increase of hydrogen ion concentration leads to the
chromogen radical scavenging reaction, whereas
~ 153 ~
under basic conditions proton dissociation
the reducing capacity of these compounds
On the other hand, all the other extracts showed
showing a significant inhibition of the DPPH radical
conditions. A higher AO activity was observed for thymol extr
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
8 wt% of thymol was significantly different from
Figure 1.8. Mean DPPH inhibition values (%) for P
letters represent significant differences at
These results showed that the AO activity of
carvacrol, possibly due to the larger steric
phenolic group; as concluded by different authors
mechanisms of action of these compounds in the DPPH
Marinova, Gordon and Raneva, 1999; Wu, Luo and Wang, 2012)
Results and Discussion. Chapter 1
under basic conditions proton dissociation in polyphenols would enhance
compounds (Pyrzynska and Pekal, 2013).
showed a noticeable AO activity,
showing a significant inhibition of the DPPH radical under the test
was observed for thymol extracts, with
the highest inhibition obtained into isooctane (42.2 ± 1.1 %). The
of the variations introduced by
AOactivity of the formulation with
of thymol was significantly different from all the other (Figure 1.8).
Mean DPPH inhibition values (%) for PPT8, PPC8 and PPTC8. Different
represent significant differences at p < 0.05.
AO activity of thymol was higher to that of
steric hindrance of the thymol
by different authors when considering the
of action of these compounds in the DPPH test (Yanishlieva,
Marinova, Gordon and Raneva, 1999; Wu, Luo and Wang, 2012). Other
Results and Discussion. Chapter 1
~ 154 ~
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
~ 155 ~
information about the real possibilities of carvacrol and thymol in active
PP-based materials. Therefore, some experiments were
carried out by using four different food simulants
between the PP-based formulations containing thymol and carvacrol at
wt% and them during 15 days.
Figure 1.9 and Figure 1.10Figure 1.10 show the
carvacrol from PPT8 and PPC8 films with
behaviour was observed for both compounds, being rapidly released
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.
Results and Discussion. Chapter 1
ies of carvacrol and thymol in active
experiments were designed and
four different food simulants and getting contact
containing thymol and carvacrol at 8
show the release of thymol and
with time, respectively. A similar
h compounds, being rapidly released from
expected increase in their release with
after approximately 120 h.
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.
Results and Discussion. Chapter 1
~ 156 ~
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
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).
Analyte (Film) Parameter Ethanol 10 % (v/v)a
Acetic acid3 % (m/v)
Carvacrol (PPC8) αap 1.38 0.07 0.96
D 1.20 0.05 1.7
Thymol (PPT8) αap 1.56 0.1 1.44
D 1.75 0.08 2.51
Migration temperature: a 40 °C;
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
) calculated from Equation 1.4 for the
release of carvacrol and thymol from PP films into different food simulants (mean ±
n=3).
Simulant
Acetic acid 3 % (m/v)a
Ethanol 95 % (v/v)a Isooctaneb
0.04 1.27 0.07 1.4 0.1
0.1 1.99 0.07 9.4 0.6
0.05 1.56 0.08 2.4 0.2
0.02 1.01 0.03 5.9 0.1
40 °C; b 20 °C
Results and Discussion. Chapter 1
~ 157 ~
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
Results and Discussion. Chapter 1
~ 158 ~
the migration of the phenolic compounds (Manzanarez-López, Soto-
Valdez, 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:
(1.6.)
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:
(1.7.)
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,
푀퐹,푡푀푃 ,표
= 푀퐹,∞푀푃,표
1 − 푒−푘 ′ 푡
푀퐹,∞푀푃,표
= 훼푎푝1 + 훼푎푝
Results and Discussion. Chapter 1
~ 159 ~
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 5.0
0,
,11
F
tF
MM
versus 5.0t 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.
Results and Discussion. Chapter 1
~ 160 ~
Figure 1.11. Plots of 5.0
5.0
0,
, 111
F
tF
MM versus
(A) and carvacrol (B) from PPT8 and PPC8 films into different food simulants.
(◊), 20 °C; acetic acid (o), 40 °C; ethanol 10 % (v/v) (□), 40 °C;
40 °C.
versus 5.0t for the migration of thymol
(A) and carvacrol (B) from PPT8 and PPC8 films into different food simulants. Isooctane
), 40 °C; and ethanol 95 %(v/v) (∆),
Results and Discussion. Chapter 1
~ 161 ~
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.
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
PPC8 PPT8
PPC8 PPT8
PP0
PP0
PPTC8
PPTC8
(a) S. aureus
(b) E. coli
Results and Discussion. Chapter 1
~ 162 ~
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
PPC4 PPC6 PPC8
PP0 n.d.
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
Results and Discussion. Chapter 1
~ 163 ~
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.
Results and Discussion. Chapter 1
~ 164 ~
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.
Results and Discussion. Chapter 1
~ 165 ~
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.
Results and Discussion. Chapter 1
~ 166 ~
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 PP-
based 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.
Results and Discussion. Chapter 1
~ 167 ~
0
1E+09
2E+09
3E+09
4E+09
5E+09
6E+09
7E+09
8E+09
9E+09
1E+10
20 21 22 23 24 25 26 27 28
Abu
ndan
ce
Time (min)
Bread stored at room temperature with PPC films
Day 0Day 2Day 5Day 10Day 15
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.
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.
Bread stored at room temperature
with PPC8 films
Days of study Peak area (x106)
0 644
2 1086
5 1443
10 1616
15 2178
Results and Discussion. Chapter 1
~ 168 ~
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.
Results and Discussion. Chapter 1
~ 169 ~
Table 1.10. Identified compounds present in the headspace of food samples packed with
PP0 films after 4 days. Bread stored at room
temperature Strawberries stored at
room temperature Strawberries refrigerated at 4 °C
Time (min) Compound Time
(min) Compound Time (min) Compound
1.4 Ethanol 3.2 Methyl isopentanoate 1.9 Hexane
5.7 2,4-dimethyl heptane 5.8 2-methyl
butylacetate 3.1 Methylbutanoate
6.6 2,4-dimethyl heptene 7.4 Methyl
hexanoate 3.9 Toluene
7.8 Isononane 10.6 Hexylacetate 6.8 Isopropyl butyrate
13.0 4-methyloctane 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
Results and Discussion. Chapter 1
~ 170 ~
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 PP-
based 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
Results and Discussion. Chapter 1
~ 171 ~
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.
Results and Discussion. Chapter 1
~ 172 ~
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.
Results and Discussion. Chapter 1
~ 173 ~
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.
Results and Discussion. Chapter 1
~ 174 ~
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, 92-
99.
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, 601-
617.
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.
Results and Discussion. Chapter 1
~ 175 ~
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
Results and Discussion. Chapter 1
~ 176 ~
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, 508-
517.
Mascheroni, E., Guillard, V., Gastaldi, E., Gontard, N., Chalier, P. (2011). Anti-
Microbial 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
Results and Discussion. Chapter 1
~ 177 ~
Eugenol Derivatives. Journal of Agricultural and Food Chemistry, 56, 3989-
3996.
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-Dihydroxy-
phenylethanol). 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.
Results and Discussion. Chapter 1
~ 178 ~
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.
Pospı Šil, 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.
Results and Discussion. Chapter 1
~ 179 ~
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.
Results and Discussion. Chapter 1
~ 180 ~
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.
Results and Discussion. Chapter 1
~ 181 ~
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, 41-
48.
Tovar, B.Z., Garcı A, 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.
Results and Discussion. Chapter 1
~ 182 ~
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, 283-
290.
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.
2 Chapter 2
Active nanocomposites based on PLA with thymol and nanomaterials for food packaging applications
Results and Discussion. Chapter 2
~ 185 ~
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.
Results and Discussion. Chapter 2
~ 187 ~
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).
Results and Discussion. Chapter 2
~ 188 ~
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). Nano-
and 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
Results and Discussion. Chapter 2
~ 189 ~
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 (Al-
Bandak and Oreopoulou, 2007; Amorati, Foti and Valgimigli, 2013;
Gyawali and Ibrahim, 2014). In fact, the presence of the hydroxyl
Results and Discussion. Chapter 2
~ 190 ~
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
Results and Discussion. Chapter 2
~ 191 ~
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, Marin-
Morales 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 Ag-
NPs (Section 2.2).
3 Section 2.1.
Active nanocomposites based on PLA with thymol and layered montmorillonite nanoclay for food
packaging applications
Results and Discussion. Chapter 2
~ 195 ~
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.
Results and Discussion. Chapter 2
~ 196 ~
Figure 2.1. General scheme of the experimental work presented in Section 2.1.
Results and Discussion. Chapter 2
~ 197 ~
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).
Results and Discussion. Chapter 2
~ 198 ~
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
Results and Discussion. Chapter 2
~ 199 ~
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 Q-
2000 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
Results and Discussion. Chapter 2
~ 200 ~
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,
Results and Discussion. Chapter 2
~ 201 ~
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 CM-
3600d 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.
(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 (UNE-
EN_20200, 2006). Materials can be considered disintegrable according to
∆퐸∗ = [(∆퐿∗)2 + (∆푎∗)2 + (∆푏∗)2]12
Results and Discussion. Chapter 2
~ 202 ~
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.
Results and Discussion. Chapter 2
~ 203 ~
(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
퐷푖푠푖푛푡푒푔푟푎푏푖푙푖푡푦 (%) = 푚푖 −푚푡
푚푖∙ 100
Results and Discussion. Chapter 2
~ 204 ~
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
Results and Discussion. Chapter 2
~ 205 ~
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:
(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
퐼(%) = 퐴퐶표푛푡푟표푙 − 퐴푆푎푚푝푙푒퐴퐶표푛푡푟표푙 ∙ 100
Results and Discussion. Chapter 2
~ 206 ~
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.
Results and Discussion. Chapter 2
~ 207 ~
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
PLA/T 5.57 ± 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
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
Results and Discussion. Chapter 2
~ 208 ~
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 365-
370 °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
Results and Discussion. Chapter 2
~ 209 ~
addition of thymol and D43B did not affect the thermal degradation
profile of the nanocomposite films.
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 non-
radical, '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){
0
50
100
0 100 200 300 400 500 600 700
Weig
ht L
oss (
wt%
)
Temperature (ºC)
PLA
PLA/D43B2.5PLA/D43B5
PLA/TPLA/T/D43B2.5
PLA/T/D43B5
-0,06
-0,04
-0,02
0,000 100 200 300 400 500 600 700
DTG
(mg
s-1)
Temperature (ºC)
PLAPLA/D43B2.5PLA/D43B5PLA/TPLA/T/D43B2.5PLA/T/D43B5
(a)
(b)
Results and Discussion. Chapter 2
~ 210 ~
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.
~ 211 ~
(a)
(b)
Figure 2.3. DSC thermograms for PLA-based films for the first heating (a) and the
second heating scan (b).
Hea
t flo
w (W
g-1
) H
eat f
low
(W g
-1)
Results and Discussion. Chapter 2
based films for the first heating (a) and the
second heating scan (b).
Results and Discussion. Chapter 2
~ 212 ~
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
Results and Discussion. Chapter 2
~ 213 ~
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).
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.
0
2000
4000
6000
8000
10000
12000
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
Inte
nsity
(cou
nts)
2θ (°)
D43BPLAPLA/TPLA/D43B2.5PLA/D43B5PLA/T/D43B2.5PLA/T/D43B5
Results and Discussion. Chapter 2
~ 214 ~
3.2.3. Morphological analysis
The dispersion of the nanoclay in the PLA matrix was also evaluated by
TEM at different magnifications (Figure 2.
delamination was observed in all cases. In fact, m
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
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
The dispersion of the nanoclay in the PLA matrix was also evaluated by
Figure 2.5). A high degree of clay
delamination was observed in all cases. In fact, micrographs obtained for
PLA/T/D43B2.5 nanocomposite showed the high degree of exfoliation
. Single dispersed clay layers (dark
randomly distributed through the PLA matrix
some regions with complete exfoliation of nanoclay
. 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
TEM images of PLA/T/D43B2.5 active nanocomposite film.
Results and Discussion. Chapter 2
~ 215 ~
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).
Results and Discussion. Chapter 2
~ 216 ~
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
PLA/T/D43B2.5 2246 ± 135b 2.4 ± 0.1d 20.1 ± 0.1 a 32.0 -0.22 -1.14 2.0
PLA/T/D43B5 2140 ± 116b 2.4 ± 0.2d 22.7 ± 1.3 a 34.4 -0.58 -1.48 4.4
a n = 5, mean ± standard deviation. b OTR·e (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)
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
Results and Discussion. Chapter 2
~ 217 ~
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
Results and Discussion. Chapter 2
~ 218 ~
distribution of the colour observed with the naked eye throughout
films (Figure 2.6) also suggests that additives were uniformly distributed
within the polymer matrix.
However, some differences in CIELab coordinat
between neat PLA and nanocomposite films were observed (
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 increased with the addition
of thymol and D43B. A yellowish-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 and 8 wt% thymol). Similar colour differences
were reported when using other active additives, such as
resveratrol, into PLA matrices, where the presence of these compounds
contributed to increase the films colour (Byun, Kim and Whiteside, 2010)
Figure 2.6. Visual observation of neat PLA and nanocomposite films.
distribution of the colour observed with the naked eye throughout the
) also suggests that additives were uniformly distributed
However, some differences in CIELab 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
ss increased with the addition
reddish tone was obtained for PLA/T
formulation, while PLA/T/D43B5 ternary nanocomposite showed the
as expected, due to the high concentrations of the
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
(Byun, Kim and Whiteside, 2010).
Visual observation of neat PLA and nanocomposite films.
Results and Discussion. Chapter 2
~ 219 ~
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).
Results and Discussion. Chapter 2
~ 220 ~
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
Results and Discussion. Chapter 2
~ 221 ~
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.
Results and Discussion. Chapter 2
~ 222 ~
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)
Results and Discussion. Chapter 2
~ 223 ~
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.
800100012001400160018002000
Tran
smita
nce (
a.u.)
Wavenumber (cm-1) PLA
Day 21 Day 7 Day 0
800100012001400160018002000
Tran
smita
nce (
a.u.)
Wavenumber (cm-1) PLA/T
Day 21 Day 7 Day 0
800100012001400160018002000
Tran
smita
nce (
a.u.)
Wavenumber (cm-1) PLA/T/D43B5
Day 21 Day 7 Day 0
(C=O) (C-O-C)
(C-C-O)
(CH(CH3)
Results and Discussion. Chapter 2
~ 224 ~
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.
Results and Discussion. Chapter 2
~ 225 ~
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.
Results and Discussion. Chapter 2
~ 226 ~
Figure 2.9. DSC curves (1st heating scan) of PLA-based films after different composting times.
Results and Discussion. Chapter 2
~ 227 ~
Figure 2.10. DSC curves (2nd heating scan) of PLA-based films after different composting times.
Results and Discussion. Chapter 2
~ 228 ~
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.
Results and Discussion. Chapter 2
~ 229 ~
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.
0
50
100
150
200
250
300
350
400
0 50 100 150 200 250 300 350 400
mg T
hym
ol kg
-1Fo
od S
imul
ant
Time (h)
PLA/TPLA/T/D43B2.5PLA/T/D43B5
Results and Discussion. Chapter 2
~ 230 ~
Figure 2.12. Normalized migration of thymol from different polymer matrices: PLA/T,
PLA/T/D43B2.5, and PLA/T/D43B5.
0
0,2
0,4
0,6
0,8
1
1,2
0 60 120 180 240 300 360
MFt
/MF∞
Time (hours)
PLA/T
0
0,2
0,4
0,6
0,8
1
0 60 120 180 240 300 360
MFt
/MF∞
Time (hours)
PLA/T/D43B2.5
0
0,2
0,4
0,6
0,8
1
0 60 120 180 240 300 360
MFt
/MF∞
Time (hours)
PLA/T/D43B5
Results and Discussion. Chapter 2
~ 231 ~
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).
(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)
(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, Sendon, 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.
푀퐹 ,푡푀푃,0
=
⎝
⎜⎛푀퐹,∞
푀푃,0
⎠
⎟⎞∙ (1 − 푒−푘′푡)
푀퐹,∞푀푃,0
= 1(1 + 훼)
훼 = 푉퐹퐾푃,퐹푉푃
Results and Discussion. Chapter 2
~ 232 ~
ii. The analysis of the partition coefficients (α and KP,F) showed that the
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):
(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 s-
1) 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
휕푐휕푡
=휕휕푥
퐷휕푐휕푥
2,
2 2 21,
2 11 exp
1F t n
nF n
M Dq tM q l
Results and Discussion. Chapter 2
~ 233 ~
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.
(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
Equation (2.8) and (2.9)
D (cm2 s-1) 3.36×10-11 4.86×10-11 2.25×10-11
RMSE 0.0773 0.0698 0.114
Equation (2.10) 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
푅푀푆퐸 = (푦푖 − ŷ푖)2푛
푛
푖=1
12
Results and Discussion. Chapter 2
~ 234 ~
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) and negative deviations for
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).
(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 values as a function of , 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.
0.5 '0.5, 0. 5
0.5,0
1 1 1F t
P
M Dt
M l
5.0
0,
,11
F
tF
MM
5.0t
Results and Discussion. Chapter 2
~ 235 ~
0 100 200 300 4000,48
0,50
0,52
0,54
0,56
[(1/
)-(M
F,t/(
MP,
0))]0.
5
t0.5 (s-0.5)
Figure 2.13. Plots of versus 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
0.5
,
,0
1 1 F t
F
MM
5.0t
Results and Discussion. Chapter 2
~ 236 ~
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
Results and Discussion. Chapter 2
~ 237 ~
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
Results and Discussion. Chapter 2
~ 238 ~
the active compound from the swelled polymer network and the intrinsic
antioxidant properties of the natural additives.
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
0
50
100
150
200
250
300
350
0
10
20
30
40
50
60
70
80
90
0 50 100 150 200 250 300 350 400
mg th
ymol
kg fo
od s
imul
ant -1
Inhi
bitio
n (%
)
Time (hours)
DPPH results in PLA/T extracts
Release of thymol from PLA/T
Results and Discussion. Chapter 2
~ 239 ~
(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.
Results and Discussion. Chapter 2
~ 240 ~
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
Results and Discussion. Chapter 2
~ 241 ~
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.
Results and Discussion. Chapter 2
~ 242 ~
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 bio-
based 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.
4 Section 2.2.
Active nanocomposites based on PLA with thymol and silver nanoparticles for food packaging
applications
Results and Discussion. Chapter 2
~ 245 ~
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.
Results and Discussion. Chapter 2
~ 246 ~
Figure 2.16. General scheme of the experimental work presented in Section 2.2.
Results and Discussion. Chapter 2
~ 247 ~
5. Experimental
5.1. Materials
Commercial poly(lactic acid) PLA-4060D (Tg = 58 °C, 11-13 wt% D-
isomer) 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 Ag-
NPs (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
Results and Discussion. Chapter 2
~ 248 ~
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).
Results and Discussion. Chapter 2
~ 249 ~
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.
Results and Discussion. Chapter 2
~ 250 ~
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.
(2.11) where ∆L*= L*standard – L*sample, ∆a* = a*standard – a*sample and ∆b*=
b*standard – b*sample
∆퐸∗ = [(∆퐿∗)2 + (∆푎∗)2 + (∆푏∗)2]12
Results and Discussion. Chapter 2
~ 251 ~
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.
(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.
푊푉푇 = 퐺 푡
퐴 (푔 ∙ ℎ−1 ∙ 푚−2)
Results and Discussion. Chapter 2
~ 252 ~
(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
푊푉푃 (푘푔 ∙ 푚 ∙ 푃푎−1 ∙ 푠−1 ∙ 푚−2) = 푊푉푇 ∙ 푥 ∙ 푒푆(푅1 − 푅2)
Results and Discussion. Chapter 2
~ 253 ~
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 Ag-
NPs 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 dog-
bone bars and films by following the ISO-20200 standard method (UNE-
EN_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
Results and Discussion. Chapter 2
~ 254 ~
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 with food
(Commission_Regulation/(EU)/No-10/2011). Double-sided, 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.
Results and Discussion. Chapter 2
~ 255 ~
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
Results and Discussion. Chapter 2
~ 256 ~
(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).
Results and Discussion. Chapter 2
~ 257 ~
(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
퐷푃푃퐻 푠푐푎푣푒푛푔푖푛푔 푎푐푡푖푣푖푡푦 (%) = 퐴퐶표푛푡푟표푙 − 퐴푆푎푚푝푙푒퐴퐶표푛푡푟표푙 ∙ 100
Results and Discussion. Chapter 2
~ 258 ~
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
~ 259 ~
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)
slight reduction in the Tmax value was observed by the addition of Ag
(p > 0.05). Regarding Tini values, a significant reduction
observed suggesting some loss in the PLA thermal stability by the addition
of Ag-NPs. Thermograms 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
PLA-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
these formulations could be processed at the same temperatures (up to
200 °C) than neat PLA without risking thermal degradation.
(a)
Results and Discussion. Chapter 2
main peak associated to the PLA thermal degradation between
(Martino, Jiménez, Ruseckaite and
2012; Burgos, Martino and Jiménez, 2013). A
value was observed by the addition of Ag-NPs
a significant reduction (p < 0.05) was
rved suggesting some loss in the PLA thermal stability by the addition
hermograms corresponding to the ternary nanocomposites
degradation step around 120 °C, which was related to the
thymol degradation as it was shown for PP-based films in Chapter 1 and
based active nanocomposites with D43B (Section 2.1). In summary,
results showed that besides the slight reduction in the
nanocomposites thermal stability by the addition of thymol and Ag-NPs,
cessed at the same temperatures (up to
200 °C) than neat PLA without risking thermal degradation.
Results and Discussion. Chapter 2
~ 260 ~
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**
PLA 56 ± 3a 324 ± 12a 363 ± 6a 3181 ± 35
PLA/Ag 56 ± 1a 317 ± 8ab 357 ± 13a 3000 ± 172
PLA/T6 50 ± 2b 327 ± 15a 358 ± 7a 3289 ± 28
PLA/T8 42 ± 1bc 316 ± 14a 353 ± 9a 2930 ± 76
PLA/Ag/T6 44 ± 1bc 284 ± 9bc 337 ± 12a 2823 ± 121
PLA/Ag/T8 41 ± 1c 288 ± 5c 336 ± 14a 2547 ± 244* (n=3; mean ± standard deviation) **(n=5; mean ± standard deviation) Tg: determined by DSC from the first heating scan at 10 °C min10 °C min-1 in N2 atmosphere. Corresponding to the 2nd degradation step.Results are represented as mean ± standard deviation, n=3. column indicate statistically significant different values (p < 0.05).
(b)
TG (a) and DTG (b) curves of neat PLA and nanocomposite injection
and thymol.
Thermal parameters and tensile properties obtained for injection moulded
samples (neat PLA and active nanocomposites).
** (MPa) εb** (%) TS** (MPa)
3181 ± 35ab 3.5 ± 0.2a 60.3 ± 8.0a
3000 ± 172bc 3.6 ± 0.2a 59.7 ± 2.9a
3289 ± 28a 2.7 ± 0.2a 52.1 ± 1.3a
2930 ± 76bc 2.6 ± 0.3b 36.4 ± 3.2b
2823 ± 121cd 3.6 ± 0.3b 36.9 ± 3.0b
2547 ± 244d 2.8 ± 0.2b 36.3 ± 2.8b
first heating scan at 10 °C min-1. Tini and Tmax: determined by TGA at degradation step.
Results are represented as mean ± standard deviation, n=3. Different superscripts within the same indicate statistically significant different values (p < 0.05).
Results and Discussion. Chapter 2
~ 261 ~
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.
Results and Discussion. Chapter 2
~ 262 ~
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).
~ 263 ~
Figure 2.19. FESEM micrographs of the surface of
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 tensile properties of neat PLA and nanocomposites were
and results are reported in Table 2.9. The addition of 1 wt% of Ag
PLA had no significant effect (p > 0.05) on the elastic modulus,
strength and elongation at break values as previously reported by other
Results and Discussion. Chapter 2
FESEM micrographs of the surface of nanocomposite injection moulded.
Cross section micrographs of PLA/Ag/T6 and PLA/Ag/T8 injection
moulded samples after processing.
he tensile properties of neat PLA and nanocomposites were evaluated
The addition of 1 wt% of 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
Results and Discussion. Chapter 2
~ 264 ~
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 Ag-
NPs 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
Results and Discussion. Chapter 2
~ 265 ~
the amorphous character of the commercial PLA used in this study. No
crystallization or melting phenomena were detected. The addition of Ag-
NPs 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
Results and Discussion. Chapter 2
~ 266 ~
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.
Results and Discussion. Chapter 2
~ 267 ~
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).
Results and Discussion. Chapter 2
~ 268 ~
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 Ag-
NPs 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
~ 269 ~
during processing. However, active nanocomposite films containing Ag
NPs 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
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
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
darkening in films when the Ag-NPs concentration is high
and Hong, 2013).
Figure 2.22. Visual observation of neat PLA and binary and ternary nanocomposite films.
Results for colour 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 (p < 0.05)
was observed in PLA films containing Ag
those with thymol (p < 0.05) when compared to the neat PLA film. In
Results and Discussion. Chapter 2
However, active nanocomposite films containing Ag-
NPs showed some darkening in the initially clear surface as well as some
is an important physical property in food
(Jamshidian, Tehrany, Imran, 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,
suggested that surface plasmon
phenomena of silver nanoparticles and phenolic compounds, such as
thymol, may modify PLA colour during processing and storage leading to
NPs concentration is high (Rhim, Wang
observation of neat PLA and binary and ternary nanocomposite films.
olour and transmittance at 500 nm of all films are shown in
. The surface colour of PLA binary and ternary films was
(p < 0.05) depending on the addition of thymol
p < 0.05) in film lightness (L-value)
was observed in PLA films containing Ag-NPs, it slightly increased in
those with thymol (p < 0.05) when compared to the neat PLA film. In
Results and Discussion. Chapter 2
~ 270 ~
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.
Samples Colour parameters Transparency
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
Results and Discussion. Chapter 2
~ 271 ~
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
Results and Discussion. Chapter 2
~ 272 ~
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 (Sánchez-
González, 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 thymol-
containing 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% α-
tocopherol (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).
Results and Discussion. Chapter 2
~ 273 ~
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
Results and Discussion. Chapter 2
~ 274 ~
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).
~ 275 ~
Figure 2.23. FTIR (a) and UV-Vis (b) spectra of PLA and active nanocomposite films.
X-ray diffraction patterns of active nanocomposite films with Ag
were clearly indicative of the presence of these nanoparticles embedded
into the polymer matrix in the films (
diffractogram of the mostly amorphous PLA only shows the characteristic
broad band around 2θ = 20º; the clear and sharp peak around 38.2º
(b)
Results and Discussion. Chapter 2
Vis (b) spectra of PLA and active nanocomposite films.
ray diffraction patterns of active nanocomposite films with Ag-NPs
were clearly indicative of the presence of these nanoparticles embedded
to the polymer matrix in the films (Figure 2.24). While the XRD
diffractogram of the mostly amorphous PLA only shows the characteristic
= 20º; the clear and sharp peak around 38.2º
Results and Discussion. Chapter 2
~ 276 ~
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.
~ 277 ~
6.5.1. Disintegrability study for injection 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 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
increase in the materials opacity to various simultaneous phenomena, such
as the formation of low molar-mass degradation by
hydrolysis and/or the changes in crystallinity in the polymer matrix
(Fukushima, Tabuani, Arena, Gennari and Camino, 2013)
general increase in the polymer and nanocomposite
place at a higher rate in their amorphous zones
in reduction of transparency and a general modification in colour of the
injected materials, as expected due to the high amorphous character of
Results and Discussion. Chapter 2
on moulded samples
The visual evaluation of the injection moulded samples at different
.
PLA and PLA nanocomposites processed by injection moulding at different
times under composting conditions at 58 °C.
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
mass degradation by-products during
hydrolysis and/or the changes in crystallinity in the polymer matrix
(Fukushima, Tabuani, Arena, Gennari and Camino, 2013). Indeed, a
general increase in the polymer and 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
Results and Discussion. Chapter 2
~ 278 ~
the PLA used in this work, as already stated by other authors
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 2.26a reports the disintegrability percentage (weight loss) as a
function of the testing 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
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 with and without thymol in their
formulations increased after 35 days (Figure 2.
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 %
disintegrability tests as required by the ISO 20200 Standard.
the PLA used in this work, as already stated by other authors (Gorrasi and
. Further results at longer testing times showed that the
physical degradation of these samples progressed with burial time
the initial morphology and general rupture
a reports the disintegrability percentage (weight loss) as a
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 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
es with and without thymol in their
Figure 2.26b).
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.
Results and Discussion. Chapter 2
~ 279 ~
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).
Results and Discussion. Chapter 2
~ 280 ~
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.
~ 281 ~
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 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
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 resulted in more degraded materials submitted
Results and Discussion. Chapter 2
FTIR spectra of PLA/Ag/T8 at different times under composting
FESEM micrographs of nanocomposite surfaces after 14 days of
. Important differences in sample
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
(Fukushima, Tabuani, Arena, Gennari and
. After 14 days, those formulations with thymol showed
important fractures up to 2 µm in width (Figure XXX). In general terms, a
ol resulted in more degraded materials submitted
Results and Discussion. Chapter 2
~ 282 ~
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 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 CO
could be also related to the action of microorganisms, which are able to
convert these low molecular weight structures into CO
(Fukushima, Abbate, Tabuani, Gennari and Camin
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
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
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
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
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
oorganisms, which are able to
convert these low molecular weight structures into CO2 and water
(Fukushima, Abbate, Tabuani, Gennari and Camino, 2009).
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).
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 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
Results and Discussion. Chapter 2
~ 283 ~
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.
Results and Discussion. Chapter 2
~ 284 ~
Figure 2.29. DSC thermograms obtained for nanocomposites processed by injection moulding at different times under composting conditions at
(first heating scan (10 °C min
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)).
Results and Discussion. Chapter 2
~ 285 ~
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,
20
25
30
35
40
45
50
55
60
0 3 6 9 12 15 18 21
Tg
(ºC
) (se
cond
hea
ting
scan
)
Time (days)PLA PLA/Ag PLA/T6 PLA/T8 PLA/Ag/T6 PLA/Ag/T8
Results and Discussion. Chapter 2
~ 286 ~
Arena, Gennari and Camino, 2013). After 4 days, all the tested materials
became brittle and just small pieces of films were recovere
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
and a general loss of transparency after 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
transparency loss and evident deformation observed in samples (
. After 4 days, all the tested materials
became brittle and just small pieces of films were 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
fter 7 days under composting
Visual appearance of neat PLA and active nanocomposite films at different
testing days under composting conditions at 58 °C.
shows the evolution of disintegrability values (%) of films vs
time (days). A progressive degradation of samples with the burial time was
by the clear whitening and
transparency loss and evident deformation observed in samples (Figure
Results and Discussion. Chapter 2
~ 287 ~
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.
Results and Discussion. Chapter 2
~ 288 ~
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
0
10
20
30
40
50
60
70
80
90
100
1 2 4 7 14
Dis
inte
grat
ion
(%)
Time (days)
PLA
PLA/Ag
PLA/T6
PLA/T8
PLA/Ag/T6
PLA/Ag/T8
a a a a ab b b b b
a a b b d dc c
a a a a a a
a a a a a a
a a a a a a
Results and Discussion. Chapter 2
~ 289 ~
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.
Results and Discussion. Chapter 2
~ 290 ~
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.
Samples Thymol and Ag-NPs migrated at 10 days 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 Ag-
NPs 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
Results and Discussion. Chapter 2
~ 291 ~
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),
(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).
퐶푡 = 퐶∞[1 − exp(−푘′푡)푑 ]
Results and Discussion. Chapter 2
~ 292 ~
0 100 200 300 400
0
10
20
30
40
0 100 200 300 400
C t (ppm
)
time (hours) 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.
(a) (b)
Results and Discussion. Chapter 2
~ 293 ~
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∞<0.60). Results are represented as the means ± standard deviation, n=3.
In order to have a deeper insight on the mechanism of thymol release, the
power law equation (Equation (2.16)) was applied, in its logarithm form
(Korsmeyer, Gurny, Doelker, Buri and Peppas, 1983)
(2.16) where k and n are fitting parameters, giving the later useful information on
the release mechanism. The validity of Equation (2.16) is restricted to
Ct/C∞ < 0.60.
The mean dissolution time (MDT), which characterizes the thymol release
rate from a given matrix to indicate the thymol-release-retarding efficiency
of the polymer (Costa, Valente, Miguel and Queiroz, 2011) can be
calculated through Equation (2.17).
퐶푡퐶∞ = 푘푡푛
Results and Discussion. Chapter 2
~ 294 ~
(2.17)
The analysis of the calculated n values (Table 2.13) showed that the
thymol migration from the active nanocomposite films did not follow a
diffusion-controlled release (so-called Fickian) but, instead, a Non-Fickian
or anomalous release. This effect occurs when the permeant mobility and
the polymer segment relaxation rates are similar. It is known that ethanol
can act as an aggressive solvent being able to be sorbed by PLA
(Manzanarez-López, Soto-Valdez, Auras and Peralta, 2011) leading to a
plasticizer-like effect (Mascheroni, Guillard, Nalin, Mora and Piergiovanni,
2010), with the consequence of molecular rearrangements caused by the
increase in mobility of the polymer chain (Mutsuga, Kawamura and
Tanamoto, 2008). In this case, it was previously reported that PLA
matrices in contact with ethanol 10 % (v/v) suffer some molecular
modifications leading to the additives release (Fortunati, Peltzer, et al,
2012).
It is worth noticing that the use of Equations (2.15) to (2.17) allows to
conclude that there is a close relationship between the fitting parameter k’
and (MDT)−1 respectively, following the function (MDT)−1 = 1.49 (0.06)
k’ (R2=0.9929), indicating the validity of both semi-empirical equations to
characterize the migration rate of thymol in these PLA matrices.
Furthermore, it could be verified that the migration rate calculated taking
into account the overall range time was moderately higher than that
obtained for short-range times, in agreement with the proposed Non-
Fickian mechanism.
However, a more accurate assessment on the migration rate should be
carried out on the basis of physical grounds. The release kinetics of
푀퐷푇 =푛
푛 + 1푘−푛−1
Results and Discussion. Chapter 2
~ 295 ~
thymol from the PLA-based films was evaluated by using the Lagergren
first-order and pseudo-second order rate equations (Aşçi, Açikel and
Açikel, 2012), which can be written, respectively, in their linear form as
(2.18)
(2.19) where Ce and Ct are the amounts of thymol migrated into the food
simulant (mg L−1) at equilibrium and at time t, respectively, and k1 and k2
are the Lagergren pseudo-first order (s−1) and pseudo-second order rate (L
mg−1 s−1) constants, respectively.
Figure 2.34 shows the representative plots of the fitting of linearized
forms of Equations (2.18) and (2.19) to experimental data. From this
analysis, joined to the fitting parameters summarized in Table 2.14, it can
be concluded that the pseudo-second order model showed higher
correlation coefficients. It was also observed that, considering the
associated imprecision, Ce values agreed well with C∞ which have been
initially estimated and reported in Table 2.13. Both facts suggest that
migration of thymol from PLA-based films follows a pseudo-second
order kinetics, which relies on the assumption that physisorption is not
the rate-limiting step (Ho, 2006), in agreement with the mechanism
described by Equation (2.16).
푙푛(퐶푒 − 퐶푡) = ln(퐶푒) − 푘1푡
푡퐶푡
=1
퐾2퐶푒2+
1퐶푒푡
Results and Discussion. Chapter 2
~ 296 ~
0,0 2,0x105 4,0x105 6,0x105 8,0x105 1,0x106
3,11
3,12
3,13
3,14
3,15
t / s
ln(C
e-Ct)
0
1x104
2x104
3x104
4x104
5x104
(t /
Ct) /
(s-1
mg-1
L)
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.
On the other hand, by comparing k2 values for the migration of thymol
from active nanocomposite films with Ag-NPs, it was observed that the
incorporation of Ag-NPs induced slowing down of the migration process
in both formulations. Perhaps due to the interactions between Ag-NPs
and thymol molecules with the polymer matrix, free space could be
reduced producing some increase in molecular mobility and consequently
easier interaction between all components (Alissawi et al, 2012; Jamshidian
et al, 2012).
Results and Discussion. Chapter 2
~ 297 ~
Table 2.14. Kinetic parameters for migration of thymol from PLA-based films by using
Equations (2.18) and (2.19).
PLA/T6 PLA/Ag/T6 PLA/T8 PLA/Ag/T8
Equation (2.18). First-order order rate equation
k1 (10-8 s-1) 3.4 0.4 1.7 0.3 3.6 0.7 2.2 0.3
Ce (mg L−1) 19 1 43 1 23 1 40 1
R2 0.8960 0.8986 0.7842 0.9384
Equation (2.19). Pseudo-second order rate equation
k2 (10-7 L mg−1 s-1) 1.7 0.2 1.2 0.2 3.1 0.3 0.9 0.1
Ce (mg L−1) 19.1 0.1 36.0 2.0 21.4 0.8 44.0 2.0
R2 0.9834 0.9824 0.9924 0.9905
The results are represented as the means ± standard deviation, n=3.
6.7. Antioxidant activity
DPPH tests were used to evaluate the AO potential of all formulations in
this work. The results obtained for all migration extracts from active
nanocomposite films at 2, 6, 12, 24, 48 hours and 5, 10 and 15 days
followed a similar pattern related to the scavenging ability on DPPH
radicals. Table 2.12 shows the results for the AO performance of all
formulations obtained after 10 days in contact with ethanol 10 % (v/v).
As expected, the scavenging activity against the DPPH radical followed a
similar behaviour than migration results. The amount of thymol present in
each extract increased with time and in consequence, the AO activity was
higher. The dependence between the amount of AO released and the
obtained scavenging activity was also observed by Delgado et al and in a
previous study in PP-based films using thymol and carvacrol as active
additives (Delgado et al, 2014).
Results and Discussion. Chapter 2
~ 298 ~
The DPPH radical scavenging ability was significantly higher in migration
extracts obtained from ternary systems than those extracted from binary
formulations (p < 0.05). The highest value was obtained for PLA/Ag/T8,
corresponding to the highest amount of thymol released. This result is in
agreement with a previous reported work dealing with bioactive
compounds, such as thymol, obtained from EOs, herbs or spices
presenting AO activity when released from the polymer matrix (Iñiguez-
Franco et al, 2012; Barbosa-Pereira et al, 2013; López-Mata et al, 2013;
Noronha, de Carvalho, Lino and Barreto, 2014).
6.8. Antibacterial activity from PLA-based films
The antibacterial activity of neat PLA and nanocomposite films was
evaluated to assess the potential of these films in antibacterial packaging
systems. Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-
positive) were selected in this study as representative bacterial strains. The
antimicrobial viability (%) was evaluated by putting in contact PLA-based
films with suspensions of each bacterial strain incubated for 3 h and 24 h
at 4, 24 and 37 °C, using neat PLA film as control (Table 2.15). 4 and 24
°C were selected as the adequate temperatures to evaluate their influence
on the antibacterial activity, taking into account that perishable food is
usually kept refrigerated at 4 °C but under transportation food is more
likely kept at higher temperatures of storage (24 °C). As expected, neat
PLA control film did not show any inhibitory activity against the studied
bacteria at the incubation times and tested temperatures.
PLA-based binary and ternary systems showed Ag and thymol dose-
dependent antibacterial activity against S.aureus 8325-A and E.coli RB
strains but with some differences. In general, a lower antibacterial effect
Results and Discussion. Chapter 2
~ 299 ~
for Escherichia coli and Staphylococcus aureus was observed regardless of
incubation times and temperatures.
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.
Formulation S. aureus 8325-4 E. coli RB
3 h 24 h 3 h 24 h
At 4 °C.
PLA/Ag 51.7 ± 5.7a 50.4 ± 4.6a 78.1 ± 6.5a 65.4 ± 5.4a
PLA/T6 61.5 ± 5.1a 87.9 ± 4.2a 96.6 ± 5.9c 97.3 ± 4.8c
PLA/T8 71.9 ± 6.0a 91.4 ± 4.9b 92.3 ± 6.8c 89.6 ± 5.6b
PLA/Ag/T6 64.2 ± 4.2a 62.6 ± 3.4a 71.5 ± 4.7a 63.9 ± 3.9a
PLA/Ag/T8 51.3 ± 3.0a 51.5 ± 2.4a 69.9 ± 3.4a 65.9 ± 2.8a
At 24 °C
PLA/Ag 51.7 ± 5.7a 50.4 ± 4.6a 78.0 ± 6.5a 65.4 ± 5.4a
PLA/T6 61.5 ± 5.1a 87.9 ± 4.2a 96.6 ± 5.9c 97.3 ± 4.8c
PLA/T8 71.9 ± 6.0a 91.4 ± 4.9b 92.3 ± 6.8c 89.6 ± 5.6b
PLA/Ag/T6 64.2 ± 4.2a 62.6 ± 3.4a 71.5 ± 4.7a 63.9 ± 3.9a
PLA/Ag/T8 51.3 ± 3.0a 51.5 ± 2.4a 69.9 ± 3.4a 65.9 ± 2.8a
At 37 °C
PLA/Ag 53.8 ± 2.4a 53.6 ± 6.1a 75.0 ± 3.1a 69.7 ± 2.7a
PLA/T6 78.7 ± 5.7b 91.4 ± 4.3c 100.0 ± 2.3c 91.2 ± 3.0c
PLA/T8 72.0 ± 12.4a 97.4 ± 14.1c 96.7 ± 3.6c 90.4 ± 4.8c
PLA/Ag/T6 61.4 ± 4.8a 67.4 ± 1.9a 72.6 ± 3.3a 75.8 ± 4.5a
PLA/Ag/T8 55.6 ± 1.5a 55.6 ± 0.7a 77.1 ± 0.9a 71.7 ± 3.9a
Data obtained are expressed as percentage of the CFU of bacteria grown on PLA film formulations to CFU of bacteria grown on PLA set as 100%. Results are represented as mean ± standard deviation, n=3. a p < 0.001. b p < 0.05. c p > 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.
Results and Discussion. Chapter 2
~ 300 ~
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 Gram-
positive 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).
Results and Discussion. Chapter 2
~ 301 ~
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 Ag-
NPs 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
Results and Discussion. Chapter 2
~ 302 ~
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
Results and Discussion. Chapter 2
~ 303 ~
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.
Results and Discussion. Chapter 2
~ 304 ~
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.
8. References
Addo Ntim, S., Thomas, T.A., Begley, T.H., Noonan, G.O. (2015). Characterisation and
Potential Migration of Silver Nanoparticles from Commercially Available
Polymeric Food Contact Materials. Food Additives & Contaminants: Part A, 32,
1003-1011.
Aguirre, A., Borneo, R., León, A.E. (2013). Antimicrobial, Mechanical and Barrier
Properties of Triticale Protein Films Incorporated with Oregano Essential Oil.
Food Bioscience, 1, 2-9.
Al-Bandak, G., Oreopoulou, V. (2007). Antioxidant Properties and Composition of
Majorana Syriaca Extracts. European Journal of Lipid Science and Technology, 109, 247-
255.
Alissawi, N., Zaporojtchenko, V., Strunskus, T., et al (2012). Tuning of the Ion Release
Properties of Silver Nanoparticles Buried under a Hydrophobic Polymer Barrier.
Journal of Nanoparticle Research, 14, 1-12.
Alix, S., Mahieu, A., Terrie, C., et al (2013). Active Pseudo-Multilayered Films from
Polycaprolactone and Starch Based Matrix for Food-Packaging Applications.
European Polymer Journal, 49, 1234-1242.
Amorati, R., Foti, M.C., Valgimigli, L. (2013). Antioxidant Activity of Essential Oils. Journal
of Agricultural and Food Chemistry, 61, 10835-10847.
Amorati, R., Valgimigli, L. (2015). Advantages and Limitations of Common Testing
Methods for Antioxidants. Free Radical Research, 49, 633-649.
Araújo, A., Botelho, G., Oliveira, M., Machado, A.V. (2014). Influence of Clay Organic
Modifier on the Thermal-Stability of PLA Based Nanocomposites. Applied Clay
Science, 88–89, 144-150.
Arrieta, M.P., López, J., Ferrándiz, S., Peltzer, M.A. (2013). Characterization of PLA-
Limonene Blends for Food Packaging Applications. Polymer Testing, 32, 760-768.
Results and Discussion. Chapter 2
~ 305 ~
Artiaga, G., Ramos, K., Ramos, L., Cámara, C., Gómez-Gómez, M. (2015). Migration and
Characterisation of Nanosilver from Food Containers by AF4-ICP-MS. Food
Chemistry, 166, 76-85.
Aşçi, Y., Açikel, Ü., Açikel, Y.S. (2012). Equilibrium, Hysteresis and Kinetics of Cadmium
Desorption from Sodium-Feldspar Using Rhamnolipid Biosurfactant.
Environmental Technology, 33, 1857-1868.
Astm D 882 - 09. Standard Test Method for Tensile Properties of Thin Plastic Sheeting.
2009
Auras, R.A., Harte, B., Selke, S., Hernández, R. (2003). Mechanical, Physical, and Barrier
Properties of Poly(Lactide) Films. Journal of Plastic Film and Sheeting, 19, 123-135.
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.
Barrere, C., Selmi, W., Hubert-Roux, M., et al (2014). Rapid Analysis of Polyester and Polyethylene Blends by Ion Mobility-Mass Spectrometry. Polymer Chemistry, 5, 3576-3582.
Beltrán, A., Valente, A.J.M., Jiménez, A., Garrigós, M.a.C. (2014). Characterization of
Poly(ε-Caprolactone)-Based Nanocomposites Containing Hydroxytyrosol for
Active Food Packaging. Journal of Agricultural and Food Chemistry, 62, 2244-2252.
Burgos, N., Martino, V.P., Jiménez, A. (2013). Characterization and Ageing Study of
Poly(Lactic Acid) Films Plasticized with Oligomeric Lactic Acid. Polymer
Degradation and Stability, 98, 651-658.
Burt, S. (2004). Essential Oils: Their Antibacterial Properties and Potential Applications in
Foods-a Review. International Journal of Food Microbiology, 94, 223-253.
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.
Campos-Requena, V.H., Rivas, B.L., Pérez, M.A., Garrido-Miranda, K.A., Pereira, E.D.
(2015). Polymer/Clay Nanocomposite Films as Active Packaging Material:
Modeling of Antimicrobial Release. European Polymer Journal, 71, 461-475.
Commission Regulation (EU) No 10/2011. Plastic Materials and Articles Intended to
Come into Contact with Food.
Costa, D., Valente, A.J.M., Miguel, M.G., Queiroz, J. (2011). Gel Network
Photodisruption: A New Strategy for the Codelivery of Plasmid DNA and
Drugs. Langmuir, 27, 13780-13789.
Results and Discussion. Chapter 2
~ 306 ~
Crank, J., (1975). The Mathematics of Diffusion, Place: Oxford University Press. Oxford
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.
De Azeredo, H.M.C. (2013). Antimicrobial Nanostructures in Food Packaging. Trends in
Food Science & Technology, 30, 56-69.
Delgado, T., Marinero, P., Asensio-S.-Manzanera, M.C., et al (2014). Antioxidant Activity
of Twenty Wild Spanish Thymus Mastichina L. Populations and Its Relation
with Their Chemical Composition. LWT - Food Science and Technology, 57, 412-
418.
Duncan, T.V. (2011). Applications of Nanotechnology in Food Packaging and Food
Safety: Barrier Materials, Antimicrobials and Sensors. Journal of Colloid and Interface
Science, 363, 1-24.
Echegoyen, Y., Nerín, C. (2013). Nanoparticle Release from Nano-Silver Antimicrobial
Food Containers. Food and Chemical Toxicology, 62, 16-22.
Efrati, R., Natan, M., Pelah, A., et al (2014). The Combined Effect of Additives and
Processing on the Thermal Stability and Controlled Release of Essential Oils in
Antimicrobial Films. Journal of Applied Polymer Science, 131, In press. doi:
10.1002/app.40564.
EFSA (2005). Opinion of the Scientific Panel on Food Additives, Flavourings, Process-Ing
Aids and Materials in Contact with Food (AFC) on a Request from the Commis-
Sion Related to a 7th List of Substances for Food Contact Materials. . EFSA
Journal. Available from: http://www.efsa.europa.eu/en/scdocs/scdoc/201a.htm, 201.
Emamifar, A., Kadivar, M., Shahedi, M., Soleimanian-Zad, S. (2011). Effect of
Nanocomposite Packaging Containing Ag and ZnO on Inactivation of
Lactobacillus Plantarum in Orange Juice. Food Control, 22, 408-413.
Emiroglu, Z.K., Yemis, G.P., Coskun, B.K., Candogan, K. (2010). Antimicrobial Activity
of Soy Edible Films Incorporated with Thyme and Oregano Essential Oils on
Fresh Ground Beef Patties. Meat Science, 86, 283-288.
Erem, A.D., Ozcan, G., Erem, H., Skrifvars, M. (2013). Antimicrobial Activity of Poly(L-
Lactide Acid)/Silver Nanocomposite Fibers. Textile Research Journal, 83, 2111-
2117.
Fabra, M.J., Talens, P., Chiralt, A. (2008). Tensile Properties and Water Vapor Permeability
of Sodium Caseinate Films Containing Oleic Acid–Beeswax Mixtures. Journal of
Food Engineering, 85, 393-400.
Results and Discussion. Chapter 2
~ 307 ~
Feng, Q.L., Wu, J., Chen, G.Q., et al (2000). A Mechanistic Study of the Antibacterial
Effect of Silver Ions on Escherichia Coli and Staphylococcus Aureus. Journal of
Biomedical Materials Research, 52, 662-668.
Fonseca, C., Ochoa, A., Ulloa, M.T., et al (2015). Poly(Lactic Acid)/TiO2 Nanocomposites
as Alternative Biocidal and Antifungal Materials. Materials Science and Engineering
C, 57, 314-320.
Fortunati, E., Armentano, I., Iannoni, A., et al (2012). New Multifunctional Poly(Lactide
Acid) Composites: Mechanical, Antibacterial, and Degradation Properties.
Journal of Applied Polymer Science, 124, 87-98.
Fortunati, E., Armentano, I., Iannoni, A., Kenny, J.M. (2010). Development and Thermal
Behaviour of Ternary PLA Matrix Composites. Polymer Degradation and Stability,
95, 2200-2206.
Fortunati, E., Armentano, I., Zhou, Q., et al (2012). Multifunctional Bionanocomposite
Films of Poly(Lactic Acid), Cellulose Nanocrystals and Silver Nanoparticles.
Carbohydrate Polymers, 87, 1596-1605.
Fortunati, E., Armentano, I., Zhou, Q., et al (2012). Microstructure and Nonisothermal
Cold Crystallization of PLA Composites Based on Silver Nanoparticles and
Nanocrystalline Cellulose. Polymer Degradation and Stability, 97, 2027-2036.
Fortunati, E., Luzi, F., Puglia, D., et al (2014). Investigation of Thermo-Mechanical,
Chemical and Degradative Properties of PLA-Limonene Films Reinforced with
Cellulose Nanocrystals Extracted from Phormium Tenax Leaves. European
Polymer Journal, 56, 77-91.
Fortunati, E., Luzi, F., Puglia, D., et al (2015). Processing of PLA Nanocomposites with
Cellulose Nanocrystals Extracted from Posidonia Oceanica Waste: Innovative
Reuse of Coastal Plant. Industrial Crops and Products, 67, 439-447.
Fortunati, E., Peltzer, M., Armentano, I., et al (2012). Effects of Modified Cellulose
Nanocrystals on the Barrier and Migration Properties of PLA Nano-
Biocomposites. Carbohydrate Polymers, 90, 948-956.
Fortunati, E., Rinaldi, S., Peltzer, M., et al (2014). Nano-Biocomposite Films with Modified
Cellulose Nanocrystals and Synthesized Silver Nanoparticles. Carbohydrate
Polymers, 101, 1122-1133.
Fukushima, K., Abbate, C., Tabuani, D., Gennari, M., Camino, G. (2009). Biodegradation
of Poly(Lactic Acid) and Its Nanocomposites. Polymer Degradation and Stability, 94,
1646-1655.
Results and Discussion. Chapter 2
~ 308 ~
Fukushima, K., Feijoo, J.L., Yang, M.-C. (2012). Abiotic Degradation of Poly(DL-Lactide),
Poly(Ɛ-Caprolactone) and Their Blends. Polymer Degradation and Stability, 97,
2347-2355.
Fukushima, K., Tabuani, D., Arena, M., Gennari, M., Camino, G. (2013). Effect of Clay
Type and Loading on Thermal, Mechanical Properties and Biodegradation of
Poly(Lactic Acid) Nanocomposites. Reactive and Functional Polymers, 73, 540-549.
Fukushima, K., Tabuani, D., Camino, G. (2009). Nanocomposites of PLA and PCL Based
on Montmorillonite and Sepiolite. Materials Science and Engineering: C, 29, 1433-
1441.
Gamez-Perez, J., Nascimento, L., Bou, J.J., et al (2011). Influence of Crystallinity on the
Fracture Toughness of Poly(Lactic Acid)/Montmorillonite Nanocomposites
Prepared by Twin-Screw Extrusion. Journal of Applied Polymer Science, 120, 896-
905.
Gonçalves, C.M.B., Tomé, L.C., Garcia, H., et al (2013). Effect of Natural and Synthetic
Antioxidants Incorporation on the Gas Permeation Properties of Poly(Lactic
Acid) Films. Journal of Food Engineering, 116, 562-571.
Gorrasi, G., Pantani, R. (2013). Effect of PLA Grades and Morphologies on Hydrolytic
Degradation at Composting Temperature: Assessment of Structural
Modification and Kinetic Parameters. Polymer Degradation and Stability, 98, 1006-
1014.
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.
Gyawali, R., Ibrahim, S.A. (2014). Natural Products as Antimicrobial Agents. Food Control,
46, 412-429.
Ho, Y.S. (2006). Review of Second-Order Models for Adsorption Systems. Journal of
Hazardous Materials, 136, 681-689.
Huang, J.Y., Li, X., Zhou, W. (2015). Safety Assessment of Nanocomposite for Food
Packaging Application. Trends in Food Science & Technology, 45, 187-199.
Hughes, J., Thomas, R., Byun, Y., Whiteside, S. (2012). Improved Flexibility of Thermally
Stable Poly-Lactic Acid (PLA). Carbohydrate Polymers, 88, 165-172.
Hwang, S.W., Shim, J.K., Selke, S.E.M., et al (2012). Poly(L-Lactic Acid) with Added α-
Tocopherol and Resveratrol: Optical, Physical, Thermal and Mechanical
Properties. Polymer International, 61, 418-425.
Results and Discussion. Chapter 2
~ 309 ~
Inkinen, S., Hakkarainen, M., Albertsson, A.C., Sodergard, A. (2011). From Lactic Acid to
Poly(Lactic Acid) (PLA): Characterization and Analysis of PLA and Its
Precursors. Biomacromolecules, 12, 523-532.
Iñiguez-Franco, F., Soto-Valdez, H., Peralta, E., et al (2012). Antioxidant Activity and
Diffusion of Catechin and Epicatechin from Antioxidant Active Films Made of
Poly(L-Lactic Acid). Journal of Agricultural and Food Chemistry, 60, 6515-6523.
Jamshidian, M., Arab Tehrany, E., Cleymand, F., et al (2012). Effects of Synthetic Phenolic
Antioxidants on Physical, Structural, Mechanical and Barrier Properties of Poly
Lactic Acid Film. Carbohydrate Polymers, 87, 1763-1773.
Jamshidian, M., Tehrany, E.A., Imran, M., Jacquot, M., Desobry, S. (2010). Poly-Lactic
Acid: Production, Applications, Nanocomposites, and Release Studies.
Comprehensive Reviews in Food Science and Food Safety, 9, 552-571.
Jokar, M., Abdul Rahman, R., Ibrahim, N.A., Abdullah, L.C., Tan, C.P. (2012). Melt
Production and Antimicrobial Efficiency of Low-Density Polyethylene (LDPE)-
Silver Nanocomposite Film. Food and Bioprocess Technology, 5, 719-728.
Kanmani, P., Rhim, J.W. (2014a). Physical, Mechanical and Antimicrobial Properties of
Gelatin Based Active Nanocomposite Films Containing Agnps and Nanoclay.
Food Hydrocolloids, 35, 644-652.
Kanmani, P., Rhim, J.W. (2014b). Physicochemical Properties of Gelatin/Silver
Nanoparticle Antimicrobial Composite Films. Food Chemistry, 148, 162-169.
Kavoosi, G., Dadfar, S.M.M., Purfard, A.M. (2013). Mechanical, Physical, Antioxidant, and
Antimicrobial Properties of Gelatin Films Incorporated with Thymol for
Potential Use as Nano Wound Dressing. Journal of Food Science, 78, E244-E250.
Khabbaz, F., Karlsson, S., Albertsson, A.C. (2000). Py-GC/MS an Effective Technique to
Characterizing of Degradation Mechanism of Poly (L-Lactide) in the Different
Environment. Journal of Applied Polymer Science, 78, 2369-2378.
Korsmeyer, R.W., Gurny, R., Doelker, E., Buri, P., Peppas, N.A. (1983). Mechanisms of
Solute Release from Porous Hydrophilic Polymers. International Journal of
Pharmaceutics, 15, 25-35.
Kumar, R., Münstedt, H. (2005). Silver Ion Release from Antimicrobial Polyamide/Silver
Composites. Biomaterials, 26, 2081-2088.
Kvítek, L., Panáček, A., Soukupová, J., et al (2008). Effect of Surfactants and Polymers on
Stability and Antibacterial Activity of Silver Nanoparticles (NPs). The Journal of
Physical Chemistry C, 112, 5825-5834.
Results and Discussion. Chapter 2
~ 310 ~
Lagaron, J.M., Lopez-Rubio, A. (2011). Nanotechnology for Bioplastics: Opportunities,
Challenges and Strategies. Trends in Food Science & Technology, 22, 611-617.
Lewitus, D., Mccarthy, S., Ophir, A., Kenig, S. (2006). The Effect of Nanoclays on the
Properties of PLLA-Modified Polymers Part 1: Mechanical and Thermal
Properties. Journal of Polymers and the Environment, 14, 171-177.
López-Mata, M., Ruiz-Cruz, S., Silva-Beltrán, N., et al (2013). Physicochemical,
Antimicrobial and Antioxidant Properties of Chitosan Films Incorporated with
Carvacrol. Molecules, 18, 13735-13753.
López-Rubio, A., Lagaron, J.M. (2010). Improvement of UV Stability and Mechanical
Properties of Biopolyesters through the Addition of β-Carotene. Polymer
Degradation and Stability, 95, 2162-2168.
Luzi, F., Fortunati, E., Puglia, D., et al (2015). Study of Disintegrability in Compost and
Enzymatic Degradation of PLA and PLA Nanocomposites Reinforced with
Cellulose Nanocrystals Extracted from Posidonia Oceanica. Polymer Degradation
and Stability, 121, 105-115.
Maneerung, T., Tokura, S., Rujiravanit, R. (2008). Impregnation of Silver Nanoparticles
into Bacterial Cellulose for Antimicrobial Wound Dressing. Carbohydrate Polymers,
72, 43-51.
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, 508-517.
Martino, V.P., Jiménez, A., Ruseckaite, R.A., Avérous, L. (2010). Structure and Properties
of Clay Nano-Biocomposites Based on Poly(Lactic Acid) Plasticized with
Polyadipates. Polymers for Advanced Technologies, 22, 2206-2213.
Martino, V.P., Ruseckaite, R.A., Jiménez, A., Averous, L. (2010). Correlation between
Composition, Structure and Properties of Poly(Lactic Acid)/Polyadipate-Based
Nano-Biocomposites. Macromolecular Materials and Engineering, 295, 551-558.
Mascheroni, E., Guillard, V., Nalin, F., Mora, L., Piergiovanni, L. (2010). Diffusivity of
Propolis Compounds in Polylactic Acid Polymer for the Development of Anti-
Microbial Packaging Films. Journal of Food Engineering, 98, 294-301.
Mastelic, J., Jerkovic, I., Blazevic, I., et al (2008). Comparative Study on the Antioxidant
and Biological Activities of Carvacrol, Thymol, and Eugenol Derivatives. Journal
of Agricultural and Food Chemistry, 56, 3989-3996.
Results and Discussion. Chapter 2
~ 311 ~
Mellinas, C., Valdés, A., Ramos, M., et al (2015). Active Edible Films: Current State and
Future Trends. Journal of Applied Polymer Science, In press. doi: 10.1002/app.42631.
Mihindukulasuriya, S.D.F., Lim, L.T. (2014). Nanotechnology Development in Food
Packaging: A Review. Trends in Food Science & Technology, 40, 149-167.
Mutsuga, M., Kawamura, Y., Tanamoto, K. (2008). Migration of Lactic Acid, Lactide and
Oligomers from Polylactide Food-Contact Materials. Food Additives &
Contaminants: Part A, 25, 1283-1290.
Nigmatullin, R., Gao, F., Konovalova, V. (2008). Polymer-Layered Silicate
Nanocomposites in the Design of Antimicrobial Materials. Journal of Materials
Science, 43, 5728-5733.
Noronha, C.M., De Carvalho, S.M., Lino, R.C., Barreto, P.L.M. (2014). Characterization of
Antioxidant Methylcellulose Film Incorporated with α-Tocopherol
Nanocapsules. Food Chemistry, 159, 529-535.
Ortiz-Vazquez, H., Shin, J., Soto-Valdez, H., Auras, R. (2011). Release of Butylated
Hydroxytoluene (BHT) from Poly(Lactic Acid) Films. Polymer Testing, 30, 463-
471.
Pagno, C.H., Costa, T.M.H., De Menezes, E.W., et al (2015). Development of Active
Biofilms of Quinoa (Chenopodium Quinoa W.) Starch Containing Gold
Nanoparticles and Evaluation of Antimicrobial Activity. Food Chemistry, 173, 755-
762.
Papadopoulou, V., Kosmidis, K., Vlachou, M., Macheras, P. (2006). On the Use of the
Weibull Function for the Discernment of Drug Release Mechanisms.
International Journal of Pharmaceutics, 309, 44-50.
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.
Paul, M.A., Delcourt, C., Alexandre, M., et al (2005). Polylactide/Montmorillonite
Nanocomposites: Study of the Hydrolytic Degradation. Polymer Degradation and
Stability, 87, 535-542.
Peelman, N., Ragaert, P., De Meulenaer, B., et al (2013). Application of Bioplastics for
Food Packaging. Trends in Food Science & Technology, 32, 128-141.
Peltzer, M., Wagner, J., Jiménez, A. (2009). Migration Study of Carvacrol as a Natural
Antioxidant in High-Density Polyethylene for Active Packaging. Food Additives
Results and Discussion. Chapter 2
~ 312 ~
& Contaminants: Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment,
26, 938 - 946.
Perricone, M., Arace, E., Corbo, M.R., Sinigaglia, M., Bevilacqua, A. (2015). Bioactivity of
Essential Oils: A Review on Their Interaction with Food Components. Frontiers
in Microbiology, 6, 76.
Persico, P., Ambrogi, V., Carfagna, C., et al (2009). Nanocomposite Polymer Films
Containing Carvacrol for Antimicrobial Active Packaging. Polymer Engineering &
Science, 49, 1447-1455.
Picard, E., Espuche, E., Fulchiron, R. (2011). Effect of an Organo-Modified
Montmorillonite on PLA Crystallization and Gas Barrier Properties. Applied Clay
Science, 53, 58-65.
Priolo, M.A., Holder, K.M., Gamboa, D., Grunlan, J.C. (2011). Influence of Clay
Concentration on the Gas Barrier of Clay–Polymer Nanobrick Wall Thin Film
Assemblies. Langmuir, 27, 12106-12114.
Proikakis, C.S., Mamouzelos, N.J., Tarantili, P.A., Andreopoulos, A.G. (2006). Swelling
and Hydrolytic Degradation of Poly(D,L-Lactic Acid) in Aqueous Solutions.
Polymer Degradation and Stability, 91, 614-619.
Qin, Y.-Y., Zhang, Z.-H., Li, L., et al (2015). Physio-Mechanical Properties of an Active
Chitosan Film Incorporated with Montmorillonite and Natural Antioxidants
Extracted from Pomegranate Rind. Journal of Food Science and Technology, 52, 1471-
1479.
Quilaqueo Gutiérrez, M., Echeverría, I., Ihl, M., Bifani, V., Mauri, A.N. (2012).
Carboxymethylcellulose–Montmorillonite Nanocomposite Films Activated with
Murta (Ugni Molinae Turcz) Leaves Extract. Carbohydrate Polymers, 87, 1495-1502.
Raquez, J.M., Habibi, Y., Murariu, M., Dubois, P. (2013). Polylactide (PLA)-Based
Nanocomposites. Progress in Polymer Science, 38, 1504-1542.
Rawi, N.F.M., Jayaraman, K., Bhattacharyya, D. (2014). Bamboo Fabric Reinforced
Polypropylene and Poly(Lactic Acid) for Packaging Applications: Impact,
Thermal, and Physical Properties. Polymer Composites, 35, 1888-1899.
Reddy, M.M., Vivekanandhan, S., Misra, M., Bhatia, S.K., Mohanty, A.K. (2013). Biobased
Plastics and Bionanocomposites: Current Status and Future Opportunities.
Progress in Polymer Science, 38, 1653-1689.
Reidy, B., Haase, A., Luch, A., Dawson, K., Lynch, I. (2013). Mechanisms of Silver
Nanoparticle Release, Transformation and Toxicity: A Critical Review of
Results and Discussion. Chapter 2
~ 313 ~
Current Knowledge and Recommendations for Future Studies and Applications.
Materials, 6, 2295-2350.
Rhim, J.W. (2013). Preparation and Characterization of Vacuum Sputter Silver Coated
PLA Film. LWT - Food Science and Technology, 54, 477-484.
Rhim, J.W., Hong, S.I., Ha, C.S. (2009). Tensile, Water Vapor Barrier and Antimicrobial
Properties of PLA/Nanoclay Composite Films. LWT - Food Science and Technology,
42, 612-617.
Rhim, J.W., Wang, L.F. (2014). Preparation and Characterization of Carrageenan-Based
Nanocomposite Films Reinforced with Clay Mineral and Silver Nanoparticles.
Applied Clay Science, 97–98, 174-181.
Rhim, J.W., Wang, L.F., Hong, S.I. (2013). Preparation and Characterization of Agar/Silver
Nanoparticles Composite Films with Antimicrobial Activity. Food Hydrocolloids,
33, 327-335.
Rukmani, A., Sundrarajan, M. (2011). Inclusion of Antibacterial Agent Thymol on β-
Cyclodextrin-Grafted Organic Cotton. Journal of Industrial Textiles, 42, 132-144.
Samsudin, H., Soto-Valdez, H., Auras, R. (2014). Poly(Lactic Acid) Film Incorporated with
Marigold Flower Extract (Tagetes Erecta) Intended for Fatty-Food Application.
Food Control, 46, 55-66.
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
Sánchez-González, L., Vargas, M., González-Martínez, C., Chiralt, A., Cháfer, M. (2011).
Use of Essential Oils in Bioactive Edible Coatings: A Review. Food Engineering
Reviews, 3, 1-16.
Sánchez-Moreno, C. (2002). Review: Methods Used to Evaluate the Free Radical
Scavenging Activity in Foods and Biological Systems. Food Science and Technology
International, 8, 121-137.
Scatto, M., Salmini, E., Castiello, S., et al (2013). Plasticized and Nanofilled Poly(Lactic
Acid)-Based Cast Films: Effect of Plasticizer and Organoclay on Processability
and Final Properties. Journal of Applied Polymer Science, 127, 4947-4956.
Scherer, R., Godoy, H.T. (2009). Antioxidant Activity Index (AAI) by the 2,2-Diphenyl-1-
Picrylhydrazyl Method. Food Chemistry, 112, 654-658.
Results and Discussion. Chapter 2
~ 314 ~
Shameli, K., Ahmad, M.B., Yunus, W.M.Z.W., et al (2010). Silver/Poly (Lactic Acid)
Nanocomposites: Preparation, Characterization, and Antibacterial Activity.
International Journal of Nanomedicine, 5, 573-579.
Sharma, V.K., Yngard, R.A., Lin, Y. (2009). Silver Nanoparticles: Green Synthesis and
Their Antimicrobial Activities. Advances in Colloid and Interface Science, 145, 83-96.
Shemesh, R., Goldman, D., Krepker, M., et al (2015). LDPE/Clay/Carvacrol
Nanocomposites with Prolonged Antimicrobial Activity. Journal of Applied
Polymer Science, 132, In press: doi: 10.1002/app.41261.
Silva, A.S., Cruz Freire, J.M., SendoN, R., Franz, R., Paseiro Losada, P. (2009). Migration
and Diffusion of Diphenylbutadiene from Packages into Foods. Journal of
Agricultural and Food Chemistry, 57, 10225-10230.
Sinha Ray, S., Yamada, K., Okamoto, M., Ueda, K. (2003). New Polylactide-Layered
Silicate Nanocomposites. 2. Concurrent Improvements of Material Properties,
Biodegradability and Melt Rheology. Polymer, 44, 857-866.
Siracusa, V., Rocculi, P., Romani, S., Rosa, M.D. (2008). Biodegradable Polymers for Food
Packaging: A Review. Trends in Food Science & Technology, 19, 634-643.
Song, H., Li, B., Lin, Q.B., Wu, H.J., Chen, Y. (2011). Migration of Silver from
Nanosilver–Polyethylene Composite Packaging into Food Simulants. Food
Additives & Contaminants: Part A, 28, 1758-1762.
Souza, P., Morales, A., Marin-Morales, M., Mei, L. (2013). PLA and Montmorilonite
Nanocomposites: Properties, Biodegradation and Potential Toxicity. Journal of
Polymers and the Environment, 21, 738-759.
Sung, S.Y., Sin, L.T., Tee, T.T., et al (2013). Antimicrobial Agents for Food Packaging
Applications. Trends in Food Science & Technology, 33, 110-123.
Tawakkal, I.S.M.A., Cran, M.J., Bigger, S.W. (2014). Effect of Kenaf Fibre Loading and
Thymol Concentration on the Mechanical and Thermal Properties of
Pla/Kenaf/Thymol Composites. Industrial Crops and Products, 61, 74-83.
Tornuk, F., Hancer, M., Sagdic, O., Yetim, H. (2015). LLDPEe Based Food Packaging
Incorporated with Nanoclays Grafted with Bioactive Compounds to Extend
Shelf Life of Some Meat Products. LWT - Food Science and Technology, 64, 540-
546.
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, 41-48.
Results and Discussion. Chapter 2
~ 315 ~
Trombetta, D., Castelli, F., Sarpietro, M.G., et al (2005). Mechanisms of Antibacterial
Action of Three Monoterpenes. Antimicrobial Agents and Chemotherapy, 49, 2474-
2478.
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
UNE-EN_20200:2006. Plastics - Determination of the Degree of Disintegration of Plastic
Materials under Simulated Composting Conditions in a Laboratory-Scale Test
(ISO 20200:2004). 2006
Valdés, A., Mellinas, A.C., Ramos, M., Garrigós, M.C., Jiménez, A. (2014). Natural
Additives and Agricultural Wastes in Biopolymer Formulations for Food
Packaging. Frontiers in Chemistry, 2, 1-10.
Vigneshwaran, N., Nachane, R.P., Balasubramanya, R.H., Varadarajan, P.V. (2006). A
Novel One-Pot ‘Green’ Synthesis of Stable Silver Nanoparticles Using Soluble
Starch. Carbohydrate Research, 341, 2012-2018.
Viuda-Martos, M., Navajas, Y.R., Zapata, E.S., Fernández-López, J., Pérez-Álvarez, J.A.
(2010). Antioxidant Activity of Essential Oils of Five Spice Plants Widely Used
in a Mediterranean Diet. Flavour and Fragrance Journal, 25, 13-19.
Wessling, C., Nielsen, T., Giacin, J.R. (2001). Antioxidant Ability of BHT and α-
Tocopherol Impregnated LDPE Film in Packaging of Oatmeal. Journal of the
Science of Food and Agriculture, 81, 194-201.
Wu, Y., Qin, Y., Yuan, M., et al (2014). Characterization of an Antimicrobial Poly(Lactic
Acid) Film Prepared with Poly(ε-Caprolactone) and Thymol for Active
Packaging. Polymers for Advanced Technologies, 25, 948-954.
Wu, Y., Yuan, M.W., Yang, J.Y., et al (2014). Physical Properties and Antimicrobial Activity
of a Poly(Lactic Acid)/Poly(Trimethylene Carbonate) Film Incorporated with
Thymol. Advanced Materials Research, 884-885, 481-484.
Yang, W., Fortunati, E., Dominici, F., Kenny, J.M., Puglia, D. (2015). Effect of Processing
Conditions and Lignin Content on Thermal, Mechanical and Degradative
Behavior of Lignin Nanoparticles/Polylactic (Acid) Bionanocomposites
Results and Discussion. Chapter 2
~ 316 ~
Prepared by Melt Extrusion and Solvent Casting. European Polymer Journal, 71,
126-139.
IV. General Conclusions
General Conclusions
~ 319 ~
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
General Conclusions
~ 320 ~
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 shelf-
life 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
General Conclusions
~ 321 ~
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 PLA-
based 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
General Conclusions
~ 322 ~
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.
