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Centro de Investigación en Alimentación y Desarrollo, A. C.
EFECTO DE LAS CARACTERÍSTICAS QUÍMICAS Y REOLÓGICAS DE LAS PECTINAS EN LA MICELARIZACIÓN DE CAROTENOIDES
Por:
Braulio Cervantes Paz
TESIS APROBADA POR LA:
UNIDAD CUAUHTÉMOC EN FISIOLOGÍA Y TECNOLOGÍA DE ALIMENTOS DE LA ZONA TEMPLADA
Como requisito parcial para obtener el grado de:
DOCTORADO EN CIENCIAS
Cd. Cuauhtémoc, Chihuahua, Enero de 2016
iii
DECLARACIÓN INSTITUCIONAL
La información generada en esta tesis es propiedad intelectual del Centro de
Investigación en Alimentación y Desarrollo, A.C. (CIAD). Se permiten y agradecen las
citas breves del material contenido en esta tesis sin permiso especial del autor, siempre y
cuando se dé crédito correspondiente. Para la reproducción parcial o total de la tesis con
fines académicos, se deberá contar con la autorización escrita del Director General del
CIAD.
La publicación en comunicaciones científicas o de divulgación popular de los
datos contenidos en esta tesis, deberá dar los créditos al CIAD, previa autorización
escrita del manuscrito en cuestión del director de tesis.
__________________________________ Dr. Pablo Wong González
Director General
iv
Este trabajo se realizó en el laboratorio de Fitoquímicos y Nutrientes del Centro
de Investigación en Alimentación y Desarrollo A.C. (Unidad Cuauhtémoc) bajo
la dirección del Dr. José de Jesús Ornelas Paz, y forma parte del proyecto
“Modificación fisicoquímica de la pectina del chile Jalapeño durante la
maduración y procesamiento térmico doméstico y su efecto en la
bioaccesibilidad de los carotenoides” (clave 103391), financiado por el Fondo
Sectorial de Investigación para la Educación (Investigación Básica SEP-
CONACYT).
v
AGRADECIMIENTOS
Al consejo Nacional de Ciencia y Tecnología (CONACYT), por la oportunidad y apoyo económico brindado para la realización de mis estudios en la obtención del grado de Maestro en Ciencias.
Al Centro de Investigación en Alimentación y Desarrollo, A. C. (CIAD), por darme la oportunidad de desarrollar y ampliar mi conocimiento académico y por contar con personal que me brindó el apoyo necesario en la realización de mi proyecto de investigación.
Al Dr. José de Jesús Ornelas Paz por su dirección, apoyo y asesorías brindadas en la realización de mi trabajo de investigación y durante la escritura de mi tesis. Por siempre estar disponible en el momento indicado para guiarme y también para llamarme la atención cuando lo necesité.
A los miembros de mi comité: Dr. Elhadi Yahia Kazuz, Dr. Alfonso Gardea Béjar y Dr. Paul Baruk Zamudio Flores, por las sugerencias, recomendaciones y comentarios tan acertados sobre mi trabajo de investigación.
A los doctores Jaime David Pérez Martínez y Jaime Reyes Hernández de la Universidad Autónoma de San Luis Potosí por permitirme realizar parte de mi trabajo en sus laboratorios y por brindarme su disponibilidad para asesorarme.
A Clau, Paty, Anita y Viki por ser esos compañeros de laboratorio con los que siempre se puede contar cuando se les necesita, pero, sobre todo, por brindarme su amistad.
A Javier Molina, Alejandro Romo, Ignacio Berlanga y Emilio Ochoa por su apoyo y disponibilidad que me brindaron, los cuales forman un granito de arena para la realización de mi trabajo en este centro de investigación.
Quiero agradecer a mi nueva familia (Claudia y Bengie) que, sin su compañía, esta travesía del Doctorado no hubiera sido posible. Gracias por ser los seres vivos que estuvieron conmigo en todo momento, en la buenas y en las malas. En especial, agradezco a mi esposa Claudia por brindarme su apoyo, asesoría, compañía, amor y cariño. Porque sin ella, esto no hubiera sido posible.
vi
Por último, pero no menos importante, agradezco infinitamente a mi mamá por darme la vida y por siempre preocuparse por mí, por ser esa señora regañona que me dio mis cinturonazos cuando me los merecía, pero también por darme una buena educación, la cual me permitió llegar hasta aquí. Agradezco a mi hermano Carlos Francisco y a mis hermanas Norma y Maru, que, aunque casi no las veo, nunca me olvido de ellas. Agradezco a mi hermana Mónica que ya no está con nosotros, pero estuvo en los momentos más difíciles de mi vida y sin su cuidado, yo no estaría aquí.
vii
DEDICATORIA
Este trabajo está dedicado a las personas más importantes en mi vida…
A mi esposa Clau que es para mí un ejemplo a seguir, pero sobre todo por tomar la decisión de pasar el resto de su vida a mi lado y compartir estos momentos de éxito,
pero también de desaliento.
TE AMO CON TODO MI CORAZÓN CLAU
A mi mamá que es la persona de la que más orgulloso me siento, por pensar siempre en mí y recordarme en todo momento, por dedicar toda su vida a mí y a mis hermanos.
Te quiero mucho mamá, gracias por todo.
A mi hermana Mónica, quien ya no está conmigo para poder agradecerle por todo lo que me dio y por dedicarme gran parte de su vida, pero sobre todo por ser mi hermana.
Te dedico este trabajo Mónica, donde quiera que te encuentres.
viii
CONTENIDO
LISTA DE COMUNICACIONES CIENTÍFICAS ..………………………………….. viii
LISTA DE FIGURAS ………………………………………………………………… ix
RESUMEN…………………………………………………………………………….. x
ABSTRACT…………………………………………………………………………… xii
INTRODUCCIÓN…………………………………………………………………….. 14
PLANTEAMIENTO DEL PROBLEMA……………………………………………... 16
RESULTADOS Y DISCUSIÓN……………………………………………………… 19
CONCLUSIONES…………………………………………………………………….. 26
REFERENCIAS………………………………………………………………………. 27
COMUNICACIONES CIENTÍFICAS
Capítulo 1. Absorption of carotenoids and mechanisms involved in their health-related
properties……………………………………………………….................
33
Capítulo 2. Factors involved in lipids emulsification process affected by fibers as
determinant mechanisms in the carotenoids absorption. An hypothesis….
86
Capítulo 3. Effect of ripening and heat processing on the physicochemical and
rheological properties of pepper pectins…………………………………..
132
Capítulo 4. Effect of pectin concentration and properties on digestive events involved
on micellarization of free and esterified carotenoids…………………….
143
Capítulo 5. Impact of the physicochemical properties of pectin on micellar and oil
phase lipid composition and carotenoid micellarization………………….
153
ix
LISTA DE FIGURAS
Figura 1 (Cap. 2) The carotenoid absorption process…………………………... 127
Figura 2 (Cap. 2) Effect of calcium-pectin interaction on lipolysis and
carotenoid micellarization……………………………………………..
128
Figura 3 (Cap. 2) General effects of BS binding by pectin on lipolysis and
carotenoid micellarization……………………………………………..
129
Figura 4 (Cap. 2) Pectin coatings on lipid droplet surface interfering on the
aggregation state, coalescence, emulsion stability and their effect on
the lipolysis……………………………………………………………
130
Figura 5 (Cap. 2) Graphical abstract…………………………………………… 131
Figura 1 (Cap. 5) Apparent viscosity of gastric and intestinal media with low
and high fiber concentration…………………………………………..
169
Figura 2 (Cap. 5) Lipid composition in oil and micellar phases of digestion
reactions with different fiber types at two concentration levels………
170
Figura 3 (Cap. 5) Micellarization of carotenoid fractions in digestion reactions
with different fiber types at two concentration levels………………...
171
x
RESUMEN
Los carotenoides son pigmentos liposolubles con efectos benéficos en la salud. Su
absorción es extremadamente baja y limita su bioactividad. El proceso de absorción
implica su transferencia de las gotas de grasa emulsificadas hacia las micelas. Solamente
los carotenoides micelarizados pueden ser absorbidos. La micelarización de carotenoides
es altamente variable, dependiendo principalmente de la matriz alimenticia que los
contiene. Bajo diversas premisas se hipotetizó que la cantidad y propiedades de las
pectinas contenidas en alimentos ricos en carotenoides limitan su micelarización a través
de diversos mecanismos involucrados en la digestión de lípidos. Esta hipótesis se intentó
demostrar tomando como modelo de estudio las pectinas y carotenoides del chile
Jalapeño. Se extrajeron y caracterizaron pectinas de chile Jalapeño verde y rojo, crudo y
procesado, permitiendo obtener pectinas con diversas propiedades. Se seleccionaron tres
pectinas con diferente peso molecular, grado de esterificación y viscosidad. Se
prepararon emulsiones aceite en agua con dos niveles (0.14% y 1%) de concentración de
pectina y aceite enriquecido con carotenoides libres y esterificados de chile Jalapeño
rojo. Las emulsiones se sometieron a un proceso de digestión in vitro para evaluar el
impacto de la cantidad y propiedades de pectina en diversos eventos involucrados en la
micelarización de carotenoides. La viscosidad del medio gastrointestinal dependió de la
concentración y propiedades de las pectinas adicionadas. Dicha viscosidad fue diferente
a la observada en soluciones modelo. El tamaño de partícula incrementó con la
concentración de pectina en los medios intestinal y micelar. Las fibras secuestraron sales
biliares, pero este efecto se rejudo al incrementar la concentración de pectina. Los
resultados sugirieron que esta capacidad de secuestro dependió del grado de
esterificación. La lipólisis, medida por titulación (PH-STAT), dependió de la
concentración y tipo de pectina. Con altas concentraciones, la lipólisis fue más tardada.
La cantidad de ácidos grasos liberados pareció estar inversamente relacionada con el
peso molecular de las pectinas. La fase micelar fue abundante en ácidos grasos libres y
xi
monoglicéridos (medidos cromatográficamente), coincidiendo con la disminución de
triglicéridos. La micelarización fue más afectada por la concentración de pectinas que
por sus características químicas y reológicas. El aumento de la concentración disminuyó
la micelarización de carotenoides. Pectinas con bajo peso molecular y bajo grado de
esterificación parecen mejorar la micelarización a través de un menor secuestro de sales
biliares. Pectinas con alto peso molecular parecen estar relacionadas con la
micelarización de β-caroteno y xantofilas esterificadas, mientras que las pectinas con un
alto grado de esterificación y alta capacidad de secuestrar sales biliares estuvieron
relacionadas con la micelarización de xantofilas libres. La viscosidad del medio
intestinal también jugó un efecto importante en la micelarización de carotenoides.
Independientemente de la cantidad y propiedades de las pectinas, la micelarización de
carotenoides fue influenciada por su polaridad (carotenoides esterificados ˂ β-caroteno ˂
xantofilas) y por la composición lipídica en la fracción micelar. La micelarización de
xantofilas libres pareció estar relacionada con el contenido de diglicéridos en la fase
micelar, mientras que la micelarización de carotenos se relacionó con los ácidos grasos
libres y monoglicéridos. En conclusión, la digestión de lípidos está directamente
relacionada con la micelarización de carotenoides; sin embargo, también depende de los
diferentes lípidos que se obtienen durante el proceso de digestión. Tanto la
concentración como las propiedades de las pectinas afectan la micelarización de
carotenoides.
Palabras clave: Micelarización de carotenoides, Digestión de lípidos, Emulsificación,
Mecanismos determinantes, Características de las pectinas
xii
ABSTRACT
Carotenoids are fat-soluble pigments with beneficial effects on human health. However,
they are poorly absorbed at the small intestine. Carotenoid absorption process involves
their transference from emulsified lipid droplets to mixed micelles during digestion.
Only micellarized carotenoids can be absorbed. The micellarization of these compounds
is highly variable, depending mainly on the food matrix. Based on several assumptions,
it was hypothesized that the quantity and properties of pectin from carotenoid-rich foods
limit the micellarization of these compounds through mechanisms involved on lipid
digestion. This hypothesis was tested using pectins and carotenoids from Jalapeño
peppers. Several pectins were extracted from raw and heat-treated Jalapeno peppers
(green and red). These pectins were diverse in physicochemical properties.
Three pectins were selected, showing an increasing molecular weight, degree of
esterification and viscosity. The pectins were added at two concentration levels to oil in
water emulsions. The oil was previously enriched with carotenes and free and esterified
xanthophylls from red Jalapeño peppers. The emulsions were subjected to in vitro
digestion to determine the impact of the amount and properties of pectin on several
events probably involved on carotenoid micellarization. The viscosity of the gastric and
intestinal media depended on the concentration and properties of added pectins and was
different to that observed in model solutions. Particle size increased with pectin
concentration in the intestinal and micellar media. Fibers bonded bile salts, but such
binding capacity decreased as pectin concentration increased. Pectin capacity to bind
bile salts was related with the degree of methyl esterification of pectin. The lipolysis,
measured by titration (pH-STAT), depended on pectin concentration and type. The
completion of lipolysis required more time under high pectin concentration conditions.
The amount of fatty acids released was inversely related to pectin molecular weight. The
micellar phase mostly contained free fatty acids and monoglycerides (measured
chromatographically). Carotenoid micellarization depended more on pectin
xiii
concentration than pectin properties. High pectin concentrations limited the carotenoid
micellarization. Pectins with low molecular weight and low degree of esterification
improved carotenoid micellarization through a reduced bile salt binding. Pectins with
high molecular weight seemed to be involved on micellarization of β-carotene and
esterified xanthophylls while pectins with a high degree of esterification and high
capacity to bind bile salts were involved on the micellarization of free xanthophylls.
Intestinal environment viscosity also played a major effect on carotenoid micellarization.
Regardless of the amount and properties of pectins, carotenoids micellarization
efficiency was also influenced by their polarity (esterified carotenoids ˂ β-carotene ˂
xanthophylls) and lipid composition in the micellar fraction. Free xanthophylls
micellarization seemed to diglycerides content in the micellar phase, while carotene
micellarization was associated to the levels of free fatty acids and monoglycerides in the
micellar phase. In conclusion, lipid digestion is directly related to the carotenoid
micellarization; however, it also depends on products of lipid digestion. The amount and
properties of pectin modulated the carotenoid micellarization.
Key words: Carotenoid micellarization, Lipid digestion, Emulsification, Determinant
mechanisms, Pectin properties.
14
INTRODUCCIÓN
Los carotenoidesson compuestos dietarios altamente bioactivos con efectos importantes
en la salud humana (Elliott, 2005). Tienen actividad pro-vitamina A, participan en el
fortalecimiento del sistema inmune, previenen la degeneración macular asociada al
envejecimiento y disminuyen el riesgo de enfermedades crónico degenerativas
(cardiovasculares, cáncer y neurodegenerativas) (Furr y Clark, 1997). Para poder ejercer
su actividad biológica, los carotenoides deben ser previamente absorbidos por el
organismo (Biehler et al., 2011). Este proceso de absorción involucra su liberación del
alimento en el que se encuentran contenidos, gracias a la masticación y movimientos
peristálticos (Low et al., 2015). Posteriormente, son incorporados en gotas de lípidos
emulsionados dentro del medio gástrico (emulsificación) (Borel et al., 1996). Una vez en
el intestino, las gotas de lípidos interactúan con las sales biliares (SB), las cuales reducen
el tamaño de las gotas e incrementan su área superficial para facilitar la acción de las
enzimas lipolíticas (Faulks y Southon, 2005; Guyton y Hall, 2001). Los productos de
digestión lipídica (ácidos grasos libres, AGL; monoglicéridos, MG; diglicéridos, DG; y
lisofosfolípidos), junto con las SB, colesterol y carotenoides, son incorporados dentro de
estructuras esféricas denominadas micelas (micelarización) (Reboul, 2013; Yonekura y
Nagao, 2007). Una vez dentro de las micelas, los carotenoides son transportados a través
del medio acuoso intestinal y posteriormente absorbidos por los enterocitos (Reboul et
al., 2013). Sin embargo, ya que solo los carotenoides micelarizados logran llegar a las
células intestinales, únicamente una pequeña fracción de carotenoides puede ser
potencialmente absorbida. Esto resulta en una baja biodisponibilidad y, por lo tanto, en
una disminución de sus acciones biológicas. Diferentes estudios in vivo han reportado
concentraciones considerablemente bajas de carotenoides (0-0.82 µmol/L) en plasma
humano y en tejidos (Rao et al., 2010, 2013; Schweiggert et al., 2014; Stracke et al.,
15
2014; Sy et al., 2012, 2013). Estos niveles bajos de biodisponibilidad se han atribuido a
diferentes factores, tales como la matriz alimentaria, grasa dietaria, fibra dietaria,
interacción entre carotenoides e interacción entre varios de los factores antes
mencionados (Castenmiller y West, 1998).
16
PLANTEAMIENTO DEL PROBLEMA
El efecto de la matriz alimentaria incluye los efectos combinados de todos los factores
de un alimento que simultáneamente promueven o reducen la biodisponibilidad
(Ornelas-Paz et al., 2008). Por ejemplo, la bioaccesibilidad de β-caroteno es altamente
variable dependiendo del fruto en el que se encuentra (mango > papaya > tomate >
zanahoria) (Schweiggert et al., 2012, 2014). Por otro lado, la maduración modifica la
cantidad y tipo de carotenoides en las frutas y vegetales debido a la biosíntesis de
carotenoides que ocurre durante transformación de cloroplastos en cromoplastos en el
transcurso de este proceso. Un incremento considerable en el contenido cualitativo y
cuantitativo de carotenoides ha sido reportado durante la maduración de frutos de chile
(Cervantes-Paz et al., 2012, 2014; Deli et al., 1996) y mango (Azevedo-Meleiro y
Rodriguez-Amaya, 2005; Ornelas-Paz et al., 2008). Una correlación positiva también ha
sido reportada entre el contenido de β-caroteno en cassava y su eficiencia de
micelarización y absorción (Thakkar et al., 2007); sin embargo, dicha relación también
depende de la polaridad de los carotenoides (Victoria-Campos et al., 2013). Por otro
lado, se ha reportado que el procesamiento térmico causa rompimiento y solubilización
de la pared celular, así como rompimiento de complejos proteína-carotenoide, por lo
tanto, reduce los efectos de la matriz del alimento y aumenta la absorción de
carotenoides (Ornelas-Paz et al., 2008; Yahia y Ornelas-Paz, 2010). Este efecto es
mediado por el tiempo, intensidad y tipo de procesamiento. Netzel et al. (2011)
reportaron que la bioaccesibilidad de carotenos de zanahoria fue 80 y 75% mayor
después de la cocción (100 °C, 10 min) y escaldado (80 °C, 10 min), respectivamente.
Uno de los efectores más importantes en la bioaccesibilidad de carotenoides podría ser la
grasa dietaria, la cual promueve la difusión de carotenoides hacia el contenido
emulsionado en el tracto gastrointestinal. La grasa dietaria promueve la secresión de
quilomicrones y consecuentemente la bioaccesibilidad de carotenoides (Guyton y Hall,
2001; Yahia y Ornelas, 2010). La concentración apical de ácidos grasos ha sido
17
positivamente asociada con la secreción de luteína y β-caroteno en estudios in vitro
(Failla et al., 2014). Además, la variabilidad en la bioaccesibilidad de carotenoides
también ha sido relacionada con el tipo de grasa (Failla et al., 2014; Gleize et al., 2013;
Huo et al., 2007; Victoria-Campos et al., 2013). La fibra dietaria podría ser el efector
con mayor influencia en la absorción de carotenoides. Diferentes tipos de fibra (pectinas,
goma de guar, alginatos, celulosa, etc.) son capaces de reducir el contenido de estos
compuestos en plasma. Diferentes estudios han demostrado que las pectinas reducen la
absorción de carotenoides en mayor grado que otros tipos de fibra (Riedl et al., 1999;
Zanutto et al., 2002). La participación de las pectinas en la micelarización y absorción de
carotenoides puede ser notable, especialmente si se considera el alto contenido de estos
polisacáridos en los alimentos vegetales, los efectos de las pectinas en la digestión de
lípidos, así como el requerimiento de lípidos y sus productos de digestión para la
absorción y transporte de carotenoides (Espinal-Ruiz et al., 2014; Faulks y Southon,
2005; Ramos-Aguilar et al., 2015; Rubio-Senent et al., 2015). Sin embargo, la
implicación del efecto de las pectinas puede ser dependiente de sus propiedades, las
cuales varían con el origen botánico y método de extracción (Ramos-Aguilar et al.,
2015). El efecto de las propiedades de las pectinas en la micelarización y absorción de
carotenoides ha sido escasamente estudiado y limitado solo a β-caroteno. Ornelas-Paz et
al. (2008) demostraron que los cambios cualitativos y cuantitativos de las pectinas
durante la maduración de frutos estuvieron relacionados con la micelarización de β-
caroteno. Recientemente, Verrijssen et al. (2014) evaluaron la micelarización de β-
caroteno en presencia de pectinas comerciales de cítricos con diferente grado de metil
esterificación y demostraron que dichas pectinas afectaron la viscosidad del medio
intestinal, la emulsión de la grasa (tamaño de partícula) y micelarización de β-caroteno.
En un estudio posterior, ellos (Verrijssen et al., 2015) demostraron que la adición de un
emulsionante cambió el efecto del grado de esterificación de las pectinas en los
productos de lipólisis y en la micelarización de β-caroteno. El efecto de las propiedades
de la pectina en la digestión de los lípidos ha recibido más atención. Algunas inferencias
sobre el efecto de las propiedades de pectina en micellarization carotenoides pueden ser
establecidas a partir de tales estudios. El peso molecular y grado de esterificación de la
pectina determinan la viscosidad de sus soluciones (Leroux et al., 2003; Ramos-Aguilar
18
et al., 2015). Variaciones en estas propiedades cambiaron la viscosidad del medio
gastrointestinal y consecuentemente la emulsión de la grasa y la lipólisis (Espinal-Ruiz,
et al., 2014). Las pectinas también pueden interactuar con las sales biliares dependiendo
de su grado de esterificación y, consecuentemente, reducir la emulsificación de la grasa
y lipólisis (Rubio-Senent et al., 2015; Xu et al., 2015). De esta manera, la emulsificación
de la grasa determina el grado de lipólisis. Estos procesos son importantes en la
micelarización de carotenoides, ya que los productos de la lipólisis, así como las sales
biliares son requeridos para la formación de micelas, las cuales son el vehículo absortivo
de los carotenoides (Faulks y Southon, 2005).
El objetivo principal del presente trabajo fue evaluar el efecto de las propiedades
químicas (grado de metil esterificación, peso molecular) y reológicas (viscosidad) de las
pectinas en los eventos involucrados en el proceso de digestión (viscosidad de medio
gastrointestinal, emulsificación de la grasa/tamaño de partícula, grado de lipólisis y
secuestro de sales biliares) y determinar la implicación de dichos eventos en la eficiencia
de micelarización de carotenoides libres y esterificados.
19
RESULTADOS Y DISCUSIÓN
En el Capítulo 1 se describieron los principales efectos benéficos de los carotenoides en
la salud humana y los mecanismos de acción involucrados. Se enfatizó en el consumo de
frutas y hortalizas (principal fuente de carotenoides) como un medio para reducir el
riesgo de padecer algunas formas de cáncer, enfermedades cardiovasculares, cataratas,
Alzheimer, entre otras. Se detalló el proceso de absorción de carotenoides, el cual se
identificó como el principal limitante de sus actividades biológicas. Este proceso
involucra su digestión, absorción y metabolismo (Fig. 16.1, Cap. 1). La incorporación de
carotenoides en micelas lipídicas es el punto crítico que determina su absorción. Se
recopiló información sobre los niveles de carotenoides en plasma y tejidos (Table 16.1,
Cap. 1), la cual permitió demostrar la baja biodisponibilidad de estos compuestos. Estos
datos mostraron una gran variabilidad. Los estudios de micelarización (bioaccesibilidad)
de carotenoides dietarios fueron también altamente variables y confirmaron la estrecha
relación entre la micelarización y absorción de estos compuestos (Table 16.2, Cap. 1).
La biodisponibilidad y bioaccesibilidad de carotenoides depende de varios factores,
incluyendo la matriz alimentaria, grasa dietaria, fibra dietaria, interacción entre
carotenoides e interacción entre varios de los factores antes mencionados. Esta última
sección del Capítulo 1 nos permitió suponer que posiblemente el factor que mayor
influencia tiene en la baja absorción de carotenoides es la fibra dietaria (principalmente
la pectina), ya que puede estar involucrada en varias etapas que determinan la absorción
de carotenoides, dando lugar al Capítulo 2.
En el Capítulo 2 se describió el proceso de absorción de carotenoides (Fig. 1, Cap. 2) y
se definió el término pectina y se describió la forma en que este nutriente altera la
digestión de lípidos. Esta alteración se efectúa mediante 1) la interacción de la pectina
con componentes involucrados en la digestión de lípidos (calcio y sales biliares); 2)
alterando la viscosidad del medio gastrointestinal; 3) promoviendo la formación de
recubrimientos alrededor de las gotas de grasa, lo cual interfiere con el estado de
agregación y coalescencia de las gotas de grasa, así como en la estabilidad de la
emulsión y, finalmente, 4) interactuando con enzimas digestivas. Puesto que la digestión
de lípidos, entre otros aspectos, es escencial para la formación de micelas, la parte
20
fundamental de este Capítulo se centró en hipotetizar que las pectinas también modulan
la micelarización y absorción de carotenoides a través de los mismos mecanísmos que
afectan la digestión de los lípidos. Se discutió la posible relación entre dichos
mecanismos y los efectos que podrían causar en el proceso de absorción de carotenoides
(Fig. 2, 3 y 4, Cap. 2). En el Capítulo 2 también se proporcionó la información necesaria
para suponer que, de todas las fibras, las pectinas son las que mayor impacto tienen en la
absorción de carotenoides y que la etapa de emulsificación es el paso determinante para
este proceso. Se propuso que las características fisicoquímicas de las pectinas (peso
molecular, grado de metil esterificación y viscosidad) son las propiedades con mayor
influencia en la micelarización y absorción de carotenoides. La hipótesis planteada en
este capítulo se trató de probar experimentalmente en los siguientes capítulos.
En el Capítulo 3 se detalla la caracterización de diferentes pectinas que fueron extraídas
a partir de chiles Jalapeños crudos, hervidos y asados, en dos diferentes estados de
madurez (verde y rojo). Las pectinas fueron extraídas de acuerdo a su solubilidad
(pectina soluble en agua: WSP, pectina soluble en agente quelante: CSP y pectina
soluble en agente alcalino: NSP). Los resultados obtenidos mostraron que el rendimiento
de pectina total (Table 2, Cap. 3) fue mayor en chiles verdes (27-31%) que en rojos (24-
28%). Este rendimiento también fue afectado por el hervido (6-9%) y el asado (13-14%).
La WSP fue la pectina más abundante (9.3-13.1%), mientras que NSP presentó los
niveles más bajos (4.6-8.3%). La CSP representó el 6.5-9.6% del contenido total de
pectina. El mayor contenido de pectina en chiles verdes, en comparación con los rojos,
fue atribuido a la degradación enzimática que ocurre durante la maduración de los
frutos, mientras que el decremento en el contenido de pectina causado por el tratamiento
térmico se atribuyó a la térmosolubilización y depolimeración de los materiales pécticos
(Christiaens et al., 2012). Las variables de color medidas en las pectinas extraídas fueron
mayores en chiles rojos que en los verdes (Table 1, Cap. 3). Este comportamiento fue
atribuido a la capacidad de las pectinas para interactuar con los pigmentos en función su
grado de esterificación y contenido de iones calcio, los cuales son dependientes de la
maduración (Holzwarth et al., 2013). La coloración también se vio afectada por el
procesamiento térmico, el cual redujo los valores de L* (11-17%) para todos los tipos de
pectina. Esta disminución pudo deberse a la demetilación de las pectinas inducida por el
21
calor y a la degradación de monosacáridos, los cuales favorecen la coloración café
(Einhorn-Stoll et al., 2014). En cuanto al contenido de proteína, NSP presentó el mayor
contenido; sin embargo, diferencias estadísticas no fueron observadas (Table 3, Cap. 3).
En chiles verdes, NSP presentó el mayor contenido de galactosa, arabinosa y ramnosa,
WSP un mayor contenido de xilosa y manosa, mientras que CSP no mostró una
tendencia clara. En chile rojos, la WSP mostró un mayor contenido de ácido
galacturónico y xilosa, CSP fue más abundante en manosa y fucosa, mientras que NSP
en galactosa, arabinosa y ramnosa (Table 4, Cap. 3). En general, el contenido de
monosacáridos neutros disminuyó con la maduración y estuvo por debajo de los niveles
reportados para pectinas extraídas de otros genotipos de chile (Popov et al., 2011). Esta
disminución fue atribuida a la degradación de pectinas durante dicho proceso (Gross y
Sams, 1984). Por otro lado, el grado de metil esterificación (Fig. 1, Cap. 3) varió desde
26 % hasta 92% y fue similar a los reportados por otros estudios (Howard et al., 1997;
Arancibia y Motsenbocker, 2006; Popov et al., 2011). El proceso de maduración
disminuyó el grado de metilación en WSP y NSP, mientras que en CSP incrementó. Este
comportamiento fue relacionado con la pérdida de la habilidad de las pectinas para
interactuar con otros componentes de la pared celular (Arancibia y Motsenbocker,
2004). El tratamiento térmico presentó un efecto diferencial. Mientras que el asado
disminuyó el grado de metilación, el hervido lo incrementó. Este comportamiento
también fue observado por Munarin et al. (2013) al aplicar un tratamiento de 121 °C
durante 15 min. Finalmente, el peso molecular (Table 5, Cap. 3) de las pecinas estuvo
relacionado con su viscosidad (Fig. 2, Cap. 3). Las pectinas extraídas de chiles crudos en
los dos estados de madurez siempre presentaron un mayor peso molecular y mayor
viscosidad que pectinas extraídas de chiles procesados. La viscosidad y el peso
molecular en los diferentes tipos de pectinas fue en el siguiente orden WSP ˃ CSP ˃
NSP. La variabilidad en el peso molecular fue atribuida a las condiciones de extracción
(Mesbahi et al., 2005; Westereng et al., 2006; Yapo, 2009), mientras que los cambios de
viscosidad se atribuyeron a propiedades de las pectinas como el contenido de calcio y
cambios en su estructura tridimensional (Lara et al., 2006; Lamikanra y Watson, 2007).
Las características evaluadas en esta investigación nos proporcionaron la información
necesaria para seleccionar tres pectinas con diferente grado de esterificación, peso
22
molecular y viscosidad, con la finalidad de evaluar su efecto en la micelarización de
carotenoides, dando lugar al Capítulo 4. Adicionalmente, en este capítulo se demostró
que las fracciones de pectinas de alimentos vegetales sufren cambios cualitativos y
cuantitativos intensos en función de la maduración y procesamiento.
En el Capítulo 4 se evaluó el efecto de la concentración y características químicas (peso
molecular y grado de esterificación) y reológicas (viscosidad) de tres diferentes pectinas
(PP1, PP2 y PP3) en los eventos digestivos involucrados en la micelarización de
carotenoides. Estas pectinas se seleccionaron de entre las extraídas durante el trabajo
experimental descrito en el Capítulo 3. Las propiedades (peso molecular, grado de metil
esterificación y viscosidad) de las pectinas seleccionadas siguieron un orden creciente o
decreciente. Se realizaron emulsiones aceite en agua que contenían una solución de
pectina al 0.14 y 1%. La cantidad de aceite de soya utilizada fue de 1.25 g y estuvo
enriquecido con carotenoides de chile Jalapeño rojo. Las emulsiones fueron sometidas a
un proceso de digestión in vitro y se evaluó su viscosidad en el medio gástrico e
intestinal, grado de lipólisis, secuestro de sales biliares, tamaño de partícula y la
micelarización de carotenoides. Los resultados de viscosidad (Fig. 1, Cap. 4) mostraron
un claro efecto causado por la concentración de pectina. Los medios gástrico e intestinal
presentaron una mayor viscosidad con 1% (0.21‒0.82 Pa.sn) que con 0.14% (0.07‒0.16
Pa.sn) de pectina. La viscosidad disminuyó al pasar del medio gástrico al intestinal. El
mismo comportamiento fue reportado por Verrijssen et al. (2014, 2015) y se atribuyó a
los cambios de pH y a la depolimerización de la pectina por la lipasa. En el medio
gástrico, PP3 presentó los valores más altos de viscosidad, mientras que PP1 exhibió la
viscosidad más elevada en el medio intestinal. Estos hallazgos demostraron que las
condiciones digestivas pudieron alterar las propiedades reológicas de las pectinas,
probablemente porque el pH de la fase gástrica favoreció la formación de geles en
presencia de PP3. La distribución del tamaño de partícula mostró una gran variabilidad
(Fig. 2, Cap. 4); sin embargo, a bajas concentraciones de pectina, el diámetro promedio
fue de 0.01-0.02 µm en los medios intestinal y micelar. Con altas concentraciones de
pectina, el tamaño de partícula fue mayor y pudo ser debido a la floculación causada por
la presión osmótica que generó una atracción entre las gotas de grasa (Espinal-Ruiz et
al., 2014). En general, PP3 presentó los tamaños de partícula más pequeños en los
23
medios intestinal y micelar, demostrando que favoreció una mayor estabilidad para las
emulsiones en comparación con las otras pectinas. Esta propiedad pudo ser relacionada
con su bajo peso molecular causando un menor número de puentes de hidrogeno y
reduciendo la floculación (Leroux et al., 2003). En cuanto al secuestro de sales biliares
(Fig. 3, Cap. 4), esta propiedad disminuyó (28-47%) con el incremento de la
concentración. Nosotros inferimos que, con altas concentraciones de pectina, la
interacción entre las cadenas de estos polímeros incrementó, limitando la interacción con
otros componentes del medio como las sales biliares. Cuando se adicionó una baja
concentración, PP1 mostró una interacción similar con taurocolato y glicodesoxicolato
(59-62%), pero más baja con taurodesoxicolato (42%). Esta interacción fue incluso
mayor que la que presentó la colestiramina (control positivo). Basados en estudios de
correlación, nosotros suponemos que la capacidad de las pectinas para secuestrar sales
biliares dependió del grado de metil esterificación. Por otro lado, el grado de hidrólisis
de lípidos (lipólisis) dependió de la concentración y tipo de pectina (Fig. 4, Cap. 4). Con
0.14% de pectina, la digestión de lípidos casi se completó durante los primeros 60
minutos; sin embargo, al incrementar la concentración, la digestión de lípidos tomó un
poco más de tiempo. Estos resultados estuvieron de acuerdo con datos reportados por
Espinal-Ruiz et al. (2014). La cantidad de ácidos grasos libres pareció estar
inversamente relacionada con el peso molecular de las pectinas (PP3 ˂ PP2 ˂ PP1).
Recientemente, Espinal-Ruiz et al. (2016) observaron un efecto similar en el que el
grado de esterificación es opuesto a la digestión de lípidos; sin embargo, futuros estudios
son necesarios para entender este efecto. Los resultados de micelarización mostraron un
mayor efecto causado por concentración de pectinas que por sus características químicas
y reológicas. La eficiencia de micelarización de carotenoides dependendió de la
polaridad de los carotenoides (carotenoides esterificados ˂ β-caroteno ˂ xantofilas) (Fig.
5, Cap. 4). Resultados similares fueron previamente reportados por otros estudios
(O´Sullivan et al., 2010; Victoria-Campos et al., 2013) y se atribuyeron a la localización
de los carotenoides dentro de las gotas de lípidos durante la digestión (Borel et al.,
1996). Con 0.14% de pectina, la micelarización de carotenoides fue mayor en presencia
de PP1, incluso en ausencia de pectina, sugiriendo que, a bajas concentraciones, las
pectinas con bajo peso molecular y grado de esterificación pueden mejorar la
24
micelarización de carotenoides. El aumento de la concentración disminuyó la
micelarización de carotenoides. Este comportamiento también fue reportado por
Ornelas-Paz et al. (2008) en digestiones con pectina de mango. Finalmente, el análisis de
componentes principales indicó que, los valores de viscosidad, lipólisis y tamaño de
partícula incrementaron a altas concentraciones de pectina (Fig. 6, Cap. 4). El análisis de
componentes también mostró que pectinas con alto peso molecular (PP1) parecen estar
relacionadas con la micelarización de β-caroteno y xantofilas mono y diesterificadas,
mientras que PP2 y PP3 con un alto grado de esterificación y alta capacidad de
secuestrar sales biliares estuvieron relacionadas con la micelarización de xantofilas
libres. Algunos estudios han demostrado por separado el efecto de la concentración y las
propiedades de las pectinas en los eventos involucrados en digestión de grasa y en la
micelarización de β-caroteno (Cheewatanakornkool et al., 2012; Espinal-Ruiz et al.,
2014; Kaltsa et al., 2014; Li et al., 2013; Rubio-Senent et al., 2015; Xu et al. 2015; Xu et
al., 2012; Zhao et al., 2015); sin embargo, ésta es la primera investigación en la que se
evalúa el efecto de todos estos factores en la subsecuente micelarización de carotenoides
libres y esterificados. Los resultados de lipólisis nos hicieron suponer que la
micelarización de carotenoides depende mayoritariamente de la composición de los
diferentes productos de la lipolisis obtenidos durante el proceso de digestión. Esta
hipótesis dio lugar al último capítulo presentado en esta tesis.
En el Capítulo 5 se evaluó el efecto de las pectinas descritas en el Capítulo 4, al 0.14 y
1%, en la composición de productos de digestión lipídica (ácidos grasos libres +
monoglicéridos: AGL+MG, diglicéridos: DG y triglicéridos: TG) y su posible relación
con la micelarización de diferentes fracciones de carotenoides. Durante la simulación del
proceso de digestión in vitro se obtuvo una fase micelar y una fase oleosa. En cada una
de estas fases se determinó la composición de lípidos empleando un método de
cromatografía de líquidos de alta resolución (CLAR) acoplado a un detector de
dispersión de luz (ELSD). La fase oleosa estuvo compuesta principalmente por TG (40-
60%), mientras que los DG y AGL+MG estuvieron en menor proporción, similar entre
ellos (Figure 2, Cap. 5). Cuando se incrementó la concentración de pectina, se observó
una ligera disminución en el porcentaje de TG y un incremento en el porcentaje de
AGL+MG, indicando una posible mayor estabilidad de las emulsiones con alta
25
concentración de pectinas y, por lo tanto, una lipolisis más eficiente (Ornelas-Paz et al.,
2010). En la fase micelar, se observaron altos porcentajes de AGL+MG, coincidiendo
con la disminución y en algunos casos, desaparición de TG. Este comportamiento fue
coherente con una lipólisis casi completa. Espinal-Ruiz et al. (2014) observaron que
emulsiones que contenían pectina presentaron porcentajes de lipólisis de 50-80%
durante 2 h de digestión y lo atribuyeron a la interacción de las pectinas con
componentes esenciales para el proceso de digestión, los cuales no estuvieron
disponibles para completar dicho proceso. Finalmente, el efecto de la concentración y
propiedades de las pectinas en la micelarización de fracciones de carotenoides siguió el
siguiente orden xantofilas libres ˃ carotenos ˃ monoésteres de xantofilas ˃ diésteres de
xantofilas (Fig. 3, Cap. 5); sin embargo, el orden fue modificado con la concentración de
pectinas. La micelarización de xantofilas libres pudo haber estado relacionado con el
contenido de DG en la fase micelar, mientras que la micelarización de carotenos se
relacionó con los porcentajes de AGL+MG en esta misma fase. Verrijssen et al. (2015)
también reportaron una relación entre la micelarización de β-caroteno y la incorporación
de ácidos grasos libres dentro de las micelas. Aquellas pectinas con bajo grado de
esterificación causaron una mayor micelarización de xantofilas mono y diesterificadas.
Este tipo de pectinas han mostrado una menor interacción con sales biliares, por lo tanto,
pudo ocurrir una mayor micelarización (Espinal-Ruiz et al., 2014; Verrijssen et al.,
2015), promoviendo una mayor incorporación de estos carotenoides dentro de las
micelas. También la viscosidad pudo haber jugado un papel importante en la
micelarización de las diferentes fracciones de carotenoides, sin embargo, esta relación
aún no es evaluada.
26
CONCLUSIONES
En términos definitivos, la digestión de lípidos está directamente relacionada con la
micelarización de carotenoides; sin embargo, la eficiencia de micelarización también
depende de la composición de lípidos que se obtienen durante el proceso de digestión.
Tanto la concentración como las propiedades químicas y reológicas de las pectinas
tienen un efecto significativo en los eventos digestivos involucrados y por consiguiente
en la eficiencia de micelarización. Las pectinas con bajo peso molecular y bajo grado de
esterificación parecen estar relacionadas con una mejor micelarización a través de un
menor secuestro de sales biliares. Las pectinas con alto peso molecular parecen estar
relacionadas con la micelarización de β-caroteno y xantofilas esterificadas, mientras que
las pectinas con un alto grado de esterificación y alta capacidad de secuestrar sales
biliares parecen tener una estrecha relación con la micelarización de xantofilas libres.
Independientemente de la cantidad y propiedades de las pectinas, la micelarización de
carotenoides fue influenciada por su polaridad. Las propiedades químicas y reológicas
de las pectinas, principalmente en el proceso de emulsificación, podrían podrían
determinar la modulación del proceso de micelarización y absorción de carotenoides.
27
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33
CAPÍTULO I
ABSORTION OF CAROTENOIDS AND MECHANISMS INVOLVED IN THEIR
HEALTH-RELATED PROPERTIES
Subcellular Biochemistry 79
Claudia Stange Editor
Carotenoids in NatureBiosynthesis, Regulation and Function
Subcellular Biochemistry
Volume 79
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Claudia Stange
Carotenoids in NatureBiosynthesis, Regulation and Function
123
Claudia StangeCentro de Biología Molecular VegetalDepartamento de BiologíaFacultad de CienciasUniversidad de ChileSantiago, Chile
ISSN 0306-0225Subcellular BiochemistryISBN 978-3-319-39124-3 ISBN 978-3-319-39126-7 (eBook)DOI 10.1007/978-3-319-39126-7
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Preface
Carotenoids are colored pigments widespread distributed in nature. These lipophilicmolecules are synthesized in plants, algae, and some yeast and bacteria. In plants,carotenoids are synthesized in photosynthetic organs as well as in fruits, flowers,seeds, and reserve roots, providing attractive yellow, orange, and red colors. Inplants and algae, carotenoids have important functional roles in photosynthesis,photomorphogenesis, and in photoprotection. They also give rise to apocarotenoids,such as the hormones abscisic acid and strigolactones, among other volatileterpenes. Additionally, they possess antioxidant properties acting as reactive oxygenspecies scavengers. In mammals, they act as provitamin A precursors and as power-ful antioxidant molecules involved in the prevention of certain types of diseases.Carotenoid biosynthesis in plants is highly regulated, although all the processesinvolved are not completely known. During the past decades, huge knowledge hasbeen published, and almost all carotenogenic genes have been identified and thosefunctions dissected as a result of molecular, genetic, and biochemical approachesutilizing different plant, yeast, and algae model systems. The information has beenused in genetic engineering for increasing abiotic stress tolerance, altering colorand the nutritional value in plants, leading to the production of novel functionalfoods. In this book, an extensive and actual review of the main topics of carotenoidbiosynthesis, regulation, and function in human health are brought together.
The first chapters provide an introduction to carotenoid biosynthesis in yeast,bacteria, and plants and a profound exposition on the structures of carotenoidmolecules. The second part covers the function and regulations of carotenoids inphotosynthesis as well as during plant, fruit, storage root, and alga development. Wealso included chapters that present an actual overview on plastids – accumulatingcarotenoids – on the epigenetic mechanisms that control carotenoid biosynthesis andon the oncoming topic regarding apocarotenoids. To finish, some chapters argueabout the metabolic engineering of carotenoids in plants and seeds that point theway of carotenoid biotechnological application. Additionally, important topics onthe effect of absorption mechanisms and carotenoids as antioxidants and vitamin Aprecursors for human nutrition were also included.
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The chapters included in each section were prepared and reviewed by experts inthe field. I would like to thank each of the authors for accepting to contribute to thisbook, for their dedicated effort providing carefully prepared manuscripts, and fortheir prompt attention to answer to requested information.
I wish to express my gratitude to the staff of Springer, in particular to Thijs vanVlijmen (Senior Publishing Editor) and to Sara Germans-Huisman (Springer SeniorEditorial Assistant) for their kind assistance and patience.
It is the aim of the editor that this book will be of benefit and reference source toanyone researching the area on carotenoid synthesis and regulation.
Santiago, Chile Claudia Stange
Contents
Part I Biosynthesis of Carotenoids
1 Carotenoid Distribution in Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Jennifer Alcaíno, Marcelo Baeza, and Víctor Cifuentes
2 Biosynthesis of Carotenoids in Plants: Enzymes and Color . . . . . . . . . . . 35Carolina Rosas-Saavedra and Claudia Stange
3 Structures and Analysis of Carotenoid Molecules . . . . . . . . . . . . . . . . . . . . . . 71Delia B. Rodriguez-Amaya
Part II Regulation of Carotenoids Biosynthesis
4 Carotenoids and Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111Hideki Hashimoto, Chiasa Uragami, and Richard J. Cogdell
5 Regulation of Carotenoid Biosynthesis in Photosynthetic Organs . . . . 141Briardo Llorente
6 Regulation of Carotenoid Biosynthesis During Fruit Development. . . 161Joanna Lado, Lorenzo Zacarías, and María Jesús Rodrigo
7 Carotenoid Biosynthesis in Daucus carota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Kevin Simpson, Ariel Cerda, and Claudia Stange
8 Carotenoids in Microalgae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219Vitalia Henríquez, Carolina Escobar, Janeth Galarza,and Javier Gimpel
9 Apocarotenoids: A New Carotenoid-Derived Pathway . . . . . . . . . . . . . . . . . 239Juan Camilo Moreno Beltran and Claudia Stange
10 Plastids and Carotenoid Accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273Li Li, Hui Yuan, Yunliu Zeng, and Qiang Xu
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11 Evidence of Epigenetic Mechanisms Affecting Carotenoids . . . . . . . . . . . 295Jacobo Arango, Jesús Beltrán, Jonathan Nuñez,and Paul Chavarriaga
Part III Carotenoids for Human Health
12 Manipulation of Carotenoid Content in Plants to ImproveHuman Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311Enriqueta Alós, Maria Jesús Rodrigo, and Lorenzo Zacarias
13 Modern Breeding and Biotechnological Approachesto Enhance Carotenoid Accumulation in Seeds . . . . . . . . . . . . . . . . . . . . . . . . . 345M.L. Federico and M.A. Schmidt
14 Carotenoids as a Source of Antioxidants in the Diet . . . . . . . . . . . . . . . . . . . . 359Ana Augusta Odorissi Xavier and Antonio Pérez-Gálvez
15 Carotenoids in Adipose Tissue Biology and Obesity . . . . . . . . . . . . . . . . . . . . 377M. Luisa Bonet, Jose A. Canas, Joan Ribot, and Andreu Palou
16 Absorption of Carotenoids and Mechanisms Involvedin Their Health-Related Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415Braulio Cervantes-Paz, Claudia I. Victoria-Campos, andJosé de Jesús Ornelas-Paz
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
Chapter 16Absorption of Carotenoids and MechanismsInvolved in Their Health-Related Properties
Braulio Cervantes-Paz, Claudia I. Victoria-Campos, andJosé de Jesús Ornelas-Paz
Abstract Carotenoids participate in the normal metabolism and function of thehuman body. They are involved in the prevention of several diseases, especiallythose related to the inflammation syndrome. Their main mechanisms of actionare associated to their potent antioxidant activity and capacity to regulate theexpression of specific genes and proteins. Recent findings suggest that carotenoidmetabolites may explain several processes where the participation of their parentcarotenoids was unclear. The health benefits of carotenoids strongly depend on theirabsorption and transformation during gastrointestinal digestion. The estimation ofthe ‘bioaccessibility’ of carotenoids through in vitro models have made possible theevaluation of the effect of a large number of factors on key stages of carotenoiddigestion and intestinal absorption. The bioaccessibility of these compounds allowsus to have a clear idea of their potential bioavailability, a term that implicitlyinvolves the biological activity of these compounds.
Keywords Bioactivity • Bioaccessibility • Absorption • Food matrix
16.1 Carotenoids and Their Health Protective Effects
Carotenoids are hydrophobic pigments constituted by eight isoprene units. They canbe acyclic or contain rings in one or both terminal groups. They have been classifiedin xanthophylls, oxygen containing structures, and carotenes, which only containhydrogen and carbon atoms in their structure (Chaps. 1, 2, and 3). The characteristicdouble-bond system in the carotenoid structure is responsible of their color andreactivity. Up to date, approximately 700 carotenoids have been identified fromnatural sources, but only 100 have been found in the typical human diet (Khachiket al. 1991). In plant foods, they are typically esterified with fatty acids, with
B. Cervantes-Paz • C.I. Victoria-Campos • J.de.J. Ornelas-Paz (!)Centro de Investigación en Alimentación y Desarrollo A. C.-Unidad Cuauhtémoc,Av. Río Conchos S/N, Parque Industrial, C.P. 31570 Cd. Cuauhtémoc, Chihuahua, Mexicoe-mail: [email protected]
© Springer International Publishing Switzerland 2016C. Stange (eds.), Carotenoids in Nature, Subcellular Biochemistry 79,DOI 10.1007/978-3-319-39126-7_16
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exception of green leafy vegetables and some fruits like kiwi (Chap. 14, Rodriguez-Amaya and Kimura 2004). Epidemiological studies have related the consume ofcarotenoid-rich foods with a high content of carotenoids in plasma and a lowerincidence of some forms of cancer (such as lung and stomach), age-related maculardegeneration, obesity and cardiovascular diseases (Chaps. 14 and 15, Kohlmeieret al. 1997; Seddon et al. 1994; van Poppel and Goldbohm 1995). However, theinvolvement of carotenoids in these effects is unclear.
16.1.1 Provitamin A Activity
The vitamin A deficiency is a public health problem in some developing countries.This deficiency can lead to xerophthalmia, anemia and weakening of the immunesystem, increasing the risk of infections. It has been estimated that carotenoids fromfruits and vegetables provide from 30 to 85% of the daily vitamin A requirements indeveloped and developing countries, respectively (Chap. 14, Bramley et al. 2000).Only the carotenoids with at least one “-ionone ring (without oxygenated groups)and a polyene side-chain of at least 11 carbons can be metabolized to vitamin A(Furr and Clark 1997). Carotenoids may be cleavage to yield retinoids throughtwo pathways, the first of them involves the cleavage of the central double bond(15,150) by the cytosolic enzyme “,“-carotene 15–150-monooxygenase (BCO1) toyield one, from “-cryptoxanthin and ’-carotene, or two, from “-carotene, moleculesof retinal, which can be reduced to retinol or oxidized to retinoic acid. The secondpathway, involves the eccentric cleavage of carotenoids to produce “-apo-carotenalsand minor fragments. Some “-apo-carotenals can be converted to “-apo-carotenoicacids and serve as precursors of retinoic acid. Others may be transported to the liverand oxidized, through a stepwise procedure, to retinal molecules (Furr and Clark1997; Harrison 2012; Failla and Chitchumroonchokchai 2005; Ornelas-Paz et al.2010). It is commonly accepted that the eccentric cleavage, also known as randomcleavage, can be performed by a non-enzymatic auto-oxidation of carotenoids.However, Kiefer et al. (2001) reported the presence of a carotene dioxygenase inmouse responsible for the asymmetric oxidative cleavage of “-carotene (BCO2)at the 90,100 double bound. The central cleavage is the main pathway of vitaminA formation from carotenoids. Nagao et al. (1996) reported that 94–100% of theconsumed “-carotene was converted in retinal. Accordingly, Barua and Olson (2000)reported that “-apo-carotenals accounted in less than 5% of the total retinoidsformed from “-carotene in intestines of rats. These bioconversion reactions mainlyoccur in the enterocytes and liver (Harrison 2012; Parker 1996). However, Lindqvistand Andersson (2004) reported the expression of BCO1 in epithelial cells from thegastrointestinal tract, hepatic parenchymal cells, pancreas, kidney, adrenal gland,skin, among other organs in humans. The bioconversion efficiency is stronglyaffected by the vitamin A status of the individual; the maximum molar proportion(1:2) for the conversion of “-carotene in retinal can be almost achieved only indeficient organisms supplemented with low doses of “-carotene (Ornelas-Paz et al.2010; van Lieshout et al. 2001; Yahia and Ornelas-Paz 2010). Other factors, such
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 417
as the bioavailability of carotenoids, the activity of “-carotene 15:150 dioxygenase,and the reducing activity of intestinal cells also affect the bioconversion efficiency(Ornelas-Paz et al. 2010; van Lieshout et al. 2001). The retinoic acid acts astranscriptional regulator of hundreds of genes, exert chemopreventive activities,promotes the growth, reproduction, cell differentiation, and a well-functioningimmune system (McGrane 2007; Yahia and Ornelas-Paz 2010).
16.1.2 Health Properties of Carotenoids Mediatedby the Regulation of the Expression of Genesand Proteins
Some epidemiological studies have reported an inverse relationship between thehigh consumption of carotenoid-rich fruits and vegetables and a low incidence ofsome forms of cancer (Eliassen et al. 2012; Giovannucci 1999; Slattery et al. 2000).This evidence for the protective effects of carotenoids against cancer is unclearsince the supplementation with pure carotenoids seems to increase the risk ofcancer in humans (Omenn et al. 1996). However, some mechanistic studies supporta protective role of these pigments on cancer risk through several mechanisms,mainly by the modulation of grown factor signaling, cell cycle progression, celldifferentiation and apoptosis (Niranjana et al. 2014; Tanaka et al. 2012). Karaset al. (2000) demonstrated that the growth stimulation of mammary cancer cellsby IGF-1 was inhibited by lycopene. The inverse association between the frequencyof consumption of cooked tomatoes (rich in lycopene) and the circulating levelsof IGF-1 and IGF-1/IGFBP-3 has also been reported in epidemiological studies(Mucci et al. 2001). The effect of carotenoids on the cell cycle arrest has been welldocumented in many in vitro studies with human cancer cell lines such as leukemia,colon, liver, breast, skin, prostate, and lung. The involvement of carotenoids on thegrowth of tumor cells seems occur at different phases of the cell cycle, accordingto findings obtained with “-carotene, lycopene, fucoxanthin, lutein, neoxanthinand “-cryptoxanthin (Niranjana et al. 2014). Interestingly, some metabolites oflycopene (apo-100-lycopenoic acid and apo-120-lycopenal) also inhibited the normalgrowth cell cycle in cancer cells from lung and prostate (Ford et al. 2011; Lianet al. 2007). Recently, Kaulmann and Bohn (2014) suggested that the reportedactivity of lycopene in the activation of the Nrf2 in different cancer cell lines (Ben-Dor et al. 2005) might be explained by the presence of apo-lycopenals, whichare more polar compounds. This conjecture explains the contradiction betweenthe lycopene hydrophobicity and the cytoplasmic location of the Nrf2. The Nrf2increases the expression of different cytoprotective genes, protecting cells fromtoxicants and carcinogens (Jaramillo and Zhang 2013). On the other hand, severalin vitro studies have also demonstrated the proapoptotic activity of carotenoids indifferent human cancer cell lines (leukemia, colon, lung, prostate, melanoma, liver,among others) (Niranjana et al. 2014). The induction of apoptosis by carotenoids
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is mainly mediated by the reduction of the mitochondrial membrane permeability,the release of mitochondrial cytochrome c, activating different caspase forms, andincreasing and reducing the Bax and Bcl-2 genes expression, respectively (Hantzet al. 2005; Palozza et al. 2003; Rokkaku et al. 2013).
Carotenoids also increase the gap junctional intercellular communication (GJIC)through mechanisms independent of their provitamin A and antioxidant activity.This communication changes the cancerous phenotype of cells. In C3H10T1/2cells, “-carotene, cantaxanthin and lycopene (10–5 M) enhanced the number ofjunctional channels through the up-regulation of Conexin 43 (Cx43) mRNA andprotein (Zhang et al. 1992). Lycopene and “-carotene (3–7 !M) also increasedthe expression of Cx43 RNA and protein in cancer cells from the oral cavity,but lycopene up-regulated GJIC more effectively than “-carotene (Livny et al.2002). More recently, it has been demonstrated that fucoxanthin (1–20 !M)increased the expression of Cx43 and Cx42 in cancer cells from the liver (SK-Hep-1) and enhanced the GJIC (Liu et al. 2009). The presence of residues of theCx43 phosphorylation competes with the gap junctional communication. Junctionalcommunication regulates the cell growth and carcinogenesis (Zhang et al. 1991;Zhang et al. 1992).
The involvement of carotenoids in the immune system is mainly based on theirprovitamin A activity. The deficiency of vitamin A and the consequent increasingrisk of infections have been well documented. However, the carotenoids themselvesalso exert an important activity in the strengthening of the immune system. Elliot(2005) suggested, according to studies in animals, that the effects of carotenoidson immune system might be explained by their capacity to stimulating the killingactivity of blood neutrophils, increasing the mitogen-induced proliferation of lym-phocytes, enhancing the antibody responses and increasing the cytochrome oxidaseand peroxidase activities in macrophages. The supplementation with “-carotene(30–60 mg/d/2 months) increased the circulating T-helper cells, cells with IL-2receptors and natural killer cells in healthy humans (Watson et al. 1991). Lycopeneinhibited the maturation of dendritic cells and reduced their T-cell stimulatorycapacity (Kim et al. 2004). Dendritic cells induce the expression of inflammatorymarkers such as cytokines and TNF-’ (Kim et al. 2004). Lutein increased the excre-tion of MMP9 (matrix metalloproteinase 9) in murine macrophages (RAW264.7)and human monocytes (U937), increasing their phagocytic ability (Lo et al. 2013).
16.1.3 Protective Effects of Carotenoids Related with TheirAntioxidant Activity
Reactive molecules (free radicals and singlet oxygen) are generated in the bodyunder normal conditions or as a consequence of external factors. These radicals arehighly reactive chemical species that contain one or more unpaired electrons. Whenthe amount of these reactive species exceeds the normal levels in the body, they may
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 419
exert harmful effects, damaging important molecules, such as proteins, DNA, lipids,and carbohydrates, causing an abnormal cell operation and various pathologies(Bramley et al. 2000; Stahl and Sies 2003). The antioxidant activity of carotenoidshas been extensively studied and associated with the conjugate double bonds in thecarotenoid structure since it constitutes a reactive electron-rich system susceptibleto react with electrophilic compounds (van den Berg et al. 2000). In animalsystems, the carotenoid capacity to quench singlet oxygen and peroxyl radicals,deactivate electronically excited sensitizer molecules and filter blue light are ofmajor importance. The three primary chemical reactions to scavenge oxidizing freeradicals by carotenoids are electron transfer (CARCROO• !ROO! CCAR•C orROOC C CAR•–), adduct formation (CARCROO• !ROOCAR•) and hydrogenatom transfer (CARCROO• !ROOHCCAR•) (Böhm et al. 2012; Edge andTruscott, 2010; van den Berg et al. 2000). The physical capacity of carotenoidsto inactivate singlet oxygen depends on the number of double bonds in theirbackbone because of at triplet energy level they are able to receive the excitationenergy from the singlet oxygen and then dissipating the energy as heat to thesurrounding media, returning to their ground state. The “-carotene, zeaxanthin,cryptoxanthin, and ’-carotene are highly active quenchers of singlet oxygen (Edgeand Truscott 2010; Stahl and Sies 2003). Carotenoids can interrupt the productionof peroxyl radicals generated during the lipid oxidation. Some harmful carotenoidradicals may be generated as intermediates in peroxidation systems; however, theinvolved chemical mechanism depends, among other factors, on the reactivity ofperoxyl radicals (Edge and Truscott 2010; El-Agamey et al. 2004; van den Berget al. 2000). The scavenging of peroxyl radicals protects cellular membranes andlipoproteins from oxidative damage (Stahl and Sies 2003). The carotenoids protectthe long-chain polyunsaturated fatty acids of the retina from the reactive oxygenspecies generated by the high-energy short wavelength visible light and reduce theformation of lipofuscin (Ma and Lin 2010). The carotenoids can be oxidized undersome conditions, such as oxidative stress, deficiency of antioxidants or high levelsof carotenoids. The “-carotene autooxidation produces epoxy-carotenoids, “-apo-carotenones and “-apo-carotenals, with some of them being precursors of vitaminA (Stahl and Sies 2003; van den Berg et al. 2000).
Lutein and zeaxanthin are accumulated in the retina and lens within the eye (Maand Lin 2010). They act as filters for the blue light, attenuating in about 40% thelight that reaches photoreceptors, retinal pigment epithelium and choriocapillaris,reducing their damage (Krinsky et al. 2003). Additionally, zeaxanthin and luteinprovide protection against photooxidation. High amounts of reactive oxygen speciesare generated in the retina by the simultaneous exposure to both light and oxygen.The high content of long-chain polyunsaturated fatty acids in the retina increasestheir vulnerability to oxidative damage. Thus, xanthophylls inhibit the peroxi-dation of membrane phospholipids and reduce the photooxidation of lipofuscinfluorophores (Schalch et al 2010; Ma and Lin 2010). The lipofuscin is a potentphotoinducible generator of reactive oxygen species that has been highly relatedto the pathogenesis of AMD (Age-related Macula Degeneration). Epidemiologicalstudies have associated the high consumption of lutein and zeaxanthin with reduced
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risk of AMD (Seddon et al. 1994; Snellen et al. 2002). The intake of lutein hasalso been associated with a reduced risk for cataracts (Lyle et al. 1999). Galeet al. (2001) reported that the plasma concentration of ’-carotene and “-carotenewas negatively correlated with the risk of nuclear cataract, whereas high plasmaconcentrations of lycopene and lutein reduced the risk of cortical and subcapsularcataract, respectively.
Epidemiological studies have shown that the consumption of carotenoid-richfruits and vegetables is associated with a lower risk of cardiovascular diseases;however, other dietary components from fruits and vegetables, like vitamin C, mightbe responsible of this protective effect (Koh et al. 2011; Ito et al. 2006; Sessoet al. 2004). The main action mechanism of carotenoids in this regard has beenassociated with their antioxidant activity. Carotenoids scavenge reactive oxygenspecies (ROS), protecting the low-density lipoproteins from oxidation, a key processin the pathogenesis of atherosclerosis. After the daily consumption of tomato juice(40 mg lycopene), carrot juice (22.3 mg “-carotene) or a liquid spinach powderpreparation (11.3 mg lutein) for two weeks, only the tomato juice reduced the lipidperoxidation in LDL of healthy men (Bub et al. 2000). Contrarily, in another studythe oxidation of LDL was inhibited by “-carotene (15 mg) but not by lycopenesupplementation (34 mg) (Dugas et al. 1999). Further work is required to evaluatethe effect of carotenoids in cardiovascular diseases.
The etiology of rheumatoid arthritis has been strongly associated with a chronicinflammation state, in which the active function of macrophages, monocytes andgranulocytes induce the formation of free radicals, which has been found in synovialfluids of patients with rheumatoid arthritis (Cerhan et al. 2003; Costenbader et al.2010; Merry et al. 1989). Thus, dietary antioxidants have been considered in theprevention and treatment of this disease. An epidemiological study reported aninverse relationship between the high intake of “-cryptoxanthin and the risk ofrheumatoid arthritis, but any relation was found for other carotenoids such as “-carotene, lycopene or lutein/zeaxanthin (Cerhan et al. 2003). Other studies neithercould relate the consumption of carotenoids with the prevention of rheumatoidarthritis (Costenbader et al. 2010; Heliövaara et al. 1994).
The pathogenesis of Alzheimer Disease (AD) has been related with oxidativestress. The brain is particularly susceptible to the oxidation due to its high metabolicactivity and demand of oxygen and because it contains abundant amounts ofpolyunsaturated fatty acids (Mecocci et al. 2002). Jiménez-Jimenéz et al. (1999)reported that AD patients showed low levels of “-carotene and vitamin A. Mecocciet al. (2002) also reported high plasmatic levels of the oxidative indicator 8-hydroxy-20-deoxyguanosine and low levels of antioxidants (zeaxanthin, “-cryptoxanthin,lycopene, and ’-carotene and “-carotene) in AD patients. High levels of phospho-lipid hydroperoxides and amyloid “-peptide (A“) have been reported in the redblood cells of AD patients (Nakagawa et al. 2011). Some in vitro and in vivo studieshave demonstrated that lutein, astaxanthin and “-carotene decreased the interactionbetween erythrocytes and A“ in cells from human and mice (Nakagawa et al. 2011).The retinoic acid may regulate genes involved in the A“ expression, such as the“-secretase enzyme, AbPP, and presenilin (Obulesu et al. 2011).
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 421
16.2 Digestion, Absorption and Metabolism
The bioactivity of carotenoids in the body depends in a first instance of theirabsorption. Only the carotenoids released from the food matrix may be available forabsorption by the intestinal epithelium. Of course, the carotenoid liberation fromthe food in the gastrointestinal tract is incomplete. Then, the carotenoids must beincorporated into the lipid phase of the emulsified gastrointestinal contents andthen to micelles. Only this reduced fraction of carotenoids can potentially reachthe enterocytes and perform a biological action. These key steps of the carotenoidabsorption process can be favored or hampered by many factors.
The majority of the studies about the health properties of carotenoids havebeen carried out in cancer cell lines or animals. These studies have showed thatcarotenoids are capable to regulate genes or proteins associated with the cancercell growth, apoptosis, cell signaling, and phagocytic activity of cells from immunesystem. They have generated invaluable clues. However, the carotenoid concen-tration that typically is assayed (up to 50 !M) does not reflect the physiologicalconcentrations of these pigments, even those reported in plasma after acute supple-mentation with carotenoids (Table 16.1). Additionally, the carotenoids added to cellcultures are dissolved in organic solvents, which may affect the normal cell functionor inhibit the carotenoid absorption in the plasmatic membrane. The incorporationmechanisms are also different to those occurring under normal conditions. On theother hand, many efforts have been done to increase the absorption of carotenoidsbut, up to date, there is not a clear evidence of that an increased absorption ratecould increase the biological action of carotenoids.
16.2.1 Digestion Process
The carotenoids must be released from the food matrix through the physical andchemical rupture of vegetable cells and intracellular compartments (Fig. 16.1).The physical disruption of the food occurs during mastication and as a consequenceof the peristaltic movements of gastrointestinal tract. The chemical disruption ofthe food is driven by digestive enzymes (amylase, gastric lipase, pepsine, etc.)and the hydrochloric acid secreted in the stomach. These processes result inthe partial liberation of carotenoids from the food matrix. Then, the carotenoidsshould be incorporated into lipid droplets dispersed in the gastrointestinal contents.Carotenoids distribute within the lipid droplet accordingly to their solubility.Carotenes and xanthophylls occupy the core and surface of these droplets, respec-tively (Furr and Clark 1997). Basically, their distribution depends on their polarity(Furr and Clark 1997). At the duodenum, the size of fat droplets is reduced bythe emulsifying action of bile salts, facilitating the action of lipolytic enzymes andthe formation of micelles, which are constituted by bile salts, phospholipids, anddigested lipids (free fatty acids, monoglycerides, carotenoids, lipophilic vitamins,
422 B. Cervantes-Paz et al.
Table16
.1Overviewof
recent
invivo
stud
iesevaluatin
gbioaccessibilityof
carotenoids
Model
Carotenoid
source
Stud
ied
carotenoids
a Adm
inistered
dose
Tested
factors
b Bioaccessibility
References
Plasmaor
serum
Other
tissues
Hum
ans
Tomatojuice
LUT,
cis-LUT,
’C,“
C,L
YC,
cis-LY
C
141mg/750g/70
kgBW
With
orwith
out
oil
0.04–2.28mg/L
Arranzetal.
(2015)
Hum
ans
Cabbage
LUT,
“C
1.6mg/300g/day
Varietie
s(black
andredcabbage)
!0.23–
5.47
mg/L
Bacchettietal.
(2014)
Hum
ans
Algae
Lutein-fortified
ferm
entedmilk
LUT
4– 120mg/100mL/day
Doselevels(low
andhigh)
0.12–
0.22
!mol/L
(2.1–2.5%)
Granado-
Lorencioetal.
(2010)
Hum
ans
Fruitjuice
LUT,
ZEA,“
C,
“CX
280–
575
!g/2
"250mL/
day
Absorption
modifiers(m
ilkandiron)
0.04–
0.43
!mol/L
(18–75
%)
Granado-
Lorencioetal.
(2009)
Hum
ans
Broccoli
LUT,
“C
7.6–
12.6
mg/200g/day
Modified
atmosphere
packaging
0.01–
0.051
!mol
Granado-
Lorencioetal.
(2008)
Hum
ans
Tomato,carrot,
spinach,
lettu
ce,
andwolfberry
LUT,
ZEA,’
C,
“C,L
YC,’
CX,
“CX
>0.75mg/day
Servingsize
(one
largemeal
ortwosm
all
meals)
0.004–
0.038
!mol/L
Goltzetal.
(2013)
Hum
ans
Tomato,carrot,
spinach,
lettu
ce,
andwolfberry
LUT,
ZEA,’
C,
“C,L
YC
25mg/serving
Amount
and
source
oftriacylglycerols
0.0013–
0.04
!mol/L
Goltzetal.
(2012)
Rats
Leafy
Vegetables
(onion
,lettuce,
spinach,
radish,
broccoli,
etc.)
LUT,
ZEA
2.69
mg/kg
diet
Food
matrixand
oiltype
4–25
pmol/m
L20–70pm
ol/g
Lakshminarayana
etal.(2007)
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 423
Hum
ans
Curly
kale
LUT,
“C,L
YC,
cis-LY
C0.003–
2.13
mg/3mL/day
Twooily
caroteno
idform
ulations
0.04–
0.28
!g/mL
0.14–0.18a.u.
Meinkeetal.
(2010)
Rats
Microalgal
biom
ass
LUT,
“C,A
ST200
!mol/rat/day
Algae
varieties
(S.p
latensis,H
.pluvialis
andB.
braunii)
!0.26–
0.48
!mol/m
L0.4–0.9
!mol/g
Rao
etal.(2013)
Rats
Algae
LUT,
“C,A
ST200
!mol/rat/day
Algae
varieties
(S.p
latensis,H
.pluvialis
andB.
braunii)
!0–
0.13
!mol/m
L!0
.02–
0.28
!mol/g
Rao
etal.(2010)
Hum
ans
Carrot,tomatoand
papaya
“C,L
YC
2.3–16.4
mg
Food
matrix
0.058–
0.61
!mol/L
Schw
eiggert
etal.(2014)
Hum
ans
Carrots
’C,“
C23.2–
24.2
mg/200g
FW
Cultiv
ation
(organicand
conventio
nal)
!0.05–
0.82
!mol/L
Strackeetal.
(2009)
Rats
Carrotp
uree
and
naturaly
oghurt
“C
0.03–0.044
mg/kg
Food
matrix
0.0067–
0.057
!mol/L
0.2–11.5
nmol/g
Syetal.(2013)
Rats
Carrots,tom
atoes,
spinachand
salm
on
“C,L
YC,L
UT,
AST
0.16–
0.33
mg/kg/day
Food
matrix
0.0029–
0.014
!mol/L
0.1–2.5nm
ol/L
Syetal.(2012)
Note:
BW
body
weight,FW
freshweight,LU
Tlutein,c
is-LUTlutein
isom
ers,AST
astaxanthin,
˛C
’-carotene,
ˇC
“-carotene,
cis-“C“-caroteneisom
ers,LY
Clycopene,cis-LYClycopene
isom
ers,˛CX’-cryptoxanthin,ˇ
CX“-cryptoxanthin
a Adm
inistereddose
isthesum
ofindividualcarotenoids
b Bioaccessibility
correspo
ndsto
theminim
umandmaxim
umvalues
ofindividu
alcaroteno
idsin
differenttreatments
c Valuesin
theparenthesisindicatetheestim
ated
bioaccessibility(%
)
424 B. Cervantes-Paz et al.
Fig. 16.1 Digestion and absorption processes of carotenoids
and cholesterol) (Guyton and Hall 2001). There is evidence of that some esterifiedxanthophylls are hydrolyzed by lipolytic enzymes mainly by the carboxyl esterlipase. The lipase and colipase have a minor participation in this regard (Breithauptet al. 2002; Chitchumroonchokchai and Failla 2006). The micelles size rangesfrom 4 to 60 nm (Parker 1996; Yonekura and Nagao 2007). The micelles transport
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 425
carotenoids through the aqueous intestinal medium and across the unstirred waterlayer adjacent to the brush border of enterocytes. This layer has an acidic nature,favoring the protonation of fatty acids and release from the micelle. This facilitatethe liberation of other lipophilic molecules, such as carotenoids, due to themicelle dissociation (Guyton and Hall 2001; Reboul 2013). Carotenoids may alsobe transported through the gastrointestinal aqueous media within unilamellar ormultilamellar vesicles of phospholipids (Reboul and Borel 2011).
16.2.2 Absorption Process
The first studies about the absorption of carotenoids suggested that they cross themembrane of enterocytes through a passive diffusion process, dependent on thecarotenoid concentration (Fig. 16.1) (Hollander et al. 1978; Moore et al. 1996).However, recent studies have suggested that cholesterol transporters are involvedin the carotenoid absorption by enterocytes (Borel 2012). Some studies on ratsand Caco-2 cells reported that the lipid transporter SR-BI, regulated the absorptionof “-carotene, lycopene and lutein (Moussa et al. 2008; Reboul et al. 2005; vanBennekum et al. 2005). This transporter protein also was associated with thepreferred absorption of xanthophylls in comparison with “-carotene in human retinalpigment epithelial cells (ARPE-19 cells) (During et al. 2008). Additionally, ithas been demonstrated that retinoic acid can repress the expression of the SR-BItransporter through the induction of the intestinal transcription factor ISX (Loboet al. 2010). Other membrane proteins of intestinal cells, such as CD36 (clusterdeterminant 36), FAT (fatty acid translocase), NPC1L (Nieman Pick C1-like 1),and the ABCG5/G8 from the ABC transporters superfamily have been associatedwith the absorption of carotenoids; however, there is not strong evidence abouttheir role in carotenoid transport (Borel 2012; Herron et al. 2006; Reboul 2013;van Bennekum et al. 2005).
Once within enterocytes, the carotenoids are transported to the Golgi apparatusand assembled in nascent chylomicrons, which are secreted into the lymphaticsystem for their transport in the bloodstream (Parker 1996). The non-provitamin Acarotenoids are transported through cytosol of enterocytes by some transporter pro-teins (Furr and Clark 1997). The CD36 has also been detected in the Golgi apparatus(Reboul and Borel 2011). Other proteins such as NPC1L1, SR-BI, or FABPs (fattyacid-binding proteins) are potentially responsible for the intracellular transport ofcarotenoids due to they are able to transport different fat-soluble nutrients as wellas by their strategic location in endosomes, lysosomes, mitochondria, cytoplasmaticlipid droplets and tubulovesicular membranes (Borel 2012; Reboul 2013). However,further genetic studies are needed to confirm their participation in the transport ofcarotenoids within enterocytes.
The carotenoids that were not assembled in chylomicrons eventually may returnto intestinal lumen (Parker 1996). Transmembrane proteins (SR-BI and ABC
426 B. Cervantes-Paz et al.
transporters) may also act in the efflux of carotenoids from enterocytes (Reboul2013). In addition to chylomicrons, it has been suggested that HDL may participatein the transport of carotenoids and retinoids from enterocytes to the lymph. Thisflux seems to be mediated by an ABCA1 transporter (Reboul 2013; Reboul andBorel 2011). Additionally, it has been reported that the expression of genes involvedin lipid absorption, metabolism and transport may be responsible of the inter-individual variation in the content of carotenoids in plasma (Borel et al. 2007; Borel2012).
Up to date, it is unclear if an exclusive or many proteins participate in thecarotenoid absorption in the intestine and other tissues; however, their involvementin the carotenoid absorption process might explain the inter-individual variationsin the efficiency of carotenoid absorption, the saturable absorption, the selec-tive absorption and the competition for absorption between different carotenoids(Reboul, 2013). Borel (2012) suggested that passive diffusion might occur withhigh levels of carotenoids while their protein-mediated transport might take place atdietary doses.
16.2.3 Metabolism
The metabolic fate of carotenoids depends on their chemical structure and nutri-tional status of individuals. Once within enterocytes, the provitamin A carotenoidsare immediately transformed in retinal, mainly by the action of BCO1. The levelof bioconversion to vitamin A depends on the nutritional status of the subject.The supplementation with “-carotene may induce carotenoderma without alter theretinol levels in plasma in individual with normal levels of vitamin A (Faulkset al. 1998). Pro- and non-pro-vitamin A carotenoids may be eccentrically cleavageby BCO2 enzymes or by auto-oxidation processes, resulting in the formationof volatile and non-volatile oxygenated cleavage products of carotenoids (Caris-Veyrat 2010). Kiefer et al. (2001) identified the “,“-carotene-90,100-oxygenase(BCO2), which produce “-apo-100-carotenal and “-ionone. The apo-carotenals maybe converted in retinaldehyde or “-apo-carotenoic acids. The “-carotenoic acids maybe precursors of retinoic acid (Failla and Chitchumroonchokchai 2005; Wang 2012).Ho et al. (2007) found “-apo-80-carotenals in plasma of a healthy man after thesupplementation with all-trans[10,100,11,110-14C]-“-carotene. Traces amounts of “-apo-carotenals (80,100,120,140) have been reported in rat intestines (Barua and Olson2000). Some eccentric cleavage products from lycopene have been found in ratssupplemented with this carotene. Gajic et al. (2006) identified apo-80-lycopenal andapo-120-lycopenal as metabolites of lycopene in rat livers. Also, apo-100-lycopenolwas reported in lungs from ferrets after the consumption of high lycopene diets(Hu et al. 2006). More recently, Kopec et al. (2010) identified apo-60, 80, 100, 120,and 140-lycopenals in plasma of humans after eight weeks of daily consumptionof tomato juice; however, they could not differentiate between metabolized and
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 427
ingested lycopenals. Other possible metabolites derived from carotenoid oxidationhave been identified in human plasma. Khachik et al. (1992) suggested that thepresence of ketocarotenoids in human plasma could be produced by the oxidation,reduction and double bond migration of lutein and zeaxanthin. In another study, itwas suggested that the presence of capsanthone in plasma from subjects who hadbeen ingested paprika juice was a consequence of the oxidation of capsanthin (Etohet al. 2000). The preferential rupture of non-provitamin A carotenoids by BCO2enzymes may be explained by variations in the intracellular localization of BCO1(cytosolic) and BCO2 (inner mitochondrial membrane) and the specific carotenoidsstorage sites within cells (Palczewski et al. 2014).
The metabolism of carotenoids in the body is unclear and subject of debatesince these molecules are highly susceptible to oxidation. The oxidation products ofcarotenoids can be generated during the extraction processes, during gastrointestinaldigestion or be natural components of foods. Regardless the source, the non-volatileapo-carotenoids and apo-lycopenoids and the volatile “-ionone products have shownchemopreventive activities such as the inhibition of cell growth, stimulation ofcell differentiation, activation of nuclear receptors, antagonize nuclear receptoractivation and induction of apoptosis (Wang 2012).
16.3 Bioaccessibility
Given the positive associations between the high consumption of fruits and veg-etables and the low incidence of different diseases, many researchers have triedto explain the protective effects of carotenoids from dietary sources. Only thecarotenoids delivered into the target organ can exert biological actions. This fractionof carotenoids has been estimated, with limitations, by the measurement of theirbioaccessibility. The bioavailability of carotenoids is defined as the fraction ofconsumed carotenoids that can be absorbed, transported, stored and/or employedin the normal biological functions. This is a complex measurement. The term‘bioaccessibility’ refers to the fraction of dietary carotenoids that is liberated fromthe food matrix during digestion, transferred into mixed micelles and/or absorbedby enterocytes and delivered in the blood stream. Only this carotenoid fraction isavailable to be used or stored by the body (Fernández-García et al. 2012). Severalin vivo and in vitro methods have been used to estimate the bioaccessibility andbioavailability of carotenoids. The isotope method consists in the administrationof physiologic doses of carotenoids labeled with stable isotopes (2H, 13C, 14C).This method provides the most reliable estimations about the absorption andmetabolism of carotenoids, allowing the discrimination of newly absorbed andexisting carotenoids and carotenoid metabolites (Failla and Chitchumroonchokchai2005; van den Berg et al. 2000; van Lieshout et al. 2003). This method has beenemployed to estimate the absorption of “-carotene, ’-carotene and “-cryptoxanthinfrom yellow and green leafy vegetables, and has been very useful to estimate
428 B. Cervantes-Paz et al.
the bioconversion efficiency of “-carotene into vitamin A (Ribaya-Mercado et al.2007; Tang et al. 2003; van Lieshout et al. 2003). However, this method is expensive.The metabolic balance technique involves the quantification of carotenoids inthe ingested meal and excreted faeces, assuming that the difference representthe amount of absorbed carotenoids (Failla and Chitchumroonchokchai 2005; vanLieshout et al. 2003). Some variants of this method are the gastrointestinal lavageand the ileostomy balance methods (van den Berg et al. 2000). The ileostomybalance involves the recuperation of digesta from ileostomic patients. This methodoffers advantages over the other balance methods since in such method thecarotenoids are not exposed to the degradative colonic microflora. Additionally, theabsorption results are comparable to those obtained measuring the plasma responseof carotenoids (Failla and Chitchumroonchokchai 2005; van den Berg et al. 2000;van Lieshout et al. 2003). The balance methods have been used to evaluate theeffects of food processing (Livny et al 2003; Unlu et al 2007) and dietary faton carotenoid absorption (Faulks et al 1997; van Loo-Bouwman et al 2010). Thequantitative plasma response of carotenoids after feeding carotenoid-rich foods isother method that has been used to estimate the bioavailability and bioaccessibilityof carotenoids. In this method, the plasmatic carotenoids are commonly monitoredduring periods that vary from 12 h to weeks (Failla and Chitchumroonchokchai2005; van den Berg et al. 2000). This technique does not allow the discriminationof newly absorbed carotenoids from those already existing in the blood stream. Itassumes that different carotenoids have similar and static rates of clearance fromplasma (Failla and Chitchumroonchokchai 2005). This is the most common methodemployed to estimate the carotenoid bioavailabity and bioaccessibility (Table 16.1).
The bioaccessibility of carotenoids may be estimated by in vitro methods. Thesetechniques include the simulation of the gastrointestinal digestion and their furtheruptake by Caco-2 cells. The in vitro methods are particularly used to determine thetransference efficiency of carotenoids to micelles. Only micellarized carotenoidscan be absorbed (Guyton and Hall, 2001; Failla and Chitchumroonchokchai 2005;Tyssandier et al. 2003). Reboul et al. (2006) reported significant correlationsbetween the in vitro bioaccessibility of carotenoids, measured as their micellariza-tion efficiency, and their plasma response in humans. Most of the observed trendsof carotenoid micellarization as a function of different factors coincide with thoseobserved under in vivo conditions. This is true for factors like the isomeric form ofcarotenoids, polarity differences, chromoplast morphology in the food matrix anddietary sources (Failla et al. 2014; Goltz et al. 2012; Reboul et al. 2006; Schweiggertet al. 2012, 2014). Thus, the estimation of the micellarization by in vitromethods hasbeen considered as valid method to estimate the potential absorption of carotenoids.
The bioaccessibility of carotenoids depends on many factors involved in keystages of their digestion and intestinal absorption, including their liberation fromthe food matrix, the emulsification of lipids (lipolysis, viscosity, lipid dropletsize), micellarization, absorption by enterocytes and secretion into the lymph. Thebioaccessibility of carotenoids from common foods is highly variable (Tables 16.1and 16.2) and depends on many factors.
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 429
Table16
.2Overviewof
recent
invitrostud
iesabou
tbioaccessibility
ofcaroteno
ids
b Bioaccessibility
Model
Carotenoid
source
Stud
ied
carotenoids
a CCTF(A
mount
offood
indigestion)
Tested
factors
Micellarizatio
nUptake(Caco-2)
References
Simulated
GId
igestio
nTo
mato
“C,L
UT,
LYC
1.8–8.4mg/100g
(2.0
g)Geographicalo
riginand
tomatotype
0.1–127%
Aherneetal.
(2009)
Simulated
GId
igestio
nOrangeand
juice
’C,“
C,L
UT,
“CX
0.18–0.27mg/100g
FW(5
g)Processing
(Orange:
choppedand
homogenized.Juice:
fresh,
flash-pasteurized
andpasteurized)
7–48
%Aschoffetal.
(2015)
Simulated
GId
igestio
nandCaco-2
cells
Spinach
“C,L
UT
33.7
!mol/L
(4.0
g)Effecto
fdivalentions
(Ca,Mg,
Zn,
Fe)
!0.01–68
%!1
.3–44.5
pmol/m
gcell
Biehler
etal.
(2011)
Simulated
GId
igestio
nOrange-fleshed
sweetp
otato
“C,cis-“C
30.5–34.4mg/100g
DW
(3.0
g)Therm
alprocessing
(fresh,b
oilin
g,homogenizationand
cooking)
andfat
16–70%
Bengtsson
etal.(2010)
Simulated
GId
igestio
nOrange-fleshed
sweetp
otato
“C,cis-“C
6.1–9.7mg/100g
DW
(3.0
g)Mechanical(cylin
ders
andslices)a
ndthermal
(boilin
g,steaming,
microwaveheating)
processing
andfat
0.5–56
%Bengtsson
etal.(2009)
Simulated
GId
igestio
nYellowpotatoes
LUT,
ZEA
0.33–1.35mg/100g
FW(0.5
g)Differentaccession
s33–71%
Burgosetal.
(2013)
Simulated
GId
igestio
nWatermelon
and
guava
“C,L
YC,L
UT
3.9–4.9mg/100g
FW(0.5
g)Fo
odmatrix
21–73%
Chandrika
etal.(2009)
(contin
ued)
430 B. Cervantes-Paz et al.
Table16
.2Overviewof
recent
invitrostud
iesabou
tbioaccessibility
ofcaroteno
ids
b Bioaccessibility
Model
Carotenoid
source
Stud
ied
carotenoids
a CCTF(A
mount
offood
indigestion)
Tested
factors
Micellarizatio
nUptake(Caco-2)
References
Simulated
GId
igestio
nFruitjuices
ZNX,“
C,
NEOC
cis-
VIO
,ZEA,
LUT,
“CX
0.024–
0.22
mg/100mL
(80g)
Processing
(therm
aland
high
pressure)o
fwhole,
skim
med,and
soymilk
beverages
4–149%
Cillaetal.
(2012)
Simulated
GId
igestio
nTo
matopu
lpLY
C(5.0
g)Processing
andfattype
(coconut,o
liveandfish
oil)
2–18
%Colleetal.
(2013)
Simulated
GId
igestio
nTo
matopu
lpLY
C4.0–5.4mg/100g
(5.0
g)Fattyp
e(cocoa
butte
r,coconu
t,palm
,oliv
e,sunfl
ower,and
fishoils)
1–5%
Colleetal.
(2012)
Simulated
GId
igestio
nTo
matopu
lpLY
C,cis-LYC
94.8–
130.0mg/100gDW
(5.0
g)
Therm
alprocessing
atdifferenttem
peratures
!12–36
%Colleetal.
(2010)
Simulated
GId
igestio
nBasil,
coriander,
dill,
mint,
parsley,
rosemary,sage,
tarragon
“C,“
CX,
ZEAC
LUT
2.0–25.8
mg/100g
(2.0
g)Fo
odmatrix
0–27
%Dalyetal.
(2010)
Simulated
GI
digestion,
synthetic
micellesand
Caco-2cells
Citrus
juices
(orange,
mandarinand
lemon
juices)
“C,“
CX,
“CX-esters
0.12–
1.64
mg/100mL
(20g)
Food
matrix
!15–40
%0.6–21
%Dhuique-
Mayer
etal.
(2007)
Simulated
GId
igestio
nBananas
and
plantains
’C,“
C,cis-“C
0.13–22mg/kg
(5.0
g)Boiledfruitsanddishes
from
plantain
and
banana
0–34
%Ekesa
etal.
(2012)
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 431
Simulated
GId
igestio
nandCaco-2
cells
Salad(tom
ato,
carrot,spinach,
lettu
ce,w
olfberry)
’C,“
C,L
YC,
LUT,
ZEA
9.1mg/100g(2.4
g)Fattyp
e(butter,olive,
cano
la,and
soyb
ean
oils)
0–57
%1.5–35.0
pmol/m
gprotein
Faillaetal.
(2014)
Simulated
GId
igestio
nandCaco-2
cells
Melon
“C
1.3–2.3mg/100gFW
Orange-fleshed
honeydew
melon
3.2%
11.6%
Fleshm
anetal.
(2011)
Simulated
GId
igestio
nYellow-fleshed
cassavaroots
“C
1.1–2.4mg/100gDW
Cooking
process
(boilin
gandfrying)
5–14
%Gom
esetal.
(2013)
Simulated
GId
igestio
nLutein-fortified
milk
LUT
4–8.2mg/100mL
(5.0
mL)
Doselevels(low
and
high)
!4–12%
Granado-
Lorencioetal.
(2010)
Simulated
GId
igestio
nFruitjuice
LUT,
ZEA,
“CX,“
C0.11–0.23mg/100mL
Absorptionmodifier
(milk
andiron)
18–75%
Granado-
Lorencioetal.
(2009)
Simulated
GId
igestio
nPeppers(A
rbol,
Chipo
tle,G
uajillo
andMorita
)
“C,“
CX,Z
EA
87.6–373.3
mg/100g
DW
(0.3
g)Varietie
s20–49%
Hervert-
Hernández
etal.
(2010)
Simulated
GId
igestio
nCarrots
“C
196.6mg/100gDW
Dryingtemperature
(70,
80,and
90ı C
)13–73%
Hiranvarachat
etal.(2012)
Simulated
GId
igestio
nCarrots
’Cand“C
(5.0
g)Cooking
andadditio
nof
oilatd
ifferentlevels
29–80%
Hornero-M
éndez
andMínguez-
Mosquera
(2007)
Simulated
GId
igestio
nSq
uash,carrot
grapefruit,
mango,
melon
papaya,
sweetp
otato,
tomato,
watermelon
’C,“
C,L
YC,
LUT,
VIO
,PE
1.33–45.1mg/100g
FW(5
mL)
Food
matrix
0–96
%Jefferyetal.
(2012)
(contin
ued)
432 B. Cervantes-Paz et al.
Table16
.2Overviewof
recent
invitrostud
iesabou
tbioaccessibility
ofcaroteno
ids
b Bioaccessibility
Model
Carotenoid
source
Stud
ied
carotenoids
a CCTF(A
mount
offood
indigestion)
Tested
factors
Micellarizatio
nUptake(Caco-2)
References
Simulated
GId
igestio
nCarrots
“Cand
cis-“C
126–158mg/100g
DW
(5.0
g)Therm
aland
mechanicalp
rocessing
(highpressure
homogenization,
thermalor
high
pressure
pasteurizatio
n)and
additio
nof
oliveoil
!10–36
%Knockaert
etal.(2012a)
Simulated
GId
igestio
nTo
matoes
LYC
(5.0
g)Therm
alandhigh
pressure
processing
!50–97
%Knockaert
etal.(2012b)
Simulated
GId
igestio
nCarrots
“C
Cooking
(gently
and
intenselycooked)a
ndmechanicalp
rocessing
(cellb
reakageand
separatio
n)
!0–19%
Lem
mens
etal.(2010)
Simulated
GId
igestio
nSp
inach,
komatsuna,
pumpkin,and
carrot
“C,L
UT
(1.5
g)Fo
odmatrixand
individualfatsandoils
!6–80%
Nagao
etal.
(2013)
Simulated
GId
igestio
nOrang
e,kiwi,red
grapefruit,
honeydew
melon,
spinach,
broccoli,
redpepp
er,sweet
potato
“C,L
YC,
LUT,
“CX,
ZEA
(2.0
g)Fo
odmatrix
2–109%
O’Connell
etal.(2007)
Simulated
GId
igestio
nandCaco-2
cells
Mango
“C
1.1–3.9mg/100g
(1.5
g)Ripeningstage,dietary
fatand
pectin
concentration
4.5–40
%13.8–19.6
pmol/m
gcell
Ornelas-Paz
etal.(2008)
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 433
Simulated
GId
igestio
nandCaco-2
cells
Belland
chili
peppers
“C,L
UT,
“CX,
ZEA
1.98–44.5mg/100g
(!2.0g)
Ripeningstages
and
varieties
6–113%
1–32
%O’Sulliv
anetal.
(2010)
Simulated
GId
igestio
nOrangecarrots,red
carrots,red
tomatoesand
atom
icredcarrots
“C,L
YC
0.8–8.5mg/100g
Matrixandthermal
processing
(water
bath
65–95
ı C;o
ilbath
95–125
ı C)
1–38
%Palm
eroetal.
(2014)
Simulated
GId
igestio
nRed,orangeand
yello
wtomatoes
LYC,—C,L
UT
0.07–4.6
mg/100g
(5.0
g)Fo
odmatrixandhigh
pressure
homogenization(20,
50,1
00MPa)
7–96
%Panozzoetal.
(2013)
Simulated
GId
igestio
nAjuicewith
orange,k
iwi,
pineapple,and
mango
’C,“
C,
cis-VIO
CNEO,
cis-ANT,
ANT,
LUT,
’CX,“
CX
0.031–0.64
mg/100mL
(200
mL)
Beverageform
ulation
(with
orwith
outm
ilk)
8–27
%Rodríguez-
Roque
etal.
(2014)
Simulated
GId
igestio
nAjuicewith
orange,k
iwi,and
pineapple
’C,“
C,
cis-VIO
CNEO,
cis-ANT,
ANT,
LUT,
’CX,“
CX
0.54
mg/100mL
(200
mL)
Beverageform
ulation
8–17
%Rodríguez-
Roque
etal.
(2013)
Simulated
GId
igestio
nandCaco-2
cells
Cou
rgette,red
pepper
andtomato
“C,L
YC,L
UT,
“CX
(2.0
g)Cooking
procedures
(boilin
g,grilling,
microwave-cooking,
andsteaming)
and
food
matrix
2–106%
!7–35%
Ryanetal.
(2008)
(contin
ued)
434 B. Cervantes-Paz et al.
Table16
.2Overviewof
recent
invitrostud
iesabou
tbioaccessibility
ofcaroteno
ids
b Bioaccessibility
Model
Carotenoid
source
Stud
ied
carotenoids
a CCTF(A
mount
offood
indigestion)
Tested
factors
Micellarizatio
nUptake(Caco-2)
References
Simulated
GId
igestio
nCarrot,mango,
papaya,and
tomato
’C,“
C,L
YC,
LUT,
“CX,
“CX-esters
0.3–4.44
mg/100g
(10g)
Food
matrixanddietary
fat(with
orwith
outo
il)!0
.4–15%
Schw
eiggert
etal.(2012)
Simulated
GId
igestio
nandCaco-2
cells
Marine
spore-form
ing
Bacillus
sp.,
carrots,
purified“C
“C
30–
44.2
!g/digestion
Food
matrix(purified
“C,carrotsandbacteria)
13–60%
6–11
%Sy
etal.
(2013)
Simulated
GId
igestio
nandCaco-2
cells
Purified
carotenoids,
carrots,
tomatoes,
spinachand
salm
on
“C,L
YC,
LUT,
AST
30–215
!g/digestion
Food
matrix
1–50
%7–11
%Sy
etal.
(2012)
GId
igestio
nsimulated
Cassava
“C,cis-“C
2.8–4.09
mg/100g
DW
(0.3g)
Styleof
processing
(raw
,boiled,
Gariand
Fufu)
!12–30
%Thakk
aretal.
(2009)
GId
igestio
nsimulated
Jalapeño
peppers
NEO,N
CR,
VIO
,LTX,
CAP-epox,
’C,“
C,C
AP,
CAP-esters,
“CX,
“CX-esters,
ZEA-esters
1.4–22.6
mg/100g
DW
(2.0g)
Processing
style
(boilin
gandgrilling),
ripening
stage(green
andred),and
fattype
(saturated
and
unsaturated)
0–308%
Victoria-
Cam
posetal.
(2013b)
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 435
GId
igestio
nsimulated
Jalapeño
peppers
NEO,N
CR,V
IO,
LTX,C
AP-epox,
’C,“
C,C
AP,
CAP-esters,
“CX,“
CX-esters,
ZEA-esters
5.4–22.5
mg/100gFW
(2.0
g)Processing
style
ripening
stageandfat
type
2–349%
Victoria-Cam
pos
etal.(2013a)
Simulated
GId
igestio
nandCaco-2
cells
Purified
carotenoids
“C,L
UT
200nm
ol/L
(15mg)
Fibertyp
e(alginate,
appleandcitrus
pectin)
41–99%
0.18–
0.41
pmol/!g
protein
Yon
ekuraand
Nagao
(2009)
FW
freshweight,DW
dryweight,LU
Tlutein,VIO
violaxanthin,cis-VIO
violaxanthin
isom
ers,
ANT
antheraxanthin,cis-ANT
antheraxanthin
isom
ers,
AST
astaxanthin,
NEO
neoxanthin;,
ZNX
zeinoxanthin,˛C
’-carotene,
ˇC
“-carotene,
cis-“C
“-caroteneisom
ers,
"C—-carotene,LY
Clycopene,cis-
LYC
lycopene
isom
ers,
PE
phytoene,PF
phytofl
uene,NCR
neochrom
e,LT
Xluteoxanthin,˛CX
’-cryptoxanthin,ˇCX
“-cryptoxanthin,ˇCX-esters
“-cryptoxanthin-esters,CAPcapsanthin,C
AP-epoxcapsanthin
5,6-epoxide,CAP-esterscapsanthin-esters,ZE
Azeaxanthin,Z
EA-esterszeaxanthin-esters
a CCTF
Carotenoidcontentintested
foods,estim
ated
asthesum
ofindividualcarotenoids
b Bioaccessibility
correspo
ndsto
theminim
umandmaxim
umvalues
ofindividu
alcaroteno
idsin
differenttreatments
436 B. Cervantes-Paz et al.
16.3.1 Food Matrix
The effect of the food matrix includes the combined effects of all factors from afood that simultaneously promote or reduce the bioavailability/bioaccessibility ofcarotenoids (Ornelas-Paz et al. 2008). The food matrix effect includes differences inthe composition and storage sites of carotenoids as well as changes in the food byripening and processing.
16.3.1.1 Chromoplast Morphology
The bioaccessibility of carotenoids from fruits is significantly higher than that ofvegetables (de Pee et al. 1998). This effect has been associated to the differentialphysical disposition of carotenoids within chromoplasts. Typically, carotenoidsare stored as a lipid solution in globular and tubular chromoplasts of maturefruits; however, they can also be accumulated as crystalline structures. They maybe complexed with proteins in chloroplasts of green vegetables (Schweiggertet al. 2012; Vásquez-Caicedo et al. 2006). During digestion, lipid bodies richin carotenoids from fruits may easily interact with the lipidic phase of the gas-trointestinal content, making them more bioaccessible and bioavailable (West andCastenmiller 1998). In contrast, crystalline forms are not completely dissolvedduring their transit through the gastrointestinal tract (de Pee et al. 1998). The invitro and in vivo bioaccessibility of “-carotene from different sources followed theorder of mango > papaya > tomato > carrot and this order was explained in terms ofdifferences in the chromoplast morphology and accumulation forms of carotenoidsin fruits and vegetables (Schweiggert et al. 2012, 2014). The difference in “-carotenebioaccessibility from mango and papaya could be consequence of differences inthe presence of carotenoids in liquid-crystalline stores in the chromoplasts of thesefoods. The results for tomato could not be explained in these terms. Ornelas-Pazet al. (2010) also demonstrated in rats that “-carotene from mango was two timesmore bioavailable than from carrots. Carrillo-Lopez et al. (2010) reported that thelevels of hepatic retinol in rats depended on the source of “-carotene, following theorder of mango > carrot > spinach > parsley.
16.3.1.2 Ripening
Ripening modifies the amount and type of carotenoids in fruits and vegeta-bles. The chloroplasts of green vegetables and immature fruits mainly containlutein, “-carotene, violaxanthin and neoxanthin. During ripening, the chloroplastsare transformed in chromoplasts with an increased biosynthesis of carotenoids(Rodriguez-Amaya and Kimura 2004; Yahia and Ornelas-Paz 2010). In somecases, the carotenoids of chloroplast serve as precursor of other carotenoids duringripening (Cervantes-Paz et al. 2012, 2014). The increase of total carotenoids during
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 437
the ripening has been reported for different fruits and vegetables. The fully ripemango, endive and lettuce have from 2.5 to 4 times more total carotenoids thanthe slightly ripe fruits or young leaves (Azevedo-Meleiro and Rodriguez-Amaya2005b; Ornelas-Paz et al. 2008). The carotenoid content in red peppers (Jalapeño,Agridulce, Bola, Szentesi Kosszarvú Paprika) is 11 to 85 times higher than ingreen peppers (Cervantes-Paz et al. 2012; Deli et al. 1996; Mínguez-Mosquera andHornero-Méndez 1994b). Of course, this behavior is not observed in all vegetablefoods, as occur in for young and mature leaves of kale or spinach (Azevedo-Meleiroand Rodriguez-Amaya 2005a, b). These qualitative and quantitative changes mayinfluence the carotenoid bioaccessibility. Thakkar et al. (2007) reported a positivecorrelation between the content of “-carotene in cassava and its efficiency ofmicellarization and uptake by Caco-2 cells.
The esterification of xanthophylls during ripening has been reported in peppers,sea buckthorn berries, bananas, kiwis, among others (Andersson et al. 2009;Cervantes-Paz et al. 2012, 2014; Mínguez-Mosquera and Hornero-Méndez, 1994a;Montefiori et al. 2009). The esterification of carotenoids reduces their polarityand bioaccessibility. After in vitro digestions of citrus juices, the micellarizationof free “-cryptoxanthin was three times higher than that of the monoesterifiedforms (Dhuique-Mayer et al. 2007). The in vitro micellarization of free zeaxanthinwas about two and seven times higher than that of mono and diesterified formsin wolfberry, orange pepper, red pepper and squash (Chitchumroonchokchai andFailla 2006). These tendencies were also seen for the uptake by Caco-2 cells inboth studies. Victoria-Campos et al. (2013a, b) reported that the micellarizationof free and esterified forms of capsanthin, antheraxanthin, mutatoxanthin, andzeaxanthin also followed the order of free >monoesterified > diesterified forms afterin vitro digestions of raw or heat-processed red peppers. The study of the in vitrobioaccessibility of different monoesterified forms of capsanthin and “-cryptoxanthinsuggests that their micellarization is influenced by the polarity provided by the fattyacid bounded to the carotenoid backbone, following an order of micellarizationefficiency of laurate >myristate > palmitate (Dhuique-Mayer et al. 2007; Victoria-Campos et al. 2013a, b). However, Breithaupt et al. (2003) demonstrated that freeand esterified forms of “-cryptoxanthin showed similar in vivo bioaccessibility.The absence of esterified carotenoids in human plasma after the consumption offruits rich in carotenoid esters suggests that only free forms are absorbed or thatsome esterases cleavage carotenoid esters (Granado et al. 1998; Wingerath et al.1995). Further studies about the digestion, absorption and metabolism of esterifiedcarotenoids are needed.
Ripening also cause fruit softening, which involve the solubilization, depoly-merization and demethylation of pectins from cell walls (de Roeck et al. 2008;Gross and Sams 1984; Redgwell et al. 1997). Pectins and other fibers couldalter the emulsification of lipids in the gastrointestinal medium and their furtherhydrolysis (Pasquier et al. 1996). These fibers are also able to interact with bilesalts, disturbing the micellarization processes (Dongowski et al. 1996; Pasquieret al. 1996). Ornelas-Paz et al. (2008) demonstrated that the bioaccessibility of“-carotene was significantly enhanced by the ripening of mango. This effect was
438 B. Cervantes-Paz et al.
associated with the quantitative and qualitative ripening-related changes of mangopectin. Victoria-Campos et al. (2013a, b) reported that ripening of peppers did notaffect the micellarization of free carotenoids; however, the ripening stage of fruitsdetermined the number and micellarization efficiency of esterified xanthophylls.The information about the effect of ripening on the bioaccessibility of carotenoidsis scarce; however, some studies suggest that qualitative and quantitative changes ofthe intrinsic pectin substances during fruit ripening play an important role. Cell wallcomposition and metabolism vary widely between plant foods.
16.3.1.3 Heat Processing
Heat processing reduces the negative effects of food matrix on carotenoid bioac-cessibility and bioavailability (Ornelas-Paz et al. 2008; Yahia & Ornelas-Paz2010). The heat processing causes the disruption of food matrix, loss of cellularintegrity and breaking of protein-carotenoid complexes. These effects may increasethe carotenoid extractability during digestion and their further bioaccessibilityand bioavailability (Yahia and Ornelas-Paz 2010). The softening of fruits by theheat processing has been associated with the solubilization, depolymerization anddemethylation of pectins (de Roeck et al 2008; Ramos-Aguilar et al. 2015; Silaet al. 2006). These heat processing mediated effects in the food matrix may varyas a function of time, intensity and type of processing. The bioaccessibility oflycopene from tomato pulp increased as the heat processing temperature rose from60 to 140 ıC, with the bioaccessibility of cis- and trans-lycopene being almost2 times higher in puree treated at 140 ıC, as compared with raw samples (Colleet al. 2010). The bioaccessibility of carotenes from carrots was 80 and 57% greaterafter cooking (100 ıC, 10 min) and blanching (80 ıC, 10 min), respectively, ascompared with raw samples (Netzel et al. 2011). Lemmens et al. (2009) reportedthat the bioaccessibility of “-carotene from carrots increased as the duration andtemperature of heat processing increased from 0 to 50 min and from 90 to 110 ıC,respectively. The heat-processing style also alters the carotenoid bioaccessibility.Bengtsston et al. (2009) reported that the bioaccessibility of “-carotene from carrotswas almost 50% lower after microwave heating in comparison with boiling andsteaming. Ryan et al. (2008) demonstrated that the bioaccessibility of “-carotenefrom boiled courgette, red pepper and tomato was higher than that of the raw,grilled, microwaved and steamed foods. Similar tendencies were observed forlutein. The bioaccessibility of lycopene from courgette was increased by grillingand microwaving. Contrarily, these treatments hindered the bioaccessibility of “-cryptoxanthin from all evaluated fruits (Ryan et al. 2008). Victoria-Campos et al.(2013b) reported that heat processing decreased the bioaccessibility of many freeand esterified carotenoids from red peppers. These studies suggest that the effect ofheat processing on carotenoid bioaccessibility depends on carotenoid type and plantfood.
The heat processing also alters the qualitative and quantitative profile ofcarotenoids. These pigments are highly thermolabile. Heat processing may
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 439
induce the trans to cis isomerization, epoxidation and degradation of carotenoids(Cervantes-Paz et al. 2012, 2014; Rodriguez-Amaya 1999). The micellarizationof 13-cis and 9-cis isomers of “-carotene is higher than that of the form all-trans(Bengtsson et al. 2009; Bechoff et al. 2009; Ekesa et al. 2012; Tyssandier et al. 2003;Victoria-Campos et al. 2013b). The micellarization and uptake of cis-lycopeneby Caco-2 cells was also higher than that of all-trans-lycopene (Failla et al. 2008).Accordingly, the greater bioaccessibility of cis-lycopene in comparison with theall-trans isomer has also been reported in vivo (Boileau et al. 1999; Cooperstoneet al. 2015; Stahl and Sies 1992). This phenomenon might be explained in termsof the higher solubility of the cis isomers of carotenoids due to the bent backbone.This might favor their transference to the micelles (Yahia and Ornelas-Paz 2010).There is scarce information about the bioaccessibility of carotenoid epoxides. It hasbeen suggested that they are not absorbed in humans (Stinco et al. 2012). Recently,Victoria-Campos et al. (2013a, b) reported the formation of capsanthin 5,6-epoxidein Jalapeño peppers as a consequence of heat processing and demonstrated thatthis compound was efficiently micellarized. However, Asai et al. (2008) reportedthat although some epoxyxanthophylls (neoxanthin and fucoxanthin) are efficientlymicellarizaced, their concentration in plasma (about 1 nmol/L) does not increaseafter the consumption of foods containing these carotenoids.
16.3.1.4 Mechanical Processing
The release of carotenoids from the food matrix is directly enhanced by the ruptureof cells and cellular compartments before the consumption of a plant food. van hetHof et al. (2000) reported that the concentration of lycopene in the triglyceride-rich lipoprotein fraction of plasma increased 32 and 62% after the ingestionof mildly and severely homogenized tomato products, respectively, as comparedwith the consumption of non-homogenized tomatoes. The concentration of “-carotene in this plasma fraction increased 5.6 and 8.2 times as a consequence ofthese homogenization levels. Livny et al. (2003) also demonstrated the “-carotenebioaccessibility from carrots puree is 50% greater than that of chopped carrots.Castenmiller et al. (1999) reported that the estimated relative bioavailability of “-carotene increased 86% after the consumption of liquefied spinach in comparison towhole leaf in healthy subjects. Recently, Aschoff et al. (2015) found that the in vitromicellarization of lutein, “-cryptoxanthin, ’-carotene, and “-carotene increasedfrom 1.3 to 3.5 times when the orange segments were replaced by orange juice inthe digestive reactions. These studies collectively indicate that homogenization stylealters the bioaccessibility of carotenoids. The new homogenization technologiessuch as high-pressure homogenization did not represent an advantage on thebioaccessibility of lycopene from tomato pulp (Colle et al. 2010). Apparently, thistechnology induces a fiber network that entraps lycopene.
440 B. Cervantes-Paz et al.
16.3.2 Dietary Fat
Dietary fat is the main effector of carotenoid bioaccessibility because it can mediateall processes involved in carotenoid absorption. Dietary fat promotes the carotenoiddiffusion from the food matrix to the emulsified content of the gastrointestinal tract.It stimulates the secretion of acid in the stomach and of bile salts and pancreaticenzymes at the duodenum, facilitating the micelle formation. Hydrolyzed lipids maycompete with carotenoids for the transporter proteins during absorption. Dietaryfat promotes the chylomicron secretion and consequently the bioaccessibility ofcarotenoids (Guyton and Hall 2001; Yahia and Ornelas 2010). Recently, Failla et al.(2014) reported that the secretion of carotenoids (lutein and “-carotene) into thebasolateral medium was positively associated with the apical concentration of fattyacids (0.5–2.0 mmol/L) in Caco-2 cell cultures. The required levels of dietary fat toenhance the micellarization of carotenoids differ for different fat types, polarity ofcarotenoids and food matrix.
To date, there is not an exact recommendation about the amount of fat that isrequired to obtain a good carotenoid absorption. In vivo studies have demonstratedthat low levels (6–12 g) of dietary fat in a meal are enough to enhance the carotenoidconcentration in plasma (Brown et al. 2004; Unlu et al. 2005). Fat contents (avocado,canola oil, soybean oil or butter) of 12 and 28 g in a meal seem to be optimalto get the highest possible plasma levels of carotenoids (’- and “-carotene, lutein,zeaxanthin, and lycopene) when these came from raw salads (Brown et al 2004;Goltz et al. 2012; Unlu et al. 2005). Roodenburg et al. (2000) did not find differencesin the plasma concentration of ’-carotene and “-carotene after the consumption ofa hot meal with 3 and 36 g of dietary fat, but lutein in plasma increased with thehighest level of fat, which could occur because of the meal contained esterifiedlutein. The optimal amount of dietary fat for the maximal absorption of carotenoidslikely depends on the food matrix and type of carotenoid in a meal. This mightexplain the variability in results from different studies.
Results from in vitro studies suggest that a percentage of fat close to 10 is enoughto get a good carotenoid micellarization. Failla et al. (2014) demonstrated that themicellarization efficiency of zeaxanthin, ’-carotene, “-carotene and lycopene from asalad puree was higher when the amount of soybean oil was increased from 1–3 to8% (Failla et al. 2014). The micellarization of “-carotene from carrots increasedslightly (!6–17%) when the digestion reaction contained 5% of olive oil, ascompared with digestions without fat; however, this variable dramatically increased(54–117%) when the amount of fat was increased to 10% (Honero-Méndez andMínguez-Mosquera 2007).
There is an effect of fat type on carotenoid bioaccessibility and bioavailability,but it is not completely understood (Colle et al. 2012). Some studies have demon-strated that monounsaturated and polyunsaturated fatty acids promote the carotenoidbioaccessibility in comparison with saturated fatty acids. Accordingly, Goltz et al.(2012) observed higher levels of carotenoids (lutein, zeaxanthin, ’-carotene, “-carotene and lycopene) in human plasma after the consumption of a carotenoid-rich
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 441
meal with canola oil (rich in C18:1) and soybean oil (rich in C18:2) than with butter(rich in C16:0 and C14:0). Failla et al. (2014) also reported that the micellarization,uptake by Caco-2 cells, and basolateral secretion of lutein, zexanthin, ’-carotene,“-carotene and lycopene were enhanced by the addition of soybean, canola andolive oils in comparison with butter. However, highly unsaturated fatty acids (C18:3,C20:4) may decrease the bioaccessibility of carotenoids (Nagao et al. 2013).
Additionally, the fatty acid chain length may also influence carotenoid micellar-ization. Huo et al. (2007) found that the micellarization of ’-carotene and “-carotenefrom a salad puree was positively associated with the chain length of fatty acids inthe order of C18:1 >C8:0 >C4:0. Nagao et al. (2013) also reported higher micel-larization of “-carotene from spinach with oleic acid (C18:1) than fatty acids withmedium-chain lengths (C6-C10). On the other hand, the increase of the carotenoidbioaccessibility as a function of fat type is clear for carotenes, lycopene andesterified xanthophylls but not for free xanthophylls (Failla et al. 2014; Gleize et al.2013; Huo et al. 2007; Roodenburg et al. 2000; Victoria-Campos et al. 2013a, b).
From these studies the positive effect of dietary fat on the bioaccessibility ofcarotenoids is evident. Apparently, mono- and di-unsaturated fatty acids with long-chain (C:18) induce greater favorable effects than saturated fatty acids with medium-chain lengths. However, further studies are needed to explain the mechanisms ofdietary fat on the micellarization and absorption processes for different carotenoids.
16.3.3 Dietary Fiber
The dietary fiber plays an important role in the bioaccessibility of carotenoids. Dif-ferent fibers (pectin, guar gum, alginate, cellulose or wheat bran) are able to reduce(33–47%) the plasma concentration of dietary carotenoids (“-carotene, lycopene,lutein), with major decreases being observed with water-soluble fibers (Riedl et al.1999). Among dietary fibers, pectin is one the most abundant polysaccharide inplant cell walls. It’s chemical properties have been strongly associated with texturaland firmness changes of different fruits (Gross and Sams 1984; Ramos-Aguilaret al. 2015; Van Buren 1979). Pectin may affect the viscosity of the gastrointestinalcontent, the lipid droplet size, the availability of bile salts and the enzymaticlipolysis of triglycerides (Pasquier et al. 1996). Rock and Swendseid (1992) reportedthat citrus pectin reduced the plasma response of “-carotene in more than 50% ascompared with the consumption of the test meal without pectin. In another study,the bioaccessibility of pure “-carotene was significantly reduced by the additionof pectin from mangoes, with the largest decrease being found with pectins fromslightly ripe mangoes in comparison with pectins from fully ripe fruits (Ornelas-Pazet al. 2008). The impact of pectin on carotenoid bioaccessibility can be regulatedby the physicochemical characteristics of pectin, which naturally vary betweendifferent fruits and during the fruit ripening (Ramos-Aguilar et al., 2015).
442 B. Cervantes-Paz et al.
In general, a detrimental effect of pectin on carotenoid bioaccessibility has beenestablished (Aschoff et al. 2015; Ornelas-Paz et al. 2008; Riedl et al. 1999; Rock andSwendseid 1992). However, this effect has quantitative and qualitative connotations.The amount of pectin has a clear negative effect of carotenoids bioaccessibility.Verrijssen et al. (2013) demonstrated that the bioaccessibility of “-carotene fromcarrots was higher (20–30%) with low amounts (1–3%) of citrus pectin thanhigh concentrations of it (3.5–5%). However, the structural characteristics of thesepolysaccharides seem to have the highest effect on carotenoid bioaccessibility.Verrijssen et al. (2014) found that citrus pectins with low esterification degree (14%)reduced the bioaccessibility of “-carotene in 40%, as compared with pectins witha high esterification degree (66–99%). Dongowski (1995) found the interactionbetween pectins and bile salts increased as the concentration, esterification degreeand molecular weight of pectins also increased; however, the degree of acetylationand amidation of pectins was inversely related with their association with bilesalts. The structural characteristics and botanical source of pectins can change theirnegative effect on carotenoid bioaccessibility. Pectins also may act as emulsifiersand fat emulsification is a key step on carotenoid bioaccessibility (Leroux et al.2003). Beet pectin seems to have higher emulsifying properties than citrus pectin.Acetylated citrus pectin showed a higher emulsifying property than the non-acetylated counterpart (Leroux et al. 2003). Citrus and beet pectins also reducedthe interfacial tension between the water and oil phases. Bonnet et al. (2005)demonstrated that some pectins (high methoxylated pectins) make more stable theoil-in-water emulsions under acidic conditions. The particle size in emulsions isreduced by pectins at intermediate pH values (pHD 5.5). These authors suggestedthat the pectins form a network that connects the oil droplets. In addition, lowesterification pectins enhanced the stability of emulsions in comparison with highlyesterified pectins (Kovacova et al. 2009). These studies collectively suggest thatpectin might increase the carotenoid bioaccessibility under some conditions.
16.3.4 Interaction Between Carotenoids
Many carotenoids typically coexist in a single food. Some studies have reportedthe competition of carotenoids to be micellarized or absorbed. Tyssandier et al.(2002) found that the chylomicron concentration of lycopene was significantlyhigher after the consumption of tomato puree than when tomato puree wasaccompanied with chopped spinach. They also observed that the response of luteinfrom spinach was reduced by the ingestion of a lycopene pill or tomato puree. Inanother study in healthy men, the supplementation with “-carotene decreased theplasma concentration of lutein in comparison with placebo, suggesting a possibleinteraction between these carotenoids (Micozzi et al. 1992). During et al. (2002)demonstrated that “-carotene reduced, in a dose-dependent fashion, the uptake of’-carotene and lycopene by Caco-2 cell layers and their subsequent basolateralsecretion. However, they (During et al. 2002) did not observe an association between
16 Absorption of Carotenoids and Mechanisms Involved in Their Health. . . 443
the uptake or transport of lutein and “-carotene. Reboul et al. (2005) reported thatmixed micelles with both lutein and “-carotene decrease the uptake of lutein (20%)in Caco-2 cells in comparison with micelles containing only lutein.
16.3.5 Interaction Between Different Factors
Some studies have demonstrated the existence of complex interactions between allfactors involved on carotenoid bioaccessibility and bioavailability. Mathematically,an interaction means that two or more factors play a role together, which is differentto the effect of individual factors (Castenmiller and West 1998). However, this typeof studies is scarce. The effect of the interaction between heat processing and dietaryfat is one of the most studied. In many cases, the positive effect of cooking in themicellarization of carotenoids is potentiated by the presence of dietary fat (Colleet al. 2013; Hornero-Méndez and Mínguez-Mosquera 2007). Hornero-Méndez andMínguez-Mosquera (2007) reported that the micellarization of carotenes increasedfrom! 29%, with raw carrots, to! 51% with cooked carrots, and up to! 80%with cooked carrots plus 10% of olive oil. Similarly, the heat treatment (120 ıCfor 20 min) increased the bioaccessibility of lycopene from tomato pulp, butgreater increments of bioaccessibility were observed when different oils (coconut,olive or fish) were added (Colle et al. 2013). Cilla et al. (2012) reported thatthe high-pressure processing (HPP) and thermal treatment (TT) decreased thebioaccessibility of carotenoids from fruit juice-milk beverages, but the addition ofskimmed milk and whole milk induce the lowest decrease in the bioaccessibility oftotal carotenoids.
Dietary fat may also increase the benefits of mechanical processing of plantfoods. Colle et al. (2013) reported that the combination of high pressure homog-enization and the addition of coconut, olive or fish oil increased the bioaccessibilityof lycopene from tomato pulp in comparison with non-treated samples and treatedsamples without lipids. Lemmens et al (2010) reported an interaction betweenthe severity of food matrix disruption and heat processing. They found that thebioaccessibility of “-carotene was higher with small particles (<160 !m) of carrotsthat had been cooked for 3 min than with particles of raw carrots or carrots cookedfor 25 min. However, with the largest carrot particles (161–6300 !m) the cookingfor 25 min increased the bioaccessibility of “-carotene, as compared with particlesof raw carrots or carrots cooked for 3 min.
The effect of the interaction of ripening, heat processing style and dietary fat typein the bioaccessibility of carotenoids from Jalapeño peppers was recently studied.Victoria-Campos et al. (2013a, b) demonstrated that the effect of dietary fat typeinfluence more the bioaccessibility of carotenoids from ripe peppers, presumablyas consequence of their highest content of esterified carotenoids, than those ofimmature fruits, where the content of esterified carotenoids is minimal. They(Victoria-Campos et al. (2013a, b) also demonstrated that the global impact of theheat processing was negative for fruits at early ripening stages, with this negative
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effect being less notable with fruits at the most advanced stages of ripening. Thiscould be due to the major thermostability of carotenoids from ripe peppers or by acompensation effect of dietary fat, which ameliorate the negative effects of the heatprocessing.
16.4 Concluding Remarks and Future Trends
In the last years, the health protective effects of carotenoids have been associatedmore to their ability to regulate the expression of genes than with their antioxidantactivity. This is of importance given the high incidence of chronic diseases as cancer,cardiovascular diseases, and type 2 diabetes. However, in vivo evidence is stillneeded.
Carotenoid metabolites such as apo-carotenoids seem exert important biologicalactivities; however, their identification is a major challenge due to their high insta-bility. Thus, future works should propose new analytical methods, and strategies toknow the proportion of bioaccessible or bioavailable metabolites that are naturallypresent in the fruit or generated during gastrointestinal digestion.
The digestive process of carotenoids is more or less known; however, thereare still some gaps of knowledge needing to be clarified, such as the digestion,hydrolysis and absorption of esterified xanthophylls as well as the carotenoiddistribution in micelles and/or vesicles in the aqueous phase.
It has been suggested that both passive diffusion and facilitate transport ofcarotenoids determine their bioavailability. However, the participation of each trans-port mechanism seems to be dependent on the intestinal carotenoid concentrationand specificity of the transporters. Genetic studies about the transmembrane andintracellular proteins might clarify the mechanisms that regulate the carotenoidtransport in cells.
Finally, plant foods contain a wide variety of carotenoids (free and esterifiedespecies). The bioaccessibility and bioavailability of carotenoids from the samematrix are differentially affected by the intrinsic properties of the food (fruit matrix,chromoplast morphology, ripening stage, etc.), processing type (mechanical andthermal), presence of some dietary components (dietary fat, phytosterols, fiber), etc.These factors do not actuate alone but also they interact each other. The interactionbetween factors should be studied in a deeper way.
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Index
AAbsorption, v, 85, 95, 97, 99, 118, 123–125,
127, 130, 131, 142, 143, 147, 329, 360,370, 379, 380, 415–444
Adiposity, 385, 388–401Algae, v, 4, 8, 15, 19, 21, 51, 53, 72, 112, 115,
117, 133, 142–145, 219–227, 229, 230,313, 318, 330, 333, 354, 422, 423
Amyloplasts, 274–276, 281–284, 286, 287,330, 349
Antioxidant activity, 8, 85, 174, 220, 228, 230,346, 361–364, 369, 371, 418–420, 444
Antioxidant capacity, 9, 299, 331, 363–365,369
Antioxidants, v, 5, 6, 8, 26, 36, 37, 87, 88,92, 174, 200, 229, 230, 278, 282, 298,299, 312, 324, 331, 335, 346, 347, 354,359–371, 379, 388, 390, 394–397, 401,419, 420
Apocarotenoids, v, 5, 37–40, 54, 80, 142, 143,151, 163, 165, 166, 173, 179, 188,239–263, 279, 280, 295, 380, 383
Astaxanthin, 4–7, 11–13, 22–26, 36, 220–223,225–230, 316, 318, 323, 328, 330, 333,346, 354–355, 384, 387, 394, 399, 401,420, 435
BBioaccessibility, 327, 370, 371, 422–423,
427–444Bioactivity, 251, 370, 421Branching, 38, 50, 177, 223, 242, 246, 247,
250–252, 254–256, 259, 260, 263, 298,301–304, 335, 351, 352
Brassica napus, 44, 303, 332, 350, 352–354
CCancer markers, 365, 418Carlactone, 38, 242, 253–256, 263“-Carotene, 4, 36, 72, 115, 145, 162, 176,
202, 220, 240, 301, 318, 349, 364,416
Carotenes, 4, 36, 72, 142, 168, 202, 220, 286,298, 323, 350, 360, 379, 415
Carotenogenesis, v, 10, 12, 16, 19, 21, 22, 38,39, 48, 148, 171, 183, 212, 221–226,297, 349
Carotenoid cleavage dioxygenases (CCDs),38–40, 151, 175, 187, 188, 202,205, 208, 240, 242–249, 255, 304,349
Carotenoids, v, 3, 35, 71, 112, 141, 162, 201,220, 240, 274, 295, 312, 346, 359,378, 415
biosynthesis, v, 12–26, 36, 39–55, 129,141–152, 161–188, 199–213, 222, 223,227–228, 247, 263, 275–279, 282, 283,285, 286, 300–302, 304, 305, 313–319,321–323, 328–332, 334, 335, 345–355
catabolism in fruits, 187–188dietary source, 359–371in fruits, 162, 173, 436gene characterization, 50, 208improvement, 318plant models, 35–56in seeds, 314, 331, 349stability, 277, 286
Carrot, 38, 44, 52, 84, 94, 96, 101, 199–213,284, 286, 313, 317, 328, 333, 334, 367,369, 420, 422, 423, 431–434, 436,438–440, 442, 443
© Springer International Publishing Switzerland 2016C. Stange (eds.), Carotenoids in Nature, Subcellular Biochemistry 79,DOI 10.1007/978-3-319-39126-7
455
456 Index
Cetocarotenoids, 36Chloroplasts, 15, 37, 40, 41, 44, 46, 47, 49, 53,
113, 143, 147, 151, 173, 182–184, 187,203, 207, 208, 211, 221–223, 227, 245,273–276, 278–283, 299, 301, 319, 321,323, 332, 335, 349, 354, 436
Chromoplasts, 40, 51, 54, 55, 78, 143, 147,151, 170, 174, 175, 178–188, 205, 207,208, 211, 240, 245, 273–277, 280–287,313, 319, 323, 328, 330, 349, 351, 428,436, 444
9-cis carotenoid cleavage dioxygenases, 240,243, 247
Citrus paradisi, 164, 168Crops fortifying, 326
DDunaliella salina, 6, 8, 221, 222, 226–229
EEnergy metabolism, 386–387Epigenetic, v, 148, 209, 280, 295–305Etioplasts, 37, 49, 147, 151, 240, 273–278E-Z isomers, 84
FFood matrix, 90, 329, 370, 421–423, 427–434,
436–440, 443Fruit ripening, 148, 162, 173, 176, 178, 179,
182, 183, 188, 248, 275, 280, 283, 285,295, 297, 299, 319, 438, 441
GGene expression, 133, 146–149, 177–182, 187,
207, 210, 211, 257, 276–278, 286, 297,299, 302, 319, 348, 349, 365, 366, 381,390, 396
Genotype-environment, 351Golden Rice, 281, 282, 314, 325, 326, 350, 351
HHaematococcus pluvialis, 6, 22, 24, 221–223,
225, 227–230, 316, 317, 323, 328, 333,354, 423
Health benefits, 4, 324, 346, 366, 370, 371
High performance liquid chromatography(HPLC) methods, 91, 92, 95, 100, 102
Histone methylation, 298Human epidemiological studies, 396–399,
401Human nutrition, v, 312–313
KKey enzymes, 39, 180, 203, 226, 277, 379
LLand plants, 53, 142–147, 147, 148, 221, 250Light-harvesting systems, 112, 113, 115, 132,
133Lutein, 5, 49, 77, 115, 146, 162, 202, 220, 276,
298, 312, 346, 364, 379, 417
MMetabolic engineering, v, 12, 51, 55, 221,
227–228, 230, 318–333, 350Metabolic regulation, 147, 176, 322, 366Microbial carotenoids, 5, 6, 9MicroRNA, 304, 352Multienzymatic complexes, 44
NNeoxhantin, 111NMR, 98, 254Nutritional important crops, 313
OObesity, 377–401, 416
PPhotosynthesis, v, 3, 4, 40, 111–134, 143, 149,
150, 152, 173, 203, 220, 222, 245, 275,278–280, 295, 301, 312, 353, 360
Pinalate, 168, 175, 177, 184, 185Plastid development, 151, 186, 283Purple photosynthetic bacteria, 112, 117–125,
128–130
QQuantitative analysis, 85–102
Index 457
RRetinoids, 37, 142, 312, 379–381, 383–385,
387, 389, 391, 392, 397, 398, 400, 401,416, 426
Root development, 203, 205, 207, 208,210–212, 247, 261
SScreening, 48, 84–85, 348, 364SDG8, 148, 298, 300–302SL synthesis, 250, 259Strigolactones (SLs), v, 38, 240, 242, 246,
249–257, 261–263, 280, 295, 301, 302,304, 312, 351
Structures, v, 4, 36, 72, 112, 142, 174, 211,222, 242, 274, 299, 323, 348, 361, 394,415
Synthesis regulation, 55, 182, 209
UUltra-high performance liquid chromatography
(UHPLC) methods, 100
VVitamin A metabolism, 379–381
XWhite adipose tissue (WAT) browning, 380,
387, 391–395
XXanthophylls, 4, 36, 77, 133, 142, 170,
207, 220, 276, 296, 315, 348, 360,380, 415
86
CAPÍTULO II
EFFECT OF PECTIN ON LIPID DIGESTION AND THEIR POSSIBLE
IMPLICATIONS ON CAROTENOID BIOAVAILABILITY AT PRE-
ADSORPTIVE STAGES. A REVIEW
Elsevier Editorial System(tm) for Food Research International Manuscript Draft Manuscript Number: Title: Effects of pectin on lipid digestion and their possible implications on carotenoid bioavailability at pre-absorptive stages, a review Article Type: SI: Carotenoids Keywords: Polysaccharides; Fiber; Lipid-soluble pigments; Bioactive compounds; Bioavailability Corresponding Author: Dr. José de Jesús Ornelas-Paz, Ph.D. Corresponding Author's Institution: Centro de Investigación en Alimentación y Desarrollo A.C. First Author: Braulio Cervantes-Paz Order of Authors: Braulio Cervantes-Paz; José de Jesús Ornelas-Paz, Ph.D.; Saul Ruiz-Cruz; Claudio Rios-Velasco; Vrani Ibarra-Junquera; Elhadi M Yahia; Alfonso A Gardea-Béjar Abstract: Pectin is an abundant polysaccharide of human diet, showing structural characteristics and functional properties that strongly dependent on food matrix (origin, type, cultivar/variety, ripening stage, style and intensity of processing, etc.). These polysaccharides exert a strong effect on lipid digestion, which is required for liberation of carotenoids from emulsified lipid droplets in the gastrointestinal content as well as for the formation of micelles, where the carotenoids must be incorporated before absorption. Only micellarized carotenoids can be absorbed and, therefore, exert protective effects on human health. The alteration of lipolysis by pectin has been explained by several mechanisms; however, they have not been linked directly to carotenoid micellarization. This paper provides an overview about the impact of pectin on lipid digestion and subsequent carotenoid micellarization, emphasizing the role of pectin properties.
Highlights
-Carotenoids exert health-related effects in humans
-The bioavailability of the same carotenoid highly varies among foods
-Pectin amount and properties are also highly variable among foods
-Pectin cause variability on carotenoid bioavailability by altering lipolysis
*Highlights (for review)
1
Effects of pectin on lipid digestion and their possible implications on 1
carotenoid bioavailability at pre-absorptive stages, a review 2
Braulio Cervantes-Paza, José de Jesús Ornelas-Paza,*, Saul Ruiz-Cruzb, Claudio 3
Rios-Velascoa, Vrani Ibarra-Junquerac, Elhadi M. Yahiad, Alfonso A. Gardea-Béjara 4
a Centro de Investigación en Alimentación y Desarrollo, A.C.-Unidad Cuauhtémoc, 5
Av. Río Conchos S/N, Parque Industrial, C.P. 31570, Cd. Cuauhtémoc, 6
Chihuahua, Mexico 7
b Instituto Tecnológico de Sonora, Departamento de Biotecnología y Ciencias 8
Alimentarias, 5 de Febrero 818 Sur. C.P. 85000. Cd. Obregón, Sonora, Mexico 9
c Universidad de Colima, Bioengineering Laboratory, Km. 9 carretera Coquimatlán-10
Colima, C.P. 28400, Coquimatlán, Colima, Mexico. 11
d Universidad Autónoma de Querétaro, Facultad de Ciencias Naturales. Avenida 12
de las Ciencias S/N, C.P. 76230. Juriquilla, Querétaro, Mexico 13
14
E-mail addresses: [email protected] (B. Cervantes-Paz), 15
[email protected] (J. J. Ornelas-Paz), [email protected] (S. Ruiz-Cruz), 16
[email protected] (C. Rios-Velasco), [email protected] (V. Ibarra-Junquera), 17
[email protected] (E.M. Yahia), [email protected] (A.A. Gardea-Béjar) 18
19
*Corresponding author. Tel/Fax: +52-625-5812920. E-mail address: 20
[email protected] (J. J. Ornelas-Paz). 21
22
*ManuscriptClick here to view linked References
2
Abstract 23
Pectin is an abundant polysaccharide in human diet, showing structural 24
characteristics and functional properties that strongly dependent on food matrix 25
(origin, type, cultivar/variety, ripening stage, style and intensity of processing, etc.). 26
These polysaccharides exert a strong effect on lipid digestion, which is required for 27
liberation of carotenoids from emulsified lipid droplets in the gastrointestinal 28
content as well as for the formation of micelles, where the carotenoids must be 29
incorporated before absorption. Only micellarized carotenoids can be absorbed 30
and, therefore, exert protective effects on human health. The alteration of lipolysis 31
by pectin has been explained by several mechanisms; however, they have not 32
been linked directly to carotenoid micellarization. This paper provides an overview 33
about the impact of pectin on lipid digestion and subsequent carotenoid 34
micellarization, emphasizing the role of pectin properties. 35
36
37
38
39
Keywords: Polysaccharides; Fiber; Lipid-soluble pigments: Bioactive compounds; 40
Bioavailability 41
42
3
1. Introduction 43
The carotenoids are lipid soluble pigments that can be found at high concentration 44
in many fruits and vegetables (Victoria-Campos et al., 2013). They confer a yellow, 45
red or orange pigmentation to many vegetable foods (Cervantes-Paz et al., 2012). 46
Several protective effects on human health have been regarded to these pigments, 47
including anti-obesity effects, strengthening of the immune system, and a reduction 48
of the risk of suffering several forms of cancer and cardiovascular diseases (Yahia 49
and Ornelas-Paz, 2010). The mechanisms involved in such effects are not clear 50
yet, but it is believed that they are a consequence of the antioxidant activity of 51
carotenoids as wells as due to their capacity to favor cell to cell communication and 52
influence gene expression (Cervantes-Paz, Victoria-Campos & Ornelas-Paz, 53
2016a). However, the biological actions of carotenoids are highly dependent, in a 54
first instance, on their intestinal absorption. One of the most relevant factors 55
limiting the bioavailability of carotenoids at the pre-absorptive stages is the food 56
matrix. This effect refers to the combined effects of all factors inherent to a food 57
that simultaneously promotes or reduces the bioavailability of carotenoids 58
(Ornelas-Paz, Failla, Yahia, & Gardea-Bejar, 2008). Fiber is one of such food 59
matrix-related factor, with pectin being one of the most abundant component of 60
fiber (Ramos-Aguilar et al., 2015). Some in vivo studies have evidenced a negative 61
effect of pectin on carotenoid bioavailability. Riedl, Linseisen, Hoffman, and 62
Wolfram (1999) demonstrated that citrus pectin reduced 42% the bioavailability of 63
β-carotene and determined that soluble fibers, like pectin, exert a higher effect than 64
the insoluble ones. Horvitz et al. (2004) attributed the low bioavailability of carrot 65
carotenoids to the high fiber content in the vegetable. Many other similar studies 66
4
demonstrated the negative effect of pectin on carotenoid bioavailability; however, 67
the mechanisms involved started to be investigated in recent years (Verrijssen, 68
Verkempinck, Christiaens, Van Loey & Hendrickx, 2015; Verrijssen et al., 2014). 69
The amount and properties of pectin in foods are highly dependent on the 70
characteristics of the vegetable matrix, like food type, cultivar/variety, ripening 71
stage, processing style/intensity, etc. (Ornelas-Paz et al. 2008; Victoria-Campos et 72
al., 2013; Ramos-Aguilar et al., 2015). These variations might explain the high 73
variability in the micellarization and bioavailability observed for the same 74
carotenoid in different foods or even in the same food type (Cervantes-Paz et al., 75
2016a). This hypothesis is reinforced for a limited number of studies that 76
demonstrated that pectin characteristics modulated carotenoid micellarization and, 77
indirectly, for many studies that demonstrated that the amount and 78
characteristics/properties of pectin alter, in different ways, the digestion of lipids, a 79
key determinant for carotenoid transport and absorption (Cervantes-Paz, et al., 80
2016b; Verrijssen et al., 2015; Yonekura & Nagao, 2007, 2009). The elucidation of 81
the mechanisms involved in this phenomenon is complex because of the main 82
structural characteristics of pectin are related each other and the study of the effect 83
of individual characteristics of pectin imply the modification of a single property 84
without altering the others, which represents a big technical challenge (Aschoff et 85
al., 2015; Espinal-Ruiz, Restrepo-Sánchez, Narváez-Cuenca, & McClements, 86
2016; Galisteo, Duarte & Zarzuelo, 2008). The purpose of this paper is to provide a 87
systematic analysis of existing evidence on the effects of pectin on lipid digestion, 88
linking such effect with the alteration of the micellarization and bioavailability of 89
dietary carotenoids. 90
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91
2. The carotenoid absorption process 92
The carotenoid absorption process involves the pigment release from food matrix 93
by food chewing, peristalsis and the disruptive activity of digestive enzymes and 94
extreme pH of gastrointestinal fluids (Low, D´Arcy & Gidley, 2015). Subsequently, 95
they are incorporated into lipid droplets of the gastric medium (Borel et al., 1996). 96
In the duodenum, the surfactant activity of bile salts (BS) causes a reduction in the 97
size of lipid droplets, increasing the surface area of the lipid phase and, therefore, 98
the area where lipolytic enzymes exert their activity (Faulks & Southon, 2005; 99
Guyton & Hall, 2001). The lipid digestion products (free fatty acids, FFA; 100
monoglycerides, MG; diglycerides, DG; and lysophospholipids), BS, cholesterol, 101
and carotenoids liberated from the lipid droplets are incorporated into micelles 102
(Reboul, 2013; Yonekura & Nagao, 2007). The hydrolysis of triglycerides (TG) from 103
the lipid droplet surface is reversible and therefore if the FFA are not micellarized 104
they might go back to the lipid droplet to form TG (Dandik & Aksoy 1992). The 105
carotenoids are transported in the micelles throughout the intestinal medium and, 106
subsequently, absorbed by the enterocytes (Reboul, 2013). Only micellarized 107
carotenoids can be absorbed, which might explain the low levels of carotenoids 108
typically found in human plasma (0-0.82 μmol/L) (Rao, Baskaran, Sarada, & 109
Ravishankar, 2013; Rao, Reddy, Baskaran, Sarada, & Ravishankar, 2010; 110
Schweiggert et al., 2014; Stracke et al., 2009; Sy et al., 2012, 2013). The lipophilic 111
nature of carotenoids makes necessary the presence of dietary fat for their 112
absorption and transport (Krinsky & Johnson, 2005). The general process for 113
6
carotenoid absorption is shown in Fig. 1. 114
115
3. Dietary pectin structure 116
Pectins are abundant and complex polysaccharides of the cell wall of plant foods 117
(Caffall & Mohnen, 2009). They are involved on cell growth, rigidity of plant tissues, 118
some defense mechanisms, ion transport regulation, permeability of the walls for 119
enzymes, and water holding capacity of cells (Caffall & Mohnen, 2009; Voragen, 120
Coenen, Verhoef, & Schols, 2009). Pectin structure comprises a backbone 121
composed by segments of homogalacturonan (HG) and rhamnogalacturonan (RG) 122
type I (RGI) and II (RGII) (Sila et al., 2009). Pectins are mainly linear polymers of 123
�D(1-4) linked D-galacturonic acid residues interrupted by 1,2-linked L-rhamnose 124
segments. Each pectin molecule is formed by hundreds of blocks, being 125
polysaccharides of very high molecular weight (MW) (Sinha & Kumria, 2001). The 126
HG are linear structures of D-galacturonic acid linked by D(1-4) bond. These 127
regions can be esterified with methanol at C-6 carboxyl or de-esterified to create a 128
block copolymer structure (Wicker et al., 2014). Methylation distribution in HG 129
varies depending on the de-esterification mechanism (Sila et al., 2009). The 130
amount of methylesters on these regions determinates the degree of methyl 131
esterification (DM) of pectin, which is also used to explain the hydrodynamic, 132
gelling, and hydration properties of these polysaccharides (Ngouémazong et al., 133
2011; Voragen et al., 2009; Ramos-Aguilar et al., 2015). Thus, the functionality of 134
pectins as gelling, thickening and stabilizing agents is highly related to their DM 135
and MW (Correding & Wicker, 2001). Regions of HG are occasionally interrupted 136
7
by neutral sugar side chains to form RGI and RGII. Additionally, other compounds 137
such as acetyl groups, ferulic acid, and proteins, can also be present in pectin 138
structure (Wicker et al., 2014). RGI are composed of [o2)-D-L-Rhap-(1o4)-DD-139
GalpA-(1o] regions. Rhamnosyl (Rha) residues of RGI are also substituted at O-4 140
by neutral sugars side chains, which are composed of galactosyl and arabinosyl 141
residues. The proportion of branched Rha residues varies from 20 to 80% 142
depending on the source of the polysaccharide (Sila et al., 2009; Voragen et al, 143
2009). Since RGI are not degraded by E-elimination events, it is believed that GalA 144
residues of RGI are not methyl esterified. However, the GalA residues in the RGI 145
backbone may be highly acetylated on positions O-2 and O-3 (Voragen et al., 146
2009). RGII are structures highly conserved in the plant kingdom and are released 147
by the action of endopolygalacturonase. Their structure contains clusters of four 148
different side chains sugar residues, such as apiose, aceric acid, 3-deoxy-lyxo-2-149
heptulosaric acid, and 3-deoxy-manno-2-octulosonic acid (Sila et al., 2009; 150
Voragen et al., 2009; Wicker et al., 2014). These side chains are attached to HG 151
with nine GalA residues, sometimes methyl esterified. RGII can be complexed with 152
boron to form a borate–diol ester, which can crosslink two HG molecules, where 153
only the apiofuranosyl residues participate in the cross-linking (Voragen et al., 154
2009). 155
156
4. Effects of pectin on lipid digestion and carotenoid bioavailability 157
4.1 The role of calcium binding by pectin 158
8
Dairy foods and green leafy vegetables are good sources of carotenoids and 159
calcium, but all of the plant foods are rich in pectin (Slavin & Lloyd). By the way, 160
the co-consumption of dairy products and plant foods is common in the human diet 161
(Hu, 2003). Pectins, as other endogenous and dietary components, can bind 162
calcium ions (Braccini & Perez, 2001; Kim & Lim, 2004; Perry, Cygan, & Mitchell, 163
2006; Rui, 2009). This interaction can lead to the formation of gels by strong 164
interactions between calcium ions and residues of galacturonic and galuronic 165
acids (Braccini & Perez, 2001). The gel formation is highly dependent on the 166
degree on methyl esterification of pectin, with low methoxylated pectins (LMP) 167
binding more calcium ions than high methoxylated pectins (HMP) as a 168
consequence of the higher number of carboxylic acid moieties in their structure 169
(Nair et al., 1987). The galacturonic acid chains of LMP tend to be loosely 170
associated each other in the presence of Ca2+, improving their ability to form gels 171
by dimerization and formation of point-like cross-links (Assifaoui et al., 2015; 172
Huynh, Lerbret, Neiers, Chambin, & Assifaoui, 2016: Fang et al., 2008). These 173
pectin-calcium complexes reduce lipid digestion by inducing a reduction of the lipid 174
droplet surface area, where lipase exerts its activity, as a consequence of lipid 175
flocculation or microgel formation (Fig. 2) (Hu, Li, Decker, & McClements, 2010). 176
Thus, the reduction of lipid digestion by these complexes might limit the 177
transference of carotenoids from lipid droplets to micelles as well as the 178
generation of lipid digestion products, which are required for the formation of 179
micelles, the absorptive vehicles of carotenoids (Yahia & Ornelas-Paz, 2010). 180
Yonekura and Nahao (2009) demonstrated that the micellarization of E-carotene 181
9
and lutein was inhibited in presence of pectins and alginates, presumably as a 182
consequence of pectin gellification. Similarly, Verrijssen et al. (2014) found a 183
significant decrease on the E–carotene micellarization as the DM of pectin 184
decreased and attributed this effect to pectin gellification. 185
The binding of calcium by pectin also might affect the carotenoid absorption in 186
other ways, independently of gel formation. Calcium has the ability to precipitate 187
the FFA, in form of insoluble calcium salts or soaps, from lipid droplet surface, 188
clearing such surface and favoring the access of lipase to TG of emulsified lipid 189
droplets (Devraj et al., 2013). Thus, the levels of free calcium in the 190
gastrointestinal medium can be reduced in presence of pectin, causing the 191
accumulation of FFA on lipid droplet surface and reducing lipid droplet digestion 192
(Fig. 2). This effect might limit the liberation of carotenoids from lipid droplets, 193
reducing their incorporation into micelles, as well as reducing the availability of 194
lipid digestion products for micelle formation and reducing carotenoid 195
bioavailability (Devraj et al., 2013). However, this reduction in the availability of 196
lipid digestion products cannot be only consequence of a limited digestion but also 197
of the precipitation of FFA as insoluble compounds (Fig. 2). The later effect seems 198
to have more relevance on carotenoid absorption, according to a limited number of 199
studies in this regard. Chai, Cooney, Franke, and Bostick (2013) observed a 200
significant decrease of serum triglyceride and carotenoids in humans 201
supplemented with elemental calcium (2.0 g/day) for 6 months, with this decrease 202
being explained by the formation of intestinal calcium-lipid complexes. Biehler et 203
al. (2011a) demonstrated that the micellarization of carotenoids was reduced by 204
10
calcium in a dose-dependent fashion, with a calcium concentration of 0.051 M 205
completely inhibiting carotenoid micellarization. They (Biehler et al., 2011a) 206
attributed such reduction/inhibition to the generation of insoluble soaps of calcium 207
with FFA and BS. In a further study, they observed that such effect also depended 208
on carotenoid structure (Biehler, Hoffmann, Krause, & Bohn, 2011b). Corte-Real et 209
al. (2016) also demonstrated that the addition of calcium inhibited the carotenoid 210
micellarization. Currently, the effect of calcium and pectin characteristics on 211
carotenoid absorption have not been determined. 212
213
4.2 Effect of the interaction of pectin with bile salts 214
The interaction of pectin and BS might modulate the carotenoid absorption since 215
BS are key components for lipid digestion and micelle formation (Fig. 3A) 216
(Cervantes-Paz et al., 2016b). The structure of BS consists of hydroxyl groups 217
(hydrophilic side), which can interact with other BS, and an ionic head 218
(hydrophobic side), which can be dissolved in water-oil interfaces (Bauer, Jakob, & 219
Mosenthin, 2005). The BS are secreted at the duodenum and accumulated on the 220
lipid droplet surface, favoring the emulsification of lipid droplets in the aqueous 221
contents, reducing the lipid droplet size and, consequently, increasing the surface 222
area of lipid phase available for lipase activity (Moghimipour, Ameri, & Handali, 223
2015). At the duodenum, BS are always in micelles under physiological normal 224
conditions (Armand et al., 1996). Thus, TG of lipid droplets are hydrolyzed to MG, 225
DG, and FFA, which diffuse from lipid surface and then are incorporated into 226
micelles along with phospholipids, which help to solubilize other lipid components 227
like cholesterol and carotenoids (McClements, 2013; Moghimipour et al., 2015). 228
11
The BS are abundant components of mixed micelles (Cohen, Thurston, 229
Chamberlin, Benedek, & Carey, 1998). Under normal conditions, BS are 230
reabsorbed in the ileum and, then, transported to the liver via enterohepatic 231
circulation (Hofmann, 2009); however, in presence of pectin, the reabsorption of 232
BS is reduced and their excretion increased, limiting the availability of BS for 233
micelle formation and lipid emulsification (Fig. 3A) (Dongowski, Huth, & Gebhardt, 234
2003). This fact could explain the low E-carotene absorption observed in BS 235
deficient children (Vanderpas et al., 1987). Olsen, Kiessling, Milley, Ross, and Lall, 236
(2005) evaluated the effect of BS in the absorption of astaxanthin in salmon and 237
observed that the addition of 2.5 g of taurocholic acid per kg of diet caused a 238
nearly 20% increase in blood astaxanthin. Hedrén et al. (2002) estimated the 239
effect of BS in the bioaccessibility of �E-carotene from carrots using in vitro 240
digestions and observed a decrease of 20% in the micellar �E-carotene when BS 241
were not added to digestion reactions. This behavior was also observed by Wright, 242
Pietrangelo, and MacNaughton, (2008), who observed a low �E-carotene 243
micellarization (less of 4%) in absence of BS. Thus, pectin reduces the availability 244
of BS, which are required for lipid emulsification, lipolysis, micelle formation, and 245
carotenoid absorption (Dongowski, Neubert, Haase, & Schnorrenberger, 1996; 246
Riedl et al., 1999). The interaction of BS with pectin depends on the botanical 247
origin and structural properties of the polysaccharide (Fig. 3B). Xu, Jiao, Yuan, 248
and Gao (2015) observed that the interaction of sodium cholate was higher with 249
citrus pectin (617 mg/g) than that of peach (376-531 mg/g). The olive pectin 250
showed an strong interaction with deoxycholate, taurocholate, and 251
12
chenodeoxycholate, while citrus pectin had a higher affinity for deoxycholic acid 252
(8.5 mM/100 mg) than for chenodeoxycholic (1.5 mM/100 mg) and cholic acids 253
(2.5 mM/100 mg) (Rodríguez-Gutiérrez, Rubio-Senent, Lama-Muñoz, García, & 254
Fernández-Bolaños, 2014; Rubio-Senent, Rodríguez-Gutiérrez, Lama-Muñoz, & 255
Fernández-Bolaños, 2015). Cheewatanakornkool et al. (2012) observed that the 256
interaction between sodium deoxycholate with apple, pomelo and citrus pectins 257
depended on pectin concentration, although at the highest pectin concentration 258
(1% w/w) the dose-dependent effect was not observed. Recently, Cervantes-Paz 259
et al. (2016b) also reported a similar effect using pepper pectins, which bound up 260
to 47% less BS when pectin concentration was increased from 0.14% to 1%. The 261
variability in the binding capacity of pectins can be attributed to differences in the 262
number of hydroxyl groups in the BS structure as well as to pectin structure; 263
although the effect of pectin structure on such interaction seem to be lower than 264
that of concentration (Fig. 3C and 3D) (Drzikova, Dongowski, Gebhardt, & Habel, 265
2005; Cervantes-Paz et al., 2016b). However, there is only one study where the 266
effect of the interaction between pectin and BS on carotenoid micellarization was 267
studied. Cervantes-Paz et al. (2016b) evaluated the effect of concentration and 268
some structural properties (MW and DM) of pectins on BS binding capacity and 269
carotenoid micellarization, finding that that the interaction between pectin and BS 270
is influenced by DM, with this interaction being stronger for medium and high DM 271
pectins. Pectins could bind BS by hydrophobic interactions, which increase with 272
the DM of pectin (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, Narváez-273
Cuenca & McClements, 2014b). Therefore, LMP and high MW pectins might favor 274
carotenoid micellarization and absorption. 275
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276
4.3 Effect of pectin-mediated modification of digestive medium viscosity 277
The viscosity of digestive medium can be altered by intermolecular interactions 278
between pectin chains due to their unordered shaped spiral structure 279
(Ngouémazong et al., 2015). This effect is highly influence by the changes of pH 280
that occur in the gastrointestinal track as well as by pectin source and proportion 281
of neutral and acid residues in pectin chains (Espinal-Ruíz et al., 2014b). Under 282
high viscosity conditions, the diffusion of lipase to the lipid droplet surface can be 283
reduced as well as the transference of digestion lipid products to the micelles (Xu 284
et al., 2014; Espinal-Ruiz et al., 2014a, 2016). The pectin-mediated modification of 285
viscosity in model oil-in-water emulsions has a significant influence on lipid 286
digestion, which is mainly dependent of the MW and DM of pectins. The viscosity 287
typically tends to decrease through gastrointestinal process (Espinal-Ruiz et al., 288
2016). Xu et al. (2014) observed that the addition of beet pectin to oil-in-water 289
emulsions retarded the release of FFA from the lipid phase and attributed this 290
effect to an impediment of pectin for the migration of lipase to the lipid droplets by 291
an alteration of media viscosity. Recently, Cervantes-Paz et al. (2016b) 292
demonstrated that the viscosity of gastrointestinal medium increased in presence 293
of pepper pectins, affecting lipolysis, but such increase depended on the DM and 294
MW of pectins. Undoubtedly, the gel formation caused by the interaction of pectin 295
and calcium ions affects the viscosity of gastrointestinal medium and, 296
subsequently, the lipolysis, diffusion of lipid digestion products and carotenoid 297
micellarization (Lofgren, Guillotin, & Hermansson, 2006; Cervantes-Paz et al., 298
2016b; Schweiggert, Mezger, Schimpf, Steingass, & Carle, 2012). These possible 299
14
mechanisms are supported by some studies in this regard. Yonekura and Nahao 300
(2009) demonstrated that the micellarization of E-carotene and lutein was inhibited 301
in presence of pectins and alginates. The high viscosity of the digestive medium 302
was proposed as a possible mechanism; however, their results did not explained 303
an incomplete hydrolysis of TG. On the other hand, Verrijssen et al. (2014) 304
observed that the pectin-mediated increase of viscosity of gastrointestinal medium 305
caused a reduction in the micellarization of β-carotene. They also observed that 306
the viscosity of gastric and intestinal media varied depending on DM of pectin. 307
Later, they demonstrated that emulsions containing different percentages of pectin 308
showed large pectin-clusters, indicating that high viscosity conditions probably 309
inhibited the lipase activity and micelle formation (Verrijssen et al., 2015). 310
Recently, Cervantes-Paz et al. (2016b) observed that pectins with high MW 311
increased the viscosity of gastrointestinal medium and micellarization of the less 312
polar carotenoids, as compared with pectins of medium and low MW. They 313
concluded that the viscosity of the gastrointestinal media is influenced by pectin 314
structure and amount but that the effect of the viscosity of the digestive medium on 315
carotenoid micellarization also depend on carotenoid speciation. Further studies 316
are need it to clarify the effect of viscosity itself on carotenoid bioavailability. 317
318
4.4 Effect of the modification of the properties of lipid droplet surface by 319
pectin 320
The composition and properties of any interfacial layer surrounding oil droplets in 321
the gastrointestinal media can strongly influence the digestion and absorption of 322
15
lipids by causing variations in the electric charge of molecules, surface tension, 323
rheology and density of the fluids (McClements, Decker, & Weiss, 2009; Bauer et 324
al., 2005; Capuano, 2016; Fave et al., 2004). These variations can alter the 325
lipolysis and modify the levels of lipid digestion products available for micelle 326
formation, and therefore, for carotenoid transport (McClements, 2013). Pectins can 327
be adsorbed around the oil droplets and form a layer that avoid the coalescence 328
and aggregation of oil droplets, preventing electrostatic and static attractions 329
between them (van der Waals and hydrophobic interactions), improving emulsion 330
stability (Fig. 4) (Dickinson, 2009; Iwanaga et al., 2008; Ngouémazong et al., 331
2015). The emulsifying activity of pectins is conferred by their structural properties, 332
including their protein and carbohydrate moieties (Leroux, Langendorff, Schick, 333
Vaishnav, & Mazoyer, 2003; Nakauma et al., 2008; Funami et al., 2011). The 334
steric stabilization is mainly promoted by the RGI while the electrostatic 335
stabilization is attributed to HG. Neutral sugar side chains of RGI regulate the 336
intermolecular interactions between adsorbed pectin chains, which form a thick 337
adsorbed hydrated layer, preventing coalescence (Funami et al., 2007, 2011). On 338
the other hand, the nonmethylated GalA residues of HG, which contain carboxylic 339
groups ionized at pH above 3.5, confers a charge distribution at pectin surface. 340
Thus, ionized pectin form an electrical layer around oil droplet, resulting in lipid 341
droplet repulsion due to mutual repulsion between functional groups (McClements, 342
2004). Zhao, Wei, Wei, Yuan and Gao (2015) observed a high stability of oil-in-343
water emulsions containing anionic polysaccharides like beet pectin. They 344
attributed such stability to the high charge density caused by polysaccharides that 345
favored electrostatic repulsion between oil droplets covered by them. Zhang, 346
16
Zhang, Zhang, Decker and McClements (2015) found that addition of pectin to oil-347
in-water emulsions improved emulsion stability; however, pectin compromised lipid 348
digestion. This might be consequence of the abundance of negative charges, 349
which caused an electrical repulsion between pectin molecules at lipid droplet 350
surface and favored a higher concentration of unabsorbed pectin that, in turn, 351
promoted lipid flocculation. In addition, they (Zhang, Zhang, Zhang, Decker, & 352
McClements, 2015) observed that oil-in-water emulsions containing HMP and LMP 353
did not show aggregation of oil droplets during gastric phase but aggregation was 354
observed during the intestinal phase. Contrarily, Espinal-Ruiz et al. (2014a) 355
observed sediments in emulsions containing pectin and since the sedimentation 356
increased with the pectin concentration, they inferred that the flocs formed by 357
these pectins were large and dense to rapidly sediment. The addition of pectin to 358
emulsions also increased the depletion between the oil droplets until the attraction 359
interactions exceeded repulsive interactions and oil droplets flocculated (Espinal-360
Ruiz et al., 2016). By the way, Qiu, Zhao, Decker and McClements, (2015) 361
observed that the addition of pectin improved stability of emulsions at low pH but 362
emulsions became unstable when the ionic strength was increased. These 363
findings suggest that pectin concentration can reverse the effect caused by pectin 364
at low concentration on the lipid droplet surface. Thus, the effect of pectin on lipid 365
aggregation and coalescence during the gastrointestinal digestion is closely 366
related to lipid digestion, and therefore, to carotenoid absorption (Verrijssen et al., 367
2014, 2015; Xia et al., 2015; Zhang et al., 2015, 2016); however, a direct 368
correlation of such event with carotenoid micellarization has not been well 369
stablished yet. Xu et al. (2014) observed that whey protein isolated-beet pectin 370
17
conjugate stabilized carotenoid-rich oil-in-water emulsions, preventing lipid droplet 371
flocculation and coalescence but generating disturbances in lipid digestion and �E-372
carotene solubilization during in vitro digestion. Verrijssen et al. (2015) suggested 373
that small oil droplets of digested oil-in-water emulsions in presence of some 374
pectins might explain the increase of lipid and�E-carotene micellarization; however, 375
other pectins might bind oil droplets, favoring the formation of larger droplet sizes 376
and delaying carotenoid micellarization. Zhang et al. (2016) demonstrated that 377
carotenoid micellarization increased as lipid droplet size decreased, presumably 378
favoring a quick micelle formation process and the subsequent carotenoid 379
micellarization. The increase of pectin concentration has also been related with the 380
formation of larger oil droplets and low carotenoid micellarization; however, a 381
contrary behavior was also observed for lipid digestion with these actions being 382
dependent of carotenoid type and structural properties of pectin (Cervantes-Paz et 383
al., 2016b). 384
385
4.5 Effect of pectin on lipase activity inhibition 386
Pectin can influence the activity of digestive enzymes, including those involved on 387
lipid digestion. Under digestive conditions, pectin can act as physical barrier 388
between substrates and digestive enzymes (McClements & Li, 2010; Mun, Decker, 389
Park, Weiss & McClements, 2006; Singh, Ye & Horne, 2009), protonate the 390
enzyme active site by the participation of the carboxylic acid residues of pectin 391
(Kumar and Chauhan, 2010), generate direct molecular interactions (electrostatic, 392
hydrogen bonding or hydrophobic) between pectin and enzymes, and compete 393
18
with substrate for the active site of the enzyme (Capuano, 2016). These pectin 394
actions can alter lipid digestion and carotenoid bioavailability and are strongly 395
dependent on pectin properties. The competence between pectin and substrates 396
for active site of enzymes depends on the methylester groups of pectin, with such 397
groups altering the polar nature of the polysacharyde and increasing pectin 398
hydrophobicity by neutralization of negative charges (Mohnen, 2008). This 399
structural characteristic of pectin seems to be highly related to the biological 400
activity of enzymes since favors the interaction between both biopolymers 401
(Benjamin, Lassé, Silcock, & Everett, 2012). Tsujita et al. (2003) observed that 402
pectin of low MW (90 KDa) strongly inhibited the lipase activity. They attributed 403
this effect to interaction of pectin with emulsified substrates, inhibiting the 404
adsorption of lipase at the emulsion interface. Contrarily, Edashige, Murakami and 405
Tsujita (2008) demonstrated that pectins with high MW (>300 KDa) caused a 406
strong inhibition of lipase activity, while low MW pectin (<300 KDa) showed a 407
lower inhibitory effect. Factors as solubility and viscosity of pectins could be 408
involved on the observed inhibition of lipases; however, the physical interference 409
of pectin between lipids and lipase was also possible (Tsujita et al., 2003). This 410
theory of the inhibition of lipase activity by pectin is reinforced by several studies, 411
which have demonstrated that the activity of other enzymes like trypsin, α-412
chymotrypsin, α-amylase, and pepsin were also inhibited by pectin (Ikeda & 413
Kusano, 1983). Several studies have shown a significant decrease in lipid 414
digestion caused by the interaction between the lipase and pectin (Cervantes-Paz 415
et al., 2016b; Espinal-Ruiz et al., 2016; Mun et al., 2006; Zhang et al., 2016, 416
2015). However, few studies have related this effect to carotenoid micellarization 417
19
or bioavailability. Verrijssen et al. (2014) found a significant decrease on the E–418
carotene micellarization when DM of pectins was reduced from 99% and 66% to 419
14%. They attributed this behavior to the embedding of oil droplets in pectin 420
clusters during digestion, inhibiting the lipase activity. Similar results were 421
presented in a further study, where they found a low E–carotene micellarization in 422
presence of pectin and phosphatidylcholine, with the low carotenoid micellarization 423
being attributed to the binding of pectin with lipid droplets, reducing lipolysis, 424
micelle formation, and carotenoid micellarization (Verrijssen et al., 2015). Yi, Lia, 425
Zhonga, and Yokoyama (2014) related the lipolysis level with a decrease of oil 426
droplet size and a high E–carotene micellarization (up to 75%). Recently, a more 427
detailed study demonstrated that high pectin concentration increased the lipolysis, 428
viscosity and particle size but decreased carotenoid micellarization. In addition, it 429
was observed that pectins with high and medium DM promoted the lipolysis and 430
slightly favored the micellarization of the more polar carotenoids (Cervantes-Paz et 431
al., 2016b). Although a great number of studies have evaluated the effect of pectin 432
on lipase activity inhibition and lipolysis, the impact of these effects on carotenoid 433
micellarization has not been clearly established. Existing evidence allow to infer 434
that although pectin can inhibit the active site of lipase, the most probably effect of 435
pectin is indirect, acting as physical barrier between lipid surface and lipase. 436
437
Conclusion 438
Many effects of pectin amount and properties on carotenoid absorption have been 439
demonstrated or inferred; however, the last consequence of all of them seems to 440
20
be lipolysis inhibition. This is particularly true if the strict and well know 441
requirement of dietary fat for carotenoid absorption is required. Most of such 442
effects probably occur at the same time although their intensity might vary 443
depending on pectin properties and concentration. Further studies are required in 444
this regard to design strategies for improving carotenoid absorption. 445
446
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Figure captions 857
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apolipoprotein B (chylomicron secretion to lymph); RE: retinyl esters. 863
864
865
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868
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873
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Fig. 4. Pectin coatings on lipid droplet surface interfering on the aggregation state, 875
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Fig. 1
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132
CAPÍTULO III
EFFECT OF RIPENING AND HEAT PROCESSING ON THE
PHYSICOCHEMICAL AND RHEOLOGICAL PROPERTIES OF PEPPER
PECTINS
Carbohydrate Polymers 115 (2015) 112–121
Contents lists available at ScienceDirect
Carbohydrate Polymers
j ourna l ho me pa g e: www.elsev ier .com/ locate /carbpol
Effect of ripening and heat processing on the physicochemical andrheological properties of pepper pectins
Olivia P. Ramos-Aguilara,b, José de Jesús Ornelas-Paza,∗, Saul Ruiz-Cruzb,Paul B. Zamudio-Floresa, Braulio Cervantes-Paza, Alfonso A. Gardea-Béjarc,Jaime D. Pérez-Martínezd, Vrani Ibarra-Junquerae, Jaime Reyes-Hernándezf
a Centro de Investigación en Alimentación y Desarrollo A.C.-Unidad Cuauhtémoc, Av. Río Conchos s/n, Parque Industrial, CP 31570 Cd. Cuauhtémoc,Chihuahua, Mexicob Instituto Tecnológico de Sonora, Departamento de Biotecnología y Ciencias Alimentarias, 5 de Febrero No. 818 Sur, CP 85000 Cd. Obregón, Sonora, Mexicoc Centro de Investigación en Alimentación y Desarrollo A.C.-Unidad Guaymas, Carretera al Varadero Nacional Km. 6.6, Col. Las Playitas, CP 85480 Guaymas,Sonora, Mexicod Universidad Autónoma de San Luis Potosí, Facultad de Ciencias Químicas, Manuel Nava No. 6, Zona Universitaria, CP 78210 San Luis Potosí, Mexicoe Universidad de Colima, Bioengineering Laboratory, Km. 9 carretera Coquimatlán-Colima, CP 28400 Coquimatlán, Colima, Mexicof Universidad Autónoma de San Luis Potosí, Facultad de Enfermería, Av. Nino Artillero No. 130, Zona Universitaria, CP 78210 San Luis Potosí, Mexico
a r t i c l e i n f o
Article history:Received 15 February 2014Received in revised form 7 August 2014Accepted 10 August 2014Available online 2 September 2014
Keywords:PolysaccharidesCell-wall materialsFunctional propertiesPhysicochemical characteristicsNew pectin sources
a b s t r a c t
Water-, chelator-, and alkali-soluble pectins were isolated from raw and heat-processed Jalapeno pep-pers (green and red) and their physiochemical and rheological properties were determined. The yield,tristimulus color, degree of methyl esterification, monosaccharide composition, molecular weights dis-tribution, and protein content depended on ripening and heat processing. The viscosity properties ofpectins were independent of ripening. The water-soluble pectin was the most abundant pectin. Pectinsfrom grilled peppers showed the lowest L* values. The alkali-soluble pectin showed the highest proteincontent. The content of xylose, rhamnose, and mannose in pectins was highly altered by tested factors.The degree of methyl esterification of pectins ranged from 26.8 to 91.6%. The peak Mw of the main fractionof tested pectins was sequentially reduced by ripening and heat processing. Pectins from raw peppersshowed the best viscosity properties.
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
The world demand for pectin is annually growing by 4–5%.Pectin is used as thickening, gelling, emulsifying, and texturizingagent in food, pharmaceutical, and cosmetic products (Mesbahi,Jamalian, & Farahnaky, 2005; Chan & Choo, 2013). Citrus peels andapple pomace remain as the most important sources of commercialpectin. New pectin sources will be necessary in the future to sat-isfy the pectin demand. Jalapeno peppers might be an alternativesource of these polysaccharides since they are highly cultivated,have a very low commercial value, unmarketable Jalapeno pep-pers are highly available due to their short shelf life, pepper wastesfrom seed-production industry are highly available, and the grow-ers are demanding new uses and adding-value strategies for thiscrop (Ornelas-Paz et al., 2012). However, little is known about
∗ Corresponding author. Tel.: +52 625 5812920x110; fax: +52 625 5812920.E-mail address: [email protected] (J.J. Ornelas-Paz).
the pectin content in peppers and its characteristics. The pectincontent in peppers depends, in a first instance, on genotype, ran-ging from 1.7 to 10.4% (DWB) (Arancibia & Motsenbocker, 2004;Bernardo, Martínez, Álvarez, Fernández, & López, 2008). However,the yield of pectin from a specific pepper genotype can be altered byripening and heat processing. Bernardo et al. (2008) demonstratedthat the pectin content of Fresno de la Vega and Benavente-LosValles peppers decreased (7–23%) during ripening. Priya, Prabhaand Tharanathan (1996) reported a considerable reduction (55%)of pectin content in Bell peppers during ripening. The ripeningalso modifies the chemical properties of pepper pectins, but thishas been scarcely studied. Arancibia and Motsenbocker (2006)demonstrated that the degree of esterification of pectin fromTabasco peppers decreased from 70 to 40% during ripening andinferred a high poligalacturonase-mediated pectin depolymeriza-tion associated with pectin methylesterase activity. The neutralmonosaccharide content in pectins from Bell pepper decreasedby 47% during ripening, with the losses of galactose (Gal) (68%)and arabinose (Ara) (43%) being the most distinctive (Priya et al.,
http://dx.doi.org/10.1016/j.carbpol.2014.08.0620144-8617/© 2014 Elsevier Ltd. All rights reserved.
O.P. Ramos-Aguilar et al. / Carbohydrate Polymers 115 (2015) 112–121 113
1996). Similar losses (42–56%) of neutral monosaccharides werereported for pectin from other genotypes of sweet and pungentpeppers during ripening (Gross & Sams, 1984). Other changes inthe physicochemical properties of pepper pectin as a function ofripening remain unknown.
The study of the effect of heat processing of peppers on yieldand physicochemical properties of their pectins is scarce. Gu,Howard, and Wagner (1999) demonstrated that the total pectincontent in several genotypes of Jalapeno peppers was reduced(11–19%) after heat processing and storage for 10 days. Similarly,Gallardo-Guerrero, Pérez-Gálvez, Aranda, Mínguez-Mosquera, andHornero-Méndez (2010) found that hot-forced air drying decreased(30–45%, approximately) the pectin content in red peppers.Howard, Burma and Wagner (1997) demonstrated that blanching(50 ◦C, 15–60 min) and subsequent pasteurization (75 ◦C, 5 min) didnot alter the esterification degree of pectin from Jalapeno peppers;however, these treatments decreased the content of several pectinfractions by 30–49%. Gallardo-Guerrero et al. (2010) found thatthe protopectin and soluble pectin are altered during hot forced-air drying of peppers. The increase of pectin solubility by someheat processing styles (pasteurization and blanching) has beenhypothesized in peppers (Howard et al., 1997). Other heat-inducedphysicochemical changes in pepper pectin remain unknown.
The modification of the chemical characteristics of pectin byripening or heat processing induces alterations in its functionalproperties, such as the rheological behavior, gelling ability, andcapacity to bind compounds (Sila, Smout, Elliot, Van Loey, &Hendrickx, 2006b). The viscosity of their solutions and the strengthof their gels increase as their molecular weight increases (Leroux,Langendorff, Schick, Vaishnav, & Mazoyer, 2003; Yapo, 2009).Pectins with high degree of methyl esterification form gels whenin acidified solutions containing a solute such as sucrose, whilepectins with low degree of methyl esterification can producegels with divalent cations (Yapo, 2009; Chan & Choo, 2013). Thedegree of methyl esterification of pectins also modulates theirbinding of cations (Khotimchenko, Kolenchenko, Khotimchenko,Khozhaenko, Kovalev, 2010). De-esterified pectins are less suscep-tible to heat-induced degradation and less soluble. The charge of thecarboxyl groups of galacturonic acid (GalA) can be influenced by pHand ionic strength of the medium, altering the three-dimensionalconformation of the polysaccharide and its ability to modify the vis-cosity (Lara, García, & Vendrell, 2006; Lamikanra & Watson, 2007).Leroux et al. (2003) found that some pectin fractions are able toimprove the emulsification of oil in water, presumably as a conse-quence of their protein content, low degree of acetyl esterification,and low molecular weight. The aim of this work was to determinethe physicochemical characteristics and viscosity of several pectintypes isolated from green and red Jalapeno peppers before and aftertwo common heat-processing styles.
2. Material and methods
2.1. Plant material and heat processing
Green and red Jalapeno peppers (Capsicum annuum L. var.Marajá) were harvested from a commercial orchard in Chihuahua,México. Only fruits free of blemishes and defects were includedin the experiment. The fruits were distributed in nine samplesfor each ripening stage. Three samples were boiled at 96 ◦C for12.3 min. Another three samples were grilled on a hot plate at210 ◦C for 13.2 min while the remaining three samples were usedas untreated controls. Each sample was individually subjected topectin extraction process. Individual fruits (raw and processed)were characterized for tristimulus color, dry matter content, firm-ness, and biometrical characteristics.
2.2. Preparation of the alcohol-insoluble residue (AIR)
The peduncle of peppers was removed. The raw peppers wereblanched at 96 ◦C for 2.5 min before pectin extraction. Triplicatepepper samples (500.0 g) from each treatment were individuallyhomogenized to puree in a kitchen blender (Taurus, Model Robot180) for 5 min and suspended in ethanol (96%, v/v) in a ratio of pep-per puree to ethanol of 1:4 (w/v). The suspension was subjectedto a more intense homogenization until total disruption of peppertissue (Homogenizer Ika T18 Basics; IKa Works Inc.). The mixturewas stirred for 2 h at 25 ◦C and then maintained overnight at 4 ◦C.The mixture was filtered using a Whatman paper (No. 3) and theretained solids were recovered. The solids were suspended in ace-tone (ratio of 1:3, w/v), stirred for 2 h at 25 ◦C, and filtered again.This step was repeated six times until complete discoloration ofsolids. The residue was lyophilized, weighed for yield calculation,and pulverized using a mortar and pestle. The dried AIR was storedin N2 atmosphere until pectin extraction.
2.3. Sequential extraction of pectic polysaccharides
Pectic polysaccharides were extracted according to Roeck,Sila, Duvetter, Van Loey, and Hendrickx (2008), with slightmodifications. The AIR was subjected to sequential extractionof pectin with distilled water (96 ◦C for 5 min), 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid monohydrate(CDTA) in 0.1 M potassium acetate (pH 6.5, 6 h at 28 ◦C), and 0.05 MNa2CO3 containing 0.02 M NaBH4 (16 h at 4 ◦C and then 6 h at 28 ◦C).The ratio of AIR to extracting solution was always 1:100 (w/v).The pH of all filtrates and retained solids was neutralized (pH 7.0)with NaOH or acetic acid. Pectic polysaccharides were precipitatedfrom each filtrate by adding 3 volumes of ethanol, and the suspen-sions were maintained overnight at room temperature. The pectinwas recovered by centrifugation (12,000 × g, 5 min at 4 ◦C) and fil-tration throughout a Whatman paper No. 541. The final residuesobtained after pectin extraction process were washed with ethanol(3 L) and then recovered by filtration. Pectins and final residueswere lyophilized, weighed, pulverized, and stored under N2 atmo-sphere. Pectins and final residues were named according to theirsolubility as water-, chelator-, and alkali-soluble pectin (WSP, CSP,and NSP) and insoluble fibers (IF), respectively. These materialswere not subjected to further purification processes and thereforethey might contain other polysaccharides. Pectins were charac-terized for their physicochemical (yield, tristimulus color, degreeof methyl esterification, monosaccharide composition, molecularweights distribution, and protein content), and rheological (viscos-ity of their solutions) properties.
2.4. GalA content and degree of methyl esterification
The GalA was liberated from pectin and quantified accordingto Ahmed and Labavitch (1978) and Filisetti-Cozzi and Carpita(1991). Pectin samples (5 mg) were subjected to acid hydrolysisfor 40 min, using concentrated H2SO4 (2 mL) and water (1 mL). Thevolume of the reaction was adjusted to 10 mL using water, andthen aliquots of each hydrolyzate (400 !L) were mixed with 4 Mpotassium sulfamate (40 !L, pH 1.6) and 75 mM sodium tetrabo-rate in concentrated H2SO4 (2.4 mL), and maintained at 96 ◦C for20 min. After cooling, 80 !L of m-hydroxydiphenyl solution (0.15%3-phenylphenol in 0.5% NaOH) or 0.5% NaOH (control reaction)were added and the GalA content was colorimetrically determinedat ! = 525 nm using a 6405 Jenway UV/Vis spectrophotometer (Jen-way Ltd., Essex, UK). The GalA quantification was performed usinga calibration curve constructed with three independent sets of dilu-tions of pure d-galacturonic acid.
114 O.P. Ramos-Aguilar et al. / Carbohydrate Polymers 115 (2015) 112–121
The methanol content in pectin was determined accordingto Voragen, Schols, and Pilnik (1986), with slight modifications.Pectins were dissolved in water at a final concentration of 1.67%(w/v). Aliquots of these solutions (600 !L) were subjected to alka-line hydrolysis by adding 7 M NaOH (100 !L) and isopropanol(700 !L) to the sample and holding the mixture for 2 h at 25 ◦C.The sample was centrifuged (2000 × g at 25 ◦C for 10 min). Aliquotsof each hydrolyzate (800 !L) were neutralized with concentratedH2SO4 (10 !L), filtered through a polyethylene membrane of0.2 !m of pore size (Millipore Corp., Bedford, MA, USA), and man-ually injected (20 !L) into a ProStar HPLC system (Varian Inc.;Walnut Creek, CA, USA), which was composed of a ternary pump(Solvent Delivery Model 9012) and a refractive index detector(Star Model 9040). The chromatographic system included a TSKgelSCX H+ (7.8 × 300 mm, 5 !m) cation-exchange column (Tosoh Bio-science LLC; Tokyo, Japan), which was kept at 40 ◦C. The mobilephase (isocratic system) was water (18.2 M!) with a flow rateof 1 mL/min. Each extract was analyzed three times. Methanolwas quantified in the samples using a calibration curve con-structed with three independent sets of dilutions of absolutemethanol. The degree of methyl esterification (DM) was calculatedas DM = [(millimoles methanol/millimoles GalA) × 100].
2.5. The monosaccharide composition of pectins
The monosaccharide composition of pectin was determinedaccording to Garna, Mabon, Wathelet, and Paquot (2004), withslight modifications. Monosaccharides were liberated by sequen-tial acid and enzymatic hydrolysis. Pectin samples (100 mg) weremixed with 0.2 M trifluoroacetic acid (5 mL) and heated then at80 ◦C for 72 h. The pH of the reaction was adjusted to 5.0 with14 M NH4OH and then 100 !L of an enzyme complex (MacerexPM complex, Enmex S.A. de C.V; México) were added. The enzy-matic hydrolysis of pectin was performed at 50 ◦C for 24 h. Theenzymatic activity was stopped by heating the sample at 96 ◦C for3 min. The mixture was cooled, filtered, and injected (20 !L) intothe HPLC system described above. Rhamnose (Rha), Ara, and fucose(Fuc) were separated using a MetaCarb H+ Plus (7.8 × 300 mm,5 !m) (Varian Inc.; Walnut Creek, CA, USA) ion-exchange column,which was kept at 58 ◦C. The mobile phase (isocratic system) was0.0085 N H2SO4 with a flow rate of 0.4 mL/min. Gal, mannose(Man), and xylose (Xyl) were separated on a Supelcogel Pb ion-exchange column (7.8 × 300 mm, 5 !m) (Sigma-Aldrich Corp.; St.Louis, MO, USA) at 70 ◦C. The mobile phase (isocratic system) waswater (18.2 M!) with a flow rate of 0.5 mL/min. Each extract wasanalyzed three times. Each monosaccharide was quantified by aconcentration–response curve constructed with three independentsets of dilutions of the reference compound.
2.6. Molecular weight of pectin
The molecular weights distribution of pectin samples wasdetermined by high performance size-exclusion chromatogra-phy (HP-SEC), according to Pérez-Martínez et al. (2013). Pectinswere dissolved in water at a final concentration of 0.5% (w/v).The solution was filtered (0.2 !m pore size; Millipore Corp.,MA, USA) and injected (20 !L) into the HPLC system describedabove. The molecular weights distribution was determined usingin series the following TSKgel columns: GMPWXL (7.8 × 300 mm,13 !m), G5000PWXL (7.8 × 300 mm, 10 !m), and G4000PWXL(7.8 × 300 mm, 10 !m) (TOSOH Bioscience; Minato-ku, Tokyo,Japan). A TSKgel PWXL guard column (6.0 × 40 mm, 12 !m) pre-ceded this column system. The columns were operated at 40 ◦C.Phosphate buffer (0.2 M, pH 6.9) was used as mobile phase at aflow rate of 0.4 mL/min. Each pectin sample was analyzed 4 timesand only the peak molecular weights of the main fractions were
considered. The molecular weights were determined relative todextrans after column calibration with seven dextran standardsfrom 25 to 670 kDa (Sigma-Aldrich; St. Louis, MO, USA).
2.7. Viscosity
Pectin samples were dissolved in water at a final concentra-tion of 2% (w/v). The viscosity of these solutions was determinedat 25 ◦C, using an AR 1500ex rheometer (TA Instruments; NewCastle, DE, USA) equipped with stainless steel parallel plate geom-etry (60 mm diameter). Shear rate ranged from 0.01 to 500 s−1 (upcurve), and from 500 to 0.01 s−1 (down curve). A gap size of 500 !mwas set. Shear rate against shear stress data were fit using thepower law model (" = k#n), and analyzed for flow behavior, n, andconsistency index, k.
2.8. Miscellaneous assays
The biometrical characteristics (weight, length, and major diam-eter), firmness, dry matter content, and tristimulus color wereindividually determined on 20–25 fruits according to Cervantes-Paz et al. (2012), in order to obtain an objective characterization ofthe pectin sources. The tristimulus color was directly determinedon triplicate samples of pectin and IF using a Minolta colorimeter(CR-300 model, Minolta Co. Ltd.; Osaka, Japan) on the basis of theCIELAB color system (L*, a*, and b*). The protein content in pectinsolutions (concentration of 0.5%, w/v) was determined using thecolorimetric (" = 595 nm) assay of Bradford (1976).
2.9. Statistical analysis
The statistical significance of differences between treatmentswas determined using an ANOVA followed by the Tukey–Kramerpost hoc test; 0.05 was the significance limit. Data analysis wasperformed using JMP statistical software (SAS Institute Inc.).
3. Results and discussion
3.1. Pectin yield
The physicochemical characteristics of tested peppers areshown in Table 1. Similar results were previously reported forJalapeno peppers as a function of ripening and heat processing(Cervantes-Paz et al., 2012). The AIR yield was statistically higher(17–18%) in green peppers than in the red counterpart (Table 2).Similarly, Inari, Yamauchi, Kato, and Takeuchi (2002) observed thatripening reduced the AIR content in Cherry tomato fruits. Boil-ing and grilling improved AIR extractability in peppers at the tworipening stages, although the impact of both processing styles wassimilar. The total pectin content, excluding non-extractable pectinof IF, represented 24–31% of AIR. The total pectin content was alsohigher in the AIR of green peppers than that of red fruits. Reductionof pectin content as a consequence of ripening has been previ-ously reported for Fresno de la Vega and Benavente-Los Vallespeppers (Bernardo et al., 2008). Ali, Chin, and Lazan (2004) alsodemonstrated that ripening reduced the total polyuronide contentin several fruits. The total pectin yield tended to be decreased byboiling (6–9%) and grilling (13–14%), but the effect of both heatprocessing styles was statistically similar in each ripening stage.Femenia et al. (2009) observed that heat processing decreased thecontent of cell-wall polysaccharides in kiwi fruit. In general, thecontent of total pectin in the AIR of tested peppers was higher thanthat reported for the AIR from several genotypes of Jalapeno, Bell,and Kulai peppers (Priya et al., 1996; Gu et al., 1999).
WSP was the most abundant pectin in tested peppers (Table 2).Pharmacological and low-viscosity properties have been regarded
O.P. Ramos-Aguilar et al. / Carbohydrate Polymers 115 (2015) 112–121 115
Table 1Physicochemical characteristics of raw and heat-treated Jalapeno peppers at two ripening stages.
Physicochemicalattribute
Green peppers Red peppers
Raw Boiled Grilled Raw Boiled Grilled
Weight (g) 25.4 ± 0.8a 24.2 ± 0.8a 18.0 ± 0.8b 27.4 ± 2.1a 24.9 ± 1.6a 19.2 ± 1.1b
Length (cm) 7.3 ± 0.1a 6.9 ± 0.2b 6.6 ± 0.1b 7.5 ± 0.2a 6.8 ± 0.2b 6.9 ± 0.2ab
Major diameter (cm) 2.9 ± 0.04a 2.6 ± 0.03b 2.6 ± 0.05b 2.9 ± 0.08a 2.7 ± 0.06ab 2.6 ± 0.06b
Dry matter (%) 9.7 ± 0.2b 9.5 ± 0.1b 12.0 ± 0.1a 12.9 ± 0.1b 13.1 ± 0.8b 16.4 ± 0.1a
Firmness (N) 37.9 ± 0.9a 9.6 ± 0.4b 6.3 ± 0.6c 32.8 ± 1.0a 9.0 ± 0.3b 4.9 ± 0.3c
Tristimulus colorL* 46.2 ± 0.8a 45.8 ± 0.7a 41.4 ± 0.4b 35.9 ± 0.5b 37.7 ± 0.4a 33.2 ± 0.5c
a* −15.8 ± 0.3c −6.9 ± 0.2b −4.2 ± 0.1a 33.4 ± 0.5b 37.1 ± 0.4a 27.3 ± 0.4c
b* 30.5 ± 0.4a 31.8 ± 0.5a 26.6 ± 0.4b 29.0 ± 0.6b 33.7 ± 0.5a 26.1 ± 0.6c
Values represent the mean of several individual measurements (n = 6–90) ± the standard error. Values in the same row for each ripening stage with different letters aresignificantly different (p < 0.05).
to this pectin type (Hamauzu & Tsujitani, 2013). The content ofWSP and NSP in the AIR of raw peppers was higher and lower,respectively, than that previously reported for the AIR of severalgenotypes (Veracruz, Mitla, Delicias, and V-H78) of Jalapeno pep-pers (Gu et al., 1999). The CSP content in AIR of raw peppers wasin the range reported for other genotypes of Jalapeno peppers (Guet al., 1999).
The distribution of percentages of pectin types was similar inthe AIR from green and red peppers (raw and processed), exceptthe % of NSP, which decreased from the green to the red stage(Table 2). The amount of WSP and CSP was also similar in the AIRfrom jujube, banana, and plantain at some ripening stages (Happi,Robert, Ronkart, Wathelet, & Paquot, 2008). The decrease of NSPmight be attributed to enzymatic degradation of pectin duringripening. Heat processing differently altered the content of eachpectin type. The WSP content was 23–40% higher in processed pep-pers that in raw fruits. In contrast, the NSP content was decreased(23–49%) by boiling and grilling. These findings suggest the heat-induced conversion of NSP into WSP. Howard et al. (1997) alsofound increases in WSP and decreases in NSP in Jalapeno peppersafter heat processing. Heat processing (100 ◦C, 30–120 min) alsocaused the conversion of NSP into WSP in carrots (Sila, Doungla,Smout, Van Loey, & Hendrickx, 2006a). This phenomenon was also
observed in broccoli and was attributed to the conversion of pro-topectin into soluble pectin by the thermo-solubilization and/or!-eliminative depolymerization of this material at high tempera-tures (Christiaens et al., 2012). In our study, the CSP from greenpeppers was not altered by heat processing while the CSP contentof red peppers was reduced (22–32%) by heat processing. Similardecreases in the content of CSP have been reported for Jalapenopeppers as a consequence of heat processing (Gu et al., 1999). Theripening-related differences in the binding force of CSP to cell wallmight explain this differential effect, with CSP from green pep-pers being more strongly bound than that from red fruits. Themonosaccharide proportion and nanostructure of CSP as a functionof ripening and heat processing could be involved in this differentialeffect (Xin et al., 2010). The IF was not altered by ripening, but itscontent was sequentially reduced by boiling and grilling, probablyas a consequence of the increase of WSP in heated fruits.
3.2. Color of pectins and IF
The tristimulus color of pectins and IF from tested peppers isshown in Table 2. The L* values of pectins were higher (5–14%) withraw red peppers than with raw green fruits, suggesting an effectof fruit ripening on pectin color. The ability of pectins to interact
Table 2The yield and tristimulus color of alcohol-insoluble residues (AIR), water soluble pectin, chelator-soluble pectin, and alkali-soluble pectin (WSP, CSP, and NSP), and insolublefibers (IF) from raw and heat-treated Jalapeno peppers at two ripening stages.
Green peppers Red peppers
Raw Boiled Grilled Raw Boiled Grilled
AIR (g/kg of peppers) 464 ± 2b 540 ± 7a 546 ± 11a 395 ± 7b 462 ± 6a 461 ± 7a
AIR composition (%)WSP 9.3 ± 1.0b 13.0 ± 0.2a 12.3 ± 0.3a 10.0 ± 0.9b 12.3 ± 0.5ab 13.1 ± 0.5a
CSP 8.8 ± 0.2a 8.0 ± 0.3a 7.8 ± 0.7a 9.6 ± 0.8a 7.5 ± 0.5ab 6.5 ± 0.1b
NSP 13.0 ± 1.1a 7.4 ± 0.3b 6.6 ± 0.3b 8.3 ± 0.7a 6.4 ± 0.2b 4.6 ± 0.1b
IF 64.8 ± 1.4a 60.9 ± 1.6a 56.8 ± 2.6a 62.4 ± 0.3a 57.5 ± 0.7a 60.7 ± 2.8a
Tristimulus color
WSP L* 75.5 ± 1.0a 77.9 ± 1.3a 63.2 ± 1.8b 86.1 ± 2.6a 75.7 ± 1.1b 55.8 ± 0.9c
a* −0.5 ± 0.2b −0.7 ± 0.1b 4.0 ± 0.2a 0.8 ± 0.4b 0.1 ± 0.2b 5.8 ± 0.1a
b* 14.8 ± 0.2a 14.7 ± 0.7a 15.0 ± 0.3a 20.2 ± 1.3a 20.5 ± 0.6a 13.7 ± 0.4b
CSP L* 82.6 ± 0.8b 87.5 ± 0.7a 73.2 ± 1.1c 92.8 ± 3.1a 85.1 ± 0.8b 71.4 ± 0.6c
a* −0.2 ± 0.1b −0.8 ± 0.04c 2.9 ± 0.1a 0.5 ± 0.3b −0.4 ± 0.1c 3.6 ± 0.1a
b* 13.2 ± 0.3b 10.4 ± 0.2c 14.6 ± 0.5a 16.1 ± 1.1a 13.0 ± 0.4b 14.6 ± 0.3ab
NSP L* 83.0 ± 1.1a 82.4 ± 0.5a 69.0 ± 1.1b 87.1 ± 2.5a 81.2 ± 1.1a 61.6 ± 2.2b
a* 0.02 ± 0.2b −1.7 ± 0.9b 3.5 ± 0.2a 0.9 ± 0.6b −0.8 ± 0.2c 5.2 ± 0.3a
b* 12.0 ± 0.3c 14.4 ± 0.2b 15.7 ± 0.2a 16.8 ± 1.0a 15.0 ± 0.5a 14.9 ± 0.5a
IF L* 86.7 ± 0.8b 89.5 ± 1.0a 78.9 ± 0.9c 88.7 ± 3.4a 86.3 ± 0.2a 65.6 ± 0.8b
a* −0.4 ± 0.1b −1.5 ± 0.0c 0.3 ± 0.1a −0.6 ± 0.1b −0.8 ± 0.1b 1.5 ± 0.1a
b* 10.8 ± 0.3b 12.7 ± 0.2a 9.9 ± 0.2c 17.9 ± 0.6a 17.4 ± 0.4a 11.5 ± 0.2b
Values represent the mean of several individual measurements (n = 3–12) ± the standard error. Values in the same row for each ripening stage with different letters aresignificantly different (p < 0.05).
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Table 3Protein content (%) in water-, chelator-, and alkali-soluble pectin (WSP, CSP, and NSP) from raw and heat-processed Jalapeno peppers at two ripening stages.
Green peppers Red peppers
Raw Boiled Grilled Raw Boiled Grilled
Pectin typeWSP 1.8 ± 0.06a 1.3 ± 0.03c 1.6 ± 0.03b 1.7 ± 0.04a 1.8 ± 0.05a 2.1 ± 0.2a
CSP 1.9 ± 0.05a 1.8 ± 0.02a 1.8 ± 0.02a 1.5 ± 0.05b 1.8 ± 0.02a 1.8 ± 0.02a
NSP 1.9 ± 0.07c 2.6 ± 0.07a 2.2 ± 0.05b 2.0 ± 0.1b 2.6 ± 0.2a 2.2 ± 0.08ab
Values represent the mean of several individual measurements (n = 9) ± the standard error. Values in the same row for each ripening stage with different letters are significantlydifferent (p < 0.05).
with pigments as a function of their esterification degree and ionscontent, which are ripening-dependent, has been previously doc-umented (Holzwarth, Korhummel, Siekmann, Carle, & Kammerer,2013). The L* values found in pectins from raw fruits were simi-lar to those reported for commercial citrus and sugar beet pectins(Mesbahi et al., 2005; Einhorn-Stoll, Kastner, & Drusch, 2014). Withgreen peppers, boiling only altered the L* value of CSP while grillingconsistently reduced the L* values (11–17%) of all pectin types.The L* values of pectins from red fruits were sequentially reducedby boiling (7–12%) and grilling (23–35%). This reduction could beattributed to heat-induced pectin demethylation and monosaccha-ride degradation, which favor the formation of unsaturated uronicacids (double bonds between C4 and C5) of brown color (Einhorn-Stoll et al., 2014). On the other hand, grilling drastically increasedthe a* values in all pectin types while boiling decreased 3-fold thea* values of CSP from green peppers and almost 2-fold those of CSPand NSP from red fruits. The a* values of tested pectins were similarto those reported for citrus and sugar beet pectins (Mesbahi et al.,2005; Einhorn-Stoll et al., 2014). Boiling decreased (19–21%) the b*values of CSP from green and red peppers, but increased (20%) thisvariable in NSP from green peppers. Grilling increased (11–31%) theb* values of CSP and NSP from green peppers, but this variable wasdecreased (32%) in WSP from red peppers. Increases in b* valueshave been attributed to the formation and degradation of unsatu-rated GalA residues (Einhorn-Stoll et al., 2014). Our b* values weresimilar to those previously reported for high/low degree of methylesterification citrus and sugar beet pectins (Mesbahi et al., 2005;Einhorn-Stoll et al., 2014). The tristimulus color of IF showed simi-lar changes to those of some pectin types as a function of ripeningand heat processing of the fruits.
The color of pectin might influence its technological applica-tions. This quality attribute depends on many factors, includingsource, extracting conditions, and methods of precipitation anddrying. Oxidized polyphenol content and demethylation of GalAhave been suggested as causes of pectin browning, becominga limiting factor for their use in light-colored foods products(Holzwarth et al., 2013; Einhorn-Stoll et al., 2014). Sudhakar andMaini (2000) reported that long-time pectin extraction processesinduce the browning of mango pectin. Lv, Wang, Wang, Li, andAdhikari (2013) obtained reddish pectin from sugar beet pulp.Sudhakar and Maini (2000) demonstrated that pectin precipitationwith aluminum chloride conferred a dull gray color to pectin whileprecipitation with alcohol led to pectin with an attractive whitecolor.
3.3. Protein content in pectin
The protein content in tested pectins is given in Table 3. TheNSP contained more protein than the other pectin types. Interest-ingly, this pectin type showed high Gal and Ara contents (Table 4).The protein-pectin complex has been proposed as a distinctivecharacteristic of pectins with a high content of Gal and arabinanchains (Nunez, Fishman, Fortis, Cooke, & Hotchkiss, 2009). Heatprocessing tended to increase the protein content in all pectins
from red peppers, although statistical differences were not foundin some cases, while the opposite was observed for pectins fromgreen peppers, except for NSP. This difference could be explainedby ripening-related alterations in the force of binding of proteinswith pectins and other cell-wall components. The protein contentslightly varied between pectins and it was into the range reportedfor pectin from peppers and other sources (Leroux et al., 2003; Yapo,2009; Popov et al., 2011). The emulsifying action of some pectinshas been attributed, in part, to their protein content (Leroux et al.,2003).
3.4. Monosaccharide composition of pectins
The monosaccharide composition of pectins from raw and heat-treated peppers is shown in Table 4. The GalA content was higherin pectin from raw red Jalapeno peppers than in that of raw greenfruits, which has been attributed to the ripening-related solubi-lization of pectin (Priya et al., 1996). WSP showed the highest GalAcontent, after pooling of all data. Similarly, Koubala et al. (2008)demonstrated that WSP from ambarella was richer in GalA thanpectins extracted with hydrochloric acid and oxalic acid. In greenpeppers, boiling increased (25%) the GalA content in WSP, but itdecreased in CSP (52%) and NSP (63%). With red peppers, bothheat-processing styles significantly increased the GalA content inCSP and NSP. The increases of GalA could be explained by theheat-induced solubilization of pectins while the GalA decreases inpectins from boiled peppers could be attributed to the leaching ofpectin or GalA into the heating solution. The GalA content in WSPfrom red fruits was not altered by heat processing.
In general, the main neutral monosaccharides in pectins wereXyl, Gal, Man, and Ara while Rha and Fuc were present at lowconcentrations, as reported for pectin from other pepper geno-types (Popov et al., 2011). The total neutral monosaccharide contentwas reduced from the green (461–553 g/kg of pectin) to the red(248–312 g/kg of pectin) stage of ripening. Priya et al., 1996 founda reduction of neutral monosaccharide content in pectin from Bellpeppers during ripening. This reduction has been attributed topectin degradation during ripening (Gross & Sams, 1984). In ourstudy, the grilling differentially altered the total neutral monosac-charide content in pectins from peppers at the two ripening stages.In pectins from raw and boiled green peppers, this content wasWSP > NSP > CSP while in pectins from grilled green peppers suchcontent was higher in NSP than in WSP and CSP. The total neutralmonosaccharide content in pectins from hot-water treated car-rots was also higher in WSP than in other pectin types from thesame matrix (De Roeck et al., 2008). The total neutral monosac-charide content in pectins from raw and boiled red peppers wasNSP > WSP > CSP, but in pectins from grilled peppers the neutralmonosaccharide content was higher in WSP than in the otherpectin types. This might be a consequence of increased pectin sol-ubilization during ripening and the high processing temperatureduring grilling. Sila et al. (2006a) demonstrated variations of neu-tral monosaccharide content in some pectin types as a function ofheat processing intensity.
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Table 4Monosaccharide composition (g/kg of pectin) of water-, chelator-, and alkali-soluble pectin (WSP, CSP, and NSP) from raw and heat-processed Jalapeno peppers at tworipening stages.
Pectin type Green peppers Red peppers
Raw Boiled Grilled Raw Boiled Grilled
Galacturonic acidWSP 401 ± 13b 500 ± 21a 371 ± 17b 776 ± 38a 746 ± 13a 837 ± 16a
CSP 691 ± 2a 330 ± 16c 517 ± 46b 359 ± 1b 576 ± 24a 639 ± 9a
NSP 684 ± 19a 251 ± 16c 416 ± 19b 208 ± 2b 585 ± 17a 571 ± 39a
XyloseWSP 156 ± 2a 114 ± 3b 101 ± 1c 6.2 ± 1.7a 10.2 ± 1.4a 9.1 ± 0.6a
CSP 46.3 ± 3.0a 36.9 ± 1.1b 15.3 ± 1.5c 3.5 ± 0.1a 3.4 ± 0.2a 2.7 ± 0.3a
NSP 1.7 ± 0.01c 8.7 ± 0.03b 53.7 ± 0.9a 15.4 ± 0.7a 5.3 ± 0.7b 5.9 ± 0.6b
GalactoseWSP 69.6 ± 1.6a 66.7 ± 0.1a 53.7 ± 0.2b 33.6 ± 0.9b 33.9 ± 0.4b 108 ± 10a
CSP 24.1 ± 2.4b 36.4 ± 4.8ab 42.6 ± 3.0a 14.4 ± 0.8b 14.6 ± 3.3b 31.1 ± 3.8a
NSP 79.3 ± 4.4a 79.8 ± 1.1a 85.5 ± 0.2a 75.3 ± 2.1b 90.8 ± 2.4a 20.5 ± 3.4c
MannoseWSP 27.1 ± 3.2a 24.8 ± 0.7a 9.5 ± 1.4b 30.5 ± 4.6a 10.7 ± 0.8b 4.2 ± 0.6b
CSP 8.4 ± 0.8c 11.3 ± 0.2b 17.1 ± 0.2a 45.8 ± 1.5a 11.5 ± 0.8b 10.2 ± 0.04b
NSP 11.6 ± 0.6c 16.7 ± 1.5b 111 ± 1a 25.2 ± 2.3a 4.9 ± 0.1b 1.8 ± 0.1b
ArabinoseWSP 20.9 ± 0.5a 20.5 ± 0.8a 16.0 ± 1.0b 18.6 ± 0.2a 14.3 ± 0.1b 14.7 ± 0.1b
CSP 11.2 ± 0.1c 16.6 ± 0.1b 17.8 ± 0.4a 16.4 ± 1.2a 17.7 ± 1.0a 18.2 ± 0.2a
NSP 23.6 ± 0.2b 25.5 ± 0.2a 26.3 ± 0.2a 18.6 ± 1.1a 22.2 ± 1.8a 23.4 ± 0.9a
RhamnoseWSP 0.6 ± 0.04a 0.5 ± 0.1a 0.4 ± 0.1a 1.1 ± 0.01a 1.0 ± 0.00a 1.0 ± 0.01b
CSP 0.9 ± 0.02a 0.9 ± 0.1a 0.8 ± 0.1a 2.0 ± 0.04a 1.8 ± 0.2a 2.0 ± 0.1a
NSP 1.3 ± 0.1a 1.1 ± 0.01b 1.0 ± 0.1b 3.7 ± 0.1a 3.7 ± 0.1a 3.5 ± 0.1a
FucoseWSP 0.26 ± 0.01a 0.30 ± 0.02a 0.31 ± 0.01a 0.37 ± 0.02a 0.37 ± 0.03a 0.33 ± 0.03a
CSP 0.35 ± 0.00b 0.45 ± 0.02a 0.42 ± 0.01a 0.87 ± 0.01a 0.68 ± 0.06b 0.64 ± 0.02b
NSP 0.40 ± 0.01a 0.28 ± 0.02b 0.24 ± 0.01b 0.51 ± 0.01a 0.45 ± 0.00b 0.41 ± 0.01c
Values represent the mean of three individual measurements ± the standard error. Values in the same row for each ripening stage with different letters are significantlydifferent (p < 0.05).
Considering only raw fruits, the content of Xyl, Gal, and Arawas reduced (88%, 29%, and 4%, respectively) in all pectin typesas the ripening stage of fruits changed from green to red whilethe Man, Rha and Fuc contents increased (115%, 143%, and 73%,respectively). Priya et al., 1996 found decreases in Gal, Ara, Xyl, andMan and increases in Rha in Bell pepper pectin during ripening. Xinet al. (2010) also found that Rha content in pectins increased duringripening of two tomato cultivars and this increase was attributed torhamnogalacturonan degradation. In pectins from green peppers,grilling significantly decreased the content of Xyl (35%), Gal (23%),Man (65%), and Ara (23%) in WSP. Boiling and grilling sequentiallyincreased the content of Gal, Man, and Ara in CSP and that of Xyland Man in NSP. The increase of neutral monosaccharide contentby heat processing has been attributed to increased heat-mediatedpectin solubilization (Sila et al., 2006a). The Rha and Fuc contentwas reduced in NSP (15–40%) by heat processing, probably as a con-sequence of pectin degradation (Femenia et al., 2009). In the case ofpectins from red peppers, grilling reduced the Rha content (9%) inWSP while the Gal content was increased (221%). Sila et al. (2006a)also demonstrated that heat processing (100 ◦C for 2 h) dramati-cally increased the Gal content in WSP from carrots, this increasebeing attributed to heat-mediated solubilization of airy regions ofpectin. Both heat-processing styles caused losses of Man (65–86%)and Ara (21–23%) in WSP, of Man (75–78%) and Fuc (22–26%) in CSP,and of Man (81–93%), Fuc (12–20%), and Xyl (62–66%) in NSP. Thesedecreases might be attributed to heat-mediated pectin degradation(Femenia et al., 2009). The Rha content in CSP from raw peppers wasnot altered by heat processing, this phenomenon being attributedto the high thermal stability of CSP, as compared with that of WSPand NSP (Sila et al., 2006a). In general, the neutral monosaccha-ride composition of tested pectins was similar to that reportedpreviously for pectin from Bell peppers and other fruits (Priyaet al., 1996). The characterization of constituent monosaccharidesof pectins is important since it influences not only their functional
but also their biological properties (Garna et al., 2004). The biolog-ical activities (immuno-modulating, anti-cancer, antioxidant andhypoglycemic) of ginseng pectin have been attributed to the highGal, Ara, and Rha contents of its rhamnogalacturonan I domain (Yuet al., 2010).
3.5. Degree of methyl esterification of pepper pectin
The DM of pectins is shown in Fig. 1. Some DM values were sim-ilar to those previously reported for pectin from Jalapeno, Bell, andTabasco peppers (Howard et al., 1997; Arancibia & Motsenbocker,2006; Popov et al., 2011). Some of our DM values were similar tothose reported for apple, citrus, sugar beet and cocoa husks pectin(Leroux et al., 2003; Chan & Choo, 2013). In general, the DM candecrease or increase during ripening, depending on fruit type andpectin methylesterase activity (Arancibia & Motsenbocker, 2006;Bédouet, Denys, Courtois, & Courtois, 2006). In this study, the ripen-ing process decreased the DM of WSP and NSP (38% and 13%,respectively), while the DM of CSP increased (153%). Ripening-related de-esterification of pectins results in cell wall looseningbecause it alters their ability to interact with cell-wall components(Arancibia & Motsenbocker, 2004), although during cell growthand development some methyl groups can be removed by pectinmethylesterase to produce free carboxyl groups in GalA residuesthat are able to interact with Ca2+ to form an structure that pre-vents the attack of polygalacturonase (Wen, Ström, Tasker, West,& Tucker, 2013). NSP showed the highest DM (Fig. 1), after pool-ing of all data, and the DM of WSP was not significantly differentto that of CSP. NSP from other sources has also been described ashighly methoxylated pectin. Heat processing affected differentlythe DM of pectins of green and red peppers, probably affectingtheir solubility, ability to interact with other components, and func-tional properties (Yen & Lin, 1998). In green peppers, the DM ofWSP and NSP was decreased by both heat treatments (17–26%
118 O.P. Ramos-Aguilar et al. / Carbohydrate Polymers 115 (2015) 112–121
Fig. 1. Degree of methyl esterification of water-, chelator-, and alkali-soluble pectin(WSP, CSP, and NSP) from raw and heat-processed Jalapeno peppers at two ripeningstages (green and red). Data represent the mean of nine replicates ± the standarderror (slim bars). Bars with different letters for each pectin type were statisticallydifferent (p < 0.05).
and 31–61%, respectively). De Roeck et al. (2008) also reporteddecreases in the DM of WSP from carrots as a consequence of highpressure/high temperature processing. Boiling increased 1.3-foldthe DM of CSP from green peppers. Similarly, Chan and Choo (2013)demonstrated that DM of cocoa husks pectin increased when thetemperature of heat treatment was increased from 50 to 95 ◦C.Munarin, Bozzini, Visai, Tanzi and Petrini (2013) demonstrated thatheating (121 ◦C at 2 atm for 15 min) decreased the degree of esteri-fication of highly methylated pectin solutions while this treatmentincreased the esterification of pectins with low degree of esteri-fication. The DM of CSP also increased during high pressure/hightemperature processing of carrot tissue (De Roeck et al., 2008). Inred peppers, the DM of CSP and NSP was dramatically decreased bygrilling (48% and 53%, respectively). Heat processing of red peppersdid not alter the DM of WSP. Decreases in the DM of WSP, CSP andNSP from guava juice have been observed after heating (95 ◦C for5 min) (Yen & Lin, 1998).
3.6. Molecular weights distribution (Mw) of pecticpolysaccharides
Tested pectins showed 1 or 2 main fractions, but other minorfractions were also observed for some pectins (Table 5). The peakMw of first fraction was considerably high, but the values observedfor the other fractions generally were similar to those previouslyreported for pepper pectins (Popov et al., 2011). Several factors areable to alter the Mw of pectin fractions, including the botanical
source and conditions used for pectin extraction (Mesbahi et al.,2005; Westereng, Yousif, Michaelsen, Knutsen, & Samuelsen, 2006;Yapo, 2009).
The Mw values observed for the first pectin fraction from greenpeppers were always higher than those of the red counterpart;however, this behavior was not clearly observed for the otherfractions (Table 5). Similarly, the Mw of pectin fractions frombanana and plantain at ripening stage 5 was higher than at stage 7(Happi et al., 2008). The reduction of Mw during ripening has beenrelated to enzymatic depolymerization of pectins by polygalactur-onases and methyl esterases, with the action of polygalacturonasesbeing dependent on methyl de-esterification of pectin (Arancibia &Motsenbocker, 2004; Happi et al., 2008).
Considering only the peak Mw of the main fraction (the mostabundant fraction) of pectins from green peppers, we infer thatCSP had a higher Mw than NSP and WSP. Similar findings havebeen observed for pectins from banana at ripening stage 5 (Happiet al., 2008). According to our findings, CSP from green fruits wasless methyl esterified than NSP and WSP, having probably morebindings with other pectin molecules by divalent ions and there-fore being less susceptible to depolymerization. This phenomenonmight explain the high Mw of pectins from green fruits. Usingthe same data analysis, on the other hand, we infer that CSP andNSP from red peppers showed similar Mw, which was higher thanthat of WSP. Similar results were observed for pectin from bananaat ripening stage 7 (Happi et al., 2008). This has been attributedto solubilization of pectins during ripening by pectin-hydrolyzingenzymes (Inari et al., 2002).
The boiling and grilling sequentially reduced the peak Mw ofall pectin fractions, with grilling causing the highest reduction(Table 5). This reduction was higher for pectin fractions from red(10–98%) than of green (8–78%) peppers. Westereng et al. (2006)demonstrated that the Mw of cabbage pectin decreased as temper-ature of the extracting solvent was raised from 50 to 100 ◦C. Theheat-induced diminishing of the Mw of pectins is a consequence oftheir degradation and solubilization (Mesbahi et al., 2005; Sila et al.,2006b). During thermal processing, the !-elimination is highlydependent on DM, pH, and the presence of ions (Sila et al., 2006b).Pectins of high DM are more susceptible to !-elimination thanthose of low DM (Sila et al., 2006b). The functionality of pectinsas gelling, thickening, and stabilizing agents in foods is relatedwith their Mw. Pectins with acetyl groups and low Mw exhibit poorgelling properties, but show good emulsion stabilizing properties(Leroux et al., 2003; Mesbahi et al., 2005). On the other hand, the Mwof pectin determines the viscosity of their solutions (Einhorn-Stollet al., 2014).
3.7. Viscosity properties of pectins
In our study, the pectins were characterized according to theviscosity and flow pattern of their solutions (Fig. 2). In general, theviscosity of solutions of all pectin types showed a decreasing trendas the shear rate increased, indicating a shear thinning behavior,mainly within the range from 0 to 100 s−1. The viscosity was almostconstant at a shear rate over 100 s−1. Vriesmann and Petkowicz(2013) also observed a major apparent viscosity from 0 to 100 s−1
for solutions of pectin from cacao pods. The shear thinning behaviorhas also been observed in solutions of apple pectin (Chen et al.,2012).
The flow behavior showed high correlation values(R2 = 0.991–0.999) with power law model. The ripening stageof fruits did not cause a significant effect on the viscosity (consis-tency index) of pectin solutions, except for those of CSP from redpeppers that decreased 24%. This was unexpected since pectinsof cell walls are enzymatically degraded during ripening, causingsignificant viscosity losses in the solutions of these polysaccharides
O.P. Ramos-Aguilar et al. / Carbohydrate Polymers 115 (2015) 112–121 119
A
0.00
0.02
0.04
0.06
0.08
0.10
0.12
B
Appa
rent
vis
cosi
ty (P
a.s)
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
C
Shear rate (1 /s)0 100 200 300 400 500 600
0.004
0.006
0.008
0.010
0.012
0.014
0.016
RGP 0.96 ± 0 .01 0.33 ± 0 .00a
GGP 0.97 ± 0.02 0.23 ± 0.00c
GRP 0.98 ± 0 .01 0.20 ± 0 .00d
sample n k (Pa.sn)
BGP 0.96 ± 0.01 0 .14 ± 0.00f
RRP 0.98 ± 0.0 1 0 .25 ± 0.00b
BRP 0. 99 ± 0.02 0.18 ± 0. 00e
sample n k (P a.sn)RGP 0.98 ± 0 .01 0. 15 ± 0.00a
BGP 0 .96 ± 0.01 0.12 ± 0 .00c
GGP 0.94 ± 0.03 0.12 ± 0.00c
RRP 0 .96 ± 0.03 0.15 ± 0 .00a
BRP 0.95 ± 0. 02 0 .14 ± 0.00b
GRP 0.94 ± 0 .01 0. 12 ± 0.00c
sample n k (Pa.sn)RGP 0.92 ± 0.01 0 .39 ± 0.03a
BGP 0. 97 ± 0.08 0.33 ± 0. 01b
GGP 0.98 ± 0 .01 0.22 ± 0 .00b
RRP 0. 94 ± 0.02 0.37 ± 0. 01a
BRP 0.96 ± 0.00 0.25 ± 0.00b
GRP 0.93 ± 0.04 0 .15 ± 0.00c
Fig. 2. Flow curves and parameters of power law model obtained from solutions of water-, chelator-, and alkali-soluble pectin ((A), (B), and (C)) from raw and heat-processedJalapeno peppers at two ripening stages. RGP: raw green pepper; BGP: boiled green pepper; GGP: grilled green pepper; RRP: raw red pepper; BRP: boiled red pepper; GRP:grilled red pepper; n: flow behavior and k: consistency index.
120 O.P. Ramos-Aguilar et al. / Carbohydrate Polymers 115 (2015) 112–121
Table 5Peak molecular weights (KDa) distribution of fractions from of water-, chelator-, and alkali-soluble pectin (WSP, CSP, and NSP) from raw and heat-processed Jalapeno peppersat two ripening stages.
Pectin type Processing style Pectin fraction (green peppers)
I II III IV V
WSP Raw 3300 ± 69a 280 ± 8 42 ± 1 24 ± 1 –Boiled 1799 ± 26b – – – –Grilled 1586 ± 53c – – – –
CSP Raw 3720 ± 73a – 202 ± 1a 27 ± 7 15 ± 4Boiled 3404 ± 77b 601 ± 77a – – –Grilled 2195 ± 5c 243 ± 29b 44 ± 1b – –
NSP Raw 2637 ± 75a 177 ± 3a – – –Boiled 2305 ± 10b 152 ± 3b – – –Grilled 2140 ± 33b 122 ± 1c 36 ± 1 – –
Pectin fraction (red peppers)WSP Raw 2743 ± 124a 1504 ± 205a 481 ± 38a 151 ± 18 66 ± 8
Boiled 1415 ± 108b 576 ± 20b 107 ± 1b – –Grilled 114 ± 1c 32 ± 2c 11 ± 2c – –
CSP Raw 2024 ± 29a 892 ± 126a 407 ± 91a 182 ± 50a 76 ± 19Boiled 1521 ± 56c 727 ± 2a 113 ± 4b 24 ± 1b –Grilled 1828 ± 32b 227 ± 13b – – –
NSP Raw 2228 ± 75a 180 ± 11a 13 ± 1 – –Boiled 1608 ± 4b 154 ± 6a – – –Grilled 1577 ± 5b 89 ± 3b – – –
Values represent the mean of four individual measurements ± the standard error. Values in the same column for each pectin type and fraction (I–V) with different letters aresignificantly different (p < 0.05).
(Happi et al., 2008; Einhorn-Stoll et al., 2014). On the other hand,the heat treatments significantly reduced the viscosity of solutionsof WSP, CSP, and NSP. Grilling caused a greater reduction (44–59%)in the viscosity of WSP solutions than boiling (15–32%). Thereduction in viscosity of NSP solutions by boiling ranged from 6.7to 20% while the losses caused by grilling were 20%. In contrast,boiling caused a higher diminishing (28–58%) of viscosity in CSPsolutions than grilling (20–30%). This could be a consequence ofthe movement of calcium ions from fruit to water (Lamikanra &Watson, 2007), changes in membrane permeability, and changesin the three-dimensional structure of fruit tissue (Lara et al., 2006).Similarly, Basanta, Ponce, Rojas and Stortz (2012) observed thatviscosity of pectin that had been extracted by boiling water waslower than that of other pectins. On the other hand, the viscosityof pectin solutions from orange peels increased when the pectinwas extracted at moderate temperatures (35–45 ◦C), but decreasedwhen the extraction temperature was raised (Guo et al., 2012).
The pectins from raw peppers showed good viscosity properties,according to the comparison of their consistency index (Fig. 2) andthose reported for commercial pectins (Moresi & Sebastiani, 2008).
4. Conclusions
This study demonstrated that pectins with specific character-istics can be obtained by selecting adequate ripening stages andheat-processing conditions of fruits, diversifying the technologicalapplications of these polysaccharides. The pectin content in pep-per was comparable to that of commercial pectin sources. Heatprocessing increased the extractability of pectin from peppers. Thechemical characteristics of isolated pectins were highly altered byripening and heat-processing style, while their viscosity proper-ties depended exclusively on processing style. The DM of pectinsvaried from low to high, depending on source, increasing theirpotential technological uses. NSP had the highest protein contentand therefore might exhibit emulsifying properties. Some pectinsshowed high Mw and therefore might form strong gels and vis-cous solutions. The physicochemical characteristics and viscosityproperties of tested pectins were similar or higher than thosereported for commercial pectins. Peppers certainly can be a new
source of commercial pectin, leading to an alternative use for thiscrop.
Acknowledgments
This research was funded by the Fondo Sectorial de Inves-tigación para la Educación (Investigación Básica SEP-CONACYT;Project Clave: 103391). The authors thank Emilio Ochoa Reyes,Ana Lourdes Ramos Aguilar, and Gustavo Iván Ventura for theirtechnical assistance.
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143
CAPÍTULO IV
EFFECT OF PECTIN CONCENTRATION AND PROPERTIES ON
DIGESTIVE EVENTS INVOLVED ON MICELLARIZATION OF FREE AND
ESTERIFIED CAROTENOIDS
Effect of pectin concentration and properties on digestive eventsinvolved on micellarization of free and esterified carotenoids
Braulio Cervantes-Paz a, Jos!e de Jesús Ornelas-Paz a, *, Jaime David P!erez-Martínez b,Jaime Reyes-Hern!andez c, Paul Baruk Zamudio-Flores a, Claudio Rios-Velasco a,Vrani Ibarra-Junquera d, Saúl Ruiz-Cruz e
a Centro de Investigaci!on en Alimentaci!on y Desarrollo A.C.-Unidad Cuauht!emoc, Av. Río Conchos S/N, Parque Industrial, C.P. 31570, Cd, Cuauht!emoc,Chihuahua, Mexicob Universidad Aut!onoma de San Luis Potosí, Facultad de Ciencias Químicas, Manuel Nava No. 6, Zona Universitaria, C.P. 78210, San Luis Potosí, Mexicoc Universidad Aut!onoma de San Luis Potosí, Facultad de Enfermería, Av. Ni~no Artillero No. 130, Zona Universitaria, C.P. 78210, San Luis Potosí, Mexicod Universidad de Colima, Bioengineering Laboratory, Km. 9 Carretera Coquimatl!an-Colima, C.P. 28400, Coquimatl!an, Colima, Mexicoe Instituto Tecnol!ogico de Sonora, Departamento de Biotecnología y Ciencias Alimentarias, 5 de Febrero 818 Sur, C.P. 85000, Cd, Obreg!on, Sonora, Mexico
a r t i c l e i n f o
Article history:Received 14 December 2015Received in revised form4 April 2016Accepted 26 April 2016Available online 28 April 2016
Keywords:Polysaccharide propertiesLipid-soluble pigmentsEmulsionBioavailabilityCarotenoid polarity
a b s t r a c t
Pectins with different chemical and rheological properties were added at two concentration levels intodigestion reactions of soybean oil enriched with a mixture of free and esterified carotenoids to studytheir effects on several events involved on carotenoid micellarization. High pectin concentrationincreased lipolysis, viscosity of the gastrointestinal medium, and particle size but decreased carotenoidmicellarization. At low concentration, pectin showed a high binding of bile salts and favored carotenoidmicellarization. Pectins with high molecular weight and viscosity in model solutions substantiallyincreased the viscosity of the intestinal media and the micellarization of the less polar carotenoids whilepectins with high and medium degree of methyl esterification promoted lipolysis, bile binding, viscosityand large sizes of lipid droplets, and slightly favored the micellarization of the more polar carotenoids.The effects of pectin properties and amount on carotenoid micellarization seemed to be related tocarotenoid speciation.
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Fruits and vegetables are the most important sources of dietarycarotenoids. These compounds act as nutrients and might beinvolved on many protective effects of human health (Krinsky &Johnson, 2005). However, the efficiency of carotenoid absorptionlimits their biological actions (Biehler, Hoffmann, Krause, & Bohn,2011). The transference of carotenoids from the food to the mixedmicelles during digestion determines their absorption since onlymicellarized carotenoids can be incorporated by the enterocytes(Faulks & Southon, 2005). The food matrix exerts the strongesteffect on carotenoid micellarization (Victoria-Campos, Ornelas-Paz,Yahia& Failla, 2013). This effect might explain the high variability inthe micellarization efficiency for most carotenoids as a function ofthe food matrix, ranging from no micellarization to the total
incorporation into the micelles (Aschoff et al., 2015a,b; O'Sullivan,Jiwan, Daly, O'Brien, & Aherne, 2010; Victoria-Campos et al.,2013). This variability has been partially attributed to differences infiber content in foods (Ornelas-Paz, Failla, Yahia, & Gardea-Bejar,2008; Riedl, Linseisen, Hoffmann, & Wolfram, 1999). Severalin vivo studies have demonstrated that the consumption of purifiedpectin reduced the carotenoid absorption and that this reduction isgenerally higher than that obtained with other dietary fibers (Riedlet al., 1999; Zanutto, Júnior, Meirelles, F!avaro, & Vannucchi, 2002).The involvement of pectin on carotenoid micellarization and ab-sorption may be high, especially if it is considered the high contentof this polysaccharide in vegetal foods, the effects of pectin on lipiddigestion as well as the requirement of lipids and their digestionproducts for carotenoid absorption and transport (Espinal-Ruiz,Parada-Alfonso, Restrepo-S!anchez, Narv!aez-Cuenca, & McCle-ments, 2014a; Faulks & Southon, 2005; Ramos-Aguilar et al., 2015;Rubio-Senent, Rodríguez-Guti!errez, Lama-Mu~noz, & Fern!andez-Bola~nos, 2015). However, such involvement might be dependent of* Corresponding author.
E-mail address: [email protected] (J.J. Ornelas-Paz).
Contents lists available at ScienceDirect
Food Hydrocolloids
journal homepage: www.elsevier .com/locate/ foodhyd
http://dx.doi.org/10.1016/j.foodhyd.2016.04.0380268-005X/© 2016 Elsevier Ltd. All rights reserved.
Food Hydrocolloids 60 (2016) 580e588
pectin properties, which vary with the botanical origin andextraction methods (Ramos-Aguilar et al., 2015). The effect ofpectin properties on carotenoid micellarization or bioavailabilityhas been scarcely studied and limited just to b-carotene and thedegree of esterification of pectin. Ornelas-Paz et al. (2008)demonstrated that the qualitative and quantitative changes ofpectin during fruit ripening were involved on b-carotene micella-rization. Recently, Verrijssen et al. (2014) studied the micellariza-tion of b-carotene in presence of commercial citrus pectin withdifferent degree of methyl esterification and demonstrated thatsuch fibers differentially affected the viscosity of the intestinalmedium, the emulsion of fat (particle size) and the micellarizationof the pigment. In a further study, they (Verrijssen Verkempinck,Christiaens, Van Loey, & Hendrickx, 2015) demonstrated that theaddition of an emulsifier changed the effect of the degree ofesterification of pectin on the micellarization of b-carotene andlipolysis products.
The effect of pectin properties on lipid digestion has receivedmore attention. Some inferences about the effect of pectin prop-erties on carotenoid micellarization can be obtained from suchstudies. The molecular weight and esterification degree of pectindetermine the viscosity of its solutions (Leroux, Langendorff,Schick, Vaishnav, & Mazoyer, 2003; Ramos-Aguilar et al., 2015).Variations in these properties changed the viscosity of the gastro-intestinal medium and, consequently, the emulsion of fat andlipolysis (Espinal-Ruiz et al., 2014a). The pectin can also bind bilesalts in an esterfication-dependent fashion, reducing the emulsifi-cation of fat and lipolysis (Rubio-Senent et al., 2015; Xu, Jiao, Yuan,& Gao, 2015). The protein content of pectin can also influence theemulsification of oil in water (Leroux et al., 2003). Thus, theemulsification of fat determines the extent of lipolysis. These pro-cesses are important on carotenoid micellarization since thelipolysis products as well as the bile salts are required for the for-mation of micelles, the absorptive vehicle of carotenoids (Faulks &Southon, 2005). The evaluation of the effect of individual propertiesof pectin in these events represents a complex technical challengesince most pectin properties are related or depend each other and,therefore, the modification of a property without the alteration ofothers is difficult. The most representative properties of pectinshould be considered together. The objective of this work was toevaluate the effect of pectin properties (degree of methyl esterifi-cation, molecular weight, and viscosity) on the viscosity ofgastrointestinal medium, fat emulsification (particle size), lipolysisand bile salts binding and determinate the involvement of suchevents and properties on the micellarization efficiency of free andesterified carotenoids.
2. Materials and methods
2.1. Materials
Cholestyramine (Chol), cellulose (Cell), bile salts (taurocholate,taurodeoxycholate, and glicodeoxycholate), lipid standards (tri-, di-and mono-glycerides and free fatty acids) and other reagents werepurchased from SigmaeAldrich (St. Louis, MO, USA). High-puritycarotenoid standards were purchased from SigmaeAldrich (St.Louis, MO, USA), Southcot Inc. (Chapel Hill, NC, USA), or Car-otenature GmbH (Lupsingen, Switzerland). Three pectins fromjalape~no peppers (PP1, PP2 and PP3) were extracted and charac-terized according to Ramos-Aguilar et al. (2015). They showedincreasing/decreasing viscosity in model solutions, molecularweight (MW) and degree of methyl esterification (DME) (PP1:k ¼ 0.37 Pa sn, MW ¼ 4942 KDa, DME ¼ 40%; PP2: k ¼ 0.25 Pa sn,MW ¼ 3580 KDa, DME ¼ 68%; PP3: k ¼ 0.15 Pa sn, MW ¼ 2421 KDa,DME ¼ 80%). The protein content of tested pectins was similar and
averaged 1.7%. Similar properties were previously reported forpepper pectins (Arancibia & Motsenbocker, 2006; Popov et al.,2011; Ramos-Aguilar et al., 2015). Soybean oil was obtained fromthe local market. A mixture of free and esterified carotenoids wasobtained from red peppers. They were extracted, identified, andquantified by HPLC-APCI-TOF-MS, according to Cervantes-Paz et al.(2012). The extract was dispersed in soybean oil and storedat "70 #C until use for in vitro digestions.
2.2. In vitro digestion
The pectins were dissolved at two concentration levels in salineby stirring for 12 h and then 1.25 mL of the carotenoid-rich soybeanoil were added and mixed until emulsification of the oil (Homog-enizer Ika T18 Basics; IKaworks Inc.). The concentration of pectin inthe final reaction was 0.14 and 1%. These emulsions were digestedin vitro. Digestions without pectin and with Cell and Chol wereincluded as control reactions. The digestions were performed ac-cording to Victoria-Campos et al. (2013). For the gastric phase, thepH of the mixture was adjusted to 2.0 using HCl and then porcinepepsin (1.6 mg/mL) was added. The gastric phase was carried out at37 #C for 1 h under reciprocal shaking (95 rpm). For the intestinalphase, the pH of the reaction was increased to 6.0 with sodiumbicarbonate and then porcine pancreatin (0.4 mg/mL) and porcinepancreatic lipase (0.8 mg/mL) were added. Taurocholate (TC),taurodeoxycholate (TDC), and glycodeoxycholate (GDC) wereadded to the reaction, providing final concentrations of 0.75, 0.45,and 0.80 mmol/L, respectively (Chitchumroonchokchai, Schwartz,& Failla, 2004). The pH was increased to 7.0 using 1N NaOH andthe reactions were incubated for 2 h at 37 #C under shaking(95 rpm). The lipolysis of soybean oil was monitored in situ duringthe intestinal phase. Aliquots of the digestion reactions were ob-tained after the completion of the gastric and intestinal phases andevaluated for viscosity. Digestate samples were used to evaluate thebinding of bile salts by pectin. Additionally, aliquots (10 mL) ofdigestate were used to recover the micellar fraction from digestateby centrifugation (15,000 g/20 min/4 #C) and filtration through amembrane of 0.22 mm of pore size (Millipore Corp., Bedford, MA,USA). The particle size distribution was determined in the digestaand micellar fraction. The composition of lipid species (FFA þ MG:Free Fatty Acids þ Monoglycerides; DG: Diglycerides; TG: Tri-glycerides) was determined in the micellar fraction. The micella-rization efficiency of carotenoids was also determined (Victoria-Campos et al., 2013).
2.3. Viscosity measurements
The viscosity of the gastric and intestinal media was measuredusing an AR 1500ex rheometer (TA Instruments; New Castle, DE,USA), equipped with the stainless steel parallel plate geometry(60 mm diameter) at 37 #C. The shear rate ranged from 0.1 to100 s"1 (up curve) and from 100 to 0.1 s"1 (down curve). Shear rateagainst shear stress data were fit using the power law model(t ¼ kgn) and analyzed for flow behavior, n, and consistency index,k.
2.4. Particle size analysis
The particle/oil droplet size distribution in the intestinal andmicellar media were measured by dynamic light scattering using aMalvern Zetasizer Nano ZS instrument (Malvern Instruments;Worcestershire, UK) with ameasuring range from 0.3 nm to 5 mm. Arefractive index for oil of 1.47 and water of 1.33 was used for thecalculation of particle sizes.
B. Cervantes-Paz et al. / Food Hydrocolloids 60 (2016) 580e588 581
2.5. Binding of bile salts
One milliliter of digestate was mixed with 9 mL of absoluteethanol at 60 !C. Themixturewas kept in repose for 1 h, centrifuged(4000 " g/4 !C/20 min), filtered (membrane of 0.22 mm of poresize), and injected (20 mL) into an Agilent 1200 HPLC system (Agi-lent Technologies Inc.; Palo Alto, CA, USA) equipped with anevaporative light scattering detector (1260 Infinity ELSD). Theseparation was carried out in a Zorbax Eclipse XDB-C18 column(4.6 " 150 mm, 5 mm) at 30 !C. The mobile phase was composed by20 mM ammonium acetate (solvent A) and methanol (solvent B),according to the following gradient: 20e100% B in 35min and 100%B for 15 min. The flow rate of the mobile phase was 1 mL/min. Thenebulization temperature was 50 !C. High-purity nitrogen at 70 !Cwas used for the evaporation at a flow-rate of 1.5 L/min. The con-centration of TC, TDC, and GDC was determined using standardcalibration curves. The bile salt binding capacity of pectins (BC, aspercentage) was calculated as: BC ¼ [(BSWF $ BSapp)/BSWF] " 100.BSWF and BSapp indicated the bile salt concentration in digestateswithout fiber and after pectin precipitation, respectively.
2.6. Lipolysis of carotenoid-rich soybean oil and lipid species inmicelles
The kinetics of triglyceride hydrolysis were followed in situ afterthe addition of porcine pancreatin and porcine pancreatic lipaseusing a pH-stat automatic titration unit (Metrohm Ltd; Herisau,Switzerland). The pH of digestate was maintained at 7.0 by titrationwith 0.05MNaOH. The volume of NaOH added to the digestionwasrecorded and used to calculate the concentration of released freefatty acids by the next equation: TA ¼ VNaOH " 0.05 " 1000, whereTA (mMol) is the total titratable acid released and VNaOH (mL) is thevolume of NaOH used to titrate the released acids during the in-testinal digestion (2 h).
Lipids from micellar fraction (2 mL) were extracted three timeswith a mixture of diethyl ether:heptane:ethanol (1:1:1 v/v; 6 mLeach time). The upper layer was recovered, the solvent evaporated,and the obtained lipids dissolved in hexane (5 mL). The extract wasfiltered and injected (20 mL) into the chromatographic systemdescribed above for the separation of bile salts. The separation oflipid species was carried out in a Luna C18 column (3.0 " 250 mm,5 mm) (Phenomenex; Torrance, CA, USA) at 30 !C. The mobile phasewas composed by a mixture of acetonitrile:methanol:water:THF:-acetic acid (75:12:8:4:1 v/v, solvent A), acetone (solvent B), andacetonitrile (solvent C) according to the following gradient: 100% Aduring the first 5 min, 96% A/3.6% B/0.4% C at min 11, 37% A/56.7% B/6.3% C atmin 24, 32% A/61.2% B/6.8% C atmin 35,18% A/73.8% B/8.2%C at min 45, 15% A/76.5% B/8.5% C at min 58.5% A/85.5% B/9.5% C atmin 60, and 100% A from min 63 to 65. The flow rate of the mobilephase was 0.7 mL/min. The lipid species were monitored using anevaporative light scattering detector (1260 Infinity ELSD). Thenebulization temperature was 30 !C. High-purity nitrogen at 70 !Cwas used for the evaporation at a flow-rate of 1.0 L/min. Thedifferent lipid species eluted at specific and well-differentiatedranges of time (FFA þ MG ¼ 0e10 min; DG ¼ 20e30 min;TG ¼ 35e55 min). The separation of FFA and MG was not achievedand therefore such lipid species were quantified together. Severalstandards of lipid species (1-oleoyl-glycerol, 1-linoleoyl-rac-glyc-erol, 1-lauroyl-rac-glycerol, glyceryl 1,3-distearate, 1.3-diolein, 1,3-dilynoleoyl-rac-glycerol, dipalmitin, glyceryl tripalmitate, glyceryltrilinoleate, glyceryl trilinolenate, glyceryl trioleate, glyceryl trido-decanoate, glyceryl tristearate, and several FFA) were used toidentify such ranges of time as well as individual lipid species.
2.7. Extraction and bioaccessibility of carotenoids
Carotenoids were extracted from soybean oil and micellarfraction using a mixture of petroleum ether/acetone (4 mL; 2:1 v/v)according to the procedure described by Victoria-Campos et al.(2013). The petroleum ether was evaporated under a slight nitro-gen flow. The residue was dissolved in acetone (2 mL), filteredthrough a membrane of 0.45 mm of pore size, and injected (20 mL)into an Agilent 1200 series HPLC system (Agilent, Palo Alto, CA,USA), equipped with a diode array detector and a C30 reversed-phase column (4.6 " 150 mm, 3 mm) (YMC Inc., Milford, MA,USA). The separation of carotenoids was conducted according toCervantes-Paz et al. (2012). The bioaccessibility of carotenoids wascalculated as the percentage of carotenoids transferred from thesoybean oil to the micellar phase.
2.8. Statistical analysis
All measurements were performed at least in triplicate. The datawere analyzed using a completely randomized design with afactorial arrangement. The statistical significance of the differencesbetween treatments was determined using an ANOVA followed bythe TukeyeKramer post hoc test; 0.05 was the significance limit.These tests were performed using the SAS statistical softwarepackage Ver. 9.0 (SAS Institute Inc.; Cary, NC, USA). The impact ofpectin properties and amount on carotenoid bioaccessibility weredetermined by principal component analysis (PCA) using MinitabVer. 17 (Minitab Inc.; State College, PA, USA).
3. Results and discusion
3.1. Effect of pectin on viscosity of gastrointestinal medium
The rheological behavior of the gastric and intestinal media isshown in Fig. 1. The apparent viscosity decreased as the shear rateincreased. This trend was more evident at shear rates below 40 s$1,indicating a shear thinning behavior, which was also reportedpreviously for digested oil-in-water emulsions containing pectinand attributed to flocculation of oil droplets, interaction betweenpectin and oil droplets, and gelation of pectin (Xu, Wang, Jiang,Yuan, & Gao, 2012). The gelation of pectin is favored by the pres-ence of proteins and ions in the gastrointestinal medium. Such gelscan be broken down as the shear rate increase, causing a decreaseof viscosity (Verrijssen et al., 2014). As expected, the pectin con-centration affected the viscosity of the digestive medium. Thegastric and intestinal media showed a higher apparent viscositywith 1% (k ¼ 0.21e0.82 Pa sn) than 0.14% (k ¼ 0.07e0.16 Pa sn) ofpectin (Fig. 1). This concentration-related effect of pectin hasrecently been reported for oil-in-water emulsions subjected to thegastric and intestinal digestion and attributed to bridging in-teractions of pectin and trapping of continuous phase by suchpolysaccharides (Kaltsa, Paximada, Mandala, & Scholten, 2014;Zhao, Wei, Wei, Yuang, & Gao, 2015).
The viscosity of the gastric medium was independent of pectintype at low concentration conditions; however, pectin type affectedsuch variable at high fiber conditions (Fig. 1). At high fiber condi-tion, PP3 conferred the highest consistency index (k¼ 0.82 Pa sn) tothe gastric medium, followed by PP2 and PP1 (k ¼ 0.22 and0.21 Pa sn, respectively). Interestingly, PP3 and PP1 showed thelowest and highest viscosity properties in model solutions,respectively. These findings demonstrated that the digestive con-ditions could alter the rheological properties exhibited by pectin inmodel solutions. This effect could be consequence of the gelation oraggregation of pectin, which are two phenomena largely mediatedby the DME of pectin and pH of the medium. The variation on DME
B. Cervantes-Paz et al. / Food Hydrocolloids 60 (2016) 580e588582
alters the overall charge of pectin molecules and, therefore, theattraction or electrostatic repulsion between pectin chains as afunction of the changes of medium pH (Logan, Wright, & Goff,2015). Thus, the low pH of gastric medium and the high DME ofPP3 were probably involved on the high viscosity of the gastricmedium since pectins with a high DME are able to form gels at lowpH (2.5e3.5) by hydrophobic interactions and hydrogen bonds(Kastner et al., 2014; L€ofgren, Guillotin, & Hermansson, 2006). Incontrast, the aggregation of pectin probably occurred in digestionreactions with PP2 and PP1. Logan et al. (2015) also observed ahigher apparent viscosity for oil-in-water emulsions containingpectin with high DME than with low DME after in vitro digestionand the observed changes of viscosity were attributed to gelationand aggregation of pectin chains. The gastric medium containinghigh concentrations of Cell and Chol showed the lowest viscosityvalues (k ¼ 0.14e0.17 Pa sn).
The intestinal digestion always decreased the apparent viscosityof the gastric medium (Fig. 1). Verrijssen et al. (2014; 2015) alsoobserved that the apparent viscosity of oil-in-water emulsionscontaining citrus pectin was reduced from the gastric(0.005e1.0 Pa s) to the intestinal (0.002e0.3 Pa s) phase of diges-tion. The neutral pH of the intestinal medium as well as thedepolymerization of pectin by pancreatin and pancreatic lipasecould be involved in such decrease of viscosity (Zhang, Alsarra, &Neau, 2002). The pectin type did not markedly affect the viscosityof the intestinal medium at both concentration levels, although the
influence of PP1 and PP2 on viscosity (k ¼ 0.23 and 0.17 Pa sn,respectively) was significantly different at high fiber concentration.High concentration of PP1 and PP3 conferred the highest viscosityto the intestinal medium. The neutral pH of the intestinal mediumprobably affected the viscosity of the gastric medium by causing thedissociation of the carboxyl groups of pectin (L€ofgren et al., 2006).
The viscosity of the media might modify the carotenoid micel-larization in several ways. Firstly, altering the diffusion of carot-enoids from oil droplets to the micelles and secondly affecting thelipolysis and, consequently, the availability of lipid digestionproducts to form micelles. Viscosity of the medium can influencethe lipolysis by affecting the diffusion of lipolytic enzymes from themedium to the lipid droplets as well as the diffusion of newlyreleased free fatty acids from the oil droplets to the medium, fa-voring or avoiding the reincorporation of such acids to the tri-glyceride backbone (Espinal-Ruiz et al., 2014a; Tsujita, Matsuura, &Okuda, 1996). The viscosity of the medium can also alter the lipiddroplet size and therefore the surface available to the hydrolyzingactivity of lipases (Pasquier et al., 1996).
3.2. Particle size distribution in digestive media
At low pectin concentration condition, the particle size in theintestinal and micellar media was independent of pectin type,ranging from 0.01 to 0.02 mm (Fig. 2). Similarly, Verrijssen et al.(2014) demonstrated that pectins with different DME (14 and99%) did not alter the particle size distribution of oil-in-wateremulsions subjected to digestion. This absence of changes in par-ticle size has been associated to highly stabilized lipid droplets inthe emulsion with minimal coalescence (Espinal-Ruiz, Restrepo-S"anchez, Narv"aez-Cuenca, & McClements, 2016). Under highpectin concentration condition, the particle sizes were higher anddifferent for intestinal and micellar media. Larger particle sizes inmedia with high pectin concentration could be caused by depletionflocculation by the high amount of pectin in the aqueous phase.Under such conditions, the dissociation of the carboxyl groups ofpectins could occur, causing the exclusion of such pectins from thenarrow region around each lipid droplet and increasing the osmoticpressure, which generated attraction between the lipid droplets(depletion force) and favored the formation of larger particles. Atlow pectin concentration, the osmotic attraction did not exceed thesteric and electrostatic repulsion between the oil droplets withscarce droplet aggregation (Espinal-Ruiz, Parada-Alfonso, Restrepo-S"anchez, Narv"aez-Cuenca, & McClements, 2014b; Espinal-Ruizet al., 2014a).
The particle size distribution in the intestinal mediumwith highconcentrations of PP1 and PP2 was centered at 0.11 and 0.27 mm,respectively. The micellar medium with such pectins showed abimodal distribution with one major class centered on 0.07 and0.9 mm and one minor class centered on 0.33 and 4.9 mm (Fig. 2).Verrijssen et al. (2015) also observed a bimodal distribution forparticle size distribution in digested oil-in-water emulsions con-taining pectin with low DME and such distribution type wasattributed to the crosslinking of pectin chains mediated by pH ofmedium aswell as by the presence of ions and proteins. The particlesize distribution in micellar medium with high concentration ofPP3 was similar (0.02 mm) to that described above for mediumwithlow pectin concentration; however, oil droplet size was 0.07 mm inthe intestinal medium. The PP3 induced the smallest particle sizesin the intestinal and micellar media, demonstrating that it favoredthe formation of stable emulsions as compared with PP1 and PP2.This property might be related with the MWof pectins, with lowerMW reducing the formation of hydrogen bounds with mediumcomponents and therefore reducing flocculation (Leroux et al.,2003). Other possible cause may be the block-wise orientation of
A)s.aP(
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Sample k (Pa.sn)WF 0.102bc 0.14%Cell 0.081c 0.14%PP1 0.071c 0.14%PP2 0.075c 0.14%PP3 0.161bc 0.14%Chol 0.083c 1%Cell 0.143bc1%PP1 0.205b1%PP2 0.215b1%PP3 0.816a 1%Chol 0.174bc
WF 0.076e0.14%Cell 0.079e 0.14%PP1 0.086e0.14%PP2 0.081e0.14%PP3 0.075e0.14%Chol 0.076e1%Cell 0.107de1%PP1 0.234a1%PP2 0.170bc1%PP3 0.196ab1%Chol 0.138cd
Sample k (Pa.sn)
Fig. 1. Viscosity of gastric (A) and intestinal (B) media of digestion reactions withoutfiber (WF, *) and with 0.14 and 1% of cellulose (Cell; B, C), pepper pectin 1 (PP1; ,), pepper pectin 2 (PP2; ,
▫
), pepper pectin 3 (PP3; , ) and cholestyramine (Chol;, ▽).
B. Cervantes-Paz et al. / Food Hydrocolloids 60 (2016) 580e588 583
hydrophilic and hydrophobic groups of galacturonic acid moleculesin PP3. In this pectin, the carboxyl groups were highly esterified andtherefore such pectin was more hydrophobic, while PP1 and PP2were more hydrophilic. It has been suggested that low DME pec-tins, like PP3, commonly show a block-wise orientation (Verrijssenet al., 2015).
The intestinal medium with Cell at low concentration exhibiteda bimodal distribution of particle size with classes of 1.3 and4.9 mm; however, at high Cell concentration the first class dis-appeared and a new class of 0.01 mm was observed. This class wasalso observed in micellar media at both Cell concentrations butmicellar medium with high Cell concentration contained an addi-tional minor class centered on 1.0 mm. The bimodal distribution ofparticle size might be consequence of the insolubility of Cell in thedigestive medium and its high length, as compared with that of oildroplets, causing the formation of networks that promoted theformation of additional sizes of lipid droplets, as reported byKalashnikova, Bizot, Cathala, and Capron (2011). In general, theparticle size decreased as the concentration of Cell increased.Similarly, Li, Al-Assaf, Fang, and Phillips (2013) demonstrated thatthe particle size of oil-in-water emulsions decreased as the con-centration of HPMC increased.
On the other hand, low concentration of Chol generated a tri-modal distribution of particle size (0.13, 1.3 and 4.9 mm) in the in-testinal medium; however, such medium showed an unimodaldistribution (0.4 mm) at high Chol concentration. The micellarmedium also showed an unimodal distribution at both Chol con-centrations but the particle size decreased from 0.15 to 0.004 mm asthe Chol concentration increased. The changes in droplet size dis-tribution might be attributed to differences in the surface interac-tion between tested fibers and oil droplets (Xu et al., 2014).Different particle sizes were observed in medium containing nofiber (0.09, 1.0 and 4.9 mm). Similarly, Espinal-Ruiz et al. (2014a)
detected several small particles in control emulsions withoutpolysaccharides (0.01 and 0.05e5.0 mm). Apparently, these parti-cles corresponded solely to mixed micelles composed by phos-pholipids, bile salts, and free fatty acids.
3.3. Binding of bile salts
The pure bile salts added to the digestion reactions (GDC, TDC,and TC) are among the most abundant of human bile(Chitchumroonchokchai et al., 2004). The bile salt binding by testedpectins decreased (28e47%) as pectin concentration increased(Fig. 3). Similarly, Cheewatanakornkool et al. (2012) also observedthat the capacity of apple pectin to bind sodium deoxycholatedecreased 20% as the pectin concentration increased from 0.1% to1%; however, such effect was not observed with citrus or pomelopectin. The causes of this phenomenon have not been explained butwe infer that under high pectin conditions the interaction betweenpectin chains increased, limiting the interaction with other com-ponents like bile salts.
At low pectin concentration, PP1 showed a similar capacity tobind TC and GDC (59e62%); however, its interaction with TDC waslower (42%). The binding capacity of the other pectins was inde-pendent of bile salt type, with PP2 showing a higher binding ca-pacity (80e83%) than PP3 (71e73%). At low concentration, thebinding capacity of these two pectins was even greater than that ofChol, which has an exceptionally high capacity to bind bile salts. Athigh concentrations of pectin, PP1 showed the lowest binding ca-pacity values (30e44%), while PP2 and PP3 presented similarbinding capacity values to TDC (51e52%) and GDC (47e50%). Cor-relation studies showed that the binding of TDC was influenced bythe MW (R2 ¼ "0.60), DME (R2 ¼ 0.68), and viscosity (R2 ¼ "0.60)of pectins. Dongowski (1995) demonstrated that the binding ofglycochenodeoxycholic acid depended on DME of pectin but such
0.14% fiber
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Fig. 2. Particle size distribution in intestinal and micellar media of digestion reactions without fiber (WF, ) and two concentrations (0.14 and 1%) of cellulose (Cell, ), pepperpectin 1 (PP1, ), pepper pectin 2 (PP2, ), pepper pectin 3 (PP3, ), and cholestyramine (Chol, ).
B. Cervantes-Paz et al. / Food Hydrocolloids 60 (2016) 580e588584
relationship was no lineal. The Chol (positive control) always pre-sented a higher bile binding capacity (52e87%) than Cell (3e21%) atboth concentrations. This effect has been attributed to the strongcapacity of Chol to bind ionic compounds like bile salts while Cell(negative control) lacks of such property (Rodríguez-Guti!errez,Rubio-Senent, Lama-Mu~noz, García, & Fern!andez-Bola~nos, 2014).
3.4. Lipolysis and lipid species incorporated into the micelles
The effect of concentration and fiber type on hydrolysis of thecarotenoid-rich soybean oil during intestinal digestion is shown inFig. 4. The amount of released FFA in digestions with 0.14% of fibershowed a steep rise during the first 60 min of digestion, whichgradually became slower with the time (Fig. 4A). This indicated thatthe oil digestion was almost completed during the first minutes ofdigestion (Espinal-Ruiz et al., 2014b). These results agreed with
those of Espinal-Ruiz et al. (2014a) and Hu, Li, Decker, andMcClements (2010), who evaluated the lipid digestion in emul-sions containing citrus pectin and observed a rapid release of FFAduring the first 5e25 min of digestion. PP3 (high DME) favored thegreatest release of FFA, followed by PP2 (medium DME) and PP1(low DME). The proportional effect of pectin DME on FFA releasingwas evident. However, Espinal-Ruiz et al. (2016) recently observedan opposite effect of DME of pectin on lipid digestion. In our study,the amount of released FFA seemed to be inversely related to theMW of tested pectins (PP3 < PP2 < PP1). The impact of the MW ofpectin on lipolysis has not been studied sufficiently. The lowestlipolysis was observed in digestions without fiber and with Cell.The digestion of beef patties and oil-in-water emulsions withoutfiber and with Cell also caused poor lipid hydrolysis (Hur, Lim, Park,& Joo, 2009). The lipolysis in digestion reactions with fibers at 1% isshown in Fig. 4B. Such fiber concentration did not affect the lipol-ysis in digestions with Cell; however, the amount of released FFA indigestions with pectin and Chol increased substantially. Thesefindings might be consequence of the trapping of newly releasedFFA by fibers and their removing from lipid droplet surfaces, wherethe lipolytic enzymes exert their activity. The quality of the surfacelipid droplets is an important factor for lipase activity (Tsujita et al.,1996). These events could probably avoided the reincorporation ofFFA into the triglycerides and allowed the progress of lipolysis(Polheim, David, Schultz, Wylie, & Johnston, 1973). Oil trapping by
Fig. 3. Binding of taurocholate (A), taurodeoxycholate (B) and glycodeoxycholate (C) indigestions without fiber (WF, ) and two concentrations (0.14 and 1%) of cellulose(Cell, ), pepper pectin 1 (PP1, ), pepper pectin 2 (PP2, ), pepper pectin 3(PP3, ), and cholestyramine (Chol, ).
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Fig. 4. Free fatty acids (FFA) released during digestion reactions without fiber (WF, )and two concentrations (0.14 and 1%) of cellulose (Cell, ), pepper pectin 1 (PP1,
), pepper pectin 2 (PP2, ), pepper pectin 3 (PP3, ), cholestyramine (Chol,).
B. Cervantes-Paz et al. / Food Hydrocolloids 60 (2016) 580e588 585
fibers also could improve the distribution of fat in the digestivereaction and, consequently, the activity of pancreatic lipase(Espinal-Ruiz et al., 2014a). On the other hand, some pectins areable to exhibit emulsifying properties, which could additionallyplay an important role on triglyceride hydrolysis (Leroux et al.,2003). At the high concentration level, the impact of the pectintype on lipolysis followed the order described above for digestionswith the low fiber concentration, confirming the roles of DME andMW of pectin on lipolysis.
The amount of lipid species incorporated into the micellesdepended on the pectin type and concentration (SupplementaryTable 1). However, the incorporation of lipid species into the mi-celles always followed the order of TG < DG < FFA þ MG. Theamount of FFA þ MG in micelles from digestion reactions withpectin represented more than 82% of total micellarized lipids.Independently of pectin concentration, PP1 and PP2 favored morethe micellarization of FFA þ MG than PP3 but PP3 always favoredmore the micellarization of DG than the other pectins. This differ-ential effect of tested pectins on lipid micellarization might beconsequence of their differences in the DME of pectins, which canfavor electrostatic repulsions between pectin chains and bile saltsin the micelles or hydrophobic interactions between pectin and theactive surface of micelles, favoring the micellarization of specificlipid species as a function of DME of pectin (Verrijssen et al., 2015).The polarity of the different lipid species and polarity of pectins,which depend on their DME, might also be involved in such phe-nomenon (Walter & Sherman, 1984).
3.5. Carotenoid bioaccessibility
In general (pooling of data), the micellarization efficiency fol-lowed the order of esterified carotenoids < b-carotene < free xan-thophylls. Similar findings have previously been reported(O'Sullivan et al., 2010; Victoria-Campos et al., 2013) and attributedto carotenoid polarity and their distribution in the lipid dropletsduring digestion (Borel et al., 1996; Victoria-Campos et al., 2013). Ingeneral, Cell and pectin at low concentration kept constant orincreased carotenoid micellarization, as compared with digestionswithout fiber (Fig. 5). At low fiber concentration, the carotenoidmicellarization was higher with PP1 than with the other pectins.The micellarization for many carotenoids with PP1 was higher thanthat observed without pectin, suggesting that at low pectin con-centration some pectins can improve carotenoid bioavailability.These findings indicated that low concentration of pectin with lowMW and DME reduce the negative effect of pectin on carotenoidmicellarization.
Pectin at high concentration significantly reduced the carot-enoid micellarizationwhile Cell increased even more such variable.Similarly, Ornelas-Paz et al. (2008) demonstrated that the micel-larization of b-carotene decreased significantly (35e43%) as theamount of pectin in the digestion reactions increased from 108 to225 mg/mL. Electrostatic and hydrogen-bonding interactions be-tween carotenoids and cellulose were probably responsible of theincreased micellarization of carotenoids in digestions with Cell(Ribas-Agusti, Van Buggenhout, Palmero, Hendrickx, & Van Loey,2014). The increase of pectin concentration in digestion reactionschanged the effect of this polymer on carotenoid micellarization. Athigh pectin concentration, the carotenoid micellarization washigher with PP3 than with the other fibers. These findingsdemonstrate that the effects of pectins on carotenoid micellariza-tion depend at the same time on pectin properties and amount. ThePCA was performed in order to determine how the pectin amountand properties affected the micellarization of carotenoids. The PCAscores showed that the first two principal components (PC1 andPC2) explained 59.9% and 17.7% of data variation. According to the
treatment distribution (Fig. 6), the main variation of data (PC1) wasexplained by pectin concentrationwhile pectin type explained PC2.This reveled the higher relevance of pectin amount than pectinproperties on carotenoid micellarization. The negative effect ofpectin concentration on carotenoid micellarization has beendemonstrated previously (Ornelas-Paz et al., 2008). The data forviscosity, released FFA, and particle size distribution in differentmedia (gastric, intestinal or micellar) were located in the left side ofthe plot, close to digestions with high pectin concentration, which,in general, increased the values of these variables in comparisonwith digestion reactions with low concentration of pectin (rightside). However, according to PC2, the PP3 at high concentrationfavored lipolysis while the effect of PP2 at high concentration wasmore related with the particle size distribution in the intestinal andmicellar phases as well as with the viscosity of gastric and intestinalmedia. In addition, the viscosity of the intestinal phase was alsocorrelated to the effect of PP1 at high concentration, probablybecause of the high MW of such pectin. The localization of PP1(both concentration levels) at the bottom of the plot supported thispositive association between viscosity and MW. The MW of pectinhas been related to the viscosity of pectin solutions (Leroux et al.,2003). Additionally, high MW of pectin (like PP1) seems to beinvolved on micellarization of b-carotene and mono and die-sterified xanthophylls, which were located on in the fourth quad-rant of the plot, close to MW and viscosity. On the other hand, thecapacity of pectins to bind TC, TDC, and GDC was located on theright side of the plot, close to digestions with low concentration ofpectin, demonstrating the involvement of low pectin concentra-tions on this variable. Bile binding data were close to PP2 and PP3,agreeing with data of Fig. 1, which showed that PP2 and PP3exhibited the greater binding capacity as compared with PP1.Furthermore, binding capacity of pectin for three bile salts wasclose to the DME, demonstrating the involving of this pectinproperty on bile binding, as suggested by Dongowski (1995). Thesefindings collectively demonstrated the involvement of pectinamount and properties on some events involved on carotenoidmicellarization.
Tested carotenoids, excepting zeaxanthin myristate, werelocated on the right side of the plot, indicating a positive associationbetween low concentrations of pectins and carotenoid bio-accessibility. However, this effect also depended on carotenoidpolarity. The effect of carotenoid polarity on micellarization effi-ciency has been demonstrated in experiments with complex foodmatrixes (Victoria-Campos et al., 2013). According to PC2, zeax-anthin and capsanthin, which were the most polar of tested xan-thophylls, were close to PP3 and PP2 (positive side) while theremaining carotenoids (less polar) were located at the bottom ofplot, close to PP1. These distributions demonstrated that themicellarization of polar carotenoids was less affected by bilebinding than that of the less polar carotenoids. This analysis alsoindicated that the capacity of pectin to bind bile was positivelyrelated to the micellarization of carotenoids but negatively relatedwith the releasing of FFA, viscosity of the gastrointestinal media,and particle size distribution. Finally, we did not find an effect of thecomposition of lipid species in micelles on carotenoid bio-accessibility. It has been reported that the micelles mainly containFFA (Martin, Nieto-Fuentes, Se~nor!ans, Reglero,& Soler-Rivas, 2010).Recently, Verrijssen et al. (2015) demonstrated that the micellari-zation of FFA favored the incorporation of b-carotene into the mi-celles. The amount of this lipid type in the micelles might beinvolved on carotenoid bioaccessibility in our study; however, theseparation of FFA and MG was not achieved and therefore the in-dividual effect of each of these lipid species could not be deter-mined. Some studies have separately demonstrated that the pectinamount and properties were involved on bile salt binding, lipid
B. Cervantes-Paz et al. / Food Hydrocolloids 60 (2016) 580e588586
droplet size distribution, viscosity and lipid digestion in oil-in-water emulsions subjected to digestion (Cheewatanakornkoolet al., 2012; Espinal-Ruiz et al., 2014a,b; Kaltsa et al., 2014; Liet al., 2013; Rubio-Senent et al., 2015; Xu et al., 2012, 2015; Zhaoet al., 2015). However, in only one of these studies such events,excepting bile binding by pectin, were related to themicellarizationof b-carotene (Verrijseen et al., 2015). In this work, the effect of theamount and properties (chemical and rheological) of pectin on suchevents were studied and related to the micellarization of severalfree and esterified carotenoids.
4. Conclusions
The amount and properties of pectin affected several eventsinvolved on carotenoid micellarization. High pectin concentrationsfavored the increase of the viscosity of the gastrointestinal mediumand larger particle sizes. On the other hand, low pectin concen-trations favored bile salt binding and the highest carotenoidmicellarization. However, the effect of pectin amount was notproportional to carotenoid micellarization. The DME and MW ofpectin influenced substantially the carotenoid micellarization,depending on carotenoid polarity. Micellarization of free caroten-oids was favored by high DME through bile salts binding capacitywhile micellarization of the less polar carotenoids was influencedby high MW and viscosity.
Fig. 5. Micellarization of carotenoids during digestion reactions without fiber (WF, ) and two concentrations (0.14 and 1%) of cellulose (Cell, ), pepper pectin 1 (PP1, ),pepper pectin 2 (PP2, ), pepper pectin 3 (PP3, ), and cholestyramine (Chol, ). Zea-Myr: Zeaxanthin Myristate; Cap-Myr: Capsanthin Myristate; Cap-Di-Myr: CapsanthinDimyristate; Cap-Lau-Myr: Capsanthin Laurate Myristate.
Fig. 6. Biplot of the principal component analysis for amount and properties of pectinand events involved on carotenoid micellarization. FFA: released free fatty acids;PS(MP): particle size in the micellar phase; PS(IP): particle size in the intestinal me-dium; visc(GP): viscosity in the gastric medium; visc(IP): viscosity in the intestinalmedium; BC(TC), BC(TDC), and BC(GDC): binding capacity to taurocholate, taur-odeoxycholate and glycodeoxycholate, respectively; zm: zeaxanthin myristate; c:capsanthin; z: zeaxanthin; aCr: a-cryptoxanthin; bCr: b-cryptoxanthin; bC: b-caro-tene; cm: capsanthin myristate; clm: capsanthin laurate myristate; cdm: capsanthin dimyristate; DME: degree methyl esterification, and MW: molecular weight; PP1, PP2,and PP3: Pepper pectin 1, 2 and 3 at two concentration levels (0.14 and 1%).
B. Cervantes-Paz et al. / Food Hydrocolloids 60 (2016) 580e588 587
Acknowledgments
This research was funded by the Fondo Sectorial de Inves-tigaci!on para la Educaci!on (Investigaci!on B!asica SEP-CONACYT;Project Clave: 103391). The authors thank Emilio Ochoa Reyes forhis technical assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.foodhyd.2016.04.038.
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153
CAPÍTULO V
IMPACT OF THE PHYSICOCHEMICAL PROPERTIES OF PECTIN ON
MICELLAR AND OIL PHASE LIPID COMPOSITION AND CAROTENOID
MICELLARIZATION
154
Impact of the physicochemical properties of pectin on micellar and oil phase lipid 1
composition and carotenoid micellarization 2
3
ABSTRACT 4
Lipids play an essential role on carotenoid micellarization but it is modulated by pectin 5
properties and amount. These effects of pectin on lipid hydrolysis during digestion and 6
their possible involvement on carotenoid micellarization are currently unclear. In this 7
study, carotenoid-rich oil was digested in vitro in presence of two concentrations of 8
pectins with different physicochemical properties. The lipid hydrolysis and carotenoids 9
micellarization were evaluated and related each other. Pectin at high concentrations 10
differentially modified the viscosity of intestinal media. TG mainly composed oil phase 11
while micellar phase was rich in FFA+MG. In general, the micellarization of carotenoids 12
was in the following order: free xanthophylls ˃ carotenes ˃ monoesterified xanthophylls 13
˃ diesterified xanthophylls; however, this order depended on pectin concentration. A 14
relationship between lipid types in the micellar phase and micellarization for some 15
carotenoid fractions was found. 16
17
Keywords: Lipid digestion; Lipolysis; Carotenoid fractions; Micellarization 18
19
155
1. Introduction 20
Pectins are polysaccharides of plant cell walls, including those of fruits and vegetables. 21
These polysaccharides are used as gelling agents, thickeners, texturizers, emulsifiers and 22
stabilizers in food systems (Sila et al., 2009). Currently, there is a growing interest in the 23
effect of pectin on lipid digestion and the effect of lipolyis on absorption of lipid soluble 24
phytochemicals (Espinal-Ruiz, Restrepo-Sánchez, Narváez-Cuenca, & McClements, 25
2016; Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, Narváez-Cuenca, & 26
McClements, 2014; Hu, Li, Decker, & McClements, 2010). Lipids play an important 27
role in human health since they facilitate the delivery and absorption of essential fatty 28
acids and lipid soluble nutrients (Failla, Chitchumronchokchai, Ferruzzi, Goltz, & 29
Campbell, 2014; McClements and Li, 2010). The absorption of lipids is modulated by 30
their hydrolysis during digestion and the products of lipid digestions are key components 31
of micelles, which are the absorptive vehicles for lipid-soluble phytochemicals. 32
Triglycerides (TG) are hydrolyzed into diglycerides (DG), monoglycerides (MG) and 33
free fatty acids (FFA), which are incorporated into micelles along with bile salts, 34
phospholipids and lipid-soluble nutrients, like carotenoids (McClements and Li, 2010; 35
Yonekura & Nagao, 2007). Carotenoids are involved on protective effects of human 36
health. Only micellarized carotenoids can be absorbed and, consequently, exert 37
biological actions (Biehler, Hoffmann, Krause, & Bohn, 2011). Carotenoid absorption is 38
improved by lipids (Failla et al., 2014). Colle, Van Buggenhout, Lemmens, Van Loey, & 39
Hendrickx (2012) demonstrated that the addition of lipids to raw tomato pulp increased 40
the in vitro bioaccessibility of lycopene. This effect depended on lipid type and amount 41
present during digestion. Huo, Ferruzzi, Schwartz & Failla (2007) observed that the 42
156
positive effect of lipids on micellarization of α-carotene, β-carotene, and lycopene 43
depended on the length of the fatty acid acyl chain but not on the unsaturation degree. 44
Victoria-Campos, Ornelas-Paz, Yahia, and Failla, (2013) showed that micellarization of 45
carotenoid esters was increased by unsaturated fat; however, the impact of fat on the 46
micellarization of free carotenoids seemed to be dependent on pigment structure. Failla 47
et al. (2014) found similar findings for β-carotene and lutein. 48
Carotenoid micellarization depend on several factors, mainly on viscosity of surrounding 49
medium and lipid digestion (TG, DG, MG, and FFA). These factors are affected by 50
pectin amount and properties, such as molecular weight (MW), degree of methyl 51
esterification (DME), and viscosity. The effect of pectin properties on lipid digestion has 52
been evaluated using PH-STAT method (Espinal-Ruiz et al., 2014; 2015; Hu et al., 53
2010). This method provided an estimation of lipid digestion, assuming that pH of 54
medium becomes acid by FFA releasing during digestion. Recently, Verrijssen, 55
Verkempinck, Christiaens, Van loey, & Hendrickx, (2015) studied the incorporation of 56
fatty acid and β-carotene to micelles affected by pectin properties. However, the 57
composition of TG, DG, MG, and FFA xfractions and their relation with carotenoids 58
micellarization was not well established. Therefore, the aim of the study was to 59
determine the role of pectin properties (DME and MW) on lipolysis (TG, DG, and 60
FFA+MG fractions) during in vitro digestion and the possible relation of lipolysis with 61
micellarization of free and esterified carotenoids. 62
Materials and Methods 63
1.1 Materials 64
157
Cholestyramine (Chol), cellulose (Cell), bile salts (taurocholate, taurodeoxycholate, and 65
glicodeoxycholate), lipid standards (tri-, di- and mono-glycerides and free fatty acids) 66
and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Three 67
pectins from jalapeño peppers (P1, P2 and P3) were extracted and characterized 68
according to Ramos-Aguilar et al. (2015). They showed similar protein content (1.7%), 69
but differences in the viscosity of model solutions, molecular weight (MW) and degree 70
of methyl esterification (DME) (P1: viscosity = 0.37 Pa.sn, MW = 4942 KDa, DME = 71
40%; P2: viscosity = 0.25 Pa.sn, MW = 3580 KDa, DME = 68%; P3: viscosity = 0.15 72
Pa.sn, MW = 2421 KDa, DME = 80%). A mixture of free and esterified carotenoids was 73
obtained from red peppers and dissolved in soybean oil purchased from local market. 74
2.2 In vitro digestion 75
The pectins were dissolved at two concentration levels in saline by stirring for 12 h and 76
then 1.25 mL of the soybean oil were added and mixed until emulsification of the oil 77
(Homogenizer Ika T18 Basics; IKa works Inc.). The concentration of pectin in the final 78
reaction was 0.14 and 1% (low and high concentration, respectively). These emulsions 79
were subjected to an in vitro digestion. Digestions without pectin and with Cell and Chol 80
were included as control reactions. The digestions were carried out according to 81
Victoria-Campos et al. (2013). For the gastric phase, the pH of the mixture was adjusted 82
to 2 using HCl and then porcine pepsin (1.6 mg/mL) was added. Digestions were 83
incubated at 37 °C for 1 h/95 rpm. For the intestinal phase, the pH of the reaction was 84
increased to 6.0 with sodium bicarbonate and then pancreatin/lipase (400/400 mg/L) 85
were added. Taurocholate taurodeoxycholate, and glycodeoxycholate were added to the 86
reaction, providing final concentrations of 0.75, 0.45, and 0.80 mmol/L, respectively 87
158
(Chitchumroonchokchai, Schwartz, & Failla 2004). The pH was increased to 7.0 using 88
1N NaOH and the reactions were incubated for 2h at above conditions. 89
After completion of the digestion process, aliquots of chyme (10 mL) were centrifuged 90
(15000g/20 min/4 °C) (centrifuge Allegra 64R, Beckman Coulter Inc., IN, USA) to 91
separate the micellar and oil phases. The micellar phase was recovered, filtered (0.22 µm 92
pore size; Millipore Corp., MA, USA) and stored under nitrogen gas at −70 °C until 93
analysis for lipids and carotenoid micellarization. 94
2.3 Apparent viscosity 95
The apparent viscosity of gastric and intestinal digestions was measured using an AR 96
1500ex rheometer (TA Instruments; New Castle, DE, USA), equipped with the stainless 97
steel parallel plate geometry (60 mm diameter) at 37 °C. The shear rate ranged from 0.1 98
to 100 s−1 (up curve) and from 100 to 0.1 s−1 (down curve). Shear rate against shear 99
stress data were fit using the power law model (τ = kγn) and analyzed for flow behavior, 100
n, and consistency index, k. 101
2.4 Determination of lipids 102
Once micellar phase was recovered, oil from tube walls was also recovered by washes 103
with hexane and adjusted to a known volume. This was the oil phase. Lipids from 104
micellar phase were extracted by adding 6 mL of diethyl ether/heptane/ethanol (1: 1: 1, 105
v/v) to 2 mL of micellar phase. The upper layer was recovered and the lower layer was 106
reextracted two times more as described above. Aliquots (20 µL) of these extracts were 107
injected into an Agilent 1200 Series HPLC system (Agilent, Palo Alto, CA, USA), 108
equipped with a 1260 infinity ELSD. The separation was carried out in a Luna C18 109
159
column (3.0 × 250 mm, 5 µm) with a C18 precolumn filter (Phenomenex). The mobile 110
phase was composed by acetonitrile (solvent A), acetone (solvent B), and 111
acetonitrile/methanol/water/THF; 75:12:8:5 (solvent C). The flow rate of the mobile 112
phase was 0.7 mL/min. The nebulization temperature was 30 °C. High-purity nitrogen at 113
70 °C was used for the evaporation at a flow-rate of 1.0 L/min. 114
2.5 Carotenoid micellarization 115
Carotenoids were extracted from micellar phase using a mixture of petroleum 116
ether/acetone (4 mL; 2:1 v/v) according to the procedure described by Victoria-Campos 117
et al. (2013). The carotenoids extracted were dissolved in acetone (2 mL), filtered 118
through a membrane of 0.45 µm of pore size and injected (20 µL) into an Agilent 1200 119
series HPLC system (Agilent, Palo Alto, CA, USA), equipped with a diode array 120
detector and a C 30 reversed-phase column (4.6 × 150 mm, 3 µm) (YMC Inc., Milford, 121
MA, USA). 122
2.6 Statistical analysis 123
All measurements were made at least in triplicate. The data were analyzed using a 124
completely randomized design with a factorial arrangement. The statistical significance 125
of the differences between treatments was determined using an ANOVA followed by the 126
Tukey-Kramer post hoc test; 0.05 was the significance limit. These tests were performed 127
using the SAS statistical software package Ver. 9.0 (SAS Institute Inc.; Cary, NC, USA). 128
2. Results and discussion 129
3.1 Apparent viscosity 130
160
The effect of concentration and type of fiber on viscosity of gastric and intestinal media 131
is shown in Figure 1. The viscosity of gastric media with high pectin concentration was 132
1.5–4 times higher than digestions with low pectin concentration. This effect was less 133
intense in the intestinal media, where viscosity was 1.7 times higher with high than with 134
low pectin concentration (Figure 1). The increases of viscosity, from 0.002 Pa.s to 0.007 135
Pa.s, has also been reported by Zhao, Wei, Wei, Yuang & Gao (2015) for emulsions 136
with 0.15% and 0.75% of bet pectin, respectively. This behavior was attributed to 137
bridging interactions of pectin and trapping of continuous phase by such polysaccharides 138
(Kaltsa, Paximada, Mandala, & Scholte, 2014). 139
The effect of pectin type on viscosity was less evident because significant differences 140
were only observed at high pectin concentration. Gastric media containing P3 presented 141
almost five times more viscosity than media with P2 and P3; however, intestinal 142
digestions containing P1 showed the highest viscosity values. A similar behavior was 143
reported by Logan Wright and Goff, (2015), who observed a higher viscosity in 144
emulsions containing low DME pectins (34%) after gastric digestion while the viscosity 145
of intestinal media was higher in emulsions containing pectins with high DME (72%). 146
The presence of methyl-esters as well as the length of galacturonan backbone of pectin 147
cause that the behavior of such polysaccharides be differently affected by pH of gastric 148
and intestinal media; thus, differences in these substituent groups could be the cause of 149
the observed behaviors. 150
3.2 Lipid composition 151
161
The lipid composition (FFA+MG, DG, and TG fractions) of oil and micellar phases is 152
shown in Figure 2. TG (40–60%) mainly composed the oil phase of digestions with low 153
pectin concentration. The content of DG and FFA+MG in such phase were similar, 154
except for emulsions with Chol. Tested pectins produced the lowest TG values (40–155
43%). At high pectin concentration, a slight decrease in the percentage of TG was 156
observed (4–6%); however, Cel and Chol caused higher decreases (13 and 41%, 157
respectively). An increase of FFA+MG fraction (5–8%) was also observed. Probably, 158
fiber at high concentrations conferred stability to the emulsion and, therefore, the 159
lipolysis was more efficient (Ornelas-Paz, Yahia, Gardea, & Failla, 2010). The 160
interfacial interaction between oil droplets and fibers was also weak, allowing the access 161
of the lipase to lipids (Mun, Decker, Park, Weiss, & McClements, 2006). 162
The micellar phase with low and high pectin concentrations showed elevated 163
percentages of FFA+MG. Media containing P1 and P2 exhibited similar content of 164
FFA+MG at low (94–95%) and high concentration (100%). The levels of FFA+MG 165
were lower in micellar phase from digestions containing P3. This indicated a lower lipid 166
digestion or incorporation into the micelles. Espinal-Ruiz et al. (2016) observed that 167
high DME pectins caused a lower release of FFA than low and medium DME pectins, as 168
P3 did it. Probably, non-polar and anionic carboxyl groups of high DME pectins 169
interacted with essential components (bile salts and calcium ions) for lipid digestion; 170
therefore, a lower releasing of FFA was observed (Espinal-Ruiz et al., 2014). The 171
micellar phase from digestions with high concentration of P1, P2, and P3 either did not 172
contain or contain low percentages of DG and TG (0.8–18%), indicating an almost 173
complete lipolysis. Espinal-Ruiz et al. (2014) observed that emulsions containing pectins 174
162
presented lipolysis of 50-80% during 2 h of digestion and such values were explained in 175
terms of the interaction of pectin with calcium ions, bile salts and phospholipids, which 176
could increase the viscosity of medium and cause accumulation of FFA in the lipid 177
droplet surface inhibiting lipolyis. In this work, the digestive media with pectin 178
presented a higher viscosity and therefore we infer that released FFA accumulated on the 179
lipid droplet surface. 180
181
3.3 Carotenoid micellarization 182
The effect of fiber type and concentration on micellarization of free xanthophylls (FX), 183
carotenes (CR), monoesterified xanthophylls (MX), and diesterified xanthophylls (DX) 184
is shown in Figure 3. The carotenoid micellarization in digestions with low pectin 185
concentration followed the order FX ˃ CR ˃ MX ˃ DX; however, this order was altered 186
by high fiber concentration. Similar findings have previously been reported (Victoria-187
Campos et al., 2013; Dhuique-Mayer et al., 2007), suggesting that the polarity of 188
carotenoids confers them a specific location into the emulsified lipid droplet, which also 189
vary their incorporation into the mixed micelles. At low pectin concentrations, the 190
highest micellarization of FX was caused by P3, followed by P2 and P1. The relative 191
content of DG in the micellar phase of such digestions followed the same order; 192
therefore, micellarization of FX and DG content in the micellar phase were related each 193
other. The micellarization of CR was similar with P1 and P2 while P3 caused lower 194
values. The micellar content CR FFA+MG followed the same order as a function of 195
pectin type (Figures 2 and 3), demonstrating that their micellarization might be related 196
163
each other. Verrijssen et al. (2015) observed a relation between the micellarization of β-197
carotene and FFA+MG, with medium DME pectin causing higher micellarization of 198
these compounds than low and high DME pectins. Their results were explained by a 199
delay of lipase action as a consequence of larger particle sizes caused by the interaction 200
of low and high DME pectins with oil droplets. However, the lack of data regarding 201
particle size in our study did not allow confirming such fact. Micellarization of MX and 202
DX was higher with P1 than P2 and P3 but a clear relation with lipid composition was 203
not found. Low DME pectin shows a low interaction with bile salts; therefore, a higher 204
formation of micelles can occur (Espinal-Ruiz et al., 2014; Verrijssen et al., 2015). Such 205
micelles could promote the incorporation of MX and DX. At high pectin concentration, 206
a clear relation between lipid composition and micellarization these carotenoid fractions 207
was no found; however, viscosity of intestinal phase could be directly related to MX 208
micellarization and inversely related to FX micellarzation in emulsions containing P1, 209
P2, and P3. 210
211
Conclusions 212
A possible link between viscosity and micellarization of different carotenoid fractions 213
might exist in digestions at high pectin concentration. Oil phase was mainly composed 214
by TG while micellar phase was rich in FFA+MG. Low DME pectin seems to favor the 215
hydrolysis of TG to FFA+MG. The relationship between lipid composition and 216
carotenoids micellarization was observed for FX and CR but not for MX and DX. The 217
relationship between lipid digestion and carotenoid micellarization was evident. 218
164
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291
292
168
Figure captions 293
294
Figure 1. Apparent viscosity of gastric and intestinal media with low and high fiber 295
concentration. Without fiber: WF ( ); cellulose: Cell ( ); pectin 1: P1 296
( ); pectin 2: P2 ( ); pectin 3: P3 ( ); cholestyramine: Chol ( ). 297
298
Figure 2. Lipid composition in oil and micellar phases of digestion reactions with 299
different fiber types at two concentration levels. Free fatty acid: FFA+MG ( ), 300
diglycerides: DG ( ), and triglycerides: TG ( ). 301
302
Figure 3. Micellarization of carotenoid fractions in digestion reactions with different 303
fiber types at two concentration levels. Free Xanthophylls ( ), Carotenes ( ), 304
Monoesterified Xanthophylls ( ), and Diesterified Xanthophylls ( ).305
158
1%0.14%
Ap
par
ent v
isco
sity
(Pa.
s)
0.0
0.2
0.4
0.6
0.8
1.0
Control
Gastric Phase
Ap
par
ent v
isco
sity
(Pa.
s)
0.00
0.05
0.10
0.15
0.20
0.25
0.30Intestinal Phase
1%0.14%Control
a aa a a
a
bb bb
a aa a a
cbc
bc
aba
Concentration
Concentration
Figure 1
161169
159
P1
WF
Fatty acids composition (%)
020406080100
Cel
lP
2P
3C
hol
P1
Cel
lP
2P
3C
hol
Low
Con
cent
ratio
nH
igh
Con
cent
ratio
n
Oil
Phas
e
Con
trol
Cel
lP
1P
2P
3C
hol
WF
Fatty acids composition (%)
020406080100
Cel
lP
1P
2P
3C
hol
Low
Con
cent
ratio
nH
igh
Con
cent
ratio
nC
ontro
lM
icel
lar P
hase
Fi
gure
2
162170
160
0.14% 1%
Mic
ella
rizat
ion
(%)
0
20
40
60
80
100
72% 71% 71% 74% 76% 75%
51% 68% 65% 68%
14%12% 12%
12% 10% 11%
12%
8%8% 7%
9% 11% 10% 9% 9% 10%31% 22% 25% 24%
5% 6% 7% 5% 5% 4% 6% 2% 2% 1%
WF Cell P1 P2 P3 Chol Cell P1 P2 P3 Chol
ConcentrationControl
Figure 3
163171
