Cromatografía

Centro de Investigación en Alimentación
y Desarrollo, A.C.
FORMACIÓN Y CARACTERIZACIÓN DE
NANOPARTÍCULAS DE LA FRACCIÓN DE ALBÚMINAS
DE SALVADO DE TRIGO POR EL MÉTODO DE
DESOLVATACIÓN
_________________________________________
Por:
Jesús Guadalupe Luna Valdez
TESIS APROBADA POR LA COORDINACIÓN DE
TECNOLOGÍA DE ALIMENTOS DE ORIGEN VEGETAL
Como requisito parcial para obtener el grado de
DOCTOR EN CIENCIAS
Hermosillo, Sonora, México Agosto de 2017

APROBACIÓN
Los miembros del comité designado para la revisión de la tesis de Jesús Guadalupe Luna
Valdez, la han encontrado satisfactoria y recomiendan que sea aceptada como requisito
parcial para obtener el grado de Doctor en Ciencias.
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DECLARACIÓN INSTITUCIONAL
3
AGRADECIMIENTOS
Al Consejo Nacional de Ciencia y Tecnología (CONACYT, México), por el apoyo

económico brindado para cubrir los gastos durante mis estudios de doctorado.
Al Centro de Investigación en Alimentación y Desarrollo (CIAD, AC), por brindarme la

oportunidad de realizar mis estudios de doctorado en dicha institución.
Al Consejo Nacional de Ciencia y Tecnología (CONACYT, México), por el

financiamiento del proyecto CB-2011/169839 a cargo del Dr. René Balandrán, para la
ejecución y desarrollo de esta tesis.
A Dios y a la Virgen María, por guiarme siempre por el buen camino.
A mi director de tesis, el Dr. René Renato Balandrán Quintana por darme la oportunidad
de formar parte de su equipo de investigación. Además, por la buena disposición que
mostró siempre para atender las dudas, comentarios e imprevistos que surgían en su
momento y sobretodo, agradezco el gran profesionalismo que siempre tuvo y con el que
trató todos los asuntos académicos, de investigación y personal con un servidor.
A mi comité de tesis por compartir sus conocimientos en cada semestre que se le

solicitó, los cuales contribuyeron en gran medida a la investigación realizada, así como a
mi formación académica. A todos ustedes gracias, Dr. René Renato Balandrán Quintana,
Dra. Ana María Mendoza Wilson, Dra. Gabriela Ramos Clamont Montfort, Dr. Agustín
Rascón Chu, Dr. José Antonio Azamar Barrios y al Dr. Tomás Jesús Madera Santana.
Además, agradezco a la Dra. Gabriela Ramos por la facilidad de usar los equipos del
Laboratorio de Bioquímica de Proteínas y Glicanos de la Coordinación de Ciencia de los
Alimentos y también al Dr. José Antonio Azamar por las facilidades brindadas en los
laboratorios de CINVESTAV─Unidad Mérida, Yucatán.
Un agradecimiento muy especial a la M.C. Dora A. Huerta Quintanilla y a la Biol. Ana

Ruth Cristobal Ramos, por los análisis de SEM-EDX, JSM-7600F. También, al M.C.
Daniel Aguilar Treviño por su asistencia técnica en los análisis de difracción de rayos X,
todos ellos personal técnico del CINVESTAV─Unidad Mérida, Yucatán.
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A la Coordinación de Programas Académicos de CIAD, por su apoyo técnico, en
especial a su personal: Argelia Marín, Verónica Araiza, Laura García, Aurora Vidal y
Héctor Galindo.
A todos los investigadores que de alguna u otra manera fueron parte y contribuyeron a
este trabajo de investigación. A los miembros de la Coordinación de Alimentos de
Origen Vegetal (CTAOV) dirigida por la Dra. Alma Rosa Islas, investigadores y
compañeros de área. A los maestros que me impartieron clases durante mi doctorado, los
doctores Alma Rosa Islas, Ana María Calderón de la Barca, Dra. Herlinda Soto, Dra.
Gabriela Ramos, Dra. Elizabeth Carvajal, Dra. Tere Gollas, Dr. René Balandrán, Dr.
Tomás Madera, Dr. Miguel Ángel Martínez, Dr. Mario Camberos, Dr. Jaime Lizardi,
Dr. Agustín Rascón, Dr. Alberto González. Además de un agradecimiento muy especial
al Dr. Ángel Huerta de la Plataforma Analítica Institucional del CIAD por su apoyo en
asesoría sobre los protocolos de preparación, obtención y disección de geles SDS-PAGE
para la identificación de proteínas mediante espectrometría de masas.
Al M.C. Jorge Mercado por el gran apoyo técnico y académico que recibí de su parte
desde el primer día en que le conocí. También por sus buenos consejos, tanto
académicos como personales.
A Lupita Chaquilla, con quien compartí los logros y fracasos que tuvimos durante todo
el tiempo que estuvimos trabajando en equipo. Además, por esa gran amistad que me
brindó desde el primer momento en que la conocí, la cual se fortaleció en el día a día el
trabajo de laboratorio. Gracias también, por todo ese cariño y aprecio que compartió con
mi familia, su familia.
A mis compañeros de laboratorio: Lupita, Javier Carmelo, Iván Torres, Jorge Zavala,
con quienes pasé momentos muy gratos durante todo el tiempo que estuve compartiendo
el laboratorio con ustedes.
Al Ing. Francisco Javier Noriega Muñoz, quien desde el principio me impulsó a

emprender esta aventura. Gracias por todo.
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DEDICATORIA
A Dios, por guiarme día a día en el buen camino y por todas sus bendiciones otorgadas.
A mis padres, Guadalupe Luna Meza y Ana María Valdez Ruelas por su apoyo
tanto económico como moral, el cual lo realizaron incondicionalmente durante toda mi
formación profesional.
A mi esposa Jazmín e hijos (José, Luna y Lupita) por su comprensión, apoyo y

paciencia durante este largo camino, el cual, estuvo lleno de aventuras.
A mi abuela, Elvira Ruelas Alcaraz, porque fue parte fundamental desde el primer
momento que salí en busca de la superación académica.
A mis hermanas Ana María e Irlanda por su apoyo en todos los aspectos y por todas las
alegrías que hemos pasado juntos.
A mi tía Ramona Reina y mi tío Alejandro Bermúdez por su grato recibimiento a mi

llegada a Hermosillo y por todas las atenciones recibidas de su parte.
A mi cuñado Arturo (el muñe) por su apoyo brindado.
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El presente trabajo fue realizado en la Coordinación de Tecnología de Alimentos de
Origen Vegetal del Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD),
bajo la dirección del Dr. René Renato Balandrán Quintana. Se contó con el apoyo
económico del Consejo Nacional de Ciencia y Tecnología (CONACYT) para el
proyecto CB-2011/169839: “Autoensamblaje de estructuras supramoleculares a partir de
péptidos liberados de la fracción de albúminas de salvado de trigo mediante proteólisis”
a cargo del Dr. René Renato Balandrán Quintana. Se reconoce la participación del
Laboratorio de Química de Materiales CINVESTAV-IPN Unidad Mérida Yucatán, y de
los laboratorios LANNBIO, proyectos FOMIX-YUCATAN 2008-108160 y CONACYT
LAB-2009-01 No. 123913, así como de la Plataforma Analítica Institucional del CIAD.
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CONTENIDO
APROBACIÓN………………………………………………………..……… 2
DECLARACIÓN INSTITUCIONAL………………………………………. 3
AGRADECIMIENTOS…………………………………………………….. 4
DEDICATORIA…………………………………………………………….. 6
CONTENIDO……………………………………………………………….. 8
RESUMEN………………………………………………………………….. 9
ABSTRACT..………………………………………………………………… 11
SINOPSIS….………………………………………………………………… 13
BIBLIOGRAFÍA……………………………………………………………. 26
1. Structural and physiochemical characterization of nanoparticles
synthesized from an aqueous extract of wheat bran by a cold-set
gelation/desolvation approach……………………………………………….. 32
2. Nanoparticle formation after mild thermal conditioning of proteins in
a wheat bran extract fractioned by size exclusion chromatography……… 42
3. FTIR analysis of thermally-induced nanoparticles from a fraction of

water soluble wheat bran proteins, to elucidate the formation
mechanism…………………………………………………………………….. 76
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RESUMEN
Se evaluó la factibilidad de sintetizar nanopartículas a partir del extracto acuoso de

salvado de trigo (EAST) mediante la técnica de desolvatación/gelación en frío. Para ello
se aplicó un pre-tratamiento térmico de 68.5 °C al EAST en solución, seguido de la
adición de CaCl2 y liofilización. Las micrografías de microscopía electrónica de barrido
(MEB) mostraron nanopartículas esféricas. El análisis químico y de espectroscopía
infrarroja indicó la naturaleza proteica de las nanoesferas, además de estar estabilizadas
por interacciones electrostáticas proteína-calcio e inmersas en una matriz de
polisacáridos. Para corroborarlo, las proteínas del EAST se purificaron parcialmente
mediante cromatografía de exclusión por tamaños (SEC), obteniéndose 6 fracciones con
masas moleculares 0.4─94 kDa. El análisis de proteína disuelta y de espectroscopía UV
e IR mostró la presencia de carbohidratos en las fracciones, y que dos de ellas estaban
compuestas por proteína, dos por una mezcla proteína-ácido ferúlico, otra por proteína
más otros compuestos no identificados y la última por ácido ferúlico libre. Cada fracción
SEC se sometió a desolvatación/gelación en frío. El análisis de MEB y la electroforesis
SDS-PAGE, mostraron que la fracción proteica 20─43 kDa es responsable de la
formación de nanoesferas con diámetros entre 190─250 nm. Estas últimas también se
observaron en la muestra control, a la que no le fue añadido Ca2+, por lo que se asumió
que el calcio no intervino en su formación sino que se produjeron por efecto del
tratamiento térmico. La ausencia de Ca2+ en las nanoesferas se verificó por
espectrometría de energía dispersiva de rayos x. Los resultados también mostraron que
las nanopartículas se formaron por la integración de nanoesferas de 30 nm de diámetro,
visibles después de un tratamiento ultrasónico. El análisis de la banda Amida I (1700-
1600 cm-1) del espectro infrarrojo de las nanopartículas mostró una disminución del 46%
de hojas β y un incremento del 33% en la formación de agregados, lo que indicó
agregación intermolecular a través de hojas β en las nanopartículas. Con base en los
resultados y el alto contenido del aminóacido cisteína reportado en las proteínas
identificadas en la fracción 20─43 kDa, se propuso el siguiente mecanismo de
formación de las nanoesferas. El tratamiento térmico indujo la formación de agregados a
través de enlaces disulfuro e interacciones moleculares tipo hoja beta, creciendo hasta
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alcanzar un diámetro de 30 nm. Posteriormente se integraron mediante interacciones
físicas (hidrofóbicas, van der Waals, electrostáticas) hasta formar los agregados
esféricos de mayor tamaño.
Palabras clave: nanopartículas, salvado de trigo, cromatografía preparativa, tecnología

de cereales, espectroscopía infrarroja, nanotecnología de proteínas.
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ABSTRACT
The feasibility of synthesizing nanoparticles from the wheat bran aqueous extract
(EAST) was evaluated by the desolvation /cold gelation technique. For this, a thermal
pretreatment of 68.5 ° C was applied to the EAST in solution, followed by the addition
of CaCl2 and lyophilization. Scanning electron microscopy (SEM) micrographs showed
spherical nanoparticles. The chemical analysis and infrared spectroscopy indicated the
protein nature of nanospheres, in addition to being stabilized by protein-calcium
interactions and immersed in a polysaccharide matrix. To corroborate it, the EAST
proteins were partially purified by size exclusion chromatography (SEC), obtaining 6
fractions with molecular masses 0.4─94 kDa. The analysis of dissolved protein and UV
and IR spectroscopies showed the presence of carbohydrates in the fractions and that
two of them were composed of protein; two for a protein-ferulic acid mixture; another
for protein plus other unidentified compounds and the last one by free ferulic acid. Each
SEC fraction was subjected to desolvation /cold gelation. SEM analysis and SDS-PAGE
electrophoresis showed that the 20─43 kDa protein fraction is responsible for the
formation of nanospheres with diameters between 190─250 nm. The latter were also
observed in the control sample, to which Ca2 + was not added, so it was assumed that
calcium did not intervene in its formation but that they were produced by the effect of
the heat treatment. The absence of Ca2+ in the nanospheres was verified by x-ray
dispersive energy spectroscopy. The results also showed that the nanoparticles were
formed by the integration of nanospheres of 30 nm diameter, visible after an ultrasonic
treatment. The analysis of the Amide I band (1700─1600 cm-1) of the infrared spectrum
of the nanoparticles showed a decrease of 46% of β-sheets and a 33% increase in
aggregate formation, which indicated intermolecular aggregation through β-sheets in the
nanoparticles. Based on the results and the high content of the amino acid cysteine
reported in the proteins identified in the 20─43 kDa fraction, the following mechanism
of nanosphere formation was proposed. The heat treatment induced the formation of
aggregates through disulfide bonds and -sheet molecular interactions, growing to a
diameter of 30 nm. Subsequently they were integrated by physical interactions
(hydrophobic, van der Waals, electrostatic) to form the larger spherical aggregates.
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Keywords: nanoparticles, wheat bran, preparative chromatography, infrared
spectroscopy, cereal technology, protein nanotechnology.
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SINOPSIS
El salvado de trigo es un subproducto de la industria molinera (Beaugrand et al., 2004)

que actualmente es subutilizado, aún y cuando contiene entre 15 y 22% de proteína
(Zhang et al., 2011). En el salvado, las proteínas se encuentran localizadas
principalmente en las células de la capa aleurona, protegidas por la pared celular
(Jerkovic, 2010), por lo que no son fácilmente digeribles, incluso por los animales. Las
albúminas y globulinas representan entre el 15-20% de la proteína total del grano de
trigo (Singh y Skerritt, 2001), de las cuales el 25% de las proteínas solubles en agua
(albúminas) se encuentran en la capa más externa, una de las siete capas principales que
conforman el salvado de trigo (Jerkovic, 2010), lo que significa que no es necesario de
un procedimiento complejo de extracción en el que se requiera el empleo de reactivos,
sino solo de agua. Es decir, las albúminas del salvado de trigo representan una fuente
importante de proteínas de bajo costo, a las cuales se les puede dar un valor agregado.
La composición química y la diversidad estructural de las albúminas de salvado de trigo
las hace candidatos para aplicaciones nanotecnológicas en las industrias alimentaria y
farmacéutica (Balandrán-Quintana et al., 2015).
Actualmente se elaboran nanopartículas a partir de fuente animal y vegetal, así como de
suero humano, las cuales tienen propiedades para encapsular y transportar fármacos y
compoeustos bioactivos (Gülseren et al., 2012; Zhang et al., 2012; Mehravar et al.,
2009). Para la elaboración de dichos nanotransportadores se emplean proteínas con
grados de pureza de al menos 90%, lo cual puede aumentar los costos de producción,
debido que se requieren de una mayor cantidad de operaciones unitarias para su
obtención. Por otro lado, también son escasos los esfuerzos para desarrollar
nanopartículas a partir de proteínas origen vegetal, a pesar de que en algunos casos estas
proteínas son más asequibles en el sentido de que forman parte de la composición de
residuos agroindustriales, además de ser más aceptables para el consumidor. Entre los
pocos ejemplos se encuentran los aislados de proteína de soya (Zhang et al., 2012), las
gliadínas de trigo (Arangoa et al., 2001; Joye et al., 2015) y las zeínas del maíz (Gomez-
Estaca et al., 2012). Por otro lado, no existen reportes sobre la elaboración de
nanopartículas a partir de proteínas que no hayan sido aisladas o purificadas. En ese
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sentido, existen fuentes proteicas de origen vegetal que no han sido consideradas, como
las albúminas del salvado de trigo, a pesar tener propiedades funcionales aceptables para
su uso en la industria de los alimentos (Idris et al., 2003). La fracción de albúminas del
salvado de trigo se caracteriza por un contenido importante de aminoácidos ácidos,
como el aspártico y el glutámico (Shewry et al., 2009), cuyos grupos carboxílicos
laterales desarrollan una carga negativa a pH mayor a 7.0. Esta característica imparte las
propiedades de solubilidad en agua de las albúminas en el estado nativo (Pace et al.,
2004), así como la susceptibilidad de formar nanopartículas a través de un enfoque de
desolvatación/gelación en frío y las posibilidades de adsorción de otras moléculas.
Además, las albúminas de salvado de trigo presentan una alta capacidad de enlaces
reversibles con moléculas hidrofóbicas, todo lo cual se considera ventajoso para la
elaboración de nanopartículas (Yedomon et al., 2013).
En el ARTÍCULO 1 de esta tesis se describe la formación de nanopartículas a partir de

extractos acuosos de salvado de trigo (EAST), mediante un enfoque de
gelación/desolvatación en frío. Este método consiste en precalentar una solución
proteica, seguida de un enfriamiento hasta temperatura ambiente y ajuste de pH a básico;
finalmente, se adicionan iones divalentes como el Ca2+ (Zhang et al., 2012). Dicho
proceso se considera como desolvatación, ya que los iones calcio compiten por las
moléculas de agua. Esto resulta en la eliminación de la capa de hidratación de las
proteínas y consecuentemente, la carga de estas se vuelve negativa debido a la presencia
en exceso de iones calcio, favoreciendo las interacciones electrostáticas proteína-calcio.
El empleo de calcio como agente de desolvatación y entrecruzante a la vez, evita el uso
de sustancias tóxicas, tales como solventes orgánicos (acetona y etanol) y glutaraldehído
para la elaboración de nanopartículas (Konan et al., 2002; Langer et al., 2003; Gülseren
et al., 2012; Yedomon et al., 2013).
En una etapa preliminar de la investigación se aplicó un tratamiento térmico de 68.5 °C
al EAST y se evaluó el efecto de diferentes concentraciones de CaCl2 y el pH sobre el
tamaño de partícula y el potencial-z de las estructuras resultantes, determinados
mediante dispersión dinámica de luz. Se determinó que la concentración de CaCl2 en el
intervalo 0.25, 0.5, 0.75, 1.0 y 1.5 M y un pH de 8, eran las condiciones más adecuadas
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para la obtención de nanopartículas. En este experimento, al agregar el CaCl2 se
obtuvieron dos fases, sobrenadante y precipitado. El contenido proteico siempre fue
mayor en los precipitados, lo cual sugirió que la agregación de proteínas fue inducida
por la desolvatación. El tamaño de los aglomerados precipitados fue directamente
proporcional a la concentración de CaCl2 en el rango 0-0.5 M, mientras que el potencial-
z se comportó de manera inversa; un efecto contrario se obtuvo en los sobrenadantes. En
un intervalo superior de concentraciones de CaCl2 (1.0-1.5 M) se obtuvieron
aglomerados hasta tres veces más grandes que las partículas de la muestra control. Esto
último probablemente se debió a una disminución en la repulsión entre partículas, y con
ello que la proximidad entre estas fuera mayor, lo que favoreció el entrecruzamiento
proteína-Ca2+.
La similitud del tamaño de partícula en la muestra control, tanto en el sobrenadante
como en el precipitado, demostró que la adición de CaCl2 favoreció la aglomeración y
que el pre-acondicionamiento no tuvo un efecto sobre la misma. Por otro lado, el
diámetro de partícula se mantuvo constante en el rango 0.25-1.0 M de CaCl2 y aumentó
significativamente en 1.5 M, lo cual pudo deberse a la mayor concentración de Ca2+ que
consecuentemente favoreció las interacciones intermoleculares. Por otro lado, la
disminución progresiva del potencial-z con respecto al aumento de la concentración del
CaCl2, corresponde con lo discutido anteriormente para el rango de concentración de
0.25-1.0 M de CaCl2 en ambas fases. No obstante, la carga en las dos fases
(sobrenadante y precipitado) a la máxima concentración evaluada, fue mayor, por lo que
no puede ser explicado en los términos antes discutidos, debido a que a tal
concentración, el número de cargas negativas restantes debería ser más baja, así que el
potencial-z tendría que ser menos negativo. Es posible que este efecto haya sido
ocasionado por un evento desconocido que inhibió a los iones calcio para formar
interacciones con proteínas, pero resultó en partículas grandes.
Después de analizar el tamaño de partícula y el potencial-z de las muestras en fresco
mediante dispersión dinámica de luz, las dos fases de cada muestra fueron congeladas y
posteriormente liofilizadas para su respectiva caracterización. Los polvos liofilizados
fueron analizados mediante microscopía electrónica de barrido (MEB), observándose la
presencia de nanopartículas de forma esférica con diámetros <100 nm en todo el rango
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de concentraciones de CaCl2 evaluado. Estas nanoestructuras se observaron inmersas en
una matriz compuesta principalmente por polisacáridos, de acuerdo con el análisis
químico. El diámetro de las esferas fue prácticamente el mismo dentro del rango 0.25-
1.0 M de CaCl2. Sin embargo, a la máxima concentración de CaCl2 evaluada, el
diámetro de partícula fue significativamente mayor debido a que se favoreció un mayor
número de interacciones intermoleculares. Estos resultados fueron similares a los
reportados por Zhang et al. (2012) quienes evaluaron el efecto del CaCl2 sobre la
formación de nanopartículas de aislados de proteína de soya; los autores encontraron que
cuanto mayor es la concentración de CaCl2 mayor es el tamaño de las partículas y la
cantidad de precipitado resultante, independientemente del pH y la concentración de
proteína. También, Gülseren et al. (2012) reportaron que un aumento de la concentración
del agente de desolvatación produjo nanopartículas de mayor tamaño.
Con el fin de analizar la estructura interna de las esferas formadas, el polvo liofilizado
de la muestra adicionada con CaCl2 0.25 M fue resuspendido en agua y sonicado durante
5 min con la intención de disgregar la matriz de polisacáridos (Czechowska-Biskup et
al., 2005). Posteriormente, la muestra fue visualizada mediante MEB en modo de
transmisión. Si bien no fue posible observar la estructura interna de las esferas, este
análisis permitió ver las nanopartículas de forma individual, las cuales formaron
estructuras con arreglo de tipo collar.
La presencia de minerales en el EAST tuvo un efecto negativo sobre la resolución de las
imágenes MEB, el cual fue mayor a la concentración 1.0 M. Este efecto pudo deberse a
la luminosidad producida por los electrones retrodispersados del Ca y otros minerales
como el P, K y Mg (Talbot y White, 2013), lo que indica que estos pudieran formar
parte de las nanoestructuras.
Debido a que a la concentración máxima de CaCl2 (1.5 M) se observó mediante MEB la
formación de lo que parecían ser cristales minerales, se realizó un análisis de difracción
de rayos-X en tres muestras seleccionadas (EAST, CaCl2 0.5 M y CaCl2 1.5 M). La
muestra EAST resultó amorfa, mientras que en la muestra CaCl2 0.5 M se detectó, por
indexación, la presencia de Na2CO3·H2O, el cual probablemente se generó al reaccionar
el sodio presente en la muestra con el CO2 atmosférico. A la concentración 1.5 M de
CaCl2 se encontró la formación de cristales tales como sylvita (KCl), brushita
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(CaHPO4·2H2O), weddellita (CaC2O4·2H2O) y fosfato de magnesio sódico
[Na6.13Mg1.44(PO4)3]. Lo anterior indica que a este nivel de concentración de CaCl2 la
nucleación y crecimiento de cristales prevaleció sobre el ensamblaje que conduce a la
formación de nanopartículas. El crecimiento de cristales es probablemente la explicación
de por qué se detectó una menor presencia de algunos minerales (Mg, P, K y Ca) en el
análisis de la composición elemental realizado mediante espectroscopía de energía
dispersiva de rayos X (EED) y la explicación de por qué el valor del potencial-z a esta
concentración de sal se volvió más negativo. Es posible que los minerales presentes
estuvieran más disponibles para el crecimiento de cristales que para el establecimiento
de las interacciones que llevan a la formación de nanopartículas, lo que resultó en un
menor número de cargas negativas neutralizadas sobre la superficie de las proteínas y así
en un potencial-z más negativo. Por lo tanto, el incremento en el tamaño de partícula se
debió a la formación de cristales y no a la aglomeración de la matriz que contenía las
nanopartículas.
Los resultados obtenidos en esta etapa de la investigación no solo permitieron establecer
los límites de concentración de CaCl2 para la obtención de nanopartículas sino mostrar
el potencial que tienen los EAST como plataformas para la mineralización inducida de
una variedad de minerales.
ARTÍCULO 2. En el artículo 1 se describió la obtención de nanopartículas a partir del

EAST mediante el método de desolvatación/gelación en frío, las cuales fueron
aparentemente estabilizadas mediante interacciones electrostáticas calcio-proteína. En
este artículo, se muestra nueva evidencia que indica que los iones calcio no participaron
en la formación de las nanopartículas, demostrando que éstas se formaron antes de la
adición de CaCl2 y que incluso no todas las proteínas que conforman el EAST
estuvieron involucradas en la formación de dichas nanoestructuras.
Se realizaron experimentos de desolvatación/gelación en frío con seis fracciones
resultantes de la separación de los extractos de salvado de trigo mediante cromatografía
de exclusión por tamaño (SEC). Cada fracción fue identificada de acuerdo al orden de
elución como pk-1, pk-Int, pk-2, pk-3, pk-4 y pk-5. Cabe destacar que la fracción pk-Int
no fue obtenida como un pico definido en el cromatograma, pero fue considerado como
17
una fracción debido a la cantidad importante de proteína que contenía. Las masas
moleculares relativas de las fracciones SEC resultaron entre 0.4-94 kDa. Estas
fracciones fueron liofilizadas y se les determinó el perfil de masas moleculares por SDS-
PAGE (electroforesis en gel de poliacrilamida con dodecilsulfato sódico). El rango de
masas moleculares (5-94 kDa) obtenida mediante electroforesis coincidió con el
encontrado en SEC. Rangos de masas moleculares similares ya han sido previamente
reportados en estudios con salvado de trigo (Jerkovic et al., 2010).
Previo a los experimentos de desolvatación/gelación en frío, a cada fracción SEC se le
determinó el contenido de proteína soluble (disueltas al 4%, peso-volumen) con el fin de
reproducir la relación calcio:proteína que resultó en la formación de nanopartículas en
los experimentos descritos en el artículo 1. La concentración de proteína osciló entre
0.04-12.6 mg/mL. Cabe destacar que la concentración de proteína en pk-4 fue muy baja
y en pk-5 no fue detectada. Es decir, la alta absorbancia registrada en el cromatograma
de SEC (Abs 280 nm) para estas dos fracciones, se debió principalmente a compuestos
fenólicos (Hromádková et al., 2013), ya que el contenido de éstos es alto en el salvado
de trigo (De Brier et al., 2015). La presencia de dichos compuestos también fue
confirmada por los barridos espectrofotométricos realizados a cada fracción en el rango
200-800 nm. De acuerdo con la evidencia obtenida, pk-Int y pk-2 resultaron estar
compuestas principalmente de proteína, pk-1 y pk-4 por una mezcla de proteína-ácido
ferúlico, pk-3 por proteína y otros compuestos no identificados, y pk-5 corresponde a
ácido ferúlico libre (Saulnier et al., 2008).
Posterior a la obtención y caracterización de las fracciones se procedió a realizar el
experimento de desolvatación/gelación en frío mediante la adición de CaCl2 bajo las
mismas condiciones empleadas en el experimento de desolvatación con EAST.
El pre-acondicionamiento térmico de las muestras SEC generó un cambio de apariencia
en pk-2 y pk-3 en los primeros dos minutos de exposición. En ambas fracciones se
formó un precipitado gelatinoso de color blanco, siendo mucho más visible en pk-2. La
gran cantidad de precipitado en pk-2 resultó interesante de analizar, por lo que se
sometió una nueva muestra (pk-2) a tratamiento térmico y el precipitado formado fue
separado mediante centrifugación. El sobrenadante fue recuperado, identificado como
pk-2CS (2) y desolvatado con CaCl2 al igual que el resto de las fracciones. La apariencia
18
del resto de las muestras no fue alterada por efecto del tratamiento térmico. Después de
la adición de CaCl2, dos fases fueron rápidamente detectadas en pk-2 y pk-3. En pk-1 y
pk-Int se obtuvo un pequeño precipitado, no así en pk-4 y pk-5. Las dos fases fueron
separadas mediante centrifugación y posteriormente liofilizadas. Todas las fracciones
desolvatadas fueron caracterizadas mediante espectroscopia infrarroja con transformada
de Fourier (FTIR), EED, SDS-PAGE y MEB.
Los espectros FTIR mostraron las bandas características de polisacáridos (región 1200-
800 cm-1) en todas las fracciones. También se encontraron las bandas más comúnmente
citadas en estudios estructurales de proteína (Pelton y McLean, 2000), es decir las
bandas Amida I (1600-1700 cm-1), Amida II (1600-1500 cm-1) y Amida III (1200-1300
cm-1) (Haris y Severcan, 1999; Barth, 2007). Además, se encontró una banda de gran
amplitud en la región de 3000-3600 cm-1, la cual corresponde a los estiramientos del
enlace O-H (Elisa et al., 2006)
En general, tanto en los sobrenadantes como en los precipitados, se encontraron las
mismas bandas. Sin embargo, en los precipitados se observó un cambio en la forma y un
ligero desplazamiento (1050 a 1017 cm-1) de la banda asignada a carbohidratos después
de la adición de CaCl2. Existe la posibilidad de que los arabinoxilanos, ácido ferúlico y
proteínas en cada una de las fracciones se encontraran formando complejos
carbohidrato-proteína mediante interacciones intermoleculares o entrecruzamientos a
través de sustituyentes de ácido ferúlico (Lapierre et al., 2001).
De forma particular, se obtuvieron los espectros IR del sobrenadante y el precipitado de
la muestra control de pk-2. Al igual que el resto de las fracciones, ambas presentaron
una banda en 1050 cm-1, la cual puede ser asignada a arabinoxilanos (Robert et al.,
2005). Además, dos bandas, una en 988 y la otra en 946 cm-1, están relacionadas con el
grado de substitución de arabinoxilanos (Robert et al., 2005), mismas que confirman la
presencia de este polisacárido. También se encontró en el espectro correspondiente a las
nanopartículas (precipitado de la muestra control de pk-2), que las bandas Amida I y
Amida II fueron más intensas y definidas en comparación con el resto de las fracciones.
También, en este mismo espectro se observó una disminución importante en la
intensidad de la banda asignada a polisacáridos, lo que sugirió que las nanopartículas
están compuestas principalmente de proteínas, a diferencia de las nanopartículas
19
obtenidas en el precipitado de la muestra desolvatada de pk-2, en la cual se obtuvo una
banda muy intensa asignada a carbohidratos. Esto sugirió que los polisacáridos presentes
en esta fracción no estaban fuertemente unidos a la fracción de proteínas involucradas en
la formación de nanopartículas, ya que estos no precipitaron con las nanoestructuras
formadas.
El análisis de composición elemental reveló la presencia de C, O, N, Na, P, S, Cl y Ca
en las fracciones. C y O fueron los elementos más abundantes debido a los altos niveles
de materia orgánica. La detección de N confirmó la presencia de proteína en las
fracciones, especialmente en las nanopartículas formadas, ya que en estas fue mayor.
Este elemento no tuvo mucha variación entre los sobrenadantes y precipitados de pk-1,
pk-Int o pk-3, lo que sugiere que las proteínas en esas fracciones no están involucradas
en la formación de nanopartículas, como fue evidente en las imágenes obtenidas en
MEB para estas tres fracciones. La presencia de Na y P probablemente se debió, en
parte, a residuos del bufer usado en la separación por SEC, mientras que el P también
podría provenir de fitatos presentes en el salvado de trigo (De Brier et al., 2015). El S
puede corresponder a residuos de aminoácidos azufrados, tal como la cisteína. En tanto,
la presencia de Cl en los sobrenadantes y precipitados proviene seguramente del agente
de desolvatación. El predominio del Ca en los precipitados de todas las fracciones, así
como la ausencia de éste en sobrenadantes, confirmó que el calcio estuvo involucrado en
los precipitados formados después de la desolvatación en esas fracciones.
Al comparar la composición de los precipitados de la muestra control de pk-2 y del
precipitado de la muestra desolvatada de pk-2, se observaron diferencias importantes
con respecto al contenido de Na, P, Cl y Ca. Es decir, las nanopartículas vistas en el
precipitado de la muestra control de pk-2 están formadas principalmente por proteínas, a
diferencia de las nanopartículas obtenidas en el precipitado desolvatado de pk-2, en el
cual se observó la presencia de estructuras coraloides, además de las nanopartículas. El
aumento en el porcentaje de S en el precipitado de la muestra control de pk-2 soporta la
presencia de proteínas, mientras que la ausencia de Ca en esta corrobora que el Ca no
participó en la formación de nanopartículas.
La morfología de las dos fases resultantes de cada fracción fue contrastada con su
muestra control. Los análisis de MEB mostraron que la muestra control de pk-1 tuvo una
20
superficie rugosa, mientras que la superficie del sobrenadante fue liso y el precipitado
presentó una morfología coraloide. En el sobrenadante y precipitado de pk-Int y pk-3, se
encontró una morfología similar a los de pk-1. Sin embargo, en la muestra control de pk-
3, se observó la presencia de estructuras esféricas y ovoides con tamaños entre 210 y
470 nm, respectivamente. Estas estructuras esféricas y ovoides, pudieron ser resultado
de la presencia de proteínas de masa molecular entre 20-31 kDa (SDS-PAGE), proteínas
de la misma masa están presentes en la muestra pk-2CP, las cuales formaron las
nanopartículas vistas en MEB. La forma de estas nanoestructuras puede deberse a la baja
concentración de estas proteínas (Ge et al., 2011).
Por otro lado, en el sobrenadante de la muestra desolvatada de pk-2, se obtuvo la
formación de partículas rocosas con diámetros entre 51-500 nm, mientras que en el
precipitado de esta misma muestra, se encontró la formación de estructuras esféricas con
diámetros entre 190-250 nm acompañadas de estructuras coraloides. Sin embargo, la
muestra control de pk-2, resultó positiva, ya que también se obtuvo la formación de
nanopartículas idénticas a las formadas en la muestra desolvatada (pk-2) con CaCl2
(precipitado). No obstante, en estas esferas no se observó la presencia de estructuras
coraloides como las vistas en el precipitado de pk-2. Debido a que en la muestra control
de pk-2 no se añadió CaCl2, la evidencia sugiere que las nanopartículas se formaron
antes de la adición de CaCl2 y que el acondicionamiento térmico fue el responsable de su
formación. Esto último se corroboró con las nuevas fases obtenidas luego de la
desolvatación de pk-2CS (2). En la imagen (MEB) del precipitado de esta muestra (pk-
2P2), se observó una morfología de tipo coral muy similar a la observada en los
precipitados de todas las fracciones evaluadas con excepción del control pk-2. Esto
significa que el calcio no está involucrado en la formación de nanopartículas, sino más
bien en la precipitación de estructuras coraloides, que de hecho podrían ser cristales
(Luna-Valdez et al., 2017).
En el ARTÍCULO 3 se analizaron los cambios conformacionales de las proteínas

involucradas en la formación de nanopartículas, generados por efecto de la temperatura.
Las nanopartículas se obtuvieron mediante un tratamiento térmico de proteínas
(albúminas) de bajo peso molecular (25-44 kDa) derivadas del salvado de trigo, las
21
cuales fueron liofilizadas y analizadas por MEB. Se obtuvieron nanopartículas de forma
esférica con diámetros entre 190-250 nm. A su vez, una porción de polvo de estas
nanopartículas fueron resuspendidas en agua deionizada y sonicadas durante 5 min, las
cuales posteriormente se analizaron por MEB en modo de transmisión. Esto permitió
distinguir que las nanoesferas están conformadas por nanopartículas de menor tamaño
(30 nm de diámetro). De acuerdo a lo reportado en la literatura, existen diferencias
importantes con respecto a los agregados proteicos por efecto de la temperatura
(albúmina de suero de bovino, β-lactoglobulina y aislados de proteína de suero) (Doi,
1993; Aymard et al., 1996; Ikeda y Morris, 2002; Lovedey, et al., 2010; Oboroceanu et
al., 2010; Lovedey et al, 2012; Borzova et al., 2016), ya que para lograr dichos
agregados se requiere del empleo de sales (Na o CaCl2) y de tiempos de inducción
térmica muy prolongados (20 h) a temperaturas de al menos 80 °C. A diferencia de las
condiciones empleadas para la obtención de las nanopartículas del presente trabajo,
donde solo fueron necesarias 3 h de inducción térmica a 68.5 °C sin el empleo de sales.
Se empleó la espectroscopía infrarroja de transformada de Fourier (FTIR) para el
análisis de la región Amida I de las proteínas (1700-1600 cm-1), ya que con esta técnica
es posible monitorear cambios sutiles en la conformación de la cadena polipeptídica
debido a que es muy sensible a cambios en los enlaces de hidrógeno (van Stokkum et al.,
1995; Natalello et al., 2005) y, por lo tanto, es útil para analizar modificaciones en la
estructura secundaria causadas por efecto de la temperatura o el pH (Vicent et al., 1984).
El análisis de la región Amida I permitió realizar una estimación cuantitativa de las
diferentes estructuras secundarias (hélices-α, hojas-β, vueltas y al azar) de las proteínas.
Dicho análisis se realizó aplicando la segunda derivada en la región 1700-1600 cm-1 del
espectro infrarrojo para resolver la banda en cuestión en dos o más señales que no son
visibles en el espectro original y poder así, identificar cada uno de los componentes de
las diferentes estructuras secundarias (Dong et al., 1992; Carbonaro y Nucara, 2010).
Para realizar lo anterior se asumió que la suma de las áreas de las conformaciones de la
Amida I, están relacionadas al total de la proteína dada (Byler y Susi, 1986; Kong y Yu,
2007). De dicho análisis resultó que las proteínas en forma nativa están compuestas
principalmente de conformaciones hojas-β con un 46%. El 18% son de giros-β y un 14%
22
a hélices-α, mientras que 16% corresponde a conformaciones aleatorias y el 5% restante
a agregados.
El cambio en la estructura secundaria de las proteínas involucradas en la formación de
nanopartículas por efecto del tratamiento térmico fue evidente, ya que la conformación
hojas-β disminuyó hasta 20% de la estructura secundaria. Estos resultados concuerdan
con lo reportado por Natalello et al. (2005), quienes reportaron una disminución del 50%
en las conformaciones hojas-β después de someter la proteína a una temperatura de 64
°C así como una disminución simultánea en hélices-α. En nuestro caso, las hélices-α
permanecieron constantes.
Por otro lado, las conformaciones de giro-β representaron el 11%, lo que significó una
disminución con respecto a las proteínas nativas. Las conformaciones aleatorias se
mantuvieron sin cambios. Además, el cambio conformacional de la estructura
secundaria fue acompañada por una banda de gran amplitud e intensidad en 1619 cm-1,
que representó el 38% y se debió a la formación de agregados intermoleculares por
hojas-β de proteínas, inducidos por el tratamiento térmico (Haris y Severcan 1999,
Seshadri et al., 1999, Yan et al., 2004, Natalello et al., 2005).
La formación de agregados por hojas-β intermoleculares puede deberse al hecho de que
el experimento se realizó a pH 8. Es decir, cuando se emplea un pH> pI de las proteínas,
la agregación procede a través de la formación de agregados ordenados relativamente
pequeños (oligómeros), los cuales se caracterizan por una cantidad considerable de
hojas-β intermoleculares, como se reveló por espectroscopia FTIR para BSA (Militello
et al., 2004).
Durante la estimación cuantitativa de las estructuras secundarias de las proteínas, se tuvo
en cuenta que los cambios conformacionales en las proteínas no solamente pueden ser
debidos al efecto de la temperatura, sino también a la pérdida de la capa de hidratación
después de la liofilización. Es decir, durante este proceso pueden producirse alteraciones
inducidas por la deshidratación en las bandas características del enlace amida. Sin
embargo, esto no excluye el uso de la espectroscopia FTIR para realizar dicha
estimación (van de Weert et al., 2001). Incluso, los posibles cambios que pudieron
haberse generado en la estructura de las proteínas por efecto de la liofilización debieron
23
haber sido mínimos ya que la morfología no fue afectada, de acuerdo a lo visto en las
imágenes de obtenidas en MEB (Griebenow y Klibanov, 1995).
Con base en los resultados obtenidos, además de información en la literatura, existe la
posibilidad de que las nanopartículas hayan sido formadas por enlaces disulfuro, debido
a la aparición de una banda de gran amplitud a 560 cm-1 vista en el espectro infrarrojo, la
cual puede ser asignada a la formación de enlaces disulfuro (Sadeghi et al., 2014). Para
soportar esto, las proteínas (20-43 kDa) que estuvieron involucradas en la formación de
nanopartículas son ricas en cisteína (Tabla 1, anexos), aminoácido que puede formar este
tipo de enlaces entre residuos vecinos (Doi, 1993). Dichas proteínas (albúminas del
salvado de trigo) han sido previamente identificadas mediante espectrometría de masas
por Chaquilla-Quilca et al. (2017) utilizando el método reportado por Huerta-Ocampo et
al. (2014). Es decir, durante el proceso de agregación térmica, las proteínas
experimentan un desdoblamiento parcial de su estructura nativa, la cual genera cambios
en la estructura tridimensional debido a la ruptura de enlaces de hidrógeno y grupos
hidrófobos no polares, lo cual causa que los grupos hidrofóbicos y los grupos SH libres
se vuelvan más expuestos y puedan formar enlaces disulfuro intermoleculares e
interacciones hidrofóbicas entre las cadenas proteicas desplegadas, resultando así en la
formación de agregados (Shimada et al., 1989; Vetri, Librizzi, Leone, & Militello,
2007).
Es probable que el mecanismo de agregación de las nanoestructuras obtenidas en este
trabajo sea el mismo que el propuesto por Aymard et al. (1996). Dicho mecanismo
consiste de agregación en dos etapas. En la primera se forman agregados de pequeñas
partículas (glóbulos) mediante intercambio disulfuro, el cual surge de monómeros
desnaturalizados que consecuentemente se asocian en dímeros. En la segunda etapa, los
glóbulos resultantes se agregan para formar estructuras fractales. De acuerdo a lo antes
mencionado, es posible considerar dicho mecanismo, ya que aparentemente las
nanoesferas están formadas por agregados más pequeños (30 nm de diámetro), que
podrían haberse formado por enlaces disulfuro (glóbulos), los cuales a su vez forman
agregados de mayor tamaño por interacciones físicas tales como las fuerzas de van der
Waals, enlaces de hidrógeno e interacciones hidrofóbicas o electrostáticas (Le Bon et al.,
1999)
24
La presencia de nanoesferas de 30 nm vistas en MEB, luego de resuspender las
nanopartículas en agua y sonicarlas durante 5 min, inclinan a considerar que las
interacciones agregado-agregado corresponden efectivamente a interacciones físicas, ya
que una parte de estas se desagregaron de las nanopartículas de mayor tamaño por efecto
del ultrasonido (Hu et al., 2013) a diferencia de las nanopartículas que no fueron
expuestas a este.
25
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31
ARTÍCULO 1
Structural and physicochemical characterization of nanoparticles

synthesized from an aqueous extract of wheat bran by a cold-set
gelation/desolvation approach
J.G. Luna-Valdez, R.R. Balandrán-Quintana, J.A. Azamar-Barrios, G. Ramos-Clamont

Montfort, A.M. Mendoza-Wilson, J.N. Mercado-Ruiz, T.J. Madera-Santana, A. Rascón-
Chu, G. Chaquilla-Quilca.
(2017)
Artículo publicado en
Food Hydrocolloids, 62:165-163
32
33
34
35
36
37
38
39
40
41
ARTÍCULO 2
Nanoparticle formation after mild thermal conditioning of proteins in a

wheat bran extract fractioned by size exclusion chromatography

Chu, G. Guadalupe Chaquilla-Quilca
(2017)
Artículo en revision en:
Biomacromolecules
42
Nanoparticle Formation after mild thermal conditioning of proteins in
a wheat bran extract fractioned by size exclusion chromatography
Authors:
Jesús G. Luna-Valdeza
René R. Balandrán-Quintanaa*
José A. Azamar-Barriosb
Gabriela Ramos-Clamont Montfortc
Ana M. Mendoza-Wilsona
Jorge N. Mercado-Ruiza
Tomás J. Madera-Santanaa
Agustín Rascón-Chua
Guadalupe Chaquilla-Quilcad
a
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de
Tecnología de Alimentos de Origen Vegetal. Carretera a La Victoria km 0.6. 83304.
Hermosillo, Sonora, México.
b
Centro de Investigación y de Estudios Avanzados del IPN, Unidad Mérida.
Departamento de Física aplicada. Carretera antigua a Progreso Km. 6. 97310. Mérida,
Yucatán. México.
c
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Ciencia
de Los Alimentos. Carretera a La Victoria km 0.6. 83304. Hermosillo, Sonora, México.
*Corresponding author: René Renato Balandrán-Quintana. Centro de Investigación en

Alimentación y Desarrollo, A.C. Coordinación de Tecnología de Alimentos de Origen
Vegetal. Carretera a La Victoria km 0.6. 83304. Hermosillo, Sonora, México. Tel. +52
662 2892400x354. e-mail: [email protected]
43
ABSTRACT
Spherical nanoparticles from protein-rich aqueous extracts of wheat bran have been
previously obtained through adaptations to the cold gelation/desolvation method.
Presumably, nanoparticles were formed by proteins, stabilized by calcium bridges and
immersed into a matrix of polysaccharides, but it was not demonstrated. In the present
work, cold gelation/desolvation experiments were performed with fractions resulting
from the separation of the wheat bran extracts by size exclusion chromatography.
Desolvated or undesolvated fractions were characterized by dispersive energy
spectroscopy, scanning electron microscopy, SDS-PAGE electrophoresis and infrared
spectroscopy. Nanoparticles of spherical shape with diameters between 190 and 250 nm
were formed from a protein fraction whose relative molecular masses were between 20
and 43 kDa. The protein nature of the nanospheres was demonstrated, but new evidence
suggests that calcium does not participate in their formation, but rather these are formed
by effect of mild heat treatment before the addition of CaCl2.
Keywords: nanostructures; cold gelation; preparative chromatography; cereal
technology; protein nanotechnology.
44
1. INTRODUCTION
The development of protein nanoparticles is a growing field with applications in the
pharmaceutical and food industries.1 Proteins are GRAS (generally recognized as safe)
substances and their functional properties of emulsion, foaming and gelling capacities,
permit their incorporation into food and pharmaceuticals.2 The chemical diversity of
proteins favors a variety of molecular interactions such as hydrophobic, electrostatic,
hydrogen bonding, van der Waals, steric repulsion and disulfide bridges. The latter
govern protein conformation and aggregation but also drive the self-assembly to
fabricate nanoparticles with uniform shape and size.3
Among the methods used to make protein nanoparticles are emulsification4 and heat-
induced gelation.5 In the former, the protein is emulsified in oil by applying vigorous
shearing force and the nanoparticles are formed at the oil/water interface. Surfactants are
used to stabilize the emulsion and a cross-linker agent is added to control the size of the
nanoparticles.6 Heat-induced gelation is a multistep mechanism based on the disruption
of the native structure of proteins when are exposed to temperatures higher than that
necessary for denaturation7. In heat-induced gelation the gel structure is driven by
polymerization of the denatured proteins, which leads to an increase in viscosity. As the
aggregation process continues, it becomes necessary to apply mechanical shear force to
limit the growth of large agglomerates.8
Another strategy to fabricate protein nanoparticles, not commonly employed, is the
adaptation of the cold gelation/desolvation method.9 In this process, a protein solution is
heated at temperatures below that of denaturation, followed by cooling to room
temperature and adjustment of pH to basicity. Finally, the protein solution is subjected to
desolvation through addition of divalent ions, usually Ca2+.10 Heat treatment and basic
45
pH make proteins to expose their negatively charged groups, favoring formation of
intra- and intermolecular Ca2+ bridges by electrostatic interactions. Thus, the repulsion
forces between proteins are eliminated and subsequent aggregation occurs.2,11 At a fixed
pH value, particle size can be controlled via the concentration of Ca2+.1 The advantages
of this method, compared to those of emulsification and heat-induced gelation, is that the
use of organic solvents and shear forces are avoided.5,12
Most of the reports on obtaining protein nanoparticles by the heat-induced gelation,
emulsification, and desolvation methods involve proteins derived from whey.13-14 Few
efforts have been directed to develop nanoparticles based on plant proteins, even though
these are more affordable and acceptable to consumers. Among the few examples are the
soy protein isolates,11,15 wheat flour gluten,16-17 zein18 and wheat bran (WB) proteins.9 In
this context, spherical nanoparticles after cold gelation/desolvation of an aqueous extract
of WB have been reported.9 The nanospheres had diameters between 20 and 100 nm and
were embedded within a matrix, so their composition and mechanism of formation were
difficult to discern. Nanoparticle formation and stabilization was attributed to proteins
and Ca2+, respectively, but the complex nature of the WB extracts raises the question of
whether other components were involved. In the present study, size exclusion
chromatography (SEC) was used to purify the proteins of aqueous WB extracts. Then,
cold gelation/desolvation assays were conducted with the SEC fractions to elucidate if
proteins were involved in nanoparticle formation.
46
2. MATERIALS AND METHODS
2.1. Materials
The WB (Triticum aestivum L.) was purchased at a commercial mill in Hermosillo,
Sonora, México. All chemicals, unless otherwise noted, were from Sigma-Aldrich
(Sigma-Aldrich, St. Louis, MO, USA). Washing and extraction of WB, as well as
reagent preparations, were performed with deionized water (18 MΩ) obtained from a
Milli-System (Bedford, MA, USA).
2.2. Aqueous extraction of WB
Prior to aqueous extraction, WB was rapidly washed by immersion in cold water and
then dried at 40 °C as previously described.19 Aqueous extraction was performed
according to the procedure reported in literature.9 Briefly, WB was mixed with water
(1:10 w/v) and extracted for 3 h at 4 °C. The extracts were filtered and the resulting
filtrate was centrifuged for 20 min; supernatant was frozen at -40 °C and lyophilized
(Labconco®, Kansas, MO, USA). The lyophilized powder was labeled as aqueous
extract of WB (AEWB) and stored at -40 °C until subsequent treatments or analysis.
2.3. Size exclusion chromatography (SEC)
2.3.1. Column preparation
The AEWB was subject to further purification by SEC using a XK 16/70 preparative
column, packed with 140 ml of Superdex 75 pg (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden). The column was equilibrated by passing 7.7 bed volumes of a buffer
47
prepared with 50 mM sodium phosphate plus 150 mM NaCl, pH 7.2. Flow rate was
maintained at 8 ml/min, using a peristaltic pump FH 100 (Thermo Fisher Scientific Inc.,
Waltham, MA, USA). Column efficiency was tested according to instructions of the
fabricant by eluting 0.7 ml of 1% acetone and collecting fractions of 0.75 ml;
absorbance at 280 nm of each fraction was measured in a UV-Vis Cary 60
spectrophotometer (Agilent Technologies Inc, Headquarters, Santa Clara, CA, USA). By
plotting Abs280 nm vs. elution volume, a peak with a symmetric factor of 1.23 was
obtained, which was within the range of optimal efficiency. The zero volume of the
column (V0) was 42.75 ml and was determined with Dextran blue 2000 (GE Healthcare
Biosciences AB, Uppsala, Sweden).
2.3.2. Preparation and separation of the AEWB by SEC
AEWB was dissolved (1:10 w/v ratio) in 50 mM sodium phosphate, 150 mM NaCl
buffer, pH 7.2, sonicated for 15 min and centrifuged at 3,000xg for 5 min. The
supernatant was vacuum filtered (0.45 μm). Then, soluble protein content of filtrate was
estimated by the Bradford method (1976) and adjusted to 3 mg/ml by dilution with the
phosphate buffer. Thus, the sample was ready to be injected into the column. The
maximum sample volume to be injected in order to obtain the highest possible yield
without compromising efficiency was 2.1 ml (1.5% of the column geometric volume).
For injections, a 3 ml syringe was used, avoiding the formation of air bubbles.
A typical SEC run consisted in injecting the AEWB and eluting with 50 mM sodium
phosphate, 150 mM NaCl buffer, pH 7.2, 1 ml/min elution rate. Volumes of 0.75 ml
were collected and the absorbance of each one was measured at 280 nm in the Cary 60
spectrophotometer. Each peak obtained in the chromatogram of elution volume versus

48
Abs280 nm, was considered a SEC fraction. The relative molecular weight of each SEC
fraction was calculated by using a standard curve constructed with proteins of known
molecular weight (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Twenty highly
reproducible SEC runs were performed and the eluates corresponding to each SEC
fraction were collected, pooled out and labeled according to their elution order as pk-
1…pk-n. The pooled SEC fractions were dialyzed in cellulose membranes (14 kDa
MWCO) immersed in deionized water (18 MΩ cm-1) for 24 h with three exchanges. The
dialysis process was done to eliminate salts from the buffer and was monitored by
measuring electrical conductivity and pH. SEC fractions were lyophilized and stored at -
40 °C after taking a sample of each one for UV spectroscopy (section 2.6).
2.4. Desolvation experiments with CaCl2
Lyophilized SEC fractions were individually dissolved in deionized water (4% w/v).
Solutions were adjusted to pH 8.0 with 1 M NaOH and then centrifuged at 5,000xg for 5
min at 4 °C. Supernatants were recovered and their soluble protein content was
estimated.20 Subsequently, supernatants were heated for 3 h in a water bath at 68.5 °C
and cooled under a stream of water to 25 °C. A volume of 0.25 ml of each solution was
individually desolvated using the necessary volume of 0.25 M CaCl2 to reach a 0.00353
mmol CaCl2/mg protein ratio (Table 1). When two phases (supernatant and precipitate)
were formed after the addition of CaCl2, these were best separated and recovered after
centrifugation at 5,000xg. Then both were frozen out, lyophilized and stored at -40 °C
until further analysis. Control samples of each fraction with no addition of CaCl 2 were
also run. When precipitation occurred in some control fractions after the thermal
49
treatment, the supernatant was recovered by centrifugation at 3,000xg for 3 min,
analyzed by SDS-PAGE as described in section 2.5 and subjected to desolvation.
2.5. Molecular mass profile (SDS-PAGE)
Proteins from AEWB and SEC fractions were resolved in 12% SDS-PAGE
polyacrylamide gel electrophoresis under reducing conditions.21 Samples containing 15
g of protein were loaded to gels in a Mini-Protean Tetra Cell (Bio Rad Laboratories
Inc., Hercules, CA, USA) at constant voltage of 80 V and 14 mA. Gels were stained
with Coomassie Brilliant Blue (G-250). Broad range markers (Bio Rad Laboratories
Inc., Hercules, CA, USA) included myosin (200 kDa), β-galactosidase (116.2 kDa),
phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa),
carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and
aprotinin (6.5 kDa). The protein migration patterns were compared with the broad range
molecular weight standards from Bio-Rad and analyzed using the Image Lab program
(Bio-Rad, Hercules, CA, USA) of the Molecular Imager Gel Doc XR+.
2.6. UV spectroscopy of SEC fractions and Fourier Transform Infrared
Spectrometry (FTIR) of desolvated SEC fractions
In order to assist in the identification of proteins and polysaccharides in SEC fractions
and desolvated samples, UV spectroscopy and FTIR spectrometry were carried out.
Before lyophilizing, samples of the SEC fractions were scanned in the UV spectral
region in the Cary 60 spectrophotometer in order to identify characteristics absorbances.
50
Additionally, lyophilized precipitates and supernatants of desolvated samples were
analyzed by FTIR. The latter was made with 50 mg of powder and data were collected in
the range of 4,000─400 cm-1 with a resolution of 2 cm-1; each spectrum was the result of
32 scans. A Nicolet FT-IR iS50 spectrophotometer (Thermo Fisher Scientific Inc.,
Waltham, MA, USA) was used.
2.7. Elemental composition
Elemental composition of desolvated SEC fractions was analyzed by X-ray dispersive
energy spectroscopy (EDS).9 Analysis was made in a JEOL JSM-7600F electron
microscope (JEOL Ltd., Tokyo, Japan), equipped with a low-angle backscattered
electron detector. Measurements were performed at 80 s with average scanned areas of
39,600 μm2. Data were reported as atomic %.
2.8. Morphology and particle size
Morphology and particle size of lyophilized supernatants and precipitates of treatment
and control samples were visualized by scanning electron microscopy (SEM). Each
sample was placed on the sample holder and then coated with palladium-gold in a
Q150R ES rotary pump ion-jet coater (Quorum Technologies Ltd., USA). SEM was
performed on a JEOL JSM-7600F electron microscope (JEOL Ltd., Tokyo, Japan) at 2.0
kV.
51
3. RESULTS AND DISCUSSION
3.1. Size exclusion chromatography
Six fractions were obtained from the SEC purification of AEWB (Figure 1A). Peaks on
the chromatogram were identified according to their elution order as pk-1, pk-Int, pk-2,
pk-3, pk-4 and pk-5. The soluble protein content of the lyophilized and reconstituted
SEC fractions (4% w/v) ranged 0.04─12.6 mg/ml, with relative molecular masses
0.4─94 kDa as seen in Table 2. The pk-Int fraction (Figure 1A) was considered as an
independent fraction because of the significant amounts of protein detected (4.78 mg/ml)
in the reconstituted lyophilisate.
Absorbance at 280 nm in the SEC fractions could be due not only to proteins, which is
more evident in fractions with low or no protein content, like pk-4 and pk-5 (Table 2).
Phenolic acids, from which ferulic acid is the most abundant in WB, are found in the
external layers22,23 frequently esterified with hemicelluloses, mainly arabinoxylans.24 To
discriminate between protein and phenolic compounds, UV spectroscopy is used.25
Typical shapes of esterified ferulic acid in the 320─340 nm region are seen in the pk-1
and pk-4 UV spectra (Figure 1B), whereas the single peak centered at 280 nm in the
other SEC fractions is assigned to protein or free phenolic compounds; in absence of
protein, the strong absorbance at 280 nm in pk-5 may be assigned to free ferulic acid.25
It is worth to note that there could also be phenolic compounds in pk-3, besides proteins,
since the more intense absorbance at 280 nm compared to that of pk-2 does not match its
protein content (Table 2). Thus, according to literature reports, protein determination
and UV spectral analysis, it is suggested that pk-Int and pk-2 are composed of protein,
although the presence of carbohydrates is not ruled out; pk-1 and pk-4 are a mix of
52
esterified ferulic acid and protein; pk-3 is protein plus free phenolic compounds and pk-
5 may be composed of free ferulic acid.25
3.2. Desolvation experiment with the SEC fractions
Figure 2A shows the aspect of prepared SEC samples prior the thermal treatment; in all
cases clear solutions were seen. Within the first 2 min of thermal conditioning (Figure
2B) both pk-2 and its control became white and gelatinous. This observation was
interesting because the temperature was not enough to favor gelation in comparison to
that reported for WB proteins26 so it was considered worth to investigate it. To do this,
the experiment was repeated with a fresh sample of pk-2 (control) and after the thermal
treatment at 68.5 °C the formation of a white precipitate occurred again, which was
separated from the supernatant by centrifuging at 3,000xg for 3 min and was identified
as pk-2CP. The supernatant was identified as pk-2CS and a sample of it was taken and
identified as pk-2CS (2) in order to be desolvated as the rest of the SEC fractions.
Aliquots of both pk-2CP and pk-2CS were subjected to SDS-PAGE as described in
section 2.5 and the remainders were lyophilized and characterized like the precipitates of
all samples. The aspect of the rest of samples was unaltered by effect of the thermal
treatment and it was maintained during the following 3 h in all cases, except pk-3 and its
control, which presented a very small fraction of precipitate with the same color and
consistency as pk-2.
After the addition of 0.25 M CaCl2 two discernible phases were quickly detected in pk-2
and pk-3 (Figure 2C) but not in pk-1, pk-Int, pk-4 or pk-5. The pk-2CS (2) sample also
formed two discernible phases after addition of CaCl2 (not shown), which were
53
identified pk-2S2 and pk-2P2 (i.e. supernatant and precipitate) and were lyophilized and
characterized like the rest of samples.
3.3. SDS-PAGE of AEWB and SEC fractions
A typical SDS-PAGE gel of AEWB and SEC fractions is shown in Figure 3A. The
molecular mass profile of AEWB was in the range 5─94 kDa, which matches well that
obtained by SEC. The sum of the bands of pk-1, pk-Int, pk-2 and pk-3 equals that of the
AEWB. No bands arose in pk-5 lane whereas two <14 kDa faint lines were observed in
pk-4, which is in accordance with the results of protein analysis (Table 2). The most
intense bands in pk-2 are between 20─30 kDa, while in pk-3 the most intense ones are
between 6─11 kDa. By the other hand, pk-Int had a range of molecular masses between
5─78 kDa.
Figure 3B shows the SDS-PAGE gels of the pk-2 fraction, the supernatant pk-2CS and
the precipitate pk-2CP. An extra lane was loaded with AEWB in order to show
reproducibility in the mass profile. If comparing the intensity of bands in pk-2, pk-2CS
and pk-2CP, it is evident that the precipitated proteins after the thermal treatment were
those of 20─43 kDa, contained in pk-2, since bands lower than 21 kDa remained in the
supernatant (pk-2CS).
3.4. FTIR
In Figure S1 and Figure S2 (Supporting Information), IR spectra of supernatants and
precipitates after desolvation of pk-1, pk-Int, pk-2 and pk-3 are shown. Those of pk-4
and pk-5 are seen in Figure S3. Spectra showed characteristic bands of proteins and
polysaccharides in the 1700─1500 cm-1 and 1200─800 cm-1 regions, respectively,27,28

54
which supports that discussed in the UV spectral analysis (section 3.1). The band
centered at 1050 cm-1 and those around 1165 cm-1 and 895 cm-1 are assigned to C-OH
bending, glycosidic linkages and -(1→4) linkages, respectively, and are characteristic
of arabinoxylans; it is not easy to distinguish ferulic acid from proteins by FTIR as both
components absorb in the same region.28 The most evident difference between IR
spectra of supernatants and precipitates is the change in shape of the 1050 cm -1 band and
its shift to 1017 cm-1. The latter can be attributed to the formation of carbohydrate-
protein complexes through intermolecular interactions or cross-linking through ferulic
acid substituents, as it is reported.29 All these components are present in the SEC
fractions (section 3.1) so the addition of Ca+2 ions could have been resulted in
carbohydrate-protein complexes through electrostatic interactions, which were reflected
in shifts of the band assigned to polysaccharides.
3.5. Particle morphology and size
In order to observe nanoparticle formation, lyophilized supernatants and precipitates of
desolvated SEC fractions were analyzed. Figure 4 shows SEM micrographs of
supernatants and precipitates of pk-1, pk-Int and pk-3 obtained after desolvation with
calcium, as well as their non-solvated controls. The pk-1 control (Figure 4A) had a
rough surface, that of supernatant was smooth (Figure 4B) and the precipitate presented
a coralloid morphology (Figure 4C).
Morphologies of control, supernatant and precipitate of pk-Int (Figure 4D-F) and pk-3
(Figure 4G-I) showed a similar pattern to that of pk-1, excepting that spherical and
ovoid shapes with sizes between 210 and 470 nm can be seen in the pk-3 control (Figure
55
4G). The formation of these nanostructures may be due to proteins of molecular mass
between 20-31 kDa (Figure 3A). Proteins of the same mass are present in pk-2CP, which
formed the nanoparticles obtained. The shape of these nanostructures may be due to the
low concentration of these proteins30 present in this fraction.
Figure 5 shows the particle morphology of supernatants and precipitates of pk-2, pk-2
control, and pk-2CS (2). The formation of rock-like particles with dimensions ranging
51─500 nm is observed in the supernatant of pk-2 (Figure 5A) whereas in the precipitate
(Figure 5B) spherical structures with diameters ranging 190─250 nm are seen, besides
some coral-like structures at the right of the image. In the precipitate of pk-2 control (pk-
2CP) spherical structures (Figure 5D) identic to those in the pk-2 precipitate (Figure 5B)
were formed, but not the coralloid structures observed at the right of Figure 5B. Since
pk-2CP comes from a control sample it is suggested that the nanoparticles were formed
prior the addition of CaCl2 and that the thermal conditioning was responsible for their
formation. The latter is corroborated with the images shown in Figure 5E and Figure 5F
which correspond to the supernatant (pk-2S2) and precipitate (pk-2P2) of the pk-2CS (2)
sample, respectively (section 3.2). It can be seen in the precipitate (Figure 5F) a coral-
like morphology very similar to that of precipitates of all the evaluated fractions,
excepting pk-2CP (Figure 5D). This confirm that added calcium is not involved in the
formation of nanoparticles but rather in the precipitation of coralloid structures, which
indeed could be crystals.9
3.6. Elemental composition of desolvated SEC fractions
The elemental composition of supernatants and precipitates of SEC fractions subject to
desolvation with CaCl2 are presented in Table S1, Table S2 and Table S3 (Supporting
56
Information). Because of the organic origin of the samples, C and O were the most
abundant elements. The presence of N is due to the protein content.
The N content of pk-3 was higher than that of pk-1 or pk-Int in both, control,
supernatant or precipitate (Table S1 of Supporting Information). This result does not
agree with the lowest protein concentration in pk-3, according to the Bradford analysis
(Table 2). However, since EDS is a surface-analyzing method, penetration of x-rays is of
just a few nanometers31 so it is suggested that proteins in pk-3 are in the surface and
hence more exposed to x-rays than in pk-1 or pk-Int. It is also interesting that in pk-1,
pk-Int or pk-3 the N content was not very different between their respective supernatants
and precipitates, which suggest that proteins in these fractions are not involved in
particle formation as was evident in Figure 4.
The absence of Ca in supernatants of pk-1, pk-Int and pk-3 (Table S1) confirms its
involving in the precipitates formed after desolvation in these fractions. Na and P are
probably due in part to remainders of phosphate buffer used in the SEC separation,
whereas P could also come from phytates present in the WB. The presence of S may
correspond to amino acid residues such as cysteine, whereas Cl in supernatants and
precipitates surely comes from CaCl2 (the desolvation agent) since it was not detected in
controls.
Elemental composition of supernatant and precipitate of pk-2 control after thermal
treatment, as well as that of pk-2 and pk-2 (2) after desolvation, is shown in Table S2
(Supporting Information). Within a same sample it is observed that the highest N content
was in the precipitate and the supernatant of the pk-2 control, in which the formation of
spherical particles apparently free of coralloid structures was observed by SEM (Figure
5D). If comparing the composition of the precipitates from pk-2 control and pk-2, an
57
evident difference in Na, P, Cl and Ca contents is seen. That is, the nanoparticles in the
pk-2 control precipitate are formed mainly by proteins, unlike those obtained in the
precipitate of desolvated pk-2, where the presence of coralloid structures was observed
in addition to nanoparticles (Figure 5B). A higher content of S is also seen, which
supports the presence of protein. The absence of Ca in the precipitate of the control
sample corroborates that Ca does not participate in the formation of nanoparticles.
Meanwhile, the elemental composition of pk-2P2 shows a higher content of Na, P and
Ca.
It is also observed in Table S2 that after desolvation and further precipitation protein is
still dissolved in the supernatant of pk-2 as suggested by the N content. However, the
latter is much higher in the pk-2 precipitate, which indicates that the nanoparticles seen
by SEM in Figure 5B are largely made up of protein. The contents of P and Ca in pk-2
and pk-2 (2) precipitates indicate that the coral-like structures seen at the right of Figure
5B and those in Figure 5F could be a form of calcium phosphate precipitated by
saturation of precursor ions once the addition of CaCl2.
CONCLUSIONS
Nanoparticles of spherical shape with sizes between 190 and 250 nm were obtained after
thermal conditioning of a SEC fraction which contained proteins with molecular mass
between 20─43 kDa estimated by SDS-PAGE. The evidence suggests that Ca did not
participate in the formation of nanoparticles, because these were formed prior to the
addition of CaCl2 by effect of the heat treatment. Further research is required to establish
the mechanism by which the nanoparticles were formed.

58
SUPPORTING INFORMATION
In supporting information file are the FTIR spectra of the desolvated SEC fractions as
well tables describing their elemental composition.
ACKNOWLEDGMENTS
To Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico, for financing the
project CB2011-169839 and the scholarship for the doctoral studies of the author Luna-
Valdez. It is acknowledged the support of the staff of the different departments of CIAD.
Thanks to the Laboratorio de Química de Materiales CINVESTAV-IPN Unidad Mérida
Yucatán and the Laboratorio Nacional de Nano y Biomateriales (LANNBIO), projects
FOMIX-YUCATAN 2008-108160 and CONACYT LAB-2009-01 N° 123913. Special
thanks to Dora Huerta-Quintana, Ana Ruth Cristobal Ramos and M.C. Daniel Aguilar
Treviño from CINVESTAV-IPN Unidad Mérida, for their technical assistance in SEM
and EDX analysis.
59
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62
Figure 1. Size exclusion chromatography (SEC) of an aqueous extract of wheat bran
(AEWB) through a column packed with Superdex 70. Protein load: 6.3 mg; flow-rate: 1
ml/min (A); UV spectra of each SEC fraction (B).
63
Figure 2. Images of the AEWB SEC fractions solutions and their respective controls.
Before thermal treatment (A); after thermal treatment (B); after desolvation with CaCl2
(C).
64
Figure 3. SDS-PAGE, under denaturing and reducing conditions of: AEWB and
fractions (A); AEWB, pk-2 fraction, pk-2CS and pk-2CP (B).
65
Figure 4. SEM images of the structures obtained after subjecting the pk-1, pk-Int and
pk-3 SEC fractions of AEWB to desolvation with CaCl2. pk-1 [control (A), supernatant
(B), precipitate (C)]; pk-Int [control (D), supernatant (E), precipitate (F)]; pk-3 [control
(G), supernatant (H), precipitate (I)].
66
Figure 5. SEM images of the morphology of supernatants and precipitates after thermal
conditioning and subjecting the p-k2 SEC fraction to desolvation with CaCl2. pk-2
desolvated with CaCl2 (A-B); pk-2CS (C); pk-2CP (D); pk-2S2 and pk-2P2 (E-F).
67
Table 1. Protein to CaCl2 ratios in each SEC fraction of AEWB subjected
to desolvation.
SEC fraction Volume Soluble protein CaCl2 mmol CaCl2/mg

and controls (ml) (mg/ml) (mmoles) protein
pk-1 control 0.250 7.74 0 0

pk-1 0.250 7.74 0.00683 0.00353
pk-Int control 0.250 4.78 0 0

pk-Int 0.250 4.78 0.00422 0.00353
pk-2 control 0.250 7.08 0 0

pk-2 0.250 7.08 0.00625 0.00353
pk-3 control 0.250 3.54 0 0

pk-3 0.250 3.54 0.00312 0.00353
68
Table 2. Molecular weight distribution of the aqueous extract of wheat bran (AEWB),
fractioned by size exclusion chromatography (SEC).
Peak Peak start Peak end Elution volume Protein Relative molecular
label (ml) (ml) (ml) (mg/ml) mass (kDa)
pk-1 41.25 49.5 45 7.74 117–73
pk-2 69.75 75.75 72.75 12.6 22–16
pk-Int 51 64.5 57.75 4.78 67-30
pk-3 82.5 90.75 85.5 3.54 11–7
pk-4 96.75 120 105.75 0.04 5-1
pk-5 129.75 144.75 137.25 nda <1

a
nd = no detected
69
Supporting information
Figure S1. FTIR spectra of supernatants after cold gelation/desolvation of the AEWB
SEC fractions with 0.25 M CaCl2.
70
Figure S2. FTIR spectra of precipitates after cold gelation/desolvation of the AEWB
SEC fractions with 0.25 M CaCl2.
71
Figure S3. FTIR spectra of pk-4 and pk-5 SEC fractions after cold gelation/desolvation
with 0.25 M CaCl2.
72
Table S1. Elemental composition (atomic %) of supernatants and precipitates of the
SEC fractions pk-1, pk-Int, and pk-3 subject to desolvation with CaCl2 after thermal
conditioning.
Controlsa Supernatants Precipitates

Element
pk-1 pk-Int pk-3 pk-1 pk-Int pk-3 pk-1 pk-Int pk-3
C 58.4 50.1 53.2 44.9 55.8 51.6 67.0 38.2 43.3
N 5.6 11 19.5 4.23 7.1 10.9 1.2 5.3 10.8
O 32.2 31.0 23.8 39.6 29.4 30.0 16.2 41.9 35.8
Na 2.4 4.1 1.4 7.29 4.4 4.1 1.1 4.7 3.1
P 1.1 2.7 1.0 2.53 1.6 1.7 5.7 4.4 3.4
S 0.0 0.1 0.8 ndb nd 0.3 Nd nd 0.2
Cl nd nd Nd 1.2 1.4 1.1 1.1 1.1 0.4
Ca nd 0.5 0.1 nd nd nd 8.6 3.6 2.6

a
pk-1, pk-Int, and pk-3 controls did not form two phases after thermal treatment
73
Table S2. Elemental composition (atomic %) of supernatants and precipitates of the pk-
2 SEC fraction of AEWB and pk-2 (2) after desolvation with CaCl2.
Eleme pk-2 control pk-2 pk-2 (2)a
nt Supernatant Precipitate Supernatant Precipitate pk-2S2 pk-2P2
C 49.0 52.8 59.1 47.4 53.2 38.5
N 9.1 19.0 3.9 14.1 4.5 nd
O 33.4 25.5 28.6 27.0 30.9 41.4
Na 5.1 1.5 5.3 2.2 6.3 4.98
P 2.8 0.7 1.7 3.8 3.2 7.32
S 0.2 0.4 ndb nd nd nd
Cl nd Nd 1.3 1.2 1.6 1.1
Ca nd nd nd 3.9 nd 6.6
a
pk-2 to which the precipitate formed after thermal treatment was removed (section 2.4)
b
nd = no detected
74
Table S3. Elemental composition of pk-4 and pk-5 SEC fractions after
preconditioning at 68.5 °C and desolvated or not (controls) with CaCl2.
pk-4 pk-5
Element
Control Desolvateda Control Desolvated
C 44.51 40.81 21.38 27.73
N 1.4 3.02 ndb nd
O 44.2 46.33 54.28 50.41
Na 5.89 5.92 15.3 16.25
Mg 0.37 0.45 nd nd
P 3.55 3.27 9.04 8.6
S ndb 0.1 nd nd
Cl nd Nd nd nd
Ca nd 0.1 nd nd
a
Two phases were no formed in pk-4 and pk-5 after adding CaCl2 so the
analysis was performed in the lyophilisate.
b
nd = no detected
75
ARTÍCULO 3
FTIR analysis of thermally-induced nanoparticles from a fraction of water soluble

wheat bran proteins, to elucidate the formation mechanism.

Chu, G.
(2017)
Artículo en revisión en:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
76
FTIR analysis of thermally-induced nanoparticles from a fraction of water soluble
wheat bran proteins, to elucidate the formation mechanism.
Authors:
Luna-Valdez, J.G.a
Balandrán-Quintana, R.R.a*
Azamar-Barrios, J.A.b
Ramos-Clamont Montfort, G.c
Mendoza-Wilson, A.M.a
Mercado-Ruiz, J.N.a
Madera-Santana, T.J.a
Rascón-Chu, A.a
a
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de
Tecnología de Alimentos de Origen Vegetal. Carretera a La Victoria km 0.6. 83304.
Hermosillo, Sonora, México.
b
Centro de Investigación y de Estudios Avanzados del IPN, Unidad Mérida.
Departamento de Física aplicada. Carretera antigua a Progreso Km. 6. 97310. Mérida,
Yucatán. México.
c
Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Ciencias
de Los Alimentos. Carretera a La Victoria km 0.6. 83304. Hermosillo, Sonora, México.
*Corresponding author: René Renato Balandrán-Quintana. Centro de Investigación en

Alimentación y Desarrollo, A.C. Coordinación de Tecnología de Alimentos de Origen
Vegetal. Carretera a La Victoria km 0.6. 83304. Hermosillo, Sonora, México. Tel. +52
662 2892400x354. E-mail: [email protected]
77
ABSTRACT
Thermal treatment of a protein-rich fraction of wheat bran resulted in the formation of
195─250 nm diameter spheres, which were the result of the aggregation of smaller
nanospheres (30 nm). The elemental analysis revealed that the nanoparticles are formed
by C, O, and N, whose elements constitute 97% while the S represented 0.45%. FTIR
spectra confirmed the protein nature of nanoparticles and the second-derivative analysis
of the Amide I band indicated that -sheet structure of proteins was reduced from 46%
to 20% after exposure to the thermal treatment, whereas that corresponding to -sheet
intermolecular aggregates increased from 5% to 38%. The overall analysis indicated that
temperature made individual proteins to exposure both thiol groups and -sheets,
favoring intermolecular interactions through disulfide bridges and -sheet aggregates
which resulted in nanospheres. Further agglomeration of nanospheres is drive by non-
specific intermolecular interactions. For first time is demonstrated that a brief thermal
treatment of proteins leads to nanospheres formation.
78
1. INTRODUCTION
Protein nanoparticles (PNPs) are of great interest to industry. In processed foods PNPs
can be used to improve nutritional value by encapsulating nutrients and bioactive
compounds labile to process conditions or even to enhance sensorial characteristics
(Foegeding & Davis, 2011; Saglam, Venema, de Vries, Sagis, & van der Linden, 2011),
whereas in pharmaceutical industry have potential as drug carriers (Boulaiz et al., 2011).
Because of their origin, the PNPs are biocompatible and biodegradable besides to have
the advantage of binding to a large number of compounds in a relatively non-specific
way (Yedomon, Fessi, & Charcosset, 2013). For example, in the manufacture of protein-
rich beverages, one of the most recurrent problems is agglomeration, which arises from
the heat treatment of foods rich in protein, which affects the texture, taste, and
appearance of these products. It is for this reason that the use of microparticles has been
proposed, with which better characteristics can be obtained. However, current
knowledge about the undesirable aggregation of proteins by effect of temperature in
concentrated systems of these macromolecules and the ways of control and prevention is
very limited, which hinders the development of products with high protein concentration
(Saglam, Venema, De Vries, Sagis, & van der Linden, 2011).
In this sense, the formation of spherical nanoparticles after cold gel/desolvation of
aqueous extracts from wheat bran has been reported (Luna-Valdez et al., 2017). These
extracts contain low molecular weight proteins (20-43 kDa) have been shown to have
the ability to form nanoparticles without the need to use reagents for their preparation,
only exposing it to a heat treatment for a very short time (Luna-Valdez et al., manuscript
in revision). These nanoparticles, being of protein sources are biocompatible,
79
biodegradable and non-antigenic, even have the peculiarity of binding to a large number
of compounds in a relatively non-specific way (Yedomon, Fessi, & Charcosset, 2013),
characteristics that are required for be used in the areas mentioned above. In addition,
they have the advantage that in these there is no possibility of toxic residues because no
organic solvents are used or cross-linking for their elaboration, as in nanoparticles made
with proteins already reported (Konan, Gurny, & Allémann, 2002; Langer et al., 2003;
Gülseren, Fang, & Corredig, 2013; Yedomon, Fessi, & Charcosset, 2013), however the
formation mechanism remained to be elucidated.
The formation of spherical nanoparticles after cold gel/desolvation of aqueous extracts
from wheat bran has been reported (Luna-Valdez et al., 2017) and demonstrated their
protein nature (Luna-Valdez et al., manuscript in revision), however the formation
mechanism remained to be elucidated.
Because the secondary structure of proteins is determinant for the way in that they are
self-assembled, a first approach to investigate the formation mechanisms of PNPs is
studying the protein conformational changes in response to process conditions. Circular
dichroism is the most used method for such a task, however the purity of proteins is
fundamental to obtain reliable results so is not appropriate for complex systems, besides
that proteins must be in solution, which excludes its use in the solid state (Kelly et al.,
2005). Other available options are x-ray crystallography and nuclear magnetic resonance
(NMR) spectroscopy, which are the most powerful techniques to analyze protein-protein
interactions and to obtain a complete three-dimensional landscape. However, these
techniques have several drawbacks. Crystallography studies require high quality
monocrystals, which are not available for most proteins, besides the procedure is very
slow. NMR spectroscopy has better flexibility but the interpretation of the NMR
80
spectrum of large proteins is very complex, so the technique is limited to small proteins
(30 kDa) (Haris & Severcan, 1999; Haris, 2013).
Another method through which is possible to monitor subtle changes in the polypeptide
backbone conformation is Fourier Transform Infrared Spectroscopy (FTIR). This tool is
very sensitive to changes in hydrogen bonds, so it is useful to analyze modifications in
the secondary structure of proteins caused by effect of temperature (van Stokkum et al.,
1995; Natalello, Ami, Brocca, Lotti, & Doglia, 2005) or pH (Vicent, Steer & Levin,
1984). In fact FTIR is successfully used in protein stability and aggregation analyses
(Carbonaro & Nucara, 2010; Miller, Bourassa, & Smith, 2013). Data on the secondary
structure of proteins predicted by FTIR is in good agreement with that obtained by X-ray
crystallography (Dong, Huang, & Caughey, 1990; Natalello, Ami, Brocca, Lotti, &
Doglia, 2005; Kong & Yu, 2007).
FTIR is based on the vibrations of atoms in a molecule. The FTIR spectrum results from
the absorption energy produced by the vibration of chemical bonds (stretching and
bending movements). Such absorption arises from the transitions between the vibrational
and rotational states of the molecule and is generated when the transition causes a
change in the dipole moment (Barth, 2007). Characteristic groups of atoms give rise to
vibrational bands centered at typical frequencies in the spectrum, regardless of the
molecule in which they are found. Because these resonant frequencies are determined by
the shape of the molecular potential energy surfaces, the atomic mass and the associated
vibronic coupling, the technique can be used for the structural and chemical
characterization of very complex mixtures (Carbonaro & Nucara, 2010).
For the quantitative estimation of different protein secondary structures (α-helix, β-
sheets, -turns, random), the Amide I region (1700─1600 cm-1) of the FTIR spectrum is
81
subjected to deconvolution or second-derivative to identify the frequency peaks of its
structural components. Then a multi-peak adjustment through a Gaussian or Lorentzian
function is made and, finally, the relative amounts of different types of secondary
structure are calculated from the areas of the plotted peaks (Dong et al., 1992; Carbonaro
& Nucara, 2010).
FTIR spectroscopy has important advantages. The spectrum of almost any biological
material can be obtained in a wide variety of environments, so is possible to analyze the
structure of proteins either in solution (aqueous and non-aqueous), suspension
(membranes and aggregates) or solid state (glass, thin films and powder), besides that a
small amount of sample is required (10 μg) regardless of protein size (Haris & Severcan,
1999; van de Weert, Haris, Hennink, & Crommelin, 2001; Carbonaro & Nucara, 2010).
In the present work, FTIR spectroscopy was used in combination with scanning electron
microscopy to investigate the formation mechanism of spherical particles that are
formed after thermal treatment of a protein fraction of wheat bran obtained by size
exclusion chromatography (SEC).
2. MATERIALS AND METHODS
2.1. Materials and nanoparticle production
Wheat bran extraction was performed according to Luna-Valdez et al. (2017) and the
aqueous extracts were further fractionated by size exclusion chromatography (SEC).
Nanoparticles were produced from an aqueous solution of the SEC fraction identified as
that containing the wheat bran proteins involved in the formation of nanoparticles
82
reported by Luna-Valdez et al. (2017). Details on SEC and nanoparticle production
methodologies are reported by Luna-Valdez et al. (manuscript in revision in the journal
Biomacromolucules). Unless otherwise noted, all reagents were purchased from Sigma
(Sigma-Aldrich, St. Louis, MO, USA).
2.2. Elemental composition
The elemental composition of the nanostructures was quantified by energy dispersive X-
ray spectroscopy (EDS) using a JEOL JSM-7600F electron microscope (JEOL Ltd.,
Tokyo, Japan) equipped with a low angle backscattered electron detector. Measurements
were performed at 80 s and the average scanned areas were 39,600 μm2 at each sample.
The results were reported as atomic percentage.
2.3. Morphology and size
Morphology and size of synthesized nanoparticles were visualized by scanning electron
microscopy (SEM) in a JEOL JSM-7600F electron microscope (JEOL Ltd., Tokyo,
Japan) at 2.0 kV. Samples were individually placed in the sample holder of the
equipment and coated with palladium-gold in a Q150R ES rotary pump ion-jet coater
(Quorum Technologies Ltd., USA). In addition, a portion of the powder nanoparticle
(50 mg) was resuspended in 1 mL of water and sonicated for 5 min at 25 °C. Then 15 μL
of this suspension was taken out and placed on the sample holder where it was allowed
to dry at room temperature (25 °C). Finally, the sample was analyzed by SEM under the
conditions mentioned above.
83
2.4. Functional group analysis (FTIR)
Functional group analysis of control sample and nanoparticles was performed using
Fourier Transform Infrared Spectrometry (FTIR). FTIR absorption spectra in the
4000─400 cm-1 range were collected in transmittance mode using a Thermo Nicolet
Nexus 670 FTIR spectrometer (Thermo Nicolet Analytical Instruments, Madison, WI).
Fifty mg of each sample were analyzed, collecting a total of 32 scans with a 2 cm -1
resolution.
2.5. FTIR data analysis
The 1700─1600 cm-1 spectral region was selected for the Amide I analysis. Each
spectrum was smoothed with an 11-point Savitzky-Golay smoothing function (Natalello,
Ami, Brocca, Lotti, & Doglia, 2005; Liu et al., 2009). The overlapping of bands was
enhanced using second-derivative calculation. The secondary structure composition was
determined by second-derivative using the OMNIC software (Liu et al., 2009) whereas
second-derivative inverted spectra were obtained multiplying by -1. Then the Gaussian
functions of the OriginPro 8.6 (OriginLab Corp., MA, USA) software were used for
multi-peak fitting. Initial band positions were taken directly from the second-derivative
spectra and then were applied to calculate the area of each component representing a
type of secondary structure. During the curve-fitting process, heights, widths and
positions of all bands were varied simultaneously, with only the peak wavenumber as
restriction (Dong et al., 1992; Murayama & Tomida, 2004).
84
3. RESULTS AND DISCUSSION
3.1. Particle morphology and size
The protein nanoparticles obtained are shown in Figure 1A. The formation of spherical
structures with diameters ranging between 190─250 nm is observed which agree as it
was reported previously (Luna-Valdez et al., 2017). Upon suspending the powder in
water and sonicate for 5 min, both size and morphology of the structures were
maintained (Figure 1B) but the presence of smaller particles is also noted, which are
better observed at higher magnification in Figure 1C. At an even greater magnification
(Figure 1D) is observed that the smaller particles were in fact nanospheres with diameter
≈ 30 nm which are assembled to form the larger spheres. It is possible that the 190─250
nm spheres were joined by weak intermolecular interactions which were disrupted by
ultrasound exposing thus the 30 nm nanospheres. Luna-Valdez et al. (2017) reported the
effect of ultrasound on making visible spherical particles formed after cold
gelation/desolvation of an aqueous extract of wheat bran.
Formation of aggregates by effect of temperature has been widely reported for proteins
such as bovine serum albumin (BSA), β-lactoglobulin, ovalbumin and whey proteins
isolates (Doi, 1993; Aymard, Gimel, Nicolai, & Durand, 1996; Ikeda & Morris, 2002;
Veerman, Sagis & Linden, 2003; Bolder, Hendrickx, Sagis & Linden, 2006; Lovedey,
Wang, Rao, Anema, Creamer & Singh, 2010; Oboroceanu, Wang, Brodkorb, Magner, &
Auty, 2010; Lovedey, Su, Rao, Anema, & Singh, 2012).
85
The morphology of those aggregates consisted of fine strands with diameters less than
10 nm, which differs from the obtained in the present work, possibly due to the
differences in process conditions since those experiments were run at pH 2, 80 °C with a
thermal induction time of 1─20 h. In most studies protein solutions had an ionic strength
in the 10─100 mM range, some using NaCl and/or CaCl2. Oboroceanu et al. (2010) and
Bolder et al. (2006) did not use salts during the thermal incubation of proteins, but even
so, fine strands aggregates were obtained. Loveday et al. (2010) and Borzova et al.,
(2016) reported the presence of a small number of spherical aggregates (15─25 nm)
intercalated within the fine strands. Unlike Loveday et al. (2010), Borzova et al., (2016)
dissolved the protein in phosphate buffer pH 7 and run the experiment at 65 °C, but in
both studies the formation of spherical nanoparticles required a thermal incubation
between 330 and 360 min, which is a very long time by comparison to the present work.
It is worth to note that although thermal treatment was 180 min, only 2 min of exposure
is enough to observe the presence of a white precipitate from which the nanoparticles are
visualized (Luna-Valdez et al., manuscript in revision).
3.2. Determination of functional groups
Figure 2A and 2B show the IR spectrum of control sample and nanoparticles obtained
by heat treatment, respectively; in both spectra, characteristic bands of proteins (1700-
1600 cm-1) and polysaccharides (1200-800 cm-1) are observed (Pelton & McLean, 2000).
The Amide I absorption occurs in the 1600─1700 cm-1 region and arises mainly from
the C=O stretching vibration of the peptide group (Haris & Severcan, 1999). The peak at
86
1600─1500 cm-1 is assigned to Amide II, which corresponds primarily to N-H bending
with a contribution from C-N stretching vibrations. In addition, one band very weak in
the region 1200-1300 cm-1 is assigned to Amide III, which is due to a complex mix of
N-H bending and C-H stretching along with deformation vibrations of C-H and N-H
(Haris & Severcan, 1999; Barth, 2007). The broad band in the 3000─3600 cm-1 region is
assigned to the O-H stretching bonds groups (Elisa, Puhl, Kadla, & Khan, 2006) and the
C-H stretch bonds appear around 2900 cm-1 (Maréchal, 2003).
When analyzing the region 1200─800 cm-1 a maximum absorption band centered at
1050 cm-1 was found, which might be assigned to the characteristic C-OH bending of
arabinoxylans, in addition to two bands at 988 and 946 cm-1 that are related to the degree
of substitution of arabinoxylans (Robert, Marquis, Barron, Guillon, & Saulnier, 2005).
In the nanoparticles spectrum (Figure 2B) a greater intensity and definition of the Amide
I, II and III bands was observed in comparison to the control sample. It is also observed
a decrease in the intensity of bands in the region 1200─800 cm-1, suggesting a lower
presence of carbohydrates in the nanoparticles. In addition, in this same spectra at 560
cm-1, a new band was observed, which can be assigned to S-S stretching (Sadeghi et al.,
2014). At the same time a decrease in intensity of the band assigned to S-H stretching
(2600-2520 cm-1) of cysteine residues (Fabian & Mäntele, 2002) was observed,
suggesting the formation of disulfide bonds in the nanoparticles.
The possibility of an overlap between the Amide bands I and II can be ruled out since
they are well defined and intense. Water vapor bands, which are easily identified as very
thin bands at 1600 and 3700 cm-1, are not seen in this spectrum (Maréchal, 2003). In
addition, sodium phosphate buffer was used as solvent for protein separation before
87
making nanoparticles, which is one of the accepted salts when analysis in the infrared
spectrum is going to be performed, since does not contain carboxylic acid groups
overlapping those of the main chain of proteins (Fabian & Vogel, 2002).
3.3. Second-derivate and estimation of protein secondary structure
Analysis of Amide I band allows detecting small conformational changes in the structure
of proteins. However, the IR bands corresponding to each conformation are very close to
each other so that they overlap. To solve individual components of each conformation,
the second-derivative method was used, which is one of the two most popularly
employed for secondary structure analysis (Kong & Yu, 2007); it is more sensitive than
deconvolution (Carbonaro & Nucara, 2010).
Figure 3 shows the second-derivative spectra of the 1700─1600 cm-1 region of both
control and nanoparticles. In the second-derivative of control (Figure 3A), 10 bands
were observed. Bands appearing in the 1680─1668 cm-1 region can be assigned to β-turn
conformations, while the bands at 1659 cm-1 and 1646 cm-1 can be attributed to α-helix
and random structures, respectively. The components at 1691 cm-1, 1638 cm-1 and 1628
cm-1 can be assigned to β-sheet structures. The band of low intensity at 1620 cm-1 can
instead be assigned to aggregates (Dong, Caughey, Caughey, Bhat & Coe, 1999; Dong,
Huang, & Caughey, 1992).
In the second-derivative spectrum of nanoparticles (Figure 3B), a decrease in the
intensity of peaks in the 1690─1670 cm-1 region, which correspond to β-turn
conformations, was observed. One band at 1664 cm-1 (not seen in the control) is also
88
observed, which is assigned to 310-helix structures. The bands at 1657 cm-1 and 1647
cm-1 are assigned to α-helical and random conformations, respectively, whereas the
bands at 1695 cm-1 and 1633 cm-1 are assigned to β-sheet structures. In addition, a band
of great amplitude and intensity at 1619 cm-1 is assigned to intermolecular β-sheets
aggregates (Dong, Huang, & Caughey, 1992; Dong, Caughey, Caughey, Bhat, & Coe,
1999).
Quantitative information on the secondary structure of proteins in both the control and
nanoparticles was obtained through a curve fitting analysis of the Amide I band and
performed as a linear combination of the components identified in the second derivative
spectrum. It is assumed that the sums of the areas of conformations of the Amide I band
are related to the total of a given protein (Byler & Susi, 1986; Kong &Yu, 2007). Figure
4 and Figure 5 show the decomposition of the Amide I band of control and
nanoparticles, respectively, in Gaussian components. The results show that the
secondary structure of proteins in control is dominated by β-sheet conformations,
representing 46%. In addition, 18% β-turn, 14% α-helix, 16% random and 5%
aggregates, was calculated.
Figure 5 shows that proteins underwent a change in their structure after being heated
since the percentage of β-sheet conformation decreased up to 21%. This result is in good
agreement with that obtained by Natalello et al. (2005), who reported a 50% decrease in
β-sheet conformations after subjecting the Candida rugosa lipase protein to 64 °C,
besides a simultaneous decrease in α-helix conformations. Unlike them, in the present
work, the α-helix remained constant
89
Meanwhile, the β-turn conformations accounted for 11%, which means a decrease in
that structure compared to the control proteins. By the other hand, the random
conformations remained unchanged. Also, the loss of β-sheets was accompanied by a
band of great amplitude and intensity at 1619 cm-1, which is represented by 38% and is
due to the formation of aggregates by intermolecular β-sheets of proteins induced by the
heat treatment (Haris & Severcan 1999; Seshadri, Khurana, & Fink, 1999; Yan, Wang,
He, & Zhou, 2004; Natalello, Ami, Brocca, Lotti, & Doglia, 2005).
The formation of aggregates by intermolecular β-sheets may be due to the experiment
was performed at pH 8. That is, when a pH>pI of the proteins is used, aggregation
proceeds through the formation of relatively small ordered aggregates (oligomers) that
are characterized by a sizeable amount of intermolecular β-sheets, as revealed by FTIR
spectroscopy for the BSA (Militello et al., 2004).
It must be considered that the results obtained on the conformational changes of the
proteins can not only be due to the effect of the temperature but also to the loss of the
hydration layer after the lyophilization process. That is, during this process dehydration-
induced alterations in the absorption characteristics of the amide bond can be produced.
Even the predicted values may not only be related to changes in secondary structure but
also to the absence or presence of protein-protein contacts and changes in hydrogen-
bonding characteristic. However, this does not exclude the use of FTIR to determine the
effect of lyophilization on protein stability (van de Weert, Haris, Hennink & Crommelin,
2001). In addition, the presence of carbohydrates in nanoparticles (Figure 2) might
protect dried proteins because these solutes bind to the dried protein, thus serving as a
water substitute, when the hydration shell of the protein is removed (Carpenter &
Crowe, 1988; Carpenter & Crowe, 1989)

90
However, the possible changes that could have been generated in the structure of
proteins by the effect of lyophilization should have been minimal, since the morphology
of the nanoparticles was not apparently affected, as can be observed when comparing
Figure 1A and Figure 1B, which correspond to lyophilized nanoparticles and to
nanoparticles that were resuspended in water and further sonicated, respectively (section
2.4).
Several investigation suggests that heating proteins above their denaturation temperature
results is aggregation, because temperature causes a molecular unfolding that favors
protein-protein intermolecular interactions, such as dipole and electrostatic as well as
thiol/disulfide exchanges (Mulvihill & Donovan, 1987; Mulvihill, Rector, & Kinsella,
1991; Anker, Standing, & Hermansson, 1999). These interactions favor the aggregation
of proteins as long as conditions of pH and temperature as well as the salt and protein
concentration are controlled. In the salt absence, the unfolded proteins can remain
separated due to the strong electrostatic repulsion, however as the salt concentration
increases the electrostatic repulsion between the charged molecules decreases. At low
salt levels, most of the surface of the protein molecules is unable to form bonds with
other proteins due to residual electrostatic repulsion between them. However, some
interactions may occur between non-polar regions of the protein whereas other domains
can be involved in hydrophobic interactions. It is suggested that these interactions form
filament structures, arguing that they bind at fixed sites at opposite ends of the molecule
(Doi, 1993). In this regard, whey proteins and β-lactoglobulin have been reported to
form gels with different structures, depending on the type and amount of salt present
during thermally induced gelation. That is, a fine stranded matrix is formed when protein
suspensions contain monovalent cation (Li+, K+, Rb+, Cs+) chlorides, sodium sulfate or
91
sodium phosphate at ionic strengths ≤0.1 M (Foegeding, Bowland & Hardin, 1995).
These morphologies have been reported by other authors (Aymard, Gimel, Nicolai, &
Durand, 1996; Ikeda & Morris, 2002; Veerman, Sagis & Linden, 2003; Lovedey, Wang,
Rao, Anema, Creamer & Singh, 2010; Lovedey, Su, Rao, Anema, & Singh, 2012). In
our case, the possibility of formation of this type of structures is unlikely, since the
proteins used for the experiments were previously dialyzed.
In cases where the salt concentration in the protein solution is high, electrostatic
repulsion between proteins is minimal, so they can form bonds at any point on their
surface. This leads to the formation of large spherical aggregates (Kitabatake & Doi,
1993). For example, when ionic strength is greater than 0.1 M, the resulting matrix is
composed of fine strands and spherical aggregates (Foegeding, Bowland & Hardin,
1995; Borzova, Markossian, Chebotareva, & Kleymenov, 2016). This possibility can
also be ruled out, due to the low presence of salts in the nanoparticles formed (Table 1).
Nevertheless, there is also the possibility that the nanoparticles have been produced by
disulfide bonds due to the appearance of a broad band at 560 cm-1 in the infrared
spectrum of nanoparticles, which display the formation of disulfide bonds (Sadeghi et
al., 2014). To support this, the proteins (20-43 kDa) that were involved in the formation
of nanoparticles are rich in cysteine, amino acid that can form these types of bonds
between neighboring residues (Doi, 1993). These proteins (wheat bran albumin) have
been previously identified by mass spectrometry by Chaquilla-Quilca et al. (2017) using
the method reported by Huerta-Ocampo et al. (2014).
During the thermal aggregation process, the proteins undergo a partial unfolding of their
native structure, which generates changes in the three-dimensional structure by
disruption of hydrogen bonds and non-polar hydrophobic groups, this causes that
92
hydrophobic groups and free-SH groups became more exposed and could form
intermolecular disulfide bond and hydrophobic interactions among the unfolded protein
chains, resulting in the formation of aggregates (Shimada et al., 1989; Vetri, Librizzi,
Leone, & Militello, 2007). It is known that the intermolecular disulfide bonds are
dependent on both pH and temperature, so is possible that the formation of aggregates
has been through this route since the experiment was run at pH 8 and 68.5 °C. Under
these conditions, the propensity of proteins to undergo irreversible thermal denaturation
increases as does the degree of thiol oxidation and subsequent polymerization
(Monahan, German, & Kinsella, 1995). In that sense, Hoffmann et al. (1994) reported
that β-lactoglobulin, dispersed in water at neutral pH and heated at 65 °C, formed
aggregates by intermolecular disulfide cross-linking, noting that non-covalent
interactions may be involved but only to a lesser extent. Similar results have been
reported by other groups (Petruccelli, & Añón, 1995; Aymard, Gimel, Nicolai, &
Durand, 1996; Bauer, Carrotta, Rischel, & Ogendal, 2000).
It is likely that the aggregation of the nanostructures obtained in the present work obeys
to the two-step mechanism proposed by Aymard et al. (1996) for the thermal
aggregation of β-lactoglobulin at pH 7. Such mechanism consists in the formation of
aggregates of small particles (globules) by disulfide interchange, which arises from
denatured monomers that are consequently associated in dimers, whose size does not
seem to depend on the initial protein concentration, temperature, and ionic strength,
under the range of conditions tested (step 1). In the second step, the globules are added
to form fractal structures. Ikeda et al. (2002) also agreed on a two-step aggregation
mechanism, reporting the formation of thermally induced aggregates (fine-strands) at pH
93
2 of β-lactoglobulin and whey protein isolates. While at pH 7, aggregates were
composed of ellipsoidal particles.
By the other hand, Oboroceanu et al. (2010) reported the fibrillary aggregation of β-
lactoglobulin at pH 2, concluding that the formation mechanism also consisted of two
steps, but unlike Aymar et al. (1996) they concluded that β-lactoglobulin fibrils consist
of polypeptide fragments bound by non-covalent intermolecular bonds and that are not
formed from intact monomers. Recently, Borzova et al. (2016) also reported BSA, pH 7,
formed aggregates by heat treatment, noting that protein unfolding resulted in the
formation of two non-native protein forms with a different propensity to aggregation.
The formation of primary aggregates occurred in a highly reactive unfolded form, while
the secondary aggregates were formed by the unfolded forms of low reactivity. The
hydrodynamic radius of the secondary aggregates was higher.
Based on the above mentioned and the morphology of the nanoparticles obtained in this
work, it is possible to consider that the nanostructures were formed by a mechanism
similar to the two-step aggregation mechanism. This mechanism coincides with that
observed in SEM (Figure 1D), because apparently the spheres are formed by smaller
aggregates (30 nm diameter), which could have been formed by disulfide bonds
(globules) and in turn, these form aggregates of greater size by physical interactions
such as van der Waals forces, hydrogen bonding, and hydrophobic or electrostatic
interactions (Le Bon, Nicolai, & Durand, 1999). The presence of nanostructures of 30
nm after resuspending the nanoparticles in water and sonicated for 5 min, makes us to
think that the aggregate-aggregate interactions effectively correspond to physical
interactions, since a part of these were disaggregated from the larger nanoparticles by
effect of ultrasound, which are observed in the surroundings of these (Figures 1B, and
94
1C), unlike nanoparticles that were not treated with ultrasound. In support of this Hu et
al. (2013) reported a decrease in no-covalent interactions of soy protein isolate in
dispersion after ultrasonic treatment.
CONCLUSION
Conformational changes experienced by wheat bran proteins involved in the formation
of nanoparticles were seen through the analysis of Amide I. The results showed that
these proteins form aggregates by intermolecular β-sheets by effect of temperature.
These aggregates were possibly consequence of the formation of globules product of the
intermolecular disulfide cross-linking. While the nanostructures seen in SEM, are
possibly the result of the connection between the globules by nonspecific physical
interactions.
ACKNOWLEDGMENTS
To Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico, for financing the
project CB2011-169839 and the scholarship for the doctoral studies of the author Luna-
Valdez. It is acknowledged the support of the staff of the different departments of CIAD.
Thanks to the Laboratorio de Química de Materiales CINVESTAV-IPN Unidad Mérida
Yucatán and the Laboratorio Nacional de Nano y Biomateriales (LANNBIO), projects
FOMIX-YUCATAN 2008-108160 and CONACYT LAB-2009-01 N° 123913 and Dora
95
Huerta Quintana, Ana Ruth Cristobal Ramos from CINVESTAV-IPN Unidad Mérida,
for their technical assistance in the STEM, and SEM-EDX analysis, respectively.
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Manuscript in revision:
Luna-Valdez, J. G., Balandrán-Quintana, R. R., Azamar-Barrios, J. A., Clamont-
Montfort, G. R., Mendoza-Wilson, A. M., Mercado-Ruiz, J. N., Madera-Santana,
T. J., Rascón-Chu, A., & Chaquilla-Quilca, G. Nanoparticle formation after mild
thermal conditioning of proteins contained in a wheat bran extract fractioned by
size exclusion chromatography (in revision in journal Biomacromolecules).
103
A B
1 μm 1 μm
X 25, 000 X 20, 000
C DX 20, 000 1 μm
100 nm 100 nm
X 50, 000 X 200,
000
Figure 1. SEM images of nanoparticles formed from water soluble proteins of wheat
bran after thermal treatment at 68.5 °C.
104
Figure 2. FTIR spectra of the control sample (A) and the nanoparticles obtained by
thermal treatment of water soluble proteins from wheat bran (B).
105
Figure 3. Second-derivative spectra of the Amide I region of control (A) and
nanoparticles obtained after thermal treatment of water soluble wheat bran proteins (B).
106
Figure 4. Decomposition of amide I band of the control proteins into Gaussian
components.
107
Figure 5. Decomposition of Amide I band of water soluble wheat bran proteins subject
to heat treatment (68.5 °C) (nanoparticles), into Gaussian components.
108
Table 1. Elemental composition of
nanoparticles.
Element (atomic %)
C 53.76
N 18.22
O 25.37
Na 1.36
P 0.62
S 0.45
109
110
ANEXO
Tabla 1. Proteínas de la fracción de albúminas de salvado de trigo involucradas en la formación de nanopartículas .
Número de banda Número de Peso molecular

Experimental e f g Porcentaje
en el gel SDS- Proteína Organismo b d Puntuación PC /CS
acceso c teórico (PM/pI ) de cisteína
PAGEa PM
10 Aldosa reductasa Triticum urartu EMS68426.1 43.5 35.95/6.87 169 6/24% 1.56
Proteína inhibidora de la
11 Triticum aestivum Q8L5C6.2 34 33.25/8.66 175 9/34% 1.6
xilanasa 1
12 Quitinasa clase II Triticum aestivum AAX83262.1 30 28.20/8.66 225 6/42% 2.69
Proteína hipotética
13 F775_32839 (similar a la Aegilops tauschii EMT26984.1 26 17.74/6.52 171 7/59% 8.75
Taumatina)
Inhibidor endógeno de alfa-
14 Triticum aestivum P16347.1 20 19.62/6.77 467 12/78% 2.2
amilasa/subtilisina
a b c
Figura 3B(pk-2) artículo 3; Números de acceso en la base de datos de proteínas NCBI; peso molecular experimental (Datos obtenidos en este trabajo mediante SDS-PAGE);
d e
peso molecular teórico y pI; resultados de M ascot reportados después de buscar en el subconjunto Viridiplantae de la base de datos de proteínas NCBI (octubre 2016, secuencias
f g
_4298025). Las puntuaciones de iones individuales> 44 indican identidad u homología extensa (p <0,05); Péptidos que coinciden; cobertura de secuencia. (Datos reportados por
Chaquilla-Quila et al., 2017).
111

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Centro de Investigación en Alimentación

Jesús Guadalupe Luna Valdez

TESIS APROBADA POR LA COORDINACIÓN DE

TECNOLOGÍA DE ALIMENTOS DE ORIGEN VEGETAL

Como requisito parcial para obtener el grado de

Hermosillo, Sonora, México Agosto de 2017

Al Consejo Nacional de Ciencia y Tecnología (CONACYT, México), por el apoyo

Al Centro de Investigación en Alimentación y Desarrollo (CIAD, AC), por brindarme la

Al Consejo Nacional de Ciencia y Tecnología (CONACYT, México), por el

A Dios y a la Virgen María, por guiarme siempre por el buen camino.

A mi comité de tesis por compartir sus conocimientos en cada semestre que se le

Un agradecimiento muy especial a la M.C. Dora A. Huerta Quintanilla y a la Biol. Ana

Al Ing. Francisco Javier Noriega Muñoz, quien desde el principio me impulsó a

A mi esposa Jazmín e hijos (José, Luna y Lupita) por su comprensión, apoyo y

A mi tía Ramona Reina y mi tío Alejandro Bermúdez por su grato recibimiento a mi

A mi cuñado Arturo (el muñe) por su apoyo brindado.

3. FTIR analysis of thermally-induced nanoparticles from a fraction of

Se evaluó la factibilidad de sintetizar nanopartículas a partir del extracto acuoso de

Palabras clave: nanopartículas, salvado de trigo, cromatografía preparativa, tecnología

El salvado de trigo es un subproducto de la industria molinera (Beaugrand et al., 2004)

En el ARTÍCULO 1 de esta tesis se describe la formación de nanopartículas a partir de

ARTÍCULO 2. En el artículo 1 se describió la obtención de nanopartículas a partir del

En el ARTÍCULO 3 se analizaron los cambios conformacionales de las proteínas

Structural and physicochemical characterization of nanoparticles

J.G. Luna-Valdez, R.R. Balandrán-Quintana, J.A. Azamar-Barrios, G. Ramos-Clamont

Food Hydrocolloids, 62:165-163

Nanoparticle formation after mild thermal conditioning of proteins in a

J.G. Luna-Valdez, R.R. Balandrán-Quintana, J.A. Azamar-Barrios, G. Ramos-Clamont

Artículo en revision en:

*Corresponding author: René Renato Balandrán-Quintana. Centro de Investigación en

previously obtained through adaptations to the cold gelation/desolvation method.

Presumably, nanoparticles were formed by proteins, stabilized by calcium bridges and

work, cold gelation/desolvation experiments were performed with fractions resulting

Desolvated or undesolvated fractions were characterized by dispersive energy

spectroscopy, scanning electron microscopy, SDS-PAGE electrophoresis and infrared

by effect of mild heat treatment before the addition of CaCl2.

Keywords: nanostructures; cold gelation; preparative chromatography; cereal

technology; protein nanotechnology.

The development of protein nanoparticles is a growing field with applications in the

proteins favors a variety of molecular interactions such as hydrophobic, electrostatic,

fabricate nanoparticles with uniform shape and size.3

nanoparticles.6 Heat-induced gelation is a multistep mechanism based on the disruption

necessary for denaturation7. In heat-induced gelation the gel structure is driven by

polymerization of the denatured proteins, which leads to an increase in viscosity. As the

aggregation process continues, it becomes necessary to apply mechanical shear force to

limit the growth of large agglomerates.8

Another strategy to fabricate protein nanoparticles, not commonly employed, is the

adaptation of the cold gelation/desolvation method.9 In this process, a protein solution is

heated at temperatures below that of denaturation, followed by cooling to room

temperature and adjustment of pH to basicity. Finally, the protein solution is subjected to

use of organic solvents and shear forces are avoided.5,12

Most of the reports on obtaining protein nanoparticles by the heat-induced gelation,

this context, spherical nanoparticles after cold gelation/desolvation of an aqueous extract

difficult to discern. Nanoparticle formation and stabilization was attributed to proteins

proteins were involved in nanoparticle formation.

The WB (Triticum aestivum L.) was purchased at a commercial mill in Hermosillo,

Milli-System (Bedford, MA, USA).

2.2. Aqueous extraction of WB