“IDENTIFICACIÓN DE METABOLITOS PRODUCTOS DE LA ...
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INSTITUTO TECNOLÓGICO DE TEPIC
“IDENTIFICACIÓN DE METABOLITOS PRODUCTOS DE LA FERMENTACIÓN COLÓNICA DE LA FRACCIÓN INDIGESTIBLE DE ALIMENTOS
FRECUENTEMENTE CONSUMIDOS POR ESCOLARES DE TEPIC, NAYARIT”
TESIS
por:
MCA. VICTOR MANUEL ZAMORA GASGA
DOCTORADO EN CIENCIAS
EN ALIMENTOS
Director:
Dra. SONIA GUADALUPE SÁYAGO AYERDI
Co-Director:
Dra. MARÍA GUADALUPE FLAVIA LOARCA PIÑA
Tepic, Nayarit, México Marzo de 2017
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CONTENIDO
Pág.
LISTA DE CUADROS v
LISTA DE FIGURAS vii
Capítulo 1. INTRODUCCIÓN 1
Capítulo 2. ANTECEDENTES 3
2.1 La dieta en México y el desarrollo de enfermedades 3
2.1.1 La dieta y el síndrome metabólico 4
2.1.2 La dieta y su relación con la obesidad 5
2.1.3 La dieta y su relación con la resistencia a la insulina 7
2.1.4 La dieta y su relación con el cáncer de colon 7
2.2 Digestión y absorción de los nutrientes de la dieta 8
2.3 Fracción Indigestible 10
2.4 El colón y la microbiota intestinal 12
2.4.1 Fermentación colónica 13
2.5 Metabolitos derivados de la fermentación colónica de carbohidratos. 14
2.5.1 Ácidos grasos de cadena corta (AGCC) 15
2.5.2 Principales AGCC y sus efectos en la salud. 16
2.6 Proteínas de la dieta y las bacterias proteolíticas 19
2.6.1 Productos de la fermentación de proteínas 19
2.7 Grasas dietéticas 21
2.8 Metabolitos microbianos derivados de polifenoles dietéticos 22
2.8.1 Metabolitos de polifenoles y su impacto sobre la salud intestinal 24
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Capítulo 3. JUSTIFICACIÓN 26
Capítulo 4. HIPÓTESIS 27
Capítulo 5. OBJETIVOS 28
5.1 Objetivo general 28
5.2 Objetivos específicos 28
Capítulo 6. METODOLOGÍA GENERAL 29
6.1 Primera Etapa 29
6.1.1 Análisis de los alimentos frecuentemente consumidos 29
6.1.1.1 Sujetos de estudio 30
6.1.1.2 Evaluación dietética 30
6.1.1.3 Datos antropométricos 31
6.1.1.4 Identificación de los patrones dietéticos en escolares 31
6.2 Segunda Etapa 33
6.2.1 Obtención de las muestras 33
6.2.2 Cuantificación de la fracción indigestible total, soluble e insoluble 33
6.2.3 Digestión gastrointestinal in vitro: Aislamiento de la fracción indigestible 35
6.2.4 Caracterización nutricional, compuestos bioactivos y actividad antioxidante en la
fracción indigestible de los menús 36
6.2.4.1 Cuantificación del Almidón Resistente (AR) 36
6.2.4.2 Análisis de los compuestos antioxidante en la fracción indigestible de los menús 36
6.2.4.3 Análisis de la capacidad antioxidante en las fracciones indigestibles 38
6.3 Tercera Etapa 40
6.3.1 Fermentación colonica in vitro de la fracción indigestible 40
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6.3.2 Análisis de componentes antioxidantes asociados en los extractos de la
fermentación 41
6.3.3 Análisis de metabolitos en la fermentación colonica in vitro de la fracción
indigestible de los menús 41
6.3.3.1 Micro extracción en fase solida de los compuestos volátiles en los extractos de
fermentación 42
6.3.3.2 Cromatografía de gases acoplada a espectrometría de masas 42
6.4 Análisis estadístico 43
Capítulo 7. RESULTADOS Y DISCUSIÓN 45
7.1 Patrones dietéticos, perfil nutricional e índice de masa corporal en escolares
mexicanos: Un estudio transversal 45
Resumen 45
7.2 Perfil de metabolitos en un sistema in vitro de fermentación colónica humana en tres
menús consumidos por escolares mexicanos durante el desayuno 63
Resumen 63
7.3 Fermentación colónica humana in vitro de la fracción indigestible aislada de menús
consumidos durante la comida: Impacto en el perfil de los metabolitos intestinales 91
Resumen 91
7.4 Efectos de los alimentos frecuentemente consumidos por escolares mexicanos en la
cena sobre el metabolismo microbiano y la capacidad antioxidante durante la
fermentación colónica in vitro 120
Resumen 120
7.5 Los metabolitos intestinales se asocian al pH y cambios en la actividad antioxidante
durante la fermentación colónica in vitro de productos procesados mexicanos de maíz. 146
Resumen 146
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Capítulo 8. CONCLUSIONES 175
Capítulo 9. BIBLIOGRAFÍA 179
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LISTA DE CUADROS Pág.
Cuadro 2.1 Prevalencia de diabetes (%) en adultos por diagnóstico previo en Encuestas
Nacionales de Salud (2000 a 2012). 7
Cuadro 2.2 Características diferenciales de la fracción indigestible y la fibra dietética. 11
Cuadro 2.3 Bacterias anaerobias predominantes en el colon y sus principales productos de
fermentación 12
Cuadro 2.4 Principales ácidos grasos de cadena corta (AGCC): Sus acciones y mecanismos
ajústalo a la izquierda para que se lea más fácil 17
Cuadro 2.5 Precursores y metabolitos polifenólicos derivados de la fermentación colónica
vitro después de 5 y 24 h de cinco muestras de alimentos. 23
Cuadro 6.1 Definiciones de los grupos de alimentos utilizados para evaluar las dietas de los
escolares del municipio de Tepic 32
Table 1 Multiple regression analysis models exploring the association of principal
components (PC) with weight and body mass index (BMI). 53
Table 2 Characteristics of Mexican schoolchildren by dietary pattern groups. 57
Table 1 Chemical composition and antioxidant compounds content, and antioxidant capacity
in the indigestible fraction (IF) isolated from breakfast menus1 73
Table 2. Short-chain fatty acids (SCFAs, mmol L-1) production at 12, 24, 48 and 72 h of in
vitro fermentation of blank, raffinose and indigestible fraction isolated from
breakfast menus (MM-B; Modified Mexican Breakfast, TM-B; Traditional Mexican
Breakfast, AM-B; Alternative Mexican Breakfast) 1. 77
Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of
blank, rafinose and indigestible fraction isolated from breakfast menus (MM-D;
Modified Mexican diet, TM-D; Traditional Mexican Diet, AM-D; Alternative
Mexican diet) analyzed by SPME–GC/MS. 89
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Table 1 Chemical composition and antioxidant compounds content, and antioxidant capacity
in the indigestible fraction (IF) isolated from lunch menus1 100
Table 2. Short-chain fatty acids (SCFAs, mmol L-1) production at 12, 24, 48 and 72 h of in
vitro fermentation of blank, raffinose and indigestible fraction isolated from lunch
menus (MM-L; Modified Mexican Lunch, TM-L; Traditional Mexican Lunch, AM-
L; Alternative Mexican Lunch) 1. 104
Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of
blank, rafinose and indigestible fraction isolated from Lunch menus (MM-L;
Modified Mexican diet, TM-L; Traditional Mexican Diet, AM-L; Alternative
Mexican diet) analyzed by SPME–GC/MS (mmol/L). 118
Table 1 Chemical composition and antioxidant compounds content, and antioxidant capacity
in the indigestible fraction (IF) isolated from dinner menus1 127
Table 2. Short-chain fatty acids (SCFAs, mmol L-1) production at 12, 24, 48 and 72 h of in
vitro fermentation of blank, raffinose and indigestible fraction isolated from dinner
menus (MM-D; Modified Mexican Dinner, TM-D; Traditional Mexican Dinner,
AM-D; Alternative Mexican Dinner) 1. 132
Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of
blank, rafinose and indigestible fraction isolated from dinner menus (MM-D;
Modified Mexican diet, TM-D; Traditional Mexican Diet, AM-D; Alternative
Mexican diet) analyzed by SPME–GC/MS. 144
Table 2. Production of short-chain fatty acids (SCFAs, mmol L-1) at 12, 24 and 48 h of in
vitro fermentation of blank, raffinose and indigestible fraction isolated from Istmo
Totopo (IT), baked corn tortilla (BCT) and traditional corn tortilla (TCT) *. 160
Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of
blank, rafinose and indigestible fraction isolated from Istmo Totopo (IF-IT), Baked
corn tortilla (IF-BCT) and traditional corn tortilla (IF-TCT) analyzed by SPME–
GC/MS (mmol/L) 173
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LISTA DE FIGURAS
Figura 2.1 Distribución porcentual de las defunciones registradas por principales causas de
muerte según sexo, hombre (░) y mujer (▓) en el Estado de Nayarit, 2009 4
Figura 2.2 Tendencias en las prevalencias de sobrepeso (□), obesidad I (░), obesidad II ( ▓
), y obesidad III (■) en adultos en el periodo 2000 a 2012. 6
Figura 2.3 Prevalencia de sobrepeso (░) y obesidad (▓) en niños y niñas de 5-11 años de
edad (1999 a 2012) en México. Fuente:Romero-Martínez y cols. (2013). 6
Figura 2.4 Las localizaciones relativas de la digestión y absorción de nutrientes en el tracto
gastrointestinal saludable. 10
Figura 2.5 Rutas de los metabolitos producidos por la interacción entre la microbiota y
metaboloma humano simplificado. 14
Figura 2.6 Alimentación cruzada por la microbiota del colon. 16
Figura 2.7 Posibles mecanismos propuestos para la prevención del cáncer por los
polifenoles dietéticos 25
Figure 1 Principal component analysis (PCA) plots. (A) Loading plots for different food
groups and (B) PCA scores plot for Mexican schoolchildren of the four principal
components. 51
Figure 2 Principal component analysis (PCA) plots. (A) Loading plots for different food
groups and (B) PCA scores plot for Mexican schoolchildren (PC 5, 6, and 7). 52
Figure 3 Mean percentage energy contribution from each food group according to dietary
pattern groups: (■) Modified Mexican Diet (n=312), (░) Traditional Mexican
Diet (n=190) and (■) Alternative Mexican Diet (n=222). 55
Figure 1 Changes in a) pH kinetic plot, b) DPPH Antiradical activity plot and c) FRAP
chelating activity plot in the extracts during in vitro colonic fermentation from (–
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) blank, (··♦··) raffinose, and indigestible fraction isolated from breakfast menus:
(-■-) Modified Mexican Breakfast, MM-B, (-▲-) Traditional Mexican
Breakfast, TM-B, and (-●-) Alternative Mexican Breakfast, AM-B at different at
different fermentation times. 75
Figure 2 Identification of Microbial metabolic pattern between the in vitro colonic
fermentation extracts of blank, raffinose and indigestible fraction isolated from
breakfast menus at different fermentation times using principal component
analysis (PCA): Loading scatter plot for a) PC1 vs. PC2; b) PC3 vs. PC4 and
PC5 vs. PC6. 79
Figure 3 Multiple regression analysis models exploring the association of microbial
metabolic pattern (Component scores) with: a) pH Values, b) DPPH antiradical
activity and c) FRAP chelating activity. 81
Figure 4 Dendrogram of hierarchical cluster analysis based on the microbial metabolic
profiles (component score) in colonic fermentation extracts of blank, raffinose,
and indigestible fraction isolated from breakfast menus (MM-B; Modified
Mexican Breakfast, TM-B; Traditional Mexican Breakfast, AM-B; Alternative
Mexican Breakfast) at different fermentation times: 12 h (T12), 24 h (T24), 48 h
(T48) and 72 h (T72). 83
Figure 1 Changes in a) pH kinetic plot, b) DPPH Antiradical activity plot and c) FRAP
chelating activity plot in the extracts during in vitro colonic fermentation from (–
) blank, (··♦··) raffinose, and indigestible fraction isolated from lunch menus: (-■-
) Modified Mexican Lunch, MM-L, (-▲-) Traditional Mexican Lunch, TM-L,
and (-●-) Alternative Mexican Lunch, AM-L at different at different
fermentation times. 102
Figure 2 Cumulative concentration (%) of volatile gut metabolites by chemical groups
between the in vitro colonic fermentation extracts of blank, rafinose and
indigestible fraction isolated from Modified Mexican Lunch (MM-L),
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Traditional Mexican Lunch (TM-L), and Alternative Mexican Lunch (AM-L) at
12 and 72 h. 106
Figure 3 Principal components plots (a) metabolites production “PC Loadings” (b) and
sample classification “PC scores” (%) during in vitro fermentation from
indigestible fraction isolated lunch menus (MM-L; Modified Mexican Lunch,
TM-L; Traditional Mexican Lunch, AM-L; Alternative Mexican Lunch). 107
Figure 1 Changes in a) pH kinetic plot, b) DPPH Antiradical activity plot and c) FRAP
chelating activity plot in the extracts during in vitro colonic fermentation from (–
) blank, (··♦··) raffinose, and indigestible fraction isolated from dinner menus: (-
■-) Modified Mexican Dinner, MM-D, (-▲-) Traditional Mexican Dinner, TM-
D, and (-●-) Alternative Mexican Dinner, AM-D at different at different
fermentation times. 130
Figure 2 Cumulative concentration (%) of volatile gut metabolites by chemical groups
between the in vitro colonic fermentation extracts of blank, rafinose and
indigestible fraction isolated from Modified Mexican Dinner (MM-D),
Traditional Mexican Dinner (TM-D), and Alternative Mexican Dinner (AM-D)
at 12 and 72 h. 134
Figure 3 Principal components plots (a) metabolites production “PC Loadings” (b) and
sample classification “PC scores” (%) during in vitro fermentation from
indigestible fraction isolated dinner menus (MM-D; Modified Mexican Dinner,
TM-D; Traditional Mexican Dinner, AM-D; Alternative Mexican Dinner). 135
Figure 4. Pearson’s R correlations between gut microbial metabolites, pH values and
antioxidant capacity (DPPH antiradical activity and FRAP chelating activity) in
extracts obtained during in vitro colonic fermentation of indigestible fraction
isolated from dinner menus (▲Increase, ▼decrease and ▬ non-correlation,
p<0.05). 136
Figure 1 Changes during in vitro colonic fermentation of (-○-) blank, (-●-) raffinose
(positive control), and indigestible fraction isolated from (-�-) Istmo Totopo, (-
x
◊-) Baked corn tortilla and (-□-) Traditional corn tortilla: a) pH values plot b)
DPPH Antiradical activity plot and b) FRAP chelating activity plot. 158
Figure 2 Cumulative concentration (%) of volatile gut metabolites by chemical groups
during in vitro colonic fermentation extracts of blank, raffinose and indigestible
fraction isolated from Istmo Totopo (IT), Baked corn tortilla (BCT) and
traditional corn tortilla (TCT) at 12 and 48 h. 162
Figure 3 Principal components plots A) metabolites production “PC Loadings” and B)
sample classification “PC scores” (%) during in vitro fermentation from
indigestible fraction isolated from Istmo Totopo (IT), Baked corn tortilla (BCT)
and Traditional Corn Tortilla (TCT). 163
Figure 4. Pearson’s R correlations between gut microbial metabolites and pH values and
antioxidant capacity (DPPH antiradical activity and FRAP chelating activity) in
extracts obtained during in vitro colonic fermentation of blank, raffinose and
indigestible fraction isolated from corn products 165
Capítulo 1. INTRODUCCIÓN
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Capítulo 1. INTRODUCCIÓN
El estilo de vida en la población mexicana ha sido objeto de grandes cambios, principalmente en
los patrones de actividad física y dieta (Abell y cols., 2008). Ambos, han contribuido a la creciente
prevalencia de obesidad, diabetes tipo II y la enfermedad cardiovascular (ECV) (Denova-Gutiérrez
y cols., 2011; Denova Gutiérrez y cols., 2010 ; Romero-Polvo y cols., 2012). México, fue uno de
los países con mayor prevalencia de sobrepeso infantil en 2010 y obesidad entre los adultos
(OECD, 2012.). Esta epidemia tiene una alta tasa de crecimiento en los niños, dando como
resultado una alta prevalencia de sobrepeso y obesidad entre los escolares de todo el país. La
Encuesta Nacional de Salud y Nutrición (ENSANUT) del 2006 menciona que en Nayarit existió
una prevalencia de sobrepeso-obesidad en escolares del 31 % (Olaiz-Fernández y cols., 2006). En
Nayarit las principales causas de muerte son las enfermedades istémicas del corazón y la diabetes
mellitus en la población adulta (INEGI, 2009); posiblemente, derivado del incremento en el
sobrepeso y obesidad de la población en los últimos años. Xu y cols. (2006), mencionan que para
generar información acerca de la relación entre estos problemas de salud pública y la dieta, se han
realizado análisis nutricionales de los alimentos y su impacto en la población. La ingesta de
semillas enteras, carencia de minerales, alimentos de alto índice glucémico y carga glucémica, se
han relacionado con estas enfermedades (Liu y cols., 2009b). Sin embargo, estos análisis se han
centrado en nutrientes individuales o alimentos específicos y, las interacciones entre
macronutrientes y micronutrientes pueden pasar por alto. De manera cotidiana la gente consume
una variedad de alimentos en complejas combinaciones de nutrientes. Para llevar a cabo una
investigación epidemiológica y nutricional que más se acerque al modelo de la experiencia
humana, los investigadores han propuesto el estudio de los hábitos alimentarios en lugar de
alimentos aislados, dando un enfoque que puede ayudar a entender con más precisión la relación
entre el consumo de alimentos y la propagación o la prevención de enfermedades crónicas
(Hoffmann y cols., 2004). Es Importante mencionar que, en México, se tienen una gran diversidad
gastronómica, donde cada Estado presenta costumbres y tradiciones culinarias únicas. Sin lugar a
dudas, varios aspectos de la dieta juegan un papel clave en la salud de la población, aspectos tales
como el contenido de grasas, carbohidratos, fibra, etc. Uno de los componentes que ha sido
ampliamente estudiado, es la fibra dietética (FD) (carbohidratos indigestibles), sin embargo, tan
Capítulo 1. INTRODUCCIÓN
2
sólo es uno de los componentes indigestibles de los alimentos, ya que existen otros componentes
que pueden no ser totalmente digeridos, a estos componentes se le conoce como fracción
indigestible (FI) y poco se conoce de los efectos en la salud de estos compuestos. El estudio de los
carbohidratos indigestible se ha enfocado tan sólo en su en la cuantificación en alimentos
individuales y hay poca información acerca de su contenido en mezclas de alimentos tal cual se
consumen sobre todo en la población mexicana. Diversos estudios han demostrado los efectos
positivos de la FD en la regulación de la respuesta glucémica, la capacidad de promover la
saciedad, la actividad prebiótica y como portador de compuestos antioxidantes (Costabile y cols.,
2008; Kim y cols., 2009). Estudios de metabolómica han demostrado que los beneficios en la
salud de la ingesta de FD, están mediados por los metabolitos producidos durante su fermentación
en el colon (Martin y cols., 2009), entre los efectos potenciales para la salud se encuentra la
producción de ácidos grasos de cadena corta (AGCC) y compuestos antioxidantes asociados a la
fibra (Hernandez-Salazar y cols., 2010). Sin embargo, no todos los metabolitos producidos durante
la fermentación colónica de la fraccipon indigestible (no sólo FD), puede estar asociados con
beneficios para la salud, y algunos de ellos pueden tener efectos adversos. Por ejemplo, el
amoníaco producto de la fermentación de proteínas, en bajas concentraciones altera la mucosa
intestinal, promueve el crecimiento de células tumorales, y facilita contraer infecciones virales
(Lin y Visek, 1991). Por tanto, es importante conocer y estudiar la dieta de la población tal como
es ingerida para tener un panorama más amplio de los efectos saludables y adversos que los
patrones dietéticos ejercen en los individuos.
Capítulo 2. ANTECEDENTES
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Capítulo 2. ANTECEDENTES
2.1 La dieta en México y el desarrollo de enfermedades
Como en muchos otros países en desarrollo, la transición epidemiológica en México se caracteriza
por una reducción de las enfermedades infecciosas y por un rápido aumento de las enfermedades
crónicas no transmisibles (Rivera y cols., 2002). Entre estas últimas, la obesidad, enfermedades
cardiovasculares (ECV), diabetes tipo II y cáncer, son los problemas de salud pública más
importantes a las que los mexicanos tienen que hacer frente en la actualidad (Olaiz y cols., 2006).
Basado en datos de varias encuestas nacionales representativas, los estudios epidemiológicos han
mostrado cambios importantes en los patrones de consumo de alimentos en los hogares en México
(Rivera y cols., 2002). Estos cambios pueden contribuir a la creciente epidemia de enfermedades
crónicas relacionadas con la nutrición. En la Figura 2.1 se muestra las principales causas de
muerte en el estado de Nayarit. Durante un corto tiempo, se ha observado un notable incremento
en el consumo de grasas, azúcares refinados y bebidas carbonatadas, así como una importante
reducción en la ingesta de frutas y verduras. Los estudios también muestran una disminución de la
actividad física recreativa, un mayor acceso a los alimentos ricos en energía y de bajo costo, así
como, una baja ingesta de micronutrientes (Hernández y cols., 2003; Rivera y cols., 2002). Los
grupos de alimentos y los análisis de nutrientes aisladas se han utilizado para describir el binomio
dieta-enfermedad. Sin embargo, los individuos y las poblaciones no consumen los nutrientes o
alimentos aislados, sino diferentes alimentos de los diferentes grupos, en combinaciones o de
acuerdo a patrones, que existen por cuestiones culturales y económicos importantes (Hu, 2002).
Los estudios de población han demostrado una asociación entre diferentes patrones alimentarios
con diversos marcadores antropométricos y bioquímicos de la obesidad además de la asociación
con el desarrollo de las enfermedades crónicas (Montonen y cols., 2005; Newby y cols., 2004;
Newby y cols., 2003).
Capítulo 2. ANTECEDENTES
4
Figura 2.1 Distribución porcentual de las defunciones registradas por principales causas de muerte
según sexo, hombre (░) y mujer (▓) en el Estado de Nayarit, 2009. Fuente: INEGI (2011)
2.1.1 La dieta y el síndrome metabólico
El síndrome metabólico (SM) es una “condición” en la que se conjugan distintos padecimientos en
el individuo como, obesidad central, dislipidemia, hiperglucemia, e hipertensión (Alberti y cols.,
2005; Reaven, 1988). Aproximadamente un tercio de los adultos en México presentan esta
condición (Aguilar-Salinas y cols., 2004), donde algunos estudios han relacionado que una mayor
ingesta de cereales integrales y frutas y verduras en la dieta diaria puede derivar en una menor
prevalencia de SM (Esmaillzadeh y cols., 2004; Sahyoun y cols., 2006). La evidencia es menos
constante en el consumo de harinas refinadas, donde los resultados han sido un tanto
contradictorios, ya que algunos estudios han reportado una asociación positiva entre la ingesta de
harinas refinadas con el SM, mientras que otros no encontraron ninguna relación. Por último, la
ingesta de refrescos y bebidas azucaradas se han asociado positivamente con el SM (Denova-
0 10 20 30 40 50
Lasdemáscausas
Enfermedadesdelhígado
Enfermedadesrespiratoriasinferiores
Accidesntesdetransporte
Enfermedadescerebrovasculares
Enfermedadesisquémicasdelcorazón
Diabetesmellitus
Tumores(neoplasias)malignos
Accidentesdetransporte
Capítulo 2. ANTECEDENTES
5
Gutiérrez y cols., 2010; Dhingra y cols., 2007). La evaluación de los hábitos alimentarios, en el
que varios alimentos o nutrientes son combinados en una variable compuesta, puede arrojar una
luz más clara sobre la relación dieta-enfermedad (Schwerin y cols., 1981; Slattery y cols., 1998),
no solo en estudios de campo sino también como parte de las metodologías del análisis de
alimentos en un laboratorio.
2.1.2 La dieta y su relación con la obesidad
La obesidad se ha convertido en una enfermedad cada vez más frecuente en todo el mundo, donde
al menos mil millones de personas presentan sobrepeso y ~ 300 millones de personas son obesas
(WHO, 2008). En México, la ENSANUT, 2006 reveló que un 70% de los adultos presentan
sobrepeso o son obesos (Olaiz-Fernández y cols., 2006) y en el 2012 según la ENSANUT
(Romero-Martínez y cols., 2013) se reportó un 71.1%. Donde las mujeres presentan mayor
tendencia hacia la obesidad y los hombres mayor tendencia hacia el sobrepeso (Ver Figura 2.2).
La obesidad del adulto se ha relacionado con numerosas enfermedades crónicas y genera altos
costos al Gobierno en salud pública. Además, la creciente prevalencia de la obesidad plantea
numerosas implicaciones negativas para la salud física y económica de muchas sociedades, y se
menciona que niños con sobrepeso u obesidad tienden al desarrollo de estas enfermedades en su
etapa adulta (Ford y Mokdad, 2008).
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2000 2006 2012 2000 2006 2012 2000 2006 2012
Total Hombres Mujeres
Porcen
taje(%
)
Capítulo 2. ANTECEDENTES
6
Figura 2.2 Tendencias en las prevalencias de sobrepeso (□), obesidad I (░), obesidad II ( ▓ ), y
obesidad III (■) en adultos en el periodo 2000 a 2012. Fuente:Romero-Martínez y cols. (2013).
En la Figura 2.3 se muestra el incremento del sobrepeso y obesidad en escolares (5-11 años)
mexicanos entre los periodos de 1999 al 2012. A pesar de considerables esfuerzos en
investigación, la etiología nutricional de la obesidad sigue siendo poco clara y controversial,
especialmente en relación con las funciones desempeñadas por las grasas dietéticas (Ebbeling y
cols., 2007; McMillan-Price y cols., 2006; Willett, 1998) y carbohidratos (Acheson, 2010). Sin
embargo, estos factores de la dieta por sí solos probablemente explican sólo una parte del efecto de
la dieta sobre la obesidad. Los hábitos alimentarios generales parecen afectar a la salud más que el
consumo de alimentos y nutrientes individuales.
Este enfoque también proporciona una imagen más precisa del consumo de alimentos en la vida
cotidiana que los estudios de nutrientes aislados, produciendo resultados que ofrecen una mayor
comprensión de los cambios dietéticos saludables y recomendaciones de salud pública (van Dam y
cols., 2002)
Figura 2.3 Prevalencia de sobrepeso (░) y obesidad (▓) en niños y niñas de 5-11 años de edad
(1999 a 2012) en México. Fuente:Romero-Martínez y cols. (2013).
0
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15
20
25
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40
1999 2006 2012 1999 2006 2012
Niños Niñas
Porcen
taje(%
)
Capítulo 2. ANTECEDENTES
7
2.1.3 La dieta y su relación con la resistencia a la insulina
La insulinoresistencia (IR) se asocia con múltiples trastornos metabólicos y aumenta el riesgo de
desarrollo de diabetes tipo II y ECV (Baxter y cols., 2006). La prevalencia mundial de diabetes ha
aumentado considerablemente en los últimos años, debido a estilos de vida cada vez menos
saludables, incluyendo dietas más desbalanceadas (Rivera y cols., 2002). En el Cuadro 2.1 se
muestra la prevalencia de diabetes mellitus en adultos mexicanos por diagnóstico previo entre los
años 2000 al 2012. En los EE.UU., la Encuesta Nacional de Examen de Salud y Nutrición
(National Health and Nutrition Examination Survey) encontró que 52.1% de los adolescentes
obesos (entre 12-19 años) presentaban IR (Lee y cols., 2006). Sin embargo, la prevalencia de IR en
niños y adolescentes mexicanos no está bien documentada.
La dieta parece desempeñar un papel clave en el desarrollo de IR. Hasta la fecha, varios aspectos
de la dieta, tales como la ingesta de grasas, cierto tipo de carbohidratos, fibra dietética, el índice
glucémico de los alimentos, la carga glucémica se han relacionado con la IR (Liu y cols., 2009a).
Estudios previos han encontrado que en los adultos un patrón alimentario caracterizado por un
consumo predominante de frutas, verduras, granos enteros, productos lácteos bajos en grasa y un
bajo consumo de grasas saturadas se asocia con un menor riesgo de IR (Baxter y cols., 2006;
Esmaillzadeh y cols., 2007). Por el contrario, se ha demostrado que los hábitos alimentarios con
altos índices glucémicos (carbohidratos fácilmente digeribles) que son bajos en granos enteros
aumentan el riesgo de IR (McNaughton y cols., 2008).
Cuadro 2.1 Prevalencia de diabetes (%) en adultos por diagnóstico previo en Encuestas
Nacionales de Salud (2000 a 2012).
Edad (años) 20 a 29 30 a 39 40 a 49 50 a 59 60 a 69 70+ Total
ENSA 2000 0.04 2.2 5.4 10 17.8 9.9 4.6
ENSANUT 2006 0.04 3.4 10.6 16.8 19.7 17.2 7.3
ENSANUT 2012 1.6 3.1 9.4 19.4 26.3 20 9.2
Fuente: Romero-Martínez y cols. (2013).
2.1.4 La dieta y su relación con el cáncer de colon
Capítulo 2. ANTECEDENTES
8
Otra de las enfermedades que hoy en día se relacionan con la dieta son algunos tipos de cáncer,
donde, el más comúnmente diagnosticados en los Estados Unidos es el cáncer colorrectal (CCR),
el cual es la tercera causa de mortalidad por cáncer en México (Campos-Vega y cols., 2010). Esta
enfermedad está definida como una neoplasia maligna a partir del crecimiento de un tumor en el
epitelio intestinal como resultado de la replicación no controlada de células dañinas que tienen la
capacidad de destruir las células adyacentes o propagar la metástasis a otros tejidos. Los factores
ambientales son de gran importancia en la relación entre la dieta y cáncer (Bingham, 1997). Las
dietas de la mayoría de los países emergentes son más altas en almidón y carbohidratos en
comparación con los países occidentales que son más altas en grasas animales y proteínas (Scott y
cols., 2012). Las diferencias en los hábitos alimenticios entre los países han aportado alguna
información útil en cuanto a cuáles son los principales factores en la asociación con el CCR. La
industrialización ha tenido un papel importante que desempeñar en el aumento de la incidencia del
CCR en los países no occidentales. En los años 50’s y 60’s en comparación con el Reino Unido,
Japón tenía un alto consumo de cereales y menor consumo de productos cárnicos en casi 60
g/persona al día y al principio de la década de 90’s esto comenzó a cambiar de forma paralela a la
ingesta de cereales, la cual cayó, mientras el consumo de carne aumentó y al mismo tiempo se
observó un incremento en la incidencia de CCR, en casi un 30% en el país Nipon en los siguientes
30 años (Key y cols., 2002). Sin embargo, los estudios epidemiológicos que vinculan la dieta y el
cáncer a menudo carecen de un marcador específico que está fuertemente asociado con el cáncer y
la modulación de la dieta por separado. Más recientemente, los vínculos entre los metabolitos
producidos a partir de la dieta y ciertos componentes de los alimentos han mostrado una
asociación con la incidencia de cáncer (Rose y cols., 2007). Los patrones dietéticos se
correlacionan positivamente e incluye la ingesta de grasas y proteínas, en concreto, de productos
cárnicos y cárnicos procesados (Rose y cols., 2007). Diversas revisiones de epidemiología y
estudios prospectivos han encontrado asociaciones entre el aumento del consumo específicamente
de proteínas de carne roja, procesada y CCR (Larsson y Wolk, 2006; Lee y cols., 2004; Norat y
cols., 2002; Sandhu y cols., 2001). La principal diferencia entre la carne roja y la carne blanca son
los niveles de hemoglobina, y más concretamente el hierro contenidos en la misma. Donde
muchos estudios han centrado el contenido de hemoglobina como un factor de riesgo para CCR a
través de la participación en la formación catalítica de compuestos de N-nitrogenados (de Vogel y
cols., 2008; Klenow y cols., 2009).
2.2 Digestión y absorción de los nutrientes de la dieta
Capítulo 2. ANTECEDENTES
9
Como se mencionó anteriormente, la dieta; tiene un gran impacto en el desarrollo de enfermedades
que afectan la salud de la población y sin duda el proceso de digestión de los nutrientes juega un
rol importante en este sentido. Las funciones de motilidad y secreción del sistema gastrointestinal
están reguladas para permitir una digestión y absorción óptima de los nutrientes presentes en los
alimentos ingeridos. La digestión de los alimentos se puede dividir en mecánica y química (Ver
Figura 2.4). La digestión mecánica se inicia con la masticación en la cavidad oral y continua en el
estómago gracias a las potentes contracciones de la zona caudal. Todos ellos permiten la división
de alimento hasta convertirlo en partículas de muy pequeño tamaño (2 mm) antes de su
vaciamiento hacia el intestino delgado. Esta división mecánica da lugar a un gran aumento en la
superficie de contacto del alimento, lo que favorece la actuación de las enzimas hidrolíticas y otras
sustancias, facilitando la digestión química (Martínez de Victoria Muñoz y cols., 2010).
La digestión química la realizan enzimas hidrolíticas presentes en la luz gastrointestinal y en el
epitelio mucosal. Estas enzimas son secretadas por distintas glándulas a la luz intestinal, donde
ejercen su función (digestión luminal), o bien se asocian a la membrana del polo apical de los
enterocitos (borde en cepillo) (digestión de membrana) (Martínez de Victoria Muñoz y cols.,
2010). Los procesos de digestión comienzan en la cavidad bucal y continúan en el estómago, pero
es en el intestino delgado donde adquieren una mayor relevancia, de hecho, las alteraciones en los
procesos de digestión sólo aparecen por malfuncionamiento de los mecanismos digestivos y de su
regulación en este segmento. Una vez terminada la digestión, los productos resultantes deben
atravesar la barrera intestinal hasta llegar al medio interno, sangre o linfa de los capilares que
irrigan las vellosidades intestinales y que se localizan en la lámina propia. Para llegar al torrente
sanguíneo o linfático, los productos de la digestión deben cruzar la capa no agitada de líquido en el
lumen, el glicocáliz, en estrecho contacto con la membrana apical del enterocito que también debe
pasar, el citoplasma enterocitario, la membrana basolateral, el espacio intercelular, la membrana
basal (Martínez de Victoria Muñoz y cols., 2010).
El transporte (absorción) de las sustancias luminales hasta el medio interno puede realizarse por
diversos mecanismos que se ponen en marcha dependiendo de sus propiedades fisicoquímicas.
Estos mecanismos son pinocitosis, difusión simple o facilitada y transporte activo. Sin embargo
existe una porción de los alimentos que no es digerida ni absorbida en el intestino delgado,
conocida como fracción indigestible, que puede alcanzar el colon y ser utilizada como sustrato por
la microflora colónica, produciendo metabolitos que han sido involucrados con la salud del
huésped (Saura-Calixto y cols., 2000).
Capítulo 2. ANTECEDENTES
10
Figura 2.4 Las localizaciones relativas de la digestión y absorción de nutrientes en el tracto gastrointestinal saludable. Fuente: Jeejeebhoy (2002)
2.3 Fracción Indigestible
La fracción indigestible (FI) se define como la parte de los “alimentos vegetales” que no se digiere
ni se absorben en el intestino delgado, llegando al colon, donde es un sustrato para la microflora
fermentativa (Saura-Calixto y cols., 2000). Como tal, comprende no sólo fibra dietética, sino
Capítulo 2. ANTECEDENTES
11
también otros compuestos de probada resistencia a la acción de enzimas digestivas tales como
almidón resistente (AR), el cual escapa de la digestión y absorción en el intestino delgado humano
en los individuos sanos (Asp y Björck, 1992; Englyst y cols., 1982) y, otras sustancias alimenticias
no incluidas en la definición de fibra dietética como proteínas resistentes, oligosacáridos, ciertos
compuestos fenólicos, etc (Cummings y Englyst, 1991). Estos compuestos además, pueden ser
fermentados por la microbiota del colon, y presentar efectos fisiológicos similares a los de la fibra
dietética (Saura-Calixto y cols., 2000). En el Cuadro 2.2 se observan las principales diferencias
entre los conceptos de fracción indigestible y fibra dietética. Este concepto surge como una
alternativa al concepto de fibra dietética; que considera en su metodología la eliminación
enzimática de la proteína y almidón.
El método para la determinación de FI combina en un solo procedimiento las metodologías para
fibra dietética (Mañas y cols., 1994) y el análisis de almidón resistente (Goñi y cols., 1996). Vale
la pena señalar que, en el método de fracción indigestible, las muestras se analizan tal como se
ingieren (fresca, hervida o frita) y las condiciones analíticas (pH, temperatura, tiempos de
incubación) están cerca de las condiciones fisiológicas. Desde un punto de vista fisiológico los
valores de la fracción indigestible representan cantidades más estrechas de las sustancias
indigestibles que podrían alcanzar el colon.
Cuadro 2.2 Características diferenciales de la fracción indigestible y la fibra dietética.
Fracción indigestible Fibra dietética
Definición Principales componentes de los alimentos indigeribles
Restringido a PNA + lignina
Condiciones analíticas Fisiológicas No fisiológicas
Preparación de la muestra Como se come Molidos y hervidos
Componentes de los residuos gravimétricos Los incluidos en la definición
PNA, Lignina y otros más (AR, PR TC)
Digestibilidad del almidón No alterada Modificada artificialmente
PNA: Polisacáridos no amiláceos, AR: Almidón resistente, PR: Proteína resistente, TC: Taninos
condensados. Fuente: (Saura-Calixto y cols., 2000)
Capítulo 2. ANTECEDENTES
12
2.4 El colón y la microbiota intestinal
El colon o intestino grueso humano es de aproximadamente 150 cm de largo con un área de
superficie (no seccionada) de 1300 cm2. La microbiota colónica consiste en varios cientos de
especies (Ver Cuadro 2.3) y, aunque hay diferencias interindividuales significativas ciertas
especies bacterianas están presentes constantemente en la mayoría de los individuos (Sekelja y
cols., 2010). La microbiota es un componente importante y constituye entre el 40 y 45% de sólidos
fecales en personas que consumen dietas occidentales (Stephen y Cummings 1980; Cabotaje et al
1990).
Cuadro 2.3 Bacterias anaerobias predominantes en el colon y sus principales productos de fermentación Productos de fermentación
Bacteria Descripción
# (Log 10/g BS heces) Nutrición C2 C3 C4 Láctico Succínico
Bacteroides Bacilos Gram (-)
11 3 Principalmente sacarolíticas, algunas especies proteolíticas
+ + - - +
Eubacteria Bacilos Gram (+)
10 7 Principalmente sacarolíticas, algunas especies proteolíticas
+ - + + -
Bifidobacteria Bacilos Gram (+)
10 2 Sacarolíticas
+ - - + -
Lactobacilos Bacilos Gram (+)
9 6 Sacarolíticas
- - - + -
Ruminococos Bacilos Gram (+)
10 2 Sacaroíiticas
+ - - - -
Peptococos Bacilos Gram (+)
10 0 Proteolíticas
+ - + + -
Peptostrep-tococos
Bacilos Gram (+)
10 1 Especies sacarolíticas y proteolíticas
+ - - + -
Clostridios Bacilos Gram (+)
9 8 Especies sacarolíticas y proteolíticas
+ + + + -
Capítulo 2. ANTECEDENTES
13
C2=Ác. Acético, C3= Ác. Propiónico, C4= Ác. Butírico. Adaptado de: Cummings y Macfarlane
(1991)
Los análisis de genes que codifican para la fracción 16S del rRNA amplificados directamente
junto con estudios de metagenómica han ayudado a definir los filotipos que son más abundantes
dentro de la microbiota fecal humana (Qin y cols., 2010; Tap y cols., 2009; Walker y cols., 2010).
Los filos bacterianos dominantes en adultos sanos son Firmicutes, Bacteroidetes y Actinobacteria,
normalmente con menor abundancia de Verrucomicrobia y Proteobacteria. Dos de las especies
bacterianas más dominantes en el intestino grueso de los individuos que consumen una dieta
occidental son las especies productoras de butirato, Faecalibacterium prausnitzii y Eubacterium
rectale (Flint y cols., 2012a), que conforman alrededor de 8% y 4% de la microbiota,
respectivamente. Este grupo de bacterias existen en condiciones relativamente estables con
respecto a los factores ambientales tales como la disponibilidad de nutrientes, pH, temperatura,
potencial óxido-reducción y el grado de anaerobiosis. La microbiota del colon está en contacto
íntimo con su huésped y toma parte en un gran número de procesos metabólicos mismos que se
dan a partir de la fermentación colónica de los componentes indigestibles de los alimentos.
2.4.1 Fermentación colónica
En el hombre, el intestino grueso recibe el material del íleon que ya ha sido digerido, los
contenidos se mezclan y son retenidos durante 6-12 h en el ciego y en el colon ascendente. A partir
de entonces, la digesta se expulsa y se pasa a través del colon transverso para su almacenamiento y
alcanza el colon descendente y su eventual elimnación. El tiempo promedio de retención del
material que entra al colon hasta su excreción es de aproximadamente 60 h en individuos del
Reino Unido (Cummings y Macfarlane, 1991). La cantidad total de material que entra en el colon
cada día es de 1.5 kg aproximadamente, mientras que el peso promedio de las heces, en el Reino
Unido, es de 120 g/d (Cummings y Macfarlane, 1991). Por lo tanto, el intestino grueso es un
sistema abierto, con nutrientes que fluyen desde el ciego, bacterias, productos metabólicos de éstas
y la fracción no utilizada en esta sección termina siendo excretada en forma de heces. La hidrólisis
anaeróbica de los carbohidratos y proteínas por bacterias se conoce convencionalmente como
fermentación colónica. En el hombre, los productos finales principales son los AGCC, ácido
acético, propiónico y butírico, los gases de H2, y CO2, amoniaco, aminas, fenoles y energía, que las
bacterias utilizan para el crecimiento y el mantenimiento de la función celular (Scott y cols.,
2012). En la Figura 2.5 se muestran las rutas metabólicas de los metabolitos producidos por la
microbiota. Desde el punto de vista del anfitrión, los productos finales de las reacciones de
Capítulo 2. ANTECEDENTES
14
fermentación son importantes, ya que se absorben en el intestino e influyen en muchos aspectos
del metabolismo.
Figura 2.5 Rutas de los metabolitos producidos por la interacción entre la microbiota y metaboloma humano simplificado. Adaptado de: Hamer y cols. (2012).
2.5 Metabolitos derivados de la fermentación colónica de carbohidratos.
Muchos carbohidratos presentes en alimentos derivados de plantas son digeridos lentamente o no
digeridos por el intestino delgado, lo que los hace disponibles a la fermentación por la microflora
del intestino grueso. Aproximadamente 40 g de carbohidratos provenientes de la dieta alcanzan el
colon cada día (Cummings y Englyst, 1991). Los principales carbohidratos son almidones
resistentes (AR), polisacáridos no amiláceos (PNA) y oligosacáridos, aunque algunos
monosacáridos y disacáridos (por ejemplo, alcoholes de azúcar). Estos carbohidratos son
degradados por las bacterias del colon a través de vías que conducen a la formación de ácido
acético, succínico, propiónico butírico y fórmico, ácido láctico, etanol, hidrógeno y CO2,
dependiendo de cada cepa y especie (Russell y cols., 2013). La formación de ácido butírico se
Capítulo 2. ANTECEDENTES
15
produce en ciertas bacterias Firmicutes, ya sea a través de la enzima quinasa del ácido butírico, (en
muchas especies de Clostridium y Coprococcus), o por la vía del butiril CoA: Acetato CoA
transferasa (Louis y Flint, 2009). Las bacterias productoras de ácido butírico como Roseburia,
Eubacterium rectale, Eubacterium hallii y Faecalibacterium prausnitzii, absorben directamente el
ácido acético externo y lo convierte a butírico (Louis y cols., 2010). El ácido acético es producido
por la mayoría de los anaerobios, incluyendo bacterias acetógenas que son capaces de realizar
acetogénesis reductiva de ácido fórmico o hidrógeno más CO2. Los productores de ácido succínico
y propiónico pertenecen al filo Bacteroidetes, pero también incluyen algunos Firmicutes. El ácido
láctico puede ser formado por muchos grupos, pero en general se convierte en ácido acético,
propiónico o butírico de un subconjunto de bacterias que utilizan ácido láctico (Flint y cols.,
2012b). La formación de los gases como H2 y CO2 varía entre especies en cultivos puros; en la
comunidad mixta encontrada en el colon estos productos son parcialmente convertidos a ácido
acético, metano y sulfuros de hidrógeno (Nakamura y cols., 2010).
2.5.1 Ácidos grasos de cadena corta (AGCC)
En la fermentación colonica ocurre un gran número de complejas interacciones derivadas de la
alimentación cruzada entre los microorganismos del colon (Ver Figura 2.6). La presencia de
carbohidratos indigestibles y la producción de AGCC pueden alterar positivamente la fisiología
del colon. Varios estudios sobre la microbiota intestinal han mostrado que la producción de AGCC
ocurre en el orden siguiente acético > propiónico > butírico, en proporciones molares de
aproximadamente 60:20:20, principalmente fermentados en el colon proximal y distal (Topping y
Clifton, 2001).
Capítulo 2. ANTECEDENTES
16
Figura 2.6 Alimentación cruzada por la microbiota del colon. Adaptado: Flint y cols. (2008)
Un aumento en la síntesis de AGCC también crea un entorno más ácido en el intestino, lo cual es
importante in vivo en términos de resistencia a la colonización contra patógenos (Roberfroid,
2000). La producción de AGCC se ve afectada por muchos factores, incluyendo la fuente de
sustrato, en particular, la composición química del sustrato fermentable, la cantidad de sustrato
disponible, su forma física (por ejemplo, tamaño de partícula, la solubilidad, la asociación con
complejos indigeribles tales como lignina), la composición de las especies de bacterias de la
microbiota, factores ecológicos (interacciones competitivas y de cooperación entre los diferentes
grupos de bacterias) y el tiempo de tránsito intestinal (Huazano-García y López, 2013). Los
AGCC se absorben rápidamente en el ciego y en el colon proximal y, se excretan en las heces sólo
del 5% al 10%. Los principales AGCC (Ácido acético, propiónico y butírico), son absorbidos en
diferentes regiones del colon. Una vez absorbidos, los AGCC se metabolizan en tres sitios
principales en el cuerpo: 1) células del epitelio en el colon proximal, donde el ácido butírico es un
sustrato importante para el mantenimiento de la energía de la microbiota; 2) las células del hígado
que metabolizan residuos de ácido butírico y propiónico usados para la gluconeogénesis, de novo
lipognénesis, donde 50 a 70% de ácido acético también es absorbido en hígado, y 3) células
musculares que generan energía a partir de la oxidación de los residuos de ácido acético (Van Loo
y cols., 1995).
2.5.2 Principales AGCC y sus efectos en la salud.
Tal como se ha mencionado anteriormente, los principales AGCC producidos en el colon son: el
ácido acético, propiónico y butírico. En el Cuadro 2.4 se presentan algunos de los efectos en la
salud de estos AGCC. El ácido acético es el principal AGCC producido en el colon, éste se
absorbe fácilmente y se transporta al hígado, y por lo tanto es el que menos se metaboliza (Cook y
Sellin, 1998). La presencia de acetil-CoA sintetasa en el citosol de tejido adiposo y glándulas
mamarias permite utilizar el ácido acético para la lipogénesis una vez que entra en la circulación
sistémica. El ácido acético es el sustrato primario para la síntesis de colesterol. También puede ser
absorbido y utilizado por los tejidos periféricos (Wong y cols., 2006). Por otro lado, el ácido
propiónico se produce a través de dos vías principales: 1) por la fijación de CO2 para formar acido
succínico, el cual posteriormente es descarboxilado (la "ruta del ácido dicarboxílico") y 2) se
forma ácido láctico y acrilato (la "vía de acrilato") (Cummings y Macfarlane, 1991). El ácido
propiónico es también un sustrato para la gluconeogénesis hepática y se ha informado de que este
Capítulo 2. ANTECEDENTES
17
ácido inhibe la síntesis de colesterol en el tejido hepático (Cheng y Lai, 2000). Por último, el ácido
butírico es el combustible preferido por las células epiteliales del colon, pero también juega un
papel importante en la regulación de la proliferación y la diferenciación celular (Topping y
Clifton, 2001). El ácido butírico es el AGCC más importante en el metabolismo de los
colonocitos, donde se metaboliza del 70 % al 90 % de ácido butírico. Éste AGCC se utiliza
preferentemente sobre el ácido propiónico y acético en una proporción de 90:30:50 (Cook y Sellin,
1998).
Cuadro 2.4 Principales ácidos grasos de cadena corta (AGCC): Sus acciones y mecanismos ajústalo a la izquierda para que se lea más fácil AGCC Sitio de acción Principio de acciones / Efectos y mecanismos propuestos.
Ácido acético Hígado,
músculo y
otros tejidos
periféricos
Metabolismo: El principal sustrato para la síntesis de colesterol
Ácido
propiónico
El tejido
adiposo y el
hígado
Metabolismo: El principal sustrato para la gluconeogénesis
(especialmente en rumiantes), inhibe HMG-CoA → disminución
de la síntesis hepática de colesterol
Anti-inflamatorios: En el intestino bajo modula la actividad de la
ciclooxigenasa, estimula GPCR41 y GPCR43; inhibe NF-κB a
través de PPARγ
Antimicrobiano: Inhibe la expresión de los genes que facilitan la
invasión y la penetración del epitelio intestinal por Salmonella
typhimurium
Mejora de la sensibilidad a la insulina: Inhibe la lipólisis y
favorece la lipogénesis en el tejido adiposo viceral (TAV) y
suprime la producción de ácidos grasos (AG) en el hígado →
bajos niveles de AG en el hígado y el plasma → menor estado
inflamatoria → reduce la resistencia a la insulina causada por la
interacción AG-inflamación
Saciedad: Promueve la saciedad a través de complejo
neuronal, endocrino, paracrino, autocrino ; influye en
Continúa Cuadro 2.4
Capítulo 2. ANTECEDENTES
18
la producción de hormonas adipocineticas, tales como la leptina,
una hormona anorexígenica potente.
Ácido butírico
Colonocitos Anticarcinogénicos: Inhibe las histonas deacetilasas (HDAC) →
hiperacetilación de histonas y mejora de la accesibilidad de los
factores de transcripción al ADN nucleosomal → regula la
expresión génica y la función celular; aumenta la actividad de la
enzima desintoxicante glutatión-S-transferasa, inhibe la
migración de células tumorales mediante la inhibición de la
expresión del factor de deterioro más acelerado (FDA) y
activación de metaloproteinasas pro-metastásicas; inhibe la
angiogénesis inducida por el tumor mediante la modulación de
las proteínas relacionadas con la angiogénesis, el factor de
crecimiento endotelial vascular (FCEV) y factor inducible de
hipoxia (FIH-1α).
Anti-inflamatorios: Suprime la activación de NF-kappa B a
través de la inhibición de la HDAC, la inhibición de la
producción de interferón-γ y / o señalización, y la regulación al
alza de receptores activados por el proliferador de peroxisomas
(PPARγ); actúa como una molécula de señalización a través de
GPR41 y GPR43
Refuerzo de las líneas de defensa del colon: Aumenta la
expresión de genes MUC2 → estimula la síntesis
de mucina; aumenta factor trébol intestinal (ITF o TFF3)
secretado por las células caliciformes intestinales
Saciedad: vía GLP-1 o el péptido YY
Adaptado de: Vipperla y O'Keefe (2012)
Aproximadamente el 95% del ácido butírico producido por las bacterias del colon es transportado
a través del epitelio, pero las concentraciones en sangre portal por lo general no son detectables
como resultado de una rápida utilización (Pryde y cols., 2002). La producción de ácido butírico
también puede ocurrir a través de la utilización de otros productos de la fermentación tales como
Continúa Cuadro 2.4
Capítulo 2. ANTECEDENTES
19
ácido acético o ácido láctico que pueden actuar como precursores del ácido butírico en la
alimentación cruzada de la microbiota colónica.
2.6 Proteínas de la dieta y las bacterias proteolíticas
Diariamente al colon llegan a diario, aproximadamente entre 12 a 18 g de proteína, esto es,
proteínas residuales de la dieta y enzimas secretadas en el intestino delgado. La proporción de
proteínas residuales de la dieta en el colon (alrededor de 10% de ingesta de proteínas) dependen de
la cantidad y tipo de proteína consumida (Cummings, 1997). El colon es un sitio activo de rotación
de proteínas que proporciona nitrógeno para el crecimiento de las bacterias sacarolíticas además de
aportar aminoácidos para la fermentación por especies sacarolíticas (Cummings y Macfarlane,
1991). Las bacterias proteolíticas predominantes identificaaos en las heces humanas son especies
de Bacteroides (especialmente del grupo B. fragilis (Macfarlane, 1995), así como Clostridium
perfringens, propionibacterias, estreptococos, bacilos y estafilococos (Macfarlane y cols., 1986).
La fermentación de aminoácidos como una fuente de energía se produce en el colon distal, donde
las fuentes de hidratos de carbono se agotan y el pH luminal es casi neutro (Hamer y cols., 2012).
Entre las bacterias fecales las especies de Bacteroides poseen actividad peptidasa muy fuerte
(Wallace y McKain, 1997) y también predominan cuando el pH es de alrededor de 6.5 (Walker y
cols., 2005), como en el colon distal. Además, varios grupos de bacterias fermentan
preferentemente aminoácidos y poseen sólo una débil actividad sacarolíticas como peptococos,
acidaminococci, veillonella, y algunas fusobacterias, eubacterias y clostridios (Macfarlane y
Macfarlane, 1997).
2.6.1 Productos de la fermentación de proteínas
La diversidad de los metabolitos resultado de la fermentación de proteínas es mayor comparados a
los metabolitos producidos por la fermentación de carbohidratos. La vía principal de la
fermentación de aminoácidos en el colon humano es la desaminación, dando lugar a la producción
de AGCC y amoniaco (Cummings y Macfarlane, 1991). Alrededor del 30% del sustrato se
convierte en ácido acético (principal AGCC), propiónico y butírico y los ácidos grasos de cadena
ramificada (AGCR) isobutírico, 2-metilbutiríco e isovaleríco. Estos AGCR están formados
Capítulo 2. ANTECEDENTES
20
principalmente de los aminoácidos de cadena ramificada valina, isoleucina y leucina,
respectivamente (Macfarlane y Macfarlane, 1997; Rasmussen y cols., 1988), y se utilizan a
menudo como marcadores fecales para la fermentación de proteínas. Concentraciones de amoníaco
fecal van del 12 a 30 mM en los seres humanos y pueden aumentar con un alto consumo de
proteínas (Hughes y cols., 2000). La mayor parte del amoníaco producido se absorbe rápidamente,
se metaboliza en el hígado produciendo urea, la cual se excreta en la orina. El amoníaco altera la
morfología de los tejidos intestinales, y puede actuar como un promotor de tumores en el intestino.
La desaminación bacteriana de los aminoácidos aromáticos conduce a la producción de los
compuestos fenólicos, p-cresol, fenilpropioníco (a partir de tirosina), fenilacético (a partir de
fenilalanina), indol propiónico e indol acético (a partir de triptófano). Las bacterias involucradas
en esta conversión incluyen especies de los géneros Clostridium, Bacteroides, Enterobacter,
Bifidobacterium y Lactobacillus. Las concentraciones de compuestos fenólicos son más altas en el
colon distal en comparación con el colon proximal (Macfarlane, 1995). Los fenoles se absorben
rápidamente en el colon, son metabolizados en el hígado y se excreta principalmente como p-
cresol en la orina (Macfarlane, 1995).
La descarboxilación de aminoácidos y péptidos conduce a la formación de una gran variedad de
aminas. El género Clostridio está vinculado a la producción de aminas, las Bifidobacterias y
Bacteroides contribuyen a su producción (Cummings y Macfarlane, 1991). Su significado
toxicológico no se entiende bien, pero las aminas podrían actuar como precursores en la formación
de nitrosaminas. Las nitrosaminas son conocidos carcinogénicos y se puede detectar en las heces
humanas. La formación de nitrosaminas gástricas ha sido bien descrita en los seres humanos y la
participación de la microbiota se ha demostrado mediante la comparación de ratas libre de
gérmenes (LG) y ratas convencionales (Massey y cols., 1988). Sin embargo, la mayoría de
bacterias capaces de producir nitrosaminas son especies aeróbicas tales como Escherichia,
Pseudomonas, Proteus, Klebsiella, sin embargo son prácticamente indetectables en el colon
(Suzuki y Mitsuoka, 1984). El sulfuro de hidrógeno (H2S) es producido por las bacterias
reductoras de sulfato (BPS, por ejemplo, Desulfovibrio spp.) a partir de azufre inorgánico de la
dieta y aminoácidos sulfatados (metionina, cisteína, cistina y taurina). Las altas concentraciones de
sulfuro en el colon pueden estar relacionadas con la enfermedad inflamatoria intestinal y colitis
ulcerosa (Cummings, 1997). La evidencia a partir de experimentos en animales LG demuestra
claramente el papel de la microbiota en la generación de estos metabolitos de proteínas. La
alimentación con una dieta alta en proteína en ratas convencionales produjo un aumento de ácidos
grasos de cadena ramificada (AGCR) en tejido del ciego, pero no en ratas LG (Lhoste y cols.,
1996). En un estudio parecido, Wikoff y cols. (2009) no detectaron fenoles e indoles, pero las
Capítulo 2. ANTECEDENTES
21
concentraciones más altas de los aminoácidos aromáticos se detectó en la sangre de los ratones LG
en comparación con los ratones convencionales.
2.7 Grasas dietéticas
La grasa de la dieta se absorbe principalmente en el intestino delgado humano, pero un estudio
reciente mostró que el 7% de los ácidos grasos de la dieta que llevan la etiqueta 13C se excreta en
las heces (Gabert y cols., 2011). Pocos estudios han investigado el efecto de la grasa de la dieta
sobre la microbiota humana y los metabolitos. El consumo de una dieta alta en grasa (DAG) en
comparación con dieta bajas en grasa (DBG) redujo significativamente las concentraciones de
AGCC en la heces, incluyendo las concentraciones de butirato y los recuentos de Bifidobacterias
(Brinkworth y cols., 2009). Sin embargo, con el fin de crear las dietas equivalentes de energía, la
dieta DAG fue baja en hidratos de carbono, mientras que la dieta DBG fue significativamente
mayor en hidratos de carbono digeribles y fibra por lo que no quedó claro si tal efecto se debió a la
presencia de los carbohidratos en mayor o menor concentración. El análisis de la microbiota de
ratones alimentados con una dieta AG para inducir la obesidad durante 12 semanas y, a
continuación un cambio a una dieta normal durante otras 10 semanas indicaron que la composición
de la microbiota del grupo de ratones obesos en la DAG era diferente, pero no fue diferente a la
del grupo de control después de 10 semanas con una dieta normal (Zhang y cols., 2012). Por lo
tanto la fuerte respuesta de la microbiota a la dieta alta en grasas puede ser reversible, lo que
indica que la microbiota responde a la dieta y no es asociada al fenotipo obeso. Los ratones obesos
eran todavía significativamente más pesados que el grupo de control, incluso después de 10
semanas de nuevo con la dieta normal. Las dietas altas en grasa causan una inflamación de bajo
grado en el intestino, debido al aumento de los niveles plasmáticos de marcadores de la
inflamación, y los niveles más altos de lipopolisacárido (LPS) (Cani y cols., 2007a). Por otro
ladoLos estudios en modelos animales han demostrado que el efecto de una dieta alta en grasa, y
libre de carbohidratos puede ser mitigado en varios parámetros de la salud mediante la adición de
prebióticos, tales como fructooligosacáridos (FOS) (Cani y cols., 2007b; Dewulf y cols., 2011) o
arabinoxilanos (Neyrinck y cols., 2011). La suplementación de la dieta AG con FOS restaura el
número de Bifidobacteria spp. mejorando la tolerancia a la glucosa y reduciendo la expresión de
citoquinas pro-inflamatorias (Cani y cols., 2007b). El consumo de una DAG condujo a una
reducción significativa en números de las especies de Roseburia, que fueron restaurados ya sea por
la administración de suplementos como arabinoxilano (Neyrinck y cols., 2011) o por quitina-
glucano de hongos (Neyrinck y cols., 2012). Los números de Bifidobacteria no solo no disminuye
en DAG en este caso, sino también se mejorara siguiendo la administración de suplementos de
Capítulo 2. ANTECEDENTES
22
arabinoxilano, y una reducción en los niveles de marcadores inflamatorios circulantes,
interleucina-6 (IL-6) y la proteína quimio atrayente de monocitos 1 (MCP-1) fue observado
(Neyrinck y cols., 2011). Los cambios positivos en los niveles de marcadores inflamatorios
circulantes, que aumentan con DAG, puede ser debido a la mejora de las funciones de barrera
intestinal mediada por el receptor de GLP-2 (Cani y cols., 2009), lo que se demostró el efecto
positivo de las DAG cuando son complementadas con FOS y arabinoxilano (Neyrinck y cols.,
2011).
2.8 Metabolitos microbianos derivados de polifenoles dietéticos El creciente interés en los polifenoles ha sido provocado por cientos de estudios que han asociado
las dietas ricas en polifenoles a varios efectos sobre la salud en los seres humanos (Cardona y
cols., 2013). Los polifenoles han sido reconocidos como promotores de la salud debido a su
actividad antioxidante. Sin embargo, en los últimos años la bioactividad de estos fotoquímicos se
ha relacionado con su biodisponibilidad y el catabolismo en los seres humanos y, se está poniendo
de manifiesto que los efectos biológicos no deben ser atribuidos a los compuestos nativos
presentes en los alimentos, sino a sus metabolitos producidos en diversos compartimentos internos
en el cuerpo humano (Manach y cols., 2005). El intestino delgado se considera una pieza clave en
la absorción y el metabolismo de polifenoles. Los polifenoles dietéticos son modificados y
absorbidos en el intestino delgado antes de entrar en la circulación sistémica, sin embargo, la
mayoría no son absorbidos eficientemente, alcanzando así el colon. En el colon, la microbiota
humana induce transformaciones drásticas que son muy diferentes de las ejercidas por las enzimas
intestinales y hepáticas. El colon alberga un ecosistema microbiano altamente complejo que
interactúa simbióticamente con el anfitrión y funciona como un biorreactor con un potencial
metabólico prácticamente ilimitado (Walle, 2004). La microbiota intestinal lleva a cabo reacciones
químicas para modificar esqueletos fenólicos y permite la absorción de una gama de metabolitos
de menor peso. Enzimas microbianas pueden hidrolizar glucósidos, glucurónidos, sulfatos, amidas,
ésteres y lactonas, además provocar la escisión de anillos, reacciones de reducción,
descarboxilación, desmetilación, y deshidroxilación (Monagas y cols., 2010). En el Cuadro 2.5 se
muestra los precursores polifenólicos y sus metabolitos derivados de la fermentación colónica in
vitro de distintos alimentos. De este modo, la biodisponibilidad de los polifenoles está empezando
a ser reconsiderada y ha sido objeto de estudio. Como un ejemplo, los ácidos clorogénicos y
flavan-3-oles producen sus espectros únicos de metabolitos colónicos, que se excretan en la orina
Capítulo 2. ANTECEDENTES
23
en cantidades sustanciales correspondientes al 29% de la ingesta de café y aproximadamente 71%
después de consumo de té verde (Manach y cols., 2004).
Cuadro 2.5 Precursores y metabolitos polifenólicos derivados de la fermentación colónica vitro después de 5 y 24 h de cinco muestras de alimentos.
Alimento Precursor fenólico Metabolito 5h 24h Chocolate negro Epicatequina, catequina, dímeros
de procianidina tipo b, trímeros de procianidina tipo b, tetrámeros de procianidina tipo b.
5-(3´,4´-dihidroxifenil)-ү-valerolactona
D D
(3,4-dihidroxifenil) ácido acético
D D
Ácido protocatéico D D ácido hidroxibenzoico D D ácido salicílico
D ND
Jugo de naranja Hexósido de delfinidina; hexósido de malonil cianidina; cianidina hexósido de dioxaloil; Hexósido de cianidina ; soforósido de cianidina, rutinósido de delfinidina ; hexósido de cuomaroil; hexósido feruloil; hexósido de ácido sináptico; hexósido de kaempferol; Narirutina; didimina; eriocitrina; diosmina, hesperidina.
Ácido dihidroferúlico D D ácido sinápico D D Ácido protocatéico D D
Salvado de Avena Ácido ferúlico; derivado de ácido ferúlico, ácido sinápico; avenantramida A; avenantramida B, ácido salicílico
Ácido dihidroferúlico D D Ácido dihidrosinápico D D
Café Ácido ferúlico; ácidos coumaroilquínico; ácidos cafeoilquínico; ácidos feruloilquínico; ácidos dicafeoilquínico, ácido salicílico
Ácido caféico D D
Ácido dihidroferúlico D D Ácido quínico D D ácido dihidrocaféico
D
D
ácido ferúlico D D
Continúa Cuadro 2.5
Capítulo 2. ANTECEDENTES
24
ácido protocatéquico D D ácido hidroxibenzoico D D
Frambuesas Hexósido de pelargonidina; Hexósido de cianidina, rutinósido de cianidina ; soforósido de cianidina ; cianidina sambubiósido ramnósido; coumaroil hexósido; cafeoil hexósido; ácido salicílico.
Ácido protocatéico D D Ácido benzoico D D
D: Detectado, ND: No detectado. Adaptado de Dall'Asta y cols. (2012)
2.8.1 Metabolitos de polifenoles y su impacto sobre la salud intestinal
Los metabolitos de los polifenoles dietéticos pueden presentar un efecto positivo en el
mantenimiento de la salud intestinal o la prevención de enfermedades como cáncer. En la Figura
2.7 se resumen los mecanismos propuestos para la prevención de cáncer de colon a partir de los
polifenoles dietéticos. Diversos estudios han demostrado los efectos que presentan los polifenoles
en la salud. Queipo-Ortuño y cols. (2012) llevaron a cabo un estudio de intervención humana y
encontraron que el consumo regular de polifenoles del vino tinto generó una disminución
significativa en los niveles plasmáticos de la presión arterial, triglicéridos y colesterol de
lipoproteínas de alta densidad. Monagas y cols. (2009), observaron que los ácidos fenólicos
dihidroxilados (ácido 3,4 – dihidroxifenil propiónico, ácido 3-hidroxifenilpropiónico y ácido 3,4-
dihidroxifenil) derivan de metabolismo microbiano de proantocianidinas presentando marcadas
propiedades antiinflamatorias in vitro, reduciendo la secreción de TNF-α (factor de necrosis
tumoral alfa), IL-1β (Interleucina 1β) y IL-6 (interleucina 6) en células mononucleares de sangre
periférica de sujetos sanos. Se ha sugerido que estos metabolitos microbianos podrían estar entre la
nueva generación de agentes terapéuticos para el tratamiento de las enfermedades
inmunoinflamatorias tales como la aterosclerosis (Monagas y cols., 2009), así como para la
amortiguación de la respuesta inflamatoria a antígenos bacterianos, que puede tener implicaciones
para enfermedades crónicas inflamatorias o autoinmunes tales como la enfermedad inflamatoria
intestinal (Tuohy y cols., 2012). Larrosa y cols. (2009) filtraron los diferentes catabolitos
microbianos de los polifenoles para evaluar su potencial antiinflamatorio in vitro, encontraron que
el ácido hidrocaféico, dihidroxifenil acético e hidroferúlico redujo la producción de prostaglandina
E2 (promotora importante de la carcinogénesis) en al menos un 50% en células de fibroblastos de
colon estimuladas con interleucina 1 beta (IL-1β). Estos resultados sugieren que los alimentos que
Capítulo 2. ANTECEDENTES
25
contienen precursores de ácido hidrocaféico (procianidinas, derivados de ácidos hidroxicinámicos,
etc) como la alcachofa, cacao, manzanas y fresas podrían ejercer actividad antiinflamatoria y
reducir la inflamación intestinal en los seres humanos.
Figura 2.7 Posibles mecanismos propuestos para la prevención del cáncer por los polifenoles dietéticos. Adaptado: Cardona y cols. (2013)
Queda claro que existe mucha bibliografía acerca del efecto de los metabolitos proveniente de
alimentos específicos en la salud, sin embargo, poco o nada se conoce de los efectos de
metabolitos generados a partir de la ingesta de patrones dietéticos de una población. Precisamente
éste es el enfoque del presente trabajo, específicamente en la salud de colon.
Capítulo 3. JUSTIFICACIÓN
26
Capítulo 3. JUSTIFICACIÓN
Las modificaciones en la actividad física y los hábitos alimentarios puede ser un factor que
contribuye a la prevalencia de sobrepeso y obesidad en la población infantil. La prevalencia de
sobrepeso y obesidad desde la infancia es un factor importante para el desarrollo de enfermedades
no transmisibles entre la población. Existe poca información de los patrones dietéticos actuales
presentes en la población infantil. Por otro lado, componentes de la fracción indigestible de los
alimentos, específicamente la fibra dietética ha asociada con una gran cantidad de beneficios
fisiológicos y existen una gran cantidad de estudios acerca de la composición, estructura y
componente asociados a la fibra dietética en alimentos aislados (un solo alimento), pero los
individuos y las poblaciones no consumen nutrientes o alimentos aislados, sino diferentes
alimentos de los diversos grupos, en combinaciones o patrones dietéticos. El interés en el papel de
la microbiota colónica humana en la salud o la enfermedad ha aumentado constantemente. A
través del proceso de fermentación, las bacterias del colon producen una amplia gama de
compuestos que pueden tener importantes implicaciones en los procesos fisiológicos de colon.
Debido a la inaccesibilidad del colon humano, los estudios in situ son difíciles y es necesaria más
información para comprender los procesos metabólicos de la microbiota. La mayoría de los
estudios que presentan este enfoque han evaluado sólo un número limitado de compuestos
orgánicos volátiles. Mayormente ácidos grasos de cadena corta (AGCC) han sido analizados a
causa de los efectos beneficiosos para el huésped, mientras que otros informaron sobre la medición
de compuestos que contienen azufre o compuestos fenólicos (fenol, indol y escatol), como
marcadores de la fermentación de proteínas. Sin embargo, poco a poco se comienza a creer que
cada metabolito tiene una importancia en la actividad biológica de todo el extracto de
fermentación. De aquí la importancia de la caracterización de los extractos que en este trabajo
proponemos.
Capítulo 4. HIPÓTESIS
27
Capítulo 4. HIPÓTESIS
La fermentación colónica de la fracción indigestible de alimentos consumidos durante el
desayuno, comida y cena producen distintos perfiles de metabolitos microbianos relacionados
directamente con los grupos alimenticios que se consumen con frecuencia en la dieta,
promoviendo cambios en pH y en el estado antioxidante afectand el estado de salud–enfermedad
de los individuos. Por lo anterior, debido a la poca información de los patrones dietéticos en la
población infantil nayarita y poco conocimiento de los efectos fisiológicos que se tenga a partir de
la ingesta de los alimentos típicos de la región, es necesario llevar a cabo un estudio que se
enfoque en estos aspectos.
Capítulo 5. OBJETIVOS
28
Capítulo 5. OBJETIVOS
5.1 Objetivo general
Determinar el perfil de metabolitos microbianos productos de la fermentación colónica de la
fracción indigestible de alimentos frecuentemente consumidos por escolares de Tepic, Nayarit.
5.2 Objetivos específicos
• Evaluar los patrones dietéticos mediante análisis multivariado y determinar los alimentos
de mayor consumo en escolares de entre 9 y 12 años del municipio de Tepic, Nayarit.
• Preparar los alimentos de mayor consumo y someterlos a un proceso de digestión
gastrointestinal in vitro para cuantificar y aislar la fracción indigestible.
• Determinar la composición nutricional y actividad antioxidante de la fracción indigestible
aisladas de los alimentos frecuentemente consumidos por los escolares.
• Realizar la fermentación colónica in vitro de la fracción indigestible e identificar los
metabolitos producidos en este proceso, así como determinar su capacidad antioxidante
Capítulo 6. METODOLOGÍA GENERAL
29
Capítulo 6. METODOLOGÍA GENERAL
Este estudio se dividió en tres etapas.
Durante la primera etapa se llevó a cabo la evaluación de una encuesta nutricional (trabajo previo)
realizada a alumnos de primaria entre 9-12 años de edad (n=724). A partir de la encuesta se realizó
el análisis de los alimentos frecuentemente consumidos, con herramientas estadísticas
multivariadas. Los alimentos con mayor consumo fueron elaborados en el laboratorio siguiendo
las condiciones culinarias propias de la región. Estos alimentos fueron sometidos a un proceso de
digestión in vitro para cuantificar y aislar la FI.
En la segunda etapa las FI aisladas fueron caracterizados parcialmente, mediante análisis de
composición química (proteínas, carbohidratos, lípidos, cenizas, humedad, almidon resistente,
compuestos bioactivos (polifenoles extraíbles y no extraíbles: taninos condensados y taninos
hidrolizables) y actividad antioxidante (AOX: DPPH, ABTS y FRAP).
La tercera etapa consistió en la simulación de la fermentación colónica in vitro de las FI aisladas y
identificación de los metabolitos producidos durante este proceso con técnicas de cromatografía de
gases acoplada a espectrometría de masas. Así mismo se determinó la capacidad antioxidante en
los extractos en los tiempos 12, 24, 48 y 72 h de fermentación.
A continuación, se describe más ampliamente la metodología que fue llevada a cabo.
6.1 Primera Etapa
6.1.1 Análisis de los alimentos frecuentemente consumidos
Este trabajo partió de una encuesta previa, realizada a escolares de escuelas primarias de la ciudad
de Tepic, Nayarit, México en el 2012. La encuesta nutricional fue para evaluar el consumo de
alimentos durante 48 h, dividiendo cada día en cinco tiempos de ingesta: Desayuno, almuerzo o
recreo, comida, merienda y cena. Los datos de estas encuestas fueron llevados al programa DIAL
“Alce Ingeniería” (Ortega y cols., 2011), diseñado para calcular, programar y modificar cualquier
Capítulo 6. METODOLOGÍA GENERAL
30
tipo de dieta. Con la información de este programa se obtuvo el perfil nutricional de los
macronutirentes (carbohidratos, lípidos y proteínas) de los 5 tiempos de ingesta. Se eligieron los
tres tiempos de ingesta con el mayor contenido energético durante el día.
6.1.1.1 Sujetos de estudio
Once escuelas públicas (10 urbanos y 1 zona suburbana) en el municipio de Tepic, Nayarit,
México fueron seleccionados al azar para participar en la encuesta. Un total de 724 escolares entre
las edades de 9 a 12 años (302 niños y 422 niñas) participaron en este estudio. Se obtuvieron la
ingesta diaria de alimentos y los datos antropométricos.
6.1.1.2 Evaluación dietética
La ingesta dietética se midió utilizando recordatorios de alimentos de 24 h en dos días no
consecutivos, donde se evaluaron las ingestas calóricas de cinco tiempos de ingesta; desayuno,
almuerzo, comida, merienda y cena. Los recordatorios de alimentos tienen las ventajas de
conceder baja presión en el encuestado, los tiempos de administración son relativamente corto y,
por lo tanto, de presentan una alta tasa de respuesta; no alteran la ingesta habitual del encuestado,
y permiten la cuantificación de los parámetros dietéticos (McPherson y cols., 2000).
Los padres ayudaron a sus hijos a completar los recordatorios de alimentos. Se registraron los
tipos de alimentos que se consumen en detalle al entrevistador; por ejemplo, el tipo de grasa con el
que se cocinó el alimento, nombre de la marca, o los constituyentes de platos combinados, etc., y
en relación con su cantidad o volumen comúnmente utilizado en casa o de otras medidas (platos,
vasos, cucharas, etc.) que se utilizaron. Las ingestas de los alimentos y nutrientes se calcularon
utilizando el software de análisis de la dieta DIAL versión 1.0 (Ortega y cols., 2012). La base de
datos de los alimentos en el software fueron modificadas utilizando tablas y recetas de alimentos
mexicanos tradicionales (De Chávez y Solano, 2010; Pérez y cols., 2008), así mismo se utilizaron
las tablas de composición química presente en las etiquetas de los productos comerciales.
Capítulo 6. METODOLOGÍA GENERAL
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6.1.1.3 Datos antropométricos
Personal capacitado midió la altura y el peso de los escolares, dichas mediciones se realizaron por
triplicado. La altura se midió utilizando un estadiómetro (Seca, 213, Hamburgo Alemania). El
peso se registró en una balanza electrónica (Seca, 803, Hamburgo Alemania) con una capacidad
de 150 kg. Los sujetos se encontraban descalzos y vestían ropas ligeras durante las mediciones. El
índice de masa corporal (IMC) fue calculado como el cociente entre el peso y el cuadro de la
estatura (Kg·m-2). Este índice se utilizó para clasificar a los escolares teniendo en cuenta un valor
de IMC <percentil 5 como bajo peso, con un percentil entre 5 a 85 como peso normal, los que
presentaron percentiles > 85 < 95 con sobrepeso y obesos a los que presentaron percentiles ≥ 95
(Barlow y Committee, 2007).
6.1.1.4 Identificación de los patrones dietéticos en escolares
Un total de 495 alimentos consumidos por los participantes fueron clasificados en 13 grupos de
acuerdo a los grupos de alimentos identificados por el software DIAL versión 1.0 (Ver Cuadro
6.1). Se realizó un análisis de componentes principales (ACP) para determinar la energía con la
que cada grupo de alimentos contribuyó a los factores extraídos. Los factores se calcularon sin
rotación y el número de factores extraídos se basaron en valores propios > 1.0 (criterio de Kaiser),
la identificación de un punto de quiebre en el gráfico de sedimentación, y la interpretabilidad (Hair
y Anderson, 2010).
Se calcularon las puntuaciones factoriales para cada individuo. Un análisis de regresión múltiple se
utilizó para determinar la relación entre los pesos, IMC y las puntuaciones de los factores
encontrados en el ACP. Además, a partir de las puntuaciones factoriales se clasificó a los escolares
en tres dietas utilizando el análisis de conglomerados con la técnica de agrupamiento K-medias,
con el objetivo de identificar los grupos de alimentos con más alto consumo de calorías y
determinar la relación entre la dieta y el sobrepeso/obesidad.
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Cuadro 6.1 Definiciones de los grupos de alimentos utilizados para evaluar las dietas de los escolares del municipio de Tepic
Grupo Alimenticio Tipo de Alimento
Cereals Cereales para el desayuno, harina de trigo, tostadas, pan blanco, pan,
galletas, pan dulce, pasta, granos de maíz, tortilla de maíz, arroz, avena.
Legumbres Soja, lentejas, garbanzos, frijoles refritos, frijoles cocidos, chicharos, ejote.
Vegetales Zanahoria, la verdolaga, tomate verde, rábano, pimiento verde, pepino, papa,
nopal, col blanca, cilantro, chile, cebolla blanca, calabaza, calabaza, brócoli.
Frutas Uva, pera, toronja, tamarindo, sandía, plátano, pitahaya, piña, pasas, naranja,
papaya, nanchi, melón, mango, manzana, limón.
Leche y productos
lácteos
Leche entera, la leche sin lactosa, leche evaporada, leche luz, yogur, batidos
de leche, crema, queso.
Carne y derivados
Carne de res, pollo, cerdo, salchicha, tocino, vientre, jamón, hígado, costilla
de res, salchichas de pavo, salchicha de cerdo, pierna de cerdo, mollejas de
pollo.
Pescados y mariscos Peces sierra, salmón, sardinas, pulpo, pargo, ostras, peces marlin, camarón
seco, camarón, atún.
Huevo Claras de huevo y huevo entero
Dulces Azúcar, piruletas, edulcorantes, pasteles, chocolate en polvo, barras de
chocolate, mermelada, caramelo gomoso, chicle, dulces con chile.
Aceites y grasas Aceite de maíz, aceite de girasol, aceite de oliva, manteca de cerdo,
mantequilla.
Bebidas Agua, café, té, refrescos, jugos naturales y procesados.
Botanas Frituras de maíz, palomitas de maíz, papas fritas, semillas de calabaza, maní,
aceitunas.
Salsas y
condimentos
Puré de tomate, la pimienta, el orégano, la salsa picante, salsa inglesa,
pipián, el vinagre, la hoja de laurel, el caldo de pollo, menta, comino, clavo,
ajo, sal.
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6.2 Segunda Etapa 6.2.1 Obtención de las muestras
De cada una de las tres dietas formadas por el análisis multivariado de los datos, se eligieron los
alimentos con mayor frecuencia de consumo en los tres principales tiempos de ingesta (Mayor
aporte de energía durante el día), y se formaron un total de 9 menús. Tomando en cuenta la
descripción dada por los entrevistados o bajo criterios típicos de la región, la elaboración de los
alimentos fue estandarizada en el laboratorio para fines científicos. Las materias primas para la
preparación de los alimentos fueron adquiridos en un supermercado local del municipio de Tepic.
Los alimentos de cada menú fueron preparados, mezclados y homogenizados utilizando un
procesador de alimentos (Nutribullet, NB-101B, China). Inmediatamente después de ser
elaborados, se determinó el contenido de humedad de las muestras por el método 925.10 de
AOAC (1990). Las muestras homogenizadas fueron colocados en bolsas con cierre hermético y
almacenadas a -80º C. Las muestras congeladas fueron secadas a vacío en una liofilizadora
(FreeZone 6, Labconco, USA). Posteriormente fueron reducidas de tamaño en un molino (Ika,
M20, USA) y tamizadas utilizando una malla con tamaño de 0.05 mm hasta obtener muestras
homogéneas, que fueron colocadas en recipientes herméticos y finalmente fueron almacenadas a -
20º C para los posteriores análisis.
6.2.2 Cuantificación de la fracción indigestible total, soluble e insoluble
Para la determinación de la fracción indigestible en las muestras se utilizó la metodología descrita
por Saura-Calixto y cols. (2000). El método emula la digestión de los alimentos en el tracto
gastrointestinal. Digestión gástrica: Se pesaron 300 mg de muestra en base seca en tubos de
centrifuga, se adicionó 10 mL de regulador de HCl-KCL (pH1.5). Se ajustó el pH de los tubos a
1.5 y se adicionaron 0.2 mL de un solución de pepsina (P-7000, Sigma Aldrich, St Louis Missouri,
USA) y buffer HCL-KCL 0.2 M (300 mg/mL). La mezcla se incubó a 40º C por 1 h con agitación
constante (80 rpm). Digestión intestinal: Se agregaron 4.5 mL de regulador de fosfato 0.1 M y se
ajustó el pH de cada tubo a 7.5. Se adicionó 1 mL de solución de pancreatina (P-1750, Sigma
Aldrich) y buffer de fosfato 0.1 M (5 mg/mL), 1 mL de solución de lipasa (L-3126, Sigma
Capítulo 6. METODOLOGÍA GENERAL
34
Aldrich) y buffer de fosfato 0.1 M (7 mg/mL) y 2 mL de una solución de extracto de bilis y buffer
de fosfatos (7 mg / mL) a cada tubo y se incubó a 37º C por 6 h con agitación constante (80 rpm).
Posteriormente, para la hidrólisis del almidón, se agregaron 9 mL de regulador tris maleato 0.1 M
y se verificó que el pH sea de 6.9 para adicionar i mL de solución de α-amilasa (A-3176, Sigma
Aldrich) y búfer tris-Maleato (120 mg/mL) y se incubó a 37º C por 16 h con agitación constante
(80 rpm). Terminado este tiempo se adicionaron 10 mL de regulador acetato de sodio y se ajustó el
pH a 4.75. A esta mezcla se le adicionaron amiloglucosidasa (A-9913, Sigma Aldrich) y se
incubaron a 60º C por 45 min. Concluidas las reacciones enzimáticas, se procedió a centrifugar las
muestras durante 15 min a 3000 rpm, recuperando los sobrenadantes. Por otro lado, los residuos
fueron lavados con 5 mL y nuevamente fueron centrifugados, recolectando los sobrenadantes en
vasos de precipitado de 100 mL. Los tubos con el residuo se colocaron durante 16 h en la estufa de
105º C para cuantificar gravimétricamente la FI insoluble de las muestras. El sobrenadante se
colocó en membrandas de diálisis (D9652-100ft avg. Ancho de 33 mm, 12400 Da, Sigma Aldrich)
previamente tratadas y lavadas con agua hirviendo durante 5 min (aproximadamente 20 cm de
cada bolsa de diálisis) y se dializó contra agua corriente por 48 h a temperatura de 25º C para
eliminar los componentes digeridos. Enseguida se transfirió el contenido de la bolsa de diálisis a
un matraz volumétrico de 50 mL y se aforó con agua destilada. Se tomaron 17 mL del volumen
anterior y se colocó en tubos de centrifuga de polipropileno con tapa, al que se le adicionó 1 mL de
ácido sulfúrico (H2SO4). Los tubos se llevaron a un baño de agua en ebullición durante 90 min.
Terminada la hidrolisis ácida, se colocaron 2 mL de la solución anterior (muestra tratada con
H2SO4) a tubos de ensaye que contenían 1 mL de reactivo DNS (ácido dinitrosalicilico) y 0.5 mL
de solución NaOH 3.9 M. Los tubos fueron mezclados en vortex. Posteriormente se preparó una
curva con glucosa como estándar (0 a 2 mg/mL). Finalmente se calentó 15 minutos en un baño de
agua a ebullición, y posterior a esto, se adicionaron 15 mL de agua destilada, para cuantificar la FI
soluble como azucares reductores con ácido dinitrosalicilico en un espectrofotómetro (Jenway,
6715, USA) a 530 nm.
La FI total se determinó como la suma de la FI insoluble más la FI soluble.
Capítulo 6. METODOLOGÍA GENERAL
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6.2.3 Digestión gastrointestinal in vitro: Aislamiento de la fracción indigestible
Cada muestra liofilizada se llevó a un proceso de digestión gastrointestinal in vitro. Esta técnica
simula las condiciones gastrointestinales fisiológicas, tomando en cuenta la acción enzimática, los
cambios de pH y la temperatura del cuerpo humano. La finalidad fue determinar la fracción que no
es digerida ni absorbida en el intestino delgado conocida como fracción indigestible (FI),
cuantificarla y aislarla para utilizarla como sustrato de la fermentación colónica. Para cuantificar la
FI se siguió la metodología descrita por Saura-Calixto y cols. (2000). Con el fin de incrementar la
cantidad de FI aislada se seguió la metodología de Saura-Calixto y cols. (2000), con las
modificaciones realizadas por Tabernero y cols. (2011). Se pesaron 9 g de cada alimento
liofilizado, los cuales fueron digeridos enzimáticamente en pasos subsecuentes simulando
condiciones fisiológica: Se utilizaron 0.6 mL de una solución de pepsina (P-7000, Sigma Aldrich)
y buffer HCL-KCL 0.2 M (300 mg/mL). Se ajustó el pH de los tubos a 1.5 y se incubó 40º C por 1
h. Posteriormente, se realizó una hidrólisis con 3 mL de solución de pancreatina (P-1750, Sigma
Aldrich) y buffer de fosfato 0.1 M (5 mg/mL). La hidrólisis de las grasas se realizó con 3 mL de
solución de lipasa (L-3126, Sigma Aldrich) y buffer de fosfato 0.1M (7 mg/mL). Además, 3 mL
de una solución de extracto de bilis y buffer de fosfatos (17.5 mg / mL) fueron colocados a cada
tubo; el pH de las muestras fue ajustado a 7.5, e incubado a 37º C por 6 h. Para la hidrólisis del
almidón, se utilizaron 3 mL de una solución con α-amilasa (A-3176, Sigma Aldrich) y búfer Tris-
Maleato (120 mg/mL) ajustando el pH de las muestras a 6.9 e incubando a 37º C por 16 h.
Finalmente 300 µL de amiloglucosidasa (A-9913, Sigma Aldrich, USA) fueron colocados en cada
tubo y se incubaran a 60º C por 45 minutos. Terminado la hidrólisis enzimática las muestras se
transfirieron a tubos de diálisis (D9652-100ft avg. Anchura plana de 47 mm, 12400 Da, Sigma
Aldrich) y permanecieron por 48 h a temperatura a 25º C para eliminar los componentes digeridos.
El retenido fue colectado, concentrado en rota vapor a 60º C y liofilizado por 72 h, y se utilizó
para los análisis posteriores: Caracterizaión de la fracción indigestible y fermentación colónica in
vitro.
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6.2.4 Caracterización nutricional, compuestos bioactivos y actividad antioxidante en la
fracción indigestible de los menús
Las fracciones indigestibles (FI) aisladas fueron caracterizadas mediante la cuantificación de
almidón resistente, la determinación de compuestos fenólicos, taninos condensados e hidrolizables
y análisis de capacidad antioxidante por los métodos FRAP, ABTS y DPPH.
6.2.4.1 Cuantificación del Almidón Resistente (AR)
El AR presente en las muestras fue determinado por el método propuestos por por Goñi y cols
(1996) con algunas modificaciones. En este trabajo se realizó la determinación a partir de 25 mg
de fracción indigestible total, los cuales fueron pesados en tubos de centrifuga, se agregaron 3 mL
de agua destilada y 3 mL de KPH 4 M, la mezcla se mantuvo con agitación constante durante 30
min a temperatura ambiente. Se agregaron 5.5 mL de HCl 2M y 3 mL de buffer de acetato de
sodio, se ajustó el pH a 4.75, se adicionaron 80 µL de amiloglucosidasa (A-9913, Sigma Aldrich,
USA) en proporción 3.8 buffer de acetato de sodio a 1 de enzima. La mezcla fue incubada a 60º C
por 45 min con agitación constante. Las muestras fueron centrifugadas a 3000 rpm por 15 min y se
recolectó el sobrenadante en un matraz aforado de 50 mL.
Para la cuantificación del AR, se tomaron 500 µL de muestras y se mezcló con 1 mL de reactivo
del reactivo GOD-POD, leyendo la absorbancia a 505 nm, utilizando una curva de glucosa entre 0-
100 mg/L. La siguiente formula se empleó para el cálculo.
% AR = glucosa µgml ∗ 0.001 ∗ 50 ∗ 0.9peso muestra (mg) ∗ 100
6.2.4.2 Análisis de los compuestos antioxidante en la fracción indigestible de los menús
Obtención de extractos
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Para estos análisis se realizó una extracción acuosa-orgánica de acuerdo con la metodología
propuesta por Pérez-Jiménez y cols. (2009). En tubos de centrifuga se pesaron 100 mg de FI
aislada y se añadieron 5 mL de una solución metabólica acidificada (metanol:agua, 50:50 v/v, HCl
2 N). Los tubos se agitaron en un orbital a temperatura ambiente durante 1 h. Los tubos se
centrifugaron a 6000 rpm durante 10 min a 4 ° C y los sobrenadantes se recuperaron en un matraz
de 25 mL . A los residuos se adicionaron 5 ml de una solución acetona-agua (70:30 v/v) y los
tubos nuevamente fueron agitados en un orbital a temperatura ambiente durante 1 h. Los tubos se
centrifugaron a 6000 rpm durante 10 min a 4 ° C y los sobrenadantes se recuperaron y mezclaron
con los sobrenadantes anteriormente recuperados. La mezcla se aforó a 10 mL con una solución
metanol/agua/HCl - acetona/agua (50:50 v/v). Los extractos fueron usados para determinar el
contenido polifenoles extraíbles (PE) y actividad antioxidante, con los métodos FRAP, ABTS, y
DPPH. Por otro lado, el residuo fue utilizado para cuantificar polifenoles no extraíbles (PNE):
Polifenoles hidrolizables (PH) y taninos condensados en las muestras.
Polifenoles extraíbles (PE) en los extractos de las fracciones indigestibles
El contenido de PE en los extractos fue determinado con el reactivo Folin-Ciocalteu utilizando la
metodología propuesta por Montreau (1972) con algunas modificaciones realizadas por Alvarez-
Parrilla y cols. (2010a). Se utilizaron 250 µL de extracto y fueron mezclados con 1000 µL de una
solución de carbonato de sodio (75 g/L), y 1250 µL del reactivo Folin–Ciocalteu (100mL/L) en
tubos de ensaye. Los tubos fueron homogenizados usando un agitador vortex. La solución fue
incubada a 50º C bajo completa obscuridad por 15 min. Pasado el tiempo de incubación, los tubos
se dejaron enfriar a temperatura ambiente y 270 µL de esta mezcla fueron colocados en pocillos de
microplacas. La absorbancia fue medida a 750 nm usando un lector de microplaca con múltiple
detección (Biotek, Synergy HT, Winooski VT, USA), utilizando el programa Gen5. Los resultados
fueron expresados en equivalente de ácido galico (EAG; mg/ g extracto bs), usando una curva
estándar de ácido gálico (0–0.2 mg/mL).
Polifenoles no extraíbles (PNE) en los extractos de las fracciones indigestibles
Capítulo 6. METODOLOGÍA GENERAL
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Como se mencionó anteriormete, los residuos fueron utilizados para determinar el contenido de
PNE en las FI aisladas.
Para los polifenoles hidrolizables (PH), los residuos fueron tratados con 4 mL de metanol
absoluto y 0.4 ml de ácido sulfúrico (H2SO4) concentrado a 85 °C por 20 h (Hartzfeld y cols.,
2002). Las muestras fueron centrifugadas (3000 rpm por 10 min), con la recuperación de los
sobrenadantes. Se realizó un lavado con 4 mL de agua destilada, el volumen final fue ajustado a 10
mL con agua destilada. El contenido plifenoles fue determinado de acuerdo al ensayo con el
reactivo de Folin-Ciocalteu, con las modificaciones realizadas por Alvarez-Parrilla y cols. (2010a),
cuya técnica fue descrita anteriormente.
Para los taninos condensados los residuos fueron tratados con 4 mL HCl/butanol/FeCl3 (5:95, v/v)
a 100º C por 3 h (Reed y cols., 1982). Las muestras fueron centrifugadas (3000 rpm por 10 min),
con la recuperación de los sobrenadantes. Se realizarán un lavado con 4 mL de HCL/butanol/FeCl3
(5:95, v/v) y el volumen final fue ajustado a 10 mL. Las muestras de TC fueron leídas a en un
espectrofotómetro a 555 nm y se compararon con vaina de algarrobo (Ceratonia siliquia) como
estándar de proantocianidinas (Nestle, Ldt., Vers-Chezl-les Blanes, Suiza). Los resultados se
expresaron como gramos equivalentes de taninos hidrolizables por gramos de FI inicial en base
humeda.
6.2.4.3 Análisis de la capacidad antioxidante en las fracciones indigestibles
Actividad Quelante: Poder antioxidante reductor del hierro (FRAP)
El ensayo FRAP se realizó de acuerdo a lo descrito por Alvarez-Parrilla y cols. (2010b). Una
solución de trabajo fue preparada por la mezcla de 25 mL Buffer de acetato (0.3 M, pH 3.6), 2.5
mL de solución de 2,4,6-tripiridil-s-Triazina (TPTZ) (10 mM TPTZ en 40 mM HCl), y 2.5 mL de
solución que contiene FeCl3 6H2O 20 mM. La solución de trabajo fue incubada a 37º C hasta antes
de ser usada. Se tomaron 24 µL de los extractos y se dejaron reaccionar con 180 µL de la solución
de trabajo FRAP. Las lecturas de absorbancia se tomaron después de 30 min de reacción
utilizando un lector de microplacas de detección múltiple (Biotek, Synergy HT, Winooski VT,
Capítulo 6. METODOLOGÍA GENERAL
39
EE.UU.) con el programa Gen5. El incremento en la absorbancia es debido a la reducción de Fe3+
por los antioxidantes y la subsecuente formación de un complejo colorido TPTA-Fe2+ medido a
una absorbancia de 595 nm. Los resultados fueron expresados en Equivalentes Trolox (ET; mM /g
FI bs) con una curva estándar usada entre 8.125x10-3 - 0.13 mM Trolox.
Actividad antiradical: Ensayo 1,1-Difenill-2-picril hidrazilo (DPPH)
El ensayo de DPPH fue realizado de acuerdo con el método propuesto por Prior y cols. (2005) con
algunas modificaciones. La solución madre se preparó disolviendo 49.29 mg DPPH con 25 ml de
metanol y después se almacenó a -20°C hasta su uso. La solución de trabajo se obtuvo mediante la
mezcla de 950 µl de solución madre con 25 ml de metanol para obtener una solución de DPPH
190 µM. Se tomaron 30 µl del extracto de la muestra y se dejaron reaccionar con 200 µl de la
solución de trabajo DPPH 190 µM La absorbancia fue medida a 517 nm, despues de 10 min de
reacción, utilizando un lector de microplacas de detección múltiple (Biotek, Synergy HT,
Winooski VT, EE.UU.) con el software Gen5. Se realizó una curva estándar entre 37,5 a 600 µM
de Trolox y los resultados se expresan en equivalentes de Trolox (ET; mM / g de FI bs).
Actividad antiradical: Ensayo del radical 2,2'-azino-bis(3-etilbenzotiazolina-acido-6-
sulfónico ( ABTS•+)
La capacidad de los extractos para neutralizar el radical ABTS•+ fue analizada utilizando la
metodología propuesta por Re y cols. (1999) con algunas modificaciones. Los radicales catiónicos
de ABTS+ fueron producidos por la reacción entre 7 Mm del reactivo ABTS y 2.45mM de
persulfato de potasio disueltos en buffer de fosfatos (0.1 M, pH 7.4). La solución fue almacenada
en total oscuridad a temperatura ambiente durante 12 a16 h con agitación constante. Antes de su
uso, la solución se diluyó con buffer de fosfatos, hasta obtener una absorbancia de 0.7 ± 0.01 a una
longitud de onda de 734 nm. La actividad antiradical se evaluó mediante la mezcla de 20 µl de los
extractos con 255 µl de ABTS a una temperatura de 30°C durante 7 minutos utilizando un lector
Capítulo 6. METODOLOGÍA GENERAL
40
de microplacas de detección múltiple (Biotek, Synergy HT) con Gen5 software. La disminución en
la absorbancia se midió a 734 nm. Se utilizó una curva estándar entre 37.5 a 600 µM de Trolox y
los resultados se expresan en equivalentes de Trolox (ET; µM / g de extracto bs).
6.3 Tercera Etapa
6.3.1 Fermentación colonica in vitro de la fracción indigestible
La FI aislada de cada muestra fueron llevadas a un sistema de fermentación colónica in vitro por
lotes, con la finalidad de evaluar la producción de metabolitos. El método utilizado fue adaptado
según la propuesta de Campos-Vega y cols. (2009). El proceso de fermentación in vitro fue
evaluado por triplicado para cada conjunto de alimentos y fue realizado a una temperatura 37º C
utilizando un baño de agua. El inóculo fecal fue proporcionado por cinco escolares nayaritas sanos
entre 9-12 años de edad, con el consentimiento de sus padres, los cuales afirmaron que los
escolares no consumieron antibióticos por al menos 3 meses antes del ensayo y que no presentaron
historial de enfermedades gastrointestinales recientes. En tubos de centrifuga estériles con
capacidad de 15 mL, fueron colocados 9 mL de medio de cultivo basal que contenía los siguientes
componentes: 2 g/L de agua peptonada, 2 g/L de extracto de levadura, 0.1 g/L de NaCl, 0.04 g/L
K2HPO4, 0.04 g/L KH2PO4, 0.01 g/L de MgSO4.7H2O, 0.01 g/L de CaCl2.2H2O, 2 g/L de
NaHCO3, 0.5 g/L de clorhidrato de L-cisteína, 0.5 g/L de sales biliares, 2 mL de tween 80, 0.2 g/L
de hematina (diluida en 5 mL de NaOH 1M) y 10 µL Vitamina K1. Los tubos sellados se
mantuvieron bajo condiciones anaerobias mediante el flujo (7.5 L/h) de una mezcla de gases que
contenía H2-CO2-N2 (10:10:80 v/v), libre de oxigeno (O2) por 12 h. El inóculo fecal fue preparado
con la mezcla de 10 g (2 g de heces por donador, 5 donadores) de heces frescas y 90 mL de buffer
de fosfato de sodio 0.1 M y se homogeneizaron en un sistema digital de alta velocidad (IKA-Ultra-
Turrax, T18, EE.UU.) durante 1 min a 6000 rpm. Los tubos con medio de cultivo basal fueron
inoculados con 1 mL de solución de heces fecales y con 100 mg de fracción indigestible aislada de
cada alimento. La rafinosa fue utilizada como un control de azúcar fermentable; también fue
Capítulo 6. METODOLOGÍA GENERAL
41
utilizado un blanco negativo, el cual correspondió al medio de cultivo más inoculo fecal. La
fermentación de ambos controles fue bajo las mismas condiciones de las muestras. Las muestras se
agitaron en vórtex durante 30 s y se determinó el pH al tiempo cero. Posteriormente se colocaron
en un baño de agua a 37ºC. Debido a que el sistema de fermentación fue por lotes, se realizaron
tres fermentaciones utilizando las fracciones indigestibles obtenidas de alimentos consumidos
durante el desayuno (Lote 1), la comida (lote 2) y la cena (lote 3). Dichas fermentaciones fueron
realizadas con inoculos diferentes provenientes de donadores distintos, pero que cumplían con las
características antes mencionadas. Durante la fermentación, el pH, la actividad antioxidantes
(DPPH y FRAP) y la producción de metabolitos orgánicos volátiles de las muestras fueron
evaluados al tiempo 12, 24, 48 y 72 h. Terminado los tiempos, los tubos de la fermentación fueron
centrifugados (Hermle Z 323 K; Wehingen, Alemania) a 4500 rpm durante 15 min a 4°C y los
sobrenadantes se dividieron en dos para realizar los siguientes análisis: A) Análisis del perfil
metabolitos por cromatografía de gases; B) Análisis de actividad antioxidante asociados a los
extractos. Los sobrenadantes fueron almacenados a -80º C hasta sus análisis.
6.3.2 Análisis de componentes antioxidantes asociados en los extractos de la fermentación
Loss sobrenadantes obtenidas después de las 12, 24, 48 y 72 h fueron utlizados para evaluar la
capacidad antioxidante por los métodos DPPH y FRAP. Las metodologías descritas en el apartado
6.1.4.5, fueron empleadas en la determinación de la capacidad antioxidante. El trolox fue utilizado
como estándar disuelto en medio de cultivo basal. Los datos fueron expresados como mMol de ET
/ g de fracción indigestible bs.
6.3.3 Análisis de metabolitos en la fermentación colonica in vitro de la fracción indigestible
de los menús
El análisis de los metabolitos productos de la fermentación de la FI de los alimentos se realizó para
determinar cuantitativamente la concentración de los tres principales ácidos grasos de cadena corta
Capítulo 6. METODOLOGÍA GENERAL
42
(ácido acético, propiónico, butírico) y semi-cuantitativamente el total de la fracción volátil
presente en los extractos. Loa anterior fue realizado utilizando la siguiente metodología:
6.3.3.1 Micro extracción en fase solida de los compuestos volátiles en los extractos de
fermentación
La micro extracción en fase sólida (MEFS) fue la técnica utilizada para la extracción-
concentración de compuestos volátiles y se realizó de acuerdo con la metodología propuesta por
Zamora-Gasga y cols. (2014). Se pesaron 500 mg de sobrenadantes en viales (20 mL). Los viales
se taparon y colocaron en el automuestreador (VT-32-20, Gerstel MPS2). La extracción se realizó
con una fibra compuesta por polidimetilsiloxano-divinilbenceno-carboxeno (PDMS/DVB/CAR)
de 2 cm. Las muestras se agitaron (250 rpm) a 45 °C durante 5 min. A continuación, la fibra
PDMS/DVB/CAR fue expuesta al espacio de cabeza, y las muestras se agitaron durante 120 min,
250 rpm a 45 °C. Posteriormente, la fibra se insertó en el puerto de inyección del equipo de
cromatografía de gases (CG) para la desorción térmica (240 ° C durante 10 min) y el análisis por
espectrometría de masas (EM). Las extracciones se realizaron por triplicado en cada tiempo de
fermentación, con un total de 60 viales en cada lote de fermentación (Un total de tres lotes). Los
análisis se realizaron en cinco corridas de doce viales por corrida y el orden de los viales fue
aleatorizado.
6.3.3.2 Cromatografía de gases acoplada a espectrometría de masas
Las muestras fueron analizadas por cromatografía de gases (CG) acoplada a espectrometría de
masas (EM) con ionización de electrones con un sistema Agilent Technologies 7890ª, utilizando
un detector selectivo de masa (Agilent Technologies 5975C VL) equipado con un MPS2 XL
automuestreador (Gerstel). La separación de las muestras se llevó acabo con una columna capilar
DB-5MS (60 m x 250 m X 0,25 micras; Agilent J & W) usando gas helio como acarreador a una
velocidad de flujo de 1 mL / min. El inyector del equipo de CG fue utilizado a una temperatura de
250°C. La fuente de EM y el cuádruplo se mantuvieron a temperaturas de 230°C y 150°C,
respectivamente. El inyector se utilizó en el modo de división máxima (splitless). La temperatura
Capítulo 6. METODOLOGÍA GENERAL
43
del horno comenzó a 40ºC por 5 min y fue programada como sigue: 5ºC / min de 40 a 200ºC, se
mantuvo a 200ºC por 2 min, más 20ºC / min hasta alcanzar 230ºC y se mantuvo a 230ºC por 15
min. El tiempo total de análisis fue de 55.5 min. La cuantificación de los AGCC en las muestras se
obtuvo a través de curvas de calibración de los ácidos acético, propiónico y butírico. Uan tentativa
identificación de los componentes volátiles se realizó mediante la comparación de los espectros de
masas de las muestras con la biblioteca de sistema de datos del programa MSD ChemStation
(Agilent G1701EAversion E.02.00.493). Se calculó la concentración relativa de todos los
metabolitos de fermentación frente al ácido acético (AGCC más volátil) como patrón interno y los
resultados se expresaron en mmol L-1 de extracto. Con el fin de comparar las diferencias en el
perfil metabólico, las concentraciones (mmol L-1) fueron estandarizadas y expresadas como
porcentajes de concentraciones relativas en cada metabolito utilizando la siguiente formula:
!" % = !" !!! !" !"#" !"#$%&'(#&(!" !!!!"#$% !" !"#$%&'(#&)) ∗ 100
Donde CR es la concentración relativa de cada metabolito
6.4 Análisis estadístico
Todos los experimentos fueron realizados por triplicado, cada alimento fue considerado como
unidad experimental. En la primera etapa, la elección de los alimentos fue realizada con un análisis
de conglomerados k-medias vía componentes principales, utilizando el programa STATISTICA,
versión 10.0 (Statsoft. Inc. 1984-2010, Tulsa, OK, EE.UU.). La caracterización nutricional,
compuestos bioactivos y actividad antioxidante de las menus y de la fracción indigestible se
analizó con un diseño de mediciones repetidas. Donde se determinó los efectos de las tres dietas
formadas (DM, DT y DA) y los tres tiempos de ingesta (desayuno, comida y cena). En la segunda
etapa, la fermentación in vitro se determinó con la combinación de un diseño factorial con
bloques. Donde los factores fueron la dieta (DM, DT y DA) y los tiempos de fermentación (12, 24
48 y 72). Cada bloque está formado por el tiempo de ingesta (desayuno, comida y cena) y las
variables de respuesta fueron, los cambios de pH, y la actividad antioxidante en los extractos. Para
la última etapa de este trabajo, se realizará un análisis de componentes principales (PCA) de las
Capítulo 6. METODOLOGÍA GENERAL
44
concentraciones (%) de los compuestos volátiles en base a los valores medios de los triplicados
utilizando el programa STATISTICA, versión 10.0 En este contexto, los casos serán las diferentes
FI de las tres dietas (DM, DT y DA), los controles positivo y negativo en cada tiempo de
fermentación (12, 24, 48 y 72 h), las variables serán los compuestos volátiles identificados y el
valor de entrada en la matriz será la concentración relativa del compuesto volátil. Los resultados
obtenidos de las determinaciones de cada etapa fueron analizados utilizando el programa
estadístico STATISTICA (Stat Soft, Inc. 1994-2010) Versión 10.0 mediante un análisis de
varianza (ANOVA), para determinar si existió efecto significativo en los los factores y una prueba
Fisher LSD se realizó para la comparación de medias con una significancia del 5% (α=0.05).
Capítulo 7. RESULTADOS Y DISCUSIÓN
45
Capítulo 7. RESULTADOS Y DISCUSIÓN
7.1 Patrones dietéticos, perfil nutricional e índice de masa corporal en escolares mexicanos:
Un estudio transversal
Resumen
México es uno de los veinte países con mayor prevalencia de sobrepeso y obesidad en el mundo.
Este problema también ocurre en la población infantil mexicana. La prevalencia de sobrepeso y
obesidad en niños mexicanos en edad escolar aumentó del 26,9% al 34,4% durante el período
1999-2012 y el 25% de los niños tiene sobrepeso (2). La dieta juega un papel importante en el
desarrollo de este trastorno. Tradicionalmente, la dieta mexicana se basa en maíz, frijoles y chiles,
que se cocinan en diferentes platos típicos, dependiendo de la región, pero han sido cada vez más
reemplazados por comidas listas para comer, comidas rápidas y bebidas endulzadas (4).
Los patrones dietéticos seguidos por los niños preadolescentes tienen influencia en su estado
nutricional no solo durante la infancia, sino también durante su adolescencia, así como, en la edad
adulta. Pocos estudios han examinado la asociación entre los patrones dietéticos y las
enfermedades no transmisibles en la población escolar mexicana, y hoy en día, está demostrado
que las dietas altas en azúcares simples pueden aumentar el riesgo de resistencia a la insulina en
niños y adolescentes (7). Por otra parte, la presión arterial diastólica, la glucosa y las
concentraciones de triglicéridos se han asociado positivamente con la ingesta de refrescos /
bebidas azucaradas y con la ingesta de grasas altas en grasas (6). Sin embargo, estos estudios se
enfocan principalmente en el área central de México y debido a la amplia diversidad culinaria del
país, se necesitan estudios complementarios en diferentes regiones.
La información sobre la relación entre los patrones dietéticos, el perfil nutricional y el índice de
masa corporal se puede utilizar para planificar una intervención nutricional eficaz para preparar las
pautas dietéticas para los niños. El objetivo de este capítulo fue identificar los patrones dietéticos
globales en la población escolar (nueve a doce años) del estado de Nayarit y determinar la relación
entre los patrones de alimentación, obesidad, perfil nutricional y tipos de alimentos consumidos.
Capítulo 7. RESULTADOS Y DISCUSIÓN
46
“Dietary patterns, nutritional profile, and body mass index in Mexican schoolchildren: A
cross-sectional study”
Victor Manuel Zamora-Gasga1, Efigenia Montalvo-González1, Guadalupe Flavia Loarca-Piña2,
Alejandra Martina Chacón-López1, Juscelino Tovar3, Sonia Guadalupe Sáyago-Ayerdi 1*
1Instituto Tecnológico de Tepic, Laboratorio Integral de Investigación en Alimentos,
División de Estudios de Posgrado, Av Instituto Tecnológico No 2595, Col Lagos del Country CP
63175, Tepic, Nayarit México.
2 Programa de Posgrado en Alimentos del Centro de la República, Facultad de Química,
Universidad Autónoma de Querétaro, Centro Universitario, Cerro de las Campanas S/N, CP
76010, Santiago de Querétaro, Querétaro México.
3Food for Health Science Centre.Lund University, MediconVillage. Lund, SE-223 81. Sweden
*Corresponding author, e-mail: [email protected]
Tel.: +52 311 211 94 00 ext 328
Summary
Childhood obesity is a serious public health problem in Mexico. In this study, childhood-specific
dietary patterns and their relationship with overweight-obesity prevalence, nutrient profiles and
Capítulo 7. RESULTADOS Y DISCUSIÓN
47
types of foods consumed were studied. A descriptive cross-sectional study of 724 randomly
selected schoolchildren between 9 to 12 years old from Nayarit State, Mexico was performed.
Data on anthropometric characteristics and food intake were recorded. Seven dietary patterns and
three specific diets were identified by multivariate analysis. A dietary pattern characterized by
high legume, snack and low beverage intake was negatively associated with weight and body mass
index. The overall overweight and obesity prevalence was 20.2% and 20.6%, respectively. Diet
type significantly influenced (p<0.05) protein, carbohydrates and fat intake but did not show
correlation with the overweight-obesity status. Simple sugars, candies, pastries and sweetened
beverages appeared in all dietary patterns. Dietary patterns in countries with a wide gastronomic
diversity should be considered to design preventive nutrition intervention programs.
Keywords: Feeding, obesity, schoolchildren, energy Intake, food habits
Resumen
La obesidad infantil es un problema de salud pública en México. En este trabajo, se estudiaron los
patrones dietéticos de escolares mexicanos y su relación con la prevalencia de sobrepeso-obesidad,
el perfil de nutrientes y los principales grupos de alimentos consumidos. Se llevó a cabo un estudio
transversal descriptivo donde participaron 724 escolares seleccionados al azar de entre 9 a 12 años
del Estado de Nayarit, México evaluándose las características antropométricas y la ingesta de
alimentos. Siete patrones dietéticos y tres dietas específicas fueron identificados mediante análisis
multivariado. Un patrón de dieta caracterizada por una alta ingesta de legumbres y aperitivos junto
con una baja ingesta de bebidas azucaradas se asoció negativamente con el peso e índice de masa
corporal. La prevalencia de sobrepeso y obesidad fue del 20.2 y 20.6%, respectivamente. El tipo
de dieta influyó significativamente (p <0.05) en la ingesta de proteínas, hidratos de carbono y el
consumo de grasas, pero no mostró correlación con la condición de sobrepeso-obesidad. Los
azúcares simples, dulces, postres y bebidas endulzadas aparecieron en todos los patrones
dietéticos. Los patrones dietéticos en los países con una gran diversidad gastronómica podrían ser
considerados de interés para diseñar programas de intervención nutricional preventiva.
Palabras clave: Alimentación, obesidad, escolares, ingesta de energía, hábitos alimenticios
Introduction
Mexico is among the twenty countries with the highest overweight and obesity prevalence in the
world. At least 69.3% of Mexican adults are overweight, and 27.2% are obese (1). This problem
also occurs in the Mexican childhood population. Overweight and obesity prevalence in school-
Capítulo 7. RESULTADOS Y DISCUSIÓN
48
aged Mexican children increased from 26.9% to 34.4% during the 1999 – 2012 period and 25% of
children are currently overweight (2). The rapid increase in the prevalence of overweight and
obesity has prompted many explanations, including genetic predisposition, altered energy
requirements, endocrine disruptors, and social, and economic factors (3). In this sense, it is clear
that diet plays an important role in the development of this disorder. Traditionally, the Mexican
diet was based on corn, beans, and chili peppers, which are cooked in different typical dishes,
depending on the region, but they have been increasingly replaced by ready to eat meals, fast
foods, as well as, commercial carbonated and sugar sweetened beverages (4). Probably, a
combination of lower real prices of food, changes in relative prices of different types of food and
increased availability of highly processed, energy-dense, micronutrient-poor foods is responsible
for such a drastic dietary change (5). In fact, Mexican schoolchildren have a high intake of fat,
sugar (sucrose and fructose), soft drinks, and processed foods (6). The dietary patterns followed by
preteen children had influence in their nutritional health not only during childhood, but also;
during their teen years, as well as, in adulthood. Few studies have examined the association
between dietary patterns and non-communicable diseases in the Mexican school population, and
nowadays; is highly demonstrated that diets high in simple sugars may increase the risk of insulin
resistance in children and adolescents (7). On the other hand, diastolic blood pressure, glucose,
and triglycerides concentrations have been positively associated with the intake of soft
drinks/sweetened beverages, and with high-fat dairy intake (6). However, these studies were
focused mainly in the central area of Mexico and due to the extensive culinary diversity in the
country, complementary studies in different regions are needed. Information about the relationship
between dietary patterns, nutritional profile, and body mass index can be used to plan an efficient
nutritional intervention to prepare dietary guidelines for children. The objective of this work was
to identify the overall dietary patterns in the schoolchildren population (nine to twelve years old)
in Nayarit State, and to determine the relationship between those dietary patterns, obesity,
nutritional profile, and types of foods consumed.
Materials and Methods.
Study population
Public Education Services of Nayarit State (SEPEN), case report form and informed consent form
approved the protocol of this descriptive cross-sectional study. Eleven public schools in the city of
Tepic, Nayarit State, Mexico were selected randomly, 724 schoolchildren (9-12 years old, 302
boys and 422 girls) participated in the study. Daily food intake and anthropometric data were
Capítulo 7. RESULTADOS Y DISCUSIÓN
49
obtained. Informed consent was obtained from children and from their parents or tutors. The
following data were collected: grade, gender, age, weight, height, and place of living.
Dietary assessment
Dietary intake was measured using two-day non-consecutive food records, where five meal food
intakes were evaluated: breakfast, mid-morning meal, lunch, afternoon snack and dinner. The
foods consumed were recorded in detail in order to obtain brand name and declared composition
of commercial items, constituents of mixed dishes, etc. The assessments of quantity/volume,
common household or other measures (plates, cups, spoons, etc.) were used. Nutrient intakes were
calculated using the DIAL dietary analysis software (8). The DIAL food database was expanded
adding the food composition of traditional Mexican foods and recipes (9, 10).
Dietary patterns identification
A total of 495 food items consumed by the participants were classified into 13 groups according to
the DIAL software as follows:
1) Cereals; breakfast cereals, wheat flour, toast, white bread, bread, biscuits, sweet bread, paste,
corn kernels, corn tortilla, rice, oats. 2) Legumes; soybeans, lentils, chickpeas, refried beans,
boiled beans, green beans, pea. 3) Vegetables; carrot, purslane, green tomato, radish, green pepper,
cucumber, potato, cactus, white cabbage, cilantro, chilli, white onion, squash, pumpkin, broccoli.
4) Fruits; Grape, pear, grapefruit, tamarind, watermelon, banana, dragon fruit, pineapple, raisins,
orange, papaya, nanchi, melon, mango, apple, lemon. 5) Milk and dairy products; whole milk,
lactose-free milk, evaporated milk, light milk, yogurt, milkshakes, cream, cheese. 6) Meat; beef,
chicken, pork, sausage, bacon, belly, ham, liver, beef rib, turkey sausage, pork sausage, pork leg,
chicken gizzards. 7) Fish and seafood; sawfish, salmon, sardines, octopus, snapper, oysters,
marlin, dry shrimp, fresh shrimp and tuna. 8) Egg; whole and white egg. 9) Sugars, sweets, and
pastries; sucrose, fructose, lollipops, artificial sweeteners, cakes, chocolate powder, chocolate bar,
jam, gummy candy, gum, candy with chili. 10) Oils and fats; corn oil, sunflower oil, olive oil, lard,
butter. 11) Beverages; purified water, coffee, tea, soda, natural and processed juices. 12) Snacks;
corn chips, popcorn, potato chips, pumpkin seeds, peanuts, olive. 13) Sauces and seasoning;
tomato puree, pepper, oregano, hot sauce, Worcestershire sauce, “pipian”, vinegar, bay leaf,
chicken broth, mint, cumin, cloves, garlic, salt.
Food group energy intakes (in kcal) were divided by the total energy intake in order to derive their
relative energy contribution to the overall diet. In order to reduce data dimensionality a principal
Capítulo 7. RESULTADOS Y DISCUSIÓN
50
component analysis (PCA) was performed to determine the energy contributed by each food group
to the extracted principal components (PC). Dietary patterns were extracted based on the
correlation matrix. PC were calculated without rotation and the number of PC was based on
eigenvalues >1.0, identification of a break point in the scree plot, and interpretability. PC scores
for each individual were calculated. Multiple regression analysis was used to test the relation
between weight, BMI and PC score. Results were reported for the final model as standardized beta
coefficient (b) and level of statistical significance (p<0.05). Furthermore, k-means cluster analysis
(KCA) with PC score was used to classify students in three diet types and to reveal meaningful
dietary patterns, as well as, to identify food groups with higher caloric intake and to determine the
relationship between diet and overweight-obesity status. Three-cluster solution was derived
through KCA with a maximum number of iterations equal to ten. Initial cluster centers were
obtained with sort distances and taking observations at constant intervals. Three clusters were
selected because they were best interpretable and to check whether PCA and KCA extracted
comparable patterns.
Anthropometric data
Trained personnel measured height and weight in scholars. Measurements were done in triplicate
and the values were an averaged. Height was measured using a stadimeter (SECA, Model 213,
Hamburg Germany) and recorded to the nearest 0.1 cm. Weight was recorded to the nearest 0.1 kg
using an electronic scale (SECA, Model 803, Hamburg Germany) with a capacity of 150 kg.
Subjects were shoeless and wore light cloths. Body mass index (BMI) was calculated and subjects
were classified as follows: BMI<5th percentile, underweight, 5 to 85th percentile, normal weight,
> 85th percentile, overweight and ≥95th percentile obese (11).
Statistical analysis
Statistica Release version 8.0 (StatSoft. Inc., Tulsa, OK, USA) was used for data analysis.
Continuous variables are presented as mean values ± standard deviation, and categorical variables
are shown as absolute frequencies. Contingency tables with calculation of χ2 test evaluated
associations between the categorical variables, while one-way analysis of variance was applied for
evaluating the associations between groups. P values < 0.05 were considered significant.
Results
Factors of schoolchildren’s dietary behaviors
Capítulo 7. RESULTADOS Y DISCUSIÓN
51
Thirteen dietary patterns emerged from PCA, but only seven had Eigenvalues > 1. This analysis
explained 70.25 % of the variance in food group intake. Figures 1A and 2A show the PCA-
loading matrix for the seven dietary patterns in the studied population.
Figure 1 Principal component analysis (PCA) plots. (A) Loading plots for different food groups
and (B) PCA scores plot for Mexican schoolchildren of the four principal components.
As higher absolute values indicate that the food variable contributes more to the development of
the principal component (PC), dietary patterns showed the following characteristics: a pattern high
in meat, oils and fats, low in cereals, milk and dairy products (PC 1);a pattern rich in vegetables,
sauces, and seasonings, and low in sugar, sweets, pastries, oils, and fats (PC 2); a pattern
-4 -3 -2 -1 0 1 2 3PC 1
-6
-4
-2
0
2
4
6
PC 2
Cereals
Legumes
Vegetables
Fruits
Milk and dairy products
Meat
Fish and seafoods
Eggs
Sugars, sweets and pastry Oils and fats
Beverages
Snacks
Sauces and seasoning
-1.0 -0.5 0.0 0.5 1.0PC 1
VE: 13.15%
-1.0
-0.5
0.0
0.5
1.0
PC 2
VE:
11.
14%
Cereals
Legumes
Vegetables
Fruits
Milk and dairy products
Meat
Fish and seafoods
Eggs
Sugars, sweets and pastry Oils and fats
Beverages
Snacks
Sauces and seasoning
CerealsLegumesVegetables
Fruits
Milk and dairyMeat
Fish and seafoods
Eggs
Sugars, sweets and pastry
Oils and fats
Beverages
Snacks
Sauces and seasoning
-1.0 -0.5 0.0 0.5 1.0PC 3
VE: 10.40%
-1.0
-0.5
0.0
0.5
1.0
PC 4
VE:
9.9
1%
CerealsLegumesVegetables
Fruits
Milk and dairyMeat
Fish and seafoods
Eggs
Sugars, sweets and pastry
Oils and fats
Beverages
Snacks
Sauces and seasoning
-5 -4 -3 -2 -1 0 1 2 3PC 3
-4
-2
0
2
4
6
PC 4
Capítulo 7. RESULTADOS Y DISCUSIÓN
52
characterized by high consumption of cereals, low consumption of vegetables, fruits, sugar,
sweets, and pastries (PC3); a pattern characterized by high consumption of beverages and snacks,
and low intake of egg (PC 4); and a pattern that represented important vegetables, fish, and
seafood consumption (PC 5), legumes and beverages (PC 6), and high consumption of legumes
and snacks, and low intake of beverages (PC 7). PCA was performed to obtain PC scores for each
child subject. PC scores were used in the multiple regression and cluster analysis.
Figure 2 Principal component analysis (PCA) plots. (A) Loading plots for different food groups
and (B) PCA scores plot for Mexican schoolchildren (PC 5, 6, and 7).
Associations between PC´s and weight or BMI were then evaluated with multiple regression
analysis; the models are presented in Table 1. PC 5 was positively associated with weight
Cereals
Legumes
Vegetables
Fruits
Milk and dairy productsMeat
Fish and seafoods
Eggs
Sugars, sweets and pastry
Oils and fats
Beverages
SnacksSauces and seasoning
-1.0 -0.5 0.0 0.5 1.0PC 5
CV: 9.33%
-1.0
-0.5
0.0
0.5
1.0
PC 6
VE:
8.2
7%
Cereals
Legumes
Vegetables
Fruits
Milk and dairy productsMeat
Fish and seafoods
Eggs
Sugars, sweets and pastry
Oils and fats
Beverages
SnacksSauces and seasoning
-4 -2 0 2 4 6 8PC 5
-4
-3
-2
-1
0
1
2
3
4
5
PC 6
-4 -3 -2 -1 0 1 2 3 4 5 6PC 7
-4
-3
-2
-1
0
1
2
3
PC 1
Cereals
Legumes
Vegetables
Fruits
Milk and dairy products
Meat
Fish and seafoods
Eggs
Sugars, sweets and pastry
Oils and fats
BeveragesSnacks
Sauces and seasoning
-1.0 -0.5 0.0 0.5 1.0
PC 7 VE: 8.05%
-1.0
-0.5
0.0
0.5
1.0
PC 1
VE:
13.
15%
Cereals
Legumes
Vegetables
Fruits
Milk and dairy products
Meat
Fish and seafoods
Eggs
Sugars, sweets and pastry
Oils and fats
BeveragesSnacks
Sauces and seasoning
Capítulo 7. RESULTADOS Y DISCUSIÓN
53
(standardized β-coefficient = 0.124, p< 0.01) and BMI (standardized β -coefficient = 0.117, p<
0.01). PC 7 was negatively associated with weight (standardized β -coefficient = -0.077, p = 0.04)
and with BMI (standardized β -coefficient = -0.073, p = 0.04). PC 4 was negatively associated
with BMI (standardized β -coefficient = -0.064 p = 0.01).
Table 1 Multiple regression analysis models exploring the association of principal components
(PC) with weight and body mass index (BMI).
Variables* Weight BMI
Standardized β-coeficient P Standardized β-coeficient P
Age (years) -0.046 0.21 -0.041 0.27
PC 1 -0.001 0.97 0.011 0.76
PC 2 0.048 0.19 0.069 0.06
PC 3 -0.009 0.81 -0.019 0.60
PC 4 -0.064 0.08 -0.103 0.01
PC 5 0.124 < 0.01 0.117 <0.01
PC 6 -0.054 0.14 -0.037 0.31
PC 7 -0.077 0.04 -0.073 0.04
* PC description; 1, high in meat, oils and fats and low in cereals, milk and dairy products; 2, rich
in vegetables, sauces and seasoning and poor of sugar, sweet, pastry and oils and fats; 3, high
consumption of cereals and low consumption of vegetables, fruits, sugar, sweets and pastry; 4,
high consumption of beverages and snacks and low intake of eggs; 5, vegetables, fish and seafood
consumption; 6, legumes and beverage intake; 7: high consumption in legumes and snacks and
low intake of beverages.
Schoolchildren diets
Using cluster analysis, the schoolchildren were divided into three categories: Cluster 1 with 312
(43.1%) subjects, cluster 2 with 190 (26.2%) and Cluster 3 with the remaining 222 (30.7%). The
PC scores are presented in Figure 1B and 2B. They were used to classify Mexican schoolchildren
into three groups. PC 1, 3 and 4, obtained the highest score in Cluster 1 (n=312), which represents
a diet high in meat, oils, cereals, snacks and beverages. Thus, Cluster 1 was labeled as “Modified
Mexican Diet”. PC 2, 6 and 7, had the highest score in cluster 2 (n=190), and was named as
“Traditional Mexican Diet” with large intake of legumes, vegetables, snacks, sauces and
Capítulo 7. RESULTADOS Y DISCUSIÓN
54
seasonings. The cluster 3 (n=222) had a high score in PC 5, with increased consumption of fish
and vegetables named as an “Alternative Mexican Diet”.
Figure 3 shows the mean energy supplied by each food group to the three diet types. In general,
the cereal group was the greatest energy source followed by meat, milk, and dairy products.
Significant differences (p<0.05) were found between each diet for these three food groups. The
higher intake of legumes was found in “Traditional Mexican Diet” (8% intake) with significant
differences (p<0.05) compared with the other diets. In “Alternative Mexican Diet”, milk and dairy
products represented the second most important energy source (24%), just below the cereals group
(26%). Energy intake from meat was more frequent than fish and seafood (8 to 17 % vs 0.5 to 2%
respectively) in all diets. The energy intake from sugars, sweets, and pastries was higher in
“Alternative Mexican Diet” (9.8%), showing significant differences (p<0.05) with “Modified and
Traditional Mexican Diets” (5.3 and 4.5% respectively), although these types of foods were
presented in all the diets. In the “Modified Mexican Diet”, beverages accounted for about 12% of
total energy intake, being significantly different (p<0.05) from the other diets. The highest levels
of fruit and vegetables intakes were observed in the “Alternative and Traditional Mexican Diets”
(4.8% and 2.7%, respectively), values that were significantly different (p< 0.05) compared with
the “Modified Mexican Diet”. Sauces and seasoning intake showed significant differences
between “Traditional Mexican Diet” (3% intake) and “Modified and Alternative Mexican Diets”
(2% intake). No significant differences (p >0.05) were observed in eggs, snacks, oil and fats
intakes among the different diet types.
Capítulo 7. RESULTADOS Y DISCUSIÓN
55
Figure 3 Mean percentage energy contribution from each food group according to dietary pattern groups: (■) Modified Mexican Diet (n=312), (░)
Traditional Mexican Diet (n=190) and (■) Alternative Mexican Diet (n=222).
Values are mean ± standard error calculated using ANOVA test (p <0.05). Means in each food group not sharing the same letter are significantly
different (p< 0.05).
Capítulo 7. RESULTADOS Y DISCUSIÓN
56
Basic characteristics of Mexican schoolchildren Characteristics of schoolchildren by dietary
pattern groups are shown in Table 2. Broadly, the prevalence of overweight was 20.2% and of
obesity was 20.6%. In girls, 2.6% showed low weight, 59.7% had normal weight, 19.4% were
overweight, and 18.2% were obese. In boys, 3.3% had low weight, 51.7% had normal weight,
21.2% were overweight, and 23.8% were obese. Sex, school grade, living place and nutritional
state did not show association with diet type (p>0.05), 43% of the whole schoolchildren sample,
40.5% of girls and 56.7% of boys respectively, consumed the “Modified Mexican Diet”. Four in
ten children with overweight or obesity consumed the “Modified Mexican Diet” and four in ten
children with low weight consumed the “Traditional Mexican Diet”. No significant differences (p
>0.05) were found between age, BMI and total energy intake. However, significant differences
were found in protein, carbohydrates, and fat intakes among the various diet types (p<0.05). Mean
energy intake in the whole-cohort of schoolchildren was 1864.80 ± 492 kcal/day. Carbohydrates
represented 50% of total energy intake (234.9 ± 72.57 g/day), protein 14% (65.51 ± 20.77 g/day),
and fat 36% (73.67± 23.66 g/day). Meals with more total energy contribution in the day were
breakfast 23% (467.44 ± 243 kcal/day), mid-morning meal 28% (568.42 ± 290 kcal/day) and
dinner 23% (452.33 ± 225 kcal/day).
Discussion
In this study, specific dietary patterns were identified in a cohort of Mexican school children living
in Nayarit State using a combination of PCA and cluster analyses and the association of each PC
with weight and BMI was evaluated. Seven dietary patterns emerged and three diet types were
identified: “Modified Mexican”, “Traditional Mexican” and “Alternative Mexican”. Positive and
negative associations between weight and BMI, and dietary pattern intake were also observed.
Interestingly, elevated BMI was associated to PC 5 (fish, moderate vegetable, oils and fats, and
low consumption of legumes, meat and sauces and seasoning), something that does not agree
completely with the generally accepted beneficial impact of vegetables or fish on health (12). This
apparent paradox may be explained by the occurrence of interactions between different food
groups in the PC. Also, both “Modified” and “Alternative” Mexican diets imply a relatively low
legume intake (3%) compared with the “Traditional Mexican Diet”, where legumes constitute 8%
of the daily intake. Thus, the two dietary patterns characterized by a low intake of legumes and
high intake of oils and fats may lead to the elevated BMI values recorded. Additionally, the form
of preparation of different dishes, both at household level and away from home, may have
influence on body mass and composition of schoolchildren.
Capítulo 7. RESULTADOS Y DISCUSIÓN
57
Table 2 Characteristics of Mexican schoolchildren by dietary pattern groups.
* Categorical variables are presented as frequencies and percentages in parentheses for diet. P
values calculated using Chi square test. † Continuous variables are presented as mean ± standard
error. p values calculated using ANOVA test.
Modified Traditional Alternative p value
n Categorical Variables*
312 (43.09) 190 (26.4) 222( 30.66) Sex
0.102
Female 171 (40.52) 109 (25.83) 142 (33.65) Male 141 (46.69) 81 (26.82) 80 (26.49) School grade
0.060
4th 75 (36.95) 65 (32.029 63 (31.03) 5th 123 (44.09) 62 (22.22) 94 (33.69) 6th 114 (47.11) 63 (26.03) 65 (26.86) Place of living
0.226
Urban 296 (43.02) 177 (25.73) 215 (31.25) Rural 16 (44.44) 13 (36.11) 7 (19.44) Nutritional state
0.700
Low weight 6 (28.57) 9 (42.86) 6 (28.57) Normal weight 177 (43.38) 105 (25.74) 126 (30.88) Overweight 61 (41.78) 39 (26.71) 46 (31.51) Obese 68 (45.64) 37 (24.83) 44 (29.53)
Continuous variables†
Age (years) 10.66 ± 0.05 10.74 ± 0.07 10.79 ± 0.06 0.318 BMI (kg/m2) 20.13 ± 0.23 19.87 ± 0.31 20.19 ± 0.28 0.727
Total Energy intake (kcal/d) 1900.29 ± 26.99 1846.26 ± 36.36 1830.79 ± 33.77 0.228
Carbohydrate intake (g) 242.15 ± 4.13 224.06 ± 5.45 234.07 ± 4.60 0.025
% Energy intake 50.89 ± 0.43 48.20 ± 0.56 51.32 ± 0.45 <0.001 Protein intake
(g) 64.31 ± 1.09 69.10 ± 1.36 64.11 ± 1.59 0.021 % Energy intake 13.59 ± 0.14 15.16 ± 0.17 14.05 ± 0.19 <0.001 Fat intake
(g) 74.94 ± 1.30 74.85 ± 1.72 70.89 ± 1.63 0.109 % Energy intake 35.52 ± 0.36 36.64 ± 0.49 34.63 ± 0.39 0.007
Capítulo 7. RESULTADOS Y DISCUSIÓN
58
Previous studies have suggested a negative influence of away-from-home food consumption on
obesity among all age groups, due to high energy, saturated and total fat content (13). A high
intake of legumes and snacks, together with low sweet beverage consumption were associated
with decreased weight and BMI. Legumes (eg, beans, lentils, chickpeas) have low fat content and
are good sources of dietary fiber and protein, and provide important levels of slowly digestible
carbohydrates (14). Several epidemiological studies have shown that dietary patterns
corresponding to high legume intake are associated with protection against heart disease (15). We
observed an association between high intake of snacks and low BMI. The energy intake from
snacks in this study ranged between 4 and 6 % total energy. This value was lower than those found
in an investigation in American children (more than 27% of total energy), although in that study
desserts and sweetened beverages were considered as snack (16). Evidently, the criteria used for
classifying different items in food groups may be of importance when establishing comparisons
with other populations. Additionally, the high consumption of whole milk and dairy products
could be associated with the lipid profile showed by Mexican children. A decrease in whole milk
intake accompanied by an increase in sugar-sweetened beverages could be contributing to obesity
development. Nonetheless, consumption of milk and dairy products low in fat with added
micronutrients could reduce obesity risk (17). Fruits and vegetables have been considered rich
sources of some essential dietary micronutrients, polyphenols and dietary fiber. Although fruits
and vegetables intake in this study may be considered low, it was not associated with increased
BMI values. On the other hand, it has long been recommended reducing the intake of red meat,
mainly due to its high content of saturated fatty acids; however, we found no relationship between
this food group and increased weight or BMI values. In this sense, lean red meat can lead to
decreased levels of ghrelin (a hormone that stimulates hunger) and increased secretion of the
hunger-reducing hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) (18). In the
present study no relationship between overweight-obesity status and diet types was found.
Multiple factors influence overweight and obesity status in children, including mothers’ obesity,
low physical activity, skipping breakfast, habitual overeating, father’s overweight and mother’s
age over 40 years (3). Recently, the R230C variant of ABCA1 (cholesterol transporter) was
associated with high circulating triglyceride and reduced high-density lipoprotein cholesterol
levels in overweight/obese Mexican schoolchildren (19). Those observations suggest a genetic
factor associated with the development of obesity in this population, although larger studies are
needed to validate this finding (19). BMI has been linked to the development of non-transmissible
diseases in childhood and children with a BMI ≥ 85th percentile and waist circumference ≥ 88.08
Capítulo 7. RESULTADOS Y DISCUSIÓN
59
cm have greater risk for prehypertension and metabolic syndrome (20). Interestingly, we did not
find a significant correlation between overweight-obesity status and the type of diets. However, we
found here that the diet type influenced the nutrient intake profile and the intake of different food
groups. Importantly, the observed differences in intake of certain food groups on the various diets
were of a reasonable magnitude (i.e. “Modified Mexican” 5.7 % higher for beverages; “Traditional
Mexican” 8 % higher in legumes, and “Alternative Mexican” 8.5 % higher in milk and dairy
products). If such differences persist after childhood they are likely to have important
consequences for the children’s health outcomes, given that dietary habits have been shown to
track over the life course. Considering this, it is important that children develop skills at early life,
which enable them to choose healthy foods in less structured environments, such as away from
home or off school setting, so that these practices can continue into adulthood (21). In Australian
schoolchildren, consumption of vegetables and fruits was higher in school days compared to non-
school days, due to the existence of school/based interventions encouraging fruit and vegetable
breaks and/or providing subsidized fruit and vegetables (22). In recent years, healthy diets and
physical activity have been promoted in Mexican education system, programs that have
contributed to control the prevalence of obesity in schoolchildren (23). However, these actions
have been conducted mainly in central Mexico, specifically, Mexico City. The information on
dietary patterns identified in this work can be used to plan a nutritional intervention and prepare
healthy dietary guidelines for specific regions, and possibly to take into account at a larger
national scale, considering the diversity in foods preparations. As far as we know, this study is the
first to examine the association between dietary patterns and overweight-obesity occurrence in
schoolchildren in western Mexico. The combination of several multivariate techniques proposed in
this work allowed to improve understanding of relationships of obesity and dietary patterns.
Several limitations need to be considered in the interpretation of present data. Besides the
relatively small sample size, dietary recalls are known to leave margin for inaccuracy. Moreover,
the cross-sectional design (two-day recall) of the study also limits the interpretation of our findings
in terms of its temporality. The classification into 13 food groups may have generalized the effects
of dietary patterns; a larger number of food groups could provide more specific information on the
influence of food items. The dietary pattern approach can create difficulties in replicating results in
other populations. Nevertheless, this approach is useful for enhancing our understanding of the
complex dietary variables involved in the development of non-communicable diseases. Further
interventional and/or longitudinal studies are necessary to confirm here-presented findings.
Capítulo 7. RESULTADOS Y DISCUSIÓN
60
Conclusion
The use of a combination of cluster analysis via principal component analysis gave seven dietary
factors and emerged three different diets: Traditional, Modified and Alternative Mexican Diets. A
dietary pattern characterized by high legume, snack and low beverage intake was negatively
associated with weight and BMI, suggesting that the Traditional Mexican dietary pattern could
lead to reduced prevalence of overweight and obesity among schoolchildren. An unclear
relationship between overweight-obesity status and diet types was found. The macronutrient intake
profile of schoolchildren is dependent on the type of diet consumed. The energy intake from
sugars, sweets, pastries and sweetened beverages appears in all dietary patterns. New policies for
the combat of overweight and obesity prevalence in Mexican schoolchildren should be consider,
including the design of nutrition intervention program customized to the dietary patterns of each
geographic zone.
Acknowledgements Special thanks to the children participants and their parents, to Public
Education Services of Nayarit State (SEPEN) and to all the teachers who readily consented to
carry out the study during school hours. VMZ-G acknowledges CONACYT, Mexico, for a
scholarship (Registration number: 253795).
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7.2 Perfil de metabolitos en un sistema in vitro de fermentación colónica humana en tres
menús consumidos por escolares mexicanos durante el desayuno
Resumen
La obesidad infantil se ha asociado con numerosos resultados negativos en el estado de salud físio
y psicológico. La mayoría de las pautas dietéticas están de acuerdo en la importancia de una
correcta distribución de las calorías durante el día, lograda principalmente por el consumo regular
de comida, almuerzo y cena (US-HHS, 2005). En México, la epidemia de obesidad entre escolares
ha resultado en políticas federales y estatales que mejoran los entornos alimentarios en las escuelas
(Levy et al., 2012). A menudo se afirma que el desayuno es la comida más importante del día.
Varios informes apoyan la asociación entre el consumo de desayuno y su calidad nutricional con
un riesgo reducido de desarrollar síndrome metabólico (Odegaard et al., 2013). Sin embargo,
teniendo en cuenta que los nutrientes o los alimentos rara vez se comen aislados, los estudios de
los patrones dietéticos o menú completo tienen un mayor número de ventajas sobre el estudio de
un único nutriente o enfoque de un solo alimento, considerando la aparición de interacciones
bioquímicas sinérgicas o antagonistas entre nutrientes, así como la existencia de diferentes fuentes
alimenticias del mismo nutriente (Barbaresko et al., 2013). La mayor parte de la información
disponible sobre los patrones dietéticos del desayuno y la salud en los escolares proviene
principalmente de estudios epidemiológicos (Karatzi et al., 2014). Sin embargo, la información
acerca de la composición de menús enteros identificados en diferentes patrones dietéticos es
escasa.
Probablemente, la combinación de análisis epidemiológico y estudios in vitro de los menús del
desayuno puedan contribuir a una mejor comprensión de los efectos que los alimentos pueden
promover en la salud. Los procesos fermentativos que tienen lugar en el intestino grueso humano
han suscitado mucho interés en las últimas décadas. Los diferentes metabolitos derivados de la
fermentación parecen tener una importante actividad fisiológica en los niveles tanto colónico como
periférico (Flint, 2016). En este capítulo se evaluó el perfil de metabolitos obtenido después de la
fermentación colónica in vitro de la fracción indigerible (FI) aislada de tres menús de desayuno
consumidos por escolares mexicanos. Se prestó especial atención en los metabolitos asociados con
cambios en el pH y la capacidad antioxidante (AOX). También se evaluó la composición general,
contenido de polifenoles extraíbles y no extraíbles, y AOX de los FI´s aisladas.
Capítulo 7. RESULTADOS Y DISCUSIÓN
64
Metabolites profile in an in vitro human colonic fermentation of three menus consumed by
Mexican schoolchildren at breakfast
Victor Manuel Zamora-Gasga1, Montalvo-González Efigenia1, Guadalupe Loarca-Piña2, Pedro
Alberto Vázquez-Landaverde3, Juscelino Tovar 4, Sonia G Sáyago-Ayerdi1*
1Instituto Tecnológico de Tepic, Laboratorio Integral de Investigación en Alimentos,
División de Estudios de Posgrado, Av Instituto Tecnológico No 2595, Col Lagos del Country CP
63175, Tepic, Nayarit México.
2 Programa de Posgrado en Alimentos del Centro de la República, Facultad de Química,
Universidad Autónoma de Querétaro, Centro Universitario, Cerro de las Campanas S/N, CP
76010, Santiago de Querétaro, Querétaro México.
3 Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada del Instituto Politécnico
Nacional, Unidad Querétaro, Cerro Blanco No. 141. Col Colinas del Cimatario, CP 76090,
Santiago de Querétaro, Querétaro México.
4 Food for Health Science Centre. Lund University, Medicon Village. Scheelevägen 2. Lund, SE-
223 81. Sweden
*Corresponding author, [email protected]
Tel.: +52 311211-940
Capítulo 7. RESULTADOS Y DISCUSIÓN
65
Highlights
• Indigestible fraction (IF) was isolated and characterized from food consumed at breakfast
• Effects of three isolated IF on gut metabolism in vitro was analyzed
• Traditional breakfast has a higher condensed and hydrolysable tannins content.
• Short chain fatty acid production was low during IF-breakfast fermentation
• Metabolite patterns were associated with pH and antioxidant capacity at fermentation
Graphical Abstract
Capítulo 7. RESULTADOS Y DISCUSIÓN
66
Abstract
Transitional food led changes in diets and lifestyles that promote childhood obesity, and Mexico is
an example of this epidemic. Breakfast is considered the most important meal of the day and the
study of a whole menu had higher number of advantages over single nutrients. The aim of this
work was to evaluate the metabolite profile of the indigestible fraction (IF) by in vitro human
colonic fermentation isolated from three different breakfast menus consumed by Mexican
schoolchildren in Tepic, State Nayarit Mexico. The menus were named as: modified, traditional,
and Alternative Mexican Breakfast (MM-B, TM-B, AM-B, respectively). The IF in all breakfast
were about 5.78 g/100 g wet basis (wb), and TM-B had 1.1 g/100 g wb for soluble IF, where beans
were included. The indigestible protein was relatively high (≈21 %) and this could contribute with
negative implications. Fermentation of menus showed a positive changed in pH only in TM-B,
which can be related with poor or low fermentation of the indigestible carbohydrates. TM-B
showed a greater pH decreased and also present a lower short chain fatty acid (SCFA) production,
but a greater relative concentration of hexanoic, heptanoic and octanoic acids. Besides, 55 volatile
compounds were detected by SPME-GC-MS and three principal components (PC) were identified.
PC1 was influenced by higher SCFA production and low fatty acid esters production. Fatty acid
ester “less than eight carbon atoms” and volatile organic acids production (PC2) resulted in a
decrease on pH and an increase on antioxidant capacity (AOX). This result suggested that total
metabolites present in the intestinal medium could affect the pH and antioxidant status in the
colon. Fermentation extracts of IF isolated from traditional breakfast presents a fermentation-time-
dependent beneficial metabolic pattern.
Keywords:
Breakfast Menus, Indigestible fraction, Antioxidant capacity, Gut metabolites.
Abbreviations:
AM-B, alternative Mexican breakfast; AOX, antioxidant capacity; CGC/MS, gas
chromatography–mass spectrometry; DP, dietary pattern; HCA, Hierarchical cluster analysis; HS-
SPME, headspace solid-phase micro-extraction; IF, Indigestible fraction; MM-B, modified
Capítulo 7. RESULTADOS Y DISCUSIÓN
67
Mexican breakfast; PCA, Principal component analysis; SCFA, short chain fatty acid; TM-B;
traditional Mexican breakfast.
1 Introduction
Childhood obesity has been associated with numerous negative health and psychological
outcomes. Its surge has been explained as consequence of globalization-related changes in diet and
lifestyle that promote positive energy balance (Malik et al., 2013). Most dietary guidelines agree
on the importance of a correct distribution of calories throughout the day, achieved principally by
a regular meal consumption of breakfast, lunch, and dinner (US-DHHS and USDA, 2015). In
Mexico, obesity epidemic among schoolchildren has resulted in federal and state policies that
improve school food environments (Levy et al., 2012). It is often stated that breakfast is the most
important meal of the day. Several reports support the association between breakfast consumption
and its nutritional quality with a reduced risk to develop metabolic syndrome (Odegaard et al.,
2013). However, taking into account that nutrients or foods are rarely eaten isolated, studies of
dietary patterns or whole menu have a higher number of advantages over the single-nutrient or
single-food approach, by considering the occurrence of synergistic or antagonistic biochemical
interactions among nutrients, as well as the existence of different food sources of the same nutrient
(Barbaresko et al., 2013). Most available information on breakfast dietary patterns (DP) and health
in schoolchildren is derived principally from epidemiological studies (Karatzi et al., 2014).
However, information is scare about the composition of whole menus identified in different
dietary patterns. Probably, the combination of epidemiological and in vitro analysis of breakfast
menus may contribute to a better understanding of the effects that food can promote on health. The
fermentative processes that take place in the human large intestine have raised much interest in the
last decades. Different fermentation-derived metabolites appear to have to important physiological
activity at both colonic and peripheral levels (Flint, 2016). In this work we evaluated the
metabolites profile obtained after in vitro colonic fermentation of the indigestible fraction (IF)
isolated from three breakfast menus consumed by Mexican schoolchildren in Tepic, State Nayarit
Mexico. Particular attention was paid to metabolites associated with changes in pH and
antioxidant capacity (AOX). The general composition, extractable and non-extractable
polyphenols content, and AOX of the isolated IFs were also evaluated
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2. Material and methods
2.1 Preparation of breakfast menus
Data sources on DP and food frequency consumption at breakfast were obtained from a nutritional
survey carried out on eleven public schools in Tepic (Nayarit State, western Mexico). These data
are being reported elsewhere (Zamora-Gasga et al., 2017). Frequently consumed foods in each DP
were used to create three breakfast menus, which comprised by the following foods: a) Modified
Mexican Breakfast (MM-B) (≈394 g total weight): one scrambled egg, three corn tortillas (90 g),
chocolate milk (whole milk 264 mL, cane sugar 7 g, chocolate powder 13 g); b) Traditional
Mexican Breakfast (TM-B) (≈ 478 g total weight): one scrambled egg, three corn tortillas (90 g),
refried beans (83 g), chocolate milk (whole milk 264 mL, cane sugar 6g, chocolate powder 11 g);
c) Alternative Mexican Breakfast (AM-B) (≈370 g total weight): one scrambled egg, two corn
tortillas (60 g), chocolate milk (whole milk 256 mL, cane sugar 10g, chocolate powder 18 g).
Individual ingredients were purchased from the local supermarket and menus were prepared in the
laboratory kitchen according to traditional regional customs. Recently prepared menus were
homogenized in a food processor (NB-101B, Nutribullet, China), frozen (-80 °C), freeze-dried
(FreeZone 6, Labconco, USA), ground, sieved through a mesh size of 500 µm, and stored at -20
°C until analysis. Each menu was prepared in triplicate.
2.2 Quantification and isolation of indigestible fraction (IF) in menus
The IF quantification was evaluated according to Saura-Calixto et al. (2000), a method that
simulates the physiological situation in the upper gastrointestinal tract. The method was run at
preparative scale, according to the modifications proposed by Tabernero et al. (2011). Insoluble
(IIF) was considered as the digestion residues pelleted by centrifugation, while those retained by
dialysis represented soluble IF (SIF); the sum of both fractions equals total IF (TIF). TIF was
collected, freeze-dried, milled (IKA M20, USA), sieved (500-micron mesh), and stored in seal
bags at -20 °C.
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2.2.2 Chemical composition of total indigestible fraction
Moisture content, ash, protein and fat were analyzed according to AOAC (1990) 925.10, 923.03,
920.87 and 920.39 methods, respectively. Total carbohydrates were evaluated by the phenol-
sulfuric method (Dubois et al., 1956). Resistant starch was assessed following a multi-enzymatic
digestion protocol (Goñi et al., 1996).
2.2.3 Antioxidant compounds analysis in the total indigestible fraction
TIF samples (250 mg) from the different menus were extracted with aqueous-organic solution
according to the methodology proposed by Pérez-Jiménez et al. (2009). Total, soluble polyphenols
(TSP) in extractable fraction were determined with the Folin–Ciocalteu’s reagent (Montreau,
1972) using a 96-well microplate reader (Biotek, Synergy HT, Winooski VT, USA) with Gen5
software, and the results were expressed as gallic acid equivalents (g GAE/ 100g IF). The residues
of the extraction were treated for non-extractable polyphenols quantification (Condensed tannins
and hydrolyzable polyphenols). Condensed tannins (CT) were assessed by the method proposed by
Reed et al. (1982); results were expressed as CT equivalents/ 100 g IF, using a carob pod
(Ceratonia siliqua) proanthocyanidin standard. Hydrolyzable polyphenols (HP) were evaluated by
Hartzfeld et al. (2002) method and results were expressed as g GAE/100 g IF.
2.2.4 Antioxidant capacity (AOX) analyses
AOX of the extractable fraction obtained above was analyzed. AOX methodologies were slightly
modified to a microplate reader (Biotek, Synergy HT, Winooski VT, USA) with Gen5 software.
1,1-Diphenyl-2-picryl hydrazyl (DPPH) antiradical activity assay was performed according to the
method of Prior et al. (2005). DDPH radical-scavenging activities were expressed as Trolox (6-
hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic) equivalent (TE; mmol /g IF db). Ferric
reducing antioxidant power (FRAP) assay was performed as described by Benzie et al. (1996).
Results are expressed as mmol of Trolox equivalents (TE: mmol /g IF db).
2.3 In vitro colonic fermentation by human microflora
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The assays were preformed using a pool of fresh fecal samples collected from five healthy
schoolchildren (9–12 years), apparently free from gastrointestinal diseases and who did not receive
antibiotic treatment during the previous 3 months. The fermentation process was developed
according to Campos Vega et al. (2009). TIF of breakfast menus was fermented at 37 °C under
anaerobic conditions. Two different controls were also conducted in parallel: a) raffinose, used as
a fermentable sugar reference that produce SCFA was incubated in medium with faeces inoculum,
and b) the faecal suspension was incubated without addition of substrate, serving as negative
control. All incubations were performed in triplicate; samples were collected at 12, 24, 48 and
72 h. and centrifuged (Hermle Z 323 K; Wehingen, Germany) (3500 × g,15 min, 4 °C).
Supernatants were placed into a 20 mL vial sealed with a magnetic cap with a poly-tetra-fluoro-
ethylene (PTFE)/silicon septum. The vials were immediately stored at −80 °C in order to minimize
any deteriorating changes in the volatile components of the samples until they were processed.
2.4 Metabolites characterization by HS-SPME- GC/MS
Supernatants from the colonic fermentation were characterized by Gas Chromatography–Mass
Spectrometry (CGC/MS) using a headspace solid-phase micro-extraction (HS-SPME) technique
according to Zamora-Gasga et al. (2015); incubation with polydimethylsiloxane-divinylbenzene-
carboxen “PDMS/DVB/CAR” fiber for 120 min, 250 rpm at 45 °C; the fiber was inserted into the
injection port of the GC system for thermal desorption (240 °C for 10 min) for GC/MS analysis.
The extractions were performed in triplicate. Five runs with twelve vials / run were performed and
the tests order was completely randomized. The volatile constituents were analyzed with an
Agilent 5975C VL mass selective detector coupled to an Agilent 7890A gas chromatograph
(Agilent Technologies, Inc., Santa Clara, CA), equipped with a DB-5MS capillary column (60 m
X 250 µm X 0.25 µm; Agilent). Sample quantification was obtained by means of acetic, propionic
and butyric acid standard curves. Tentative identification of the volatile components was done
comparing the mass spectra of the samples with the data system library MSD ChemStation
software (Agilent G1701EAversion E.02.00.493). Relative concentration of all fermentation
metabolites versus acetic acid as internal standard was calculated and the results were expressed in
mmol L-1.
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2.5 Statistical analysis
Data were subjected to one-way ANOVA/Fisher's LSD test (Levene's test, p≥0.05, Shapiro–Wilk
W test, p≥0.05, n = 9) and to independent-samples Kruskal–Wallis non-parametric test/Multiple
comparisons of mean ranks for all test groups (Levene's test, p<0.05, Shapiro–Wilk W test,
p<0.05, n = 9). Two-way ANOVA with a post hoc Fisher's LSD test was used to determine the
effect of the substrate type and fermentation time on pH changes and antioxidant capacity
(Substrate type × fermentation time). Principal component analysis (PCA) of fermentation
metabolites was performed based on the mean values of triplicates. Components were calculated
without rotation and the number of extracted factors were based on eigenvalues >1.0 and
explained variance (%) >70. A secondary analysis based on multiple lineal regression analysis was
used to identify which metabolic profiling (Component score) where associated with pH and
antioxidant capacity (DPPH and FRAP methods). In order to identify which samples (blank,
raffinose, and samples IF) showed similar metabolic profiles, a hierarchical cluster analysis (HCA)
was realized. Single Linkage amalgamation method of clustering and the Euclidean distance
measure were used. All analyzes were performed using STATISTICA software, version 10.0
(StatSoft. Inc. 1984–2007, Tulsa, OK, USA).
3. Results and Discussion
3.1 Characterization of indigestible fraction in breakfast menus
Moisture, IF content, and chemical composition of complete menus are shown in Table 1. No
significant differences in moisture content were observed between menus; this probably reflects
the similar volume amount of milk included in the various menus (≈250 mL). Small but significant
differences (p<0.05) in IF content were found between menus (6.11, 6.22 and 5.03g /100 g wet
basis (wb) for MM-B, TM-B and AM-B respectively). The insoluble IF (IIF) contents in MM-B
and TM-B (5.60, 5.13 g/100 g wb) were not significantly different. TM-B presented the highest
Soluble IF (SIF) value (1.10 g/100 g menu wb). It is worth noting that these menus included
refried beans. Cooked beans are good sources of dietary fiber (DF), with contents as high as 59
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mg/g dry basis (DB) insoluble DF (Dueñas et al., 2016). Ash content in the IFs showed significant
differences (p<0.05), with values ranging from 0.19 to 0.25 g/100 g menus wb. Has been reported
that some minerals can be poorly absorbed in the human small intestine because of the presence of
certain inhibitory compounds like phytates, CT, and DF (present in cereal- and legumes-
containing breakfast), although they may be absorbed in colon (Raes et al., 2014). On the other
hand, TM-B and MM-B exhibited the highest indigestible carbohydrate content, with 0.89 g / 100
g menu wb, which was about three times higher (p<0.05) than in AM-B (0.34 g / 100 g menu wb).
Similarly, the resistant starch (RS) content was significantly (p<0.05) higher in IF isolated from
TM-B, with values that were 30 and 60% higher than in MM-B and AM-B, respectively. These
results could be attributed to the presence of appreciable levels of indigestible carbohydrates, as
DF and RS, in corn tortilla and beans (Sáyago Ayerdi et al., 2014). IF of menus showed a high
IF fat content of 3.30, 3.32 and 3.24 g/100 g menu wb, in MM-B, TM-B and AM-B, respectively.
The ratio between fat/indigestible carbohydrate content in menus was 9.5, 4.5 and 3.7 w/w (AM-
B, MM-B and TM-B respectively). Protein content was relatively high (1.30, 1.42 and 1.07 g/100
g menu wb for MM-B, TM-B and AM-B, respectively) with significant differences (p<0.05).
These values may have negative implications to the intestinal health in schoolchildren, as diets
high in protein and low in indigestible carbohydrates have been reported to result in decreased
colonic production of fecal cancer-protective metabolites (e.g. butyric acid) and increased
concentrations of hazardous compounds (e.g. N-nitroso compounds) (Russell et al., 2011).
3.2 Antioxidant compounds in the indigestible fraction
The levels of TSP, HP, and CT present in IF of the studied menus are summarized in Table 1. TSP
associated to IF were released in the aqueous-organic extraction system and showed chelating
AOX, but no DPPH radical scavenging activity was observed (Table 1). Chelating activity was
higher (21.25 mmol TE/g IF DW) in extracts from IF isolated from MM-B menus and lower in
TM-B (16.82 mmol TE/g IF DW). Besides polyphenols, other constituents present in IF from the
breakfasts may affect AOX. The presence of egg yolk components like phosvitin, phospholipids,
carotenoids and free aromatic amino acids may contribute to the overall antioxidant capacity of the
samples (Remanan et al., 2014); however; the concentration of these compounds in IF from food
mixtures has not been studied. TM-B showed a high CT content (2.15 g/100 g IF DW),
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significantly greater than in MM-B (0.57 g/100 g IF Dw), although not different from the level
measured in IF from AM-B (0.92 g/100 g IF DW). TM-B exhibited the highest HP content (2.15
g/100 g IF DW) among the studied breakfasts. The presence of beans in TM-B could be largely
responsible for the high non-extractable polyphenols contents, as they are abundant in this legume
(Ramírez-Jiménez et al., 2015).
Table 1 Chemical composition and antioxidant compounds content, and antioxidant capacity in
the indigestible fraction (IF) isolated from breakfast menus1
Parameter Menus2
MM-B TM-B AM-B Moisture content (g/100g menu ) 73.26 ± 0.37a 74.13 ± 0.10a 73.62 ± 0.40a IF (g/100g menu wb) Insoluble IF 5.60 ± 0.22b 5.13 ± 0.21ab 4.57 ± 0.10a Soluble IF 0.51 ± 0.06a 1.10 ± 0.14b 0.46 ± 0.06a Total IF 3 6.11 ± 0.24b 6.22 ± 0.35b 5.03 ± 0.09a IF Nutritional Composition (g/ 100g menu wb) Ash 0.25 ± 0.01a 0.24 ± 0.01a 0.19 ± 0.01b Indigestible Carbohydrates 0.73 ± 0.01a 0.89 ± 0.07a 0.34 ± 0.05b Resistant Starch 0.008 ± 0.001a 0.012 ± 0.001b 0.004 ± 0.001c Lipids 3.30 ± 0.04a 3.32 ± 0.18a 3.24 ± 0.04a Proteins 4 1.30 ± 0.05a 1.42 ± 0.09a 1.07 ± 0.02b IF Antioxidant Compounds (g/ 100g IF DB) Total soluble polyphenols 1.16 ± 0.03a 0.94 ± 0.02ab 0.93 ± 0.02b Condensed Tannins 0.57 ± 0.02a 2.15 ± 0.13b 0.92 ± 0.04ba Hydrolyzable polyphenols 1.57 ± 0.01a 2.15 ± 0.02c 1.78 ± 0.02b IF Antioxidant Capacity (mmol TE/g FI DB) DPPH ND ND ND FRAP 21.25 ± 1.24b 16.82 ± 0.16a 19.23 ± 0.15ab
1Values are mean ± standard error (n = 3); Wet basis (wb); Dry basis (db); No Detected (ND).
Means in rows marked with different letters indicate significant difference (p <0.05). 2 Menus;
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MM-B: Modified Mexican Breakfast, TM-B: Traditional Mexican Breakfast, AM-B: Alternative
Mexican Breakfast. 3 Total IF = Sum of soluble IF + insoluble IF. 4 Conversion factor: % N x 6.25.
Also, interactions between indigestible proteins and polyphenols presents in food matrix may
avoid the absorption of the polyphenols in the small intestine, increasing its content in the IF
(Mullen et al., 2009).
3.3 Changes in pH and antioxidant capacity during in vitro colonic fermentation
The progress of the fermentation process was followed measuring pH changes in each sample
during 72 h (Figure 1a). Significant differences (p < 0.05) were found among the substrates. The
largest decrease in pH was raffinose, but among the IF from breakfast menus was observed for
TM-B after 72 h (pH= 6.07). The pH variations between 0 and 72 h of fermentation were -0.02,
0.48, and -0.07 for MM-B, TM-B, and AM-B menus, respectively. Only the TM-B menu showed
a positive change in pH. Such increased pH values can be related to poor or limited fermentation
of the indigestible carbohydrates present in the substrate (Kettle et al., 2015). AOX evaluated by
DPPH and FRAP assays (Figure 1b and c), showed time- dependence and significant differences
(p <0.05). It is noteworthy that DPPH activity (less than 40 mmol TE/g IF) was detected in
fermentation extracts but not in aqueous-organic extracts. In terms of FRAP, MM-B (16.31 mmol
TE/g IF DW) showed significant differences respect to TM-B (8.13 mmol TE/g IF db) and AM-B
(10.79 mmol TE/g IF DW) at 48 h. However, the AM-B menu had the highest FRAP activity at 72
h (10.85 mmol TE/g IF DW). Microbial metabolites can influence AOX in fermentation extracts.
It has been reported that the presence of 4-vinylguaiacol (microbial metabolite of ferulic acid)
decreases AOX of fermentation extracts from arabino-xylanoligosaccharides (Snelders et al.,
2014). Recently, in vitro antioxidant effects of urolithin A (colonic metabolite of ellagic acid) on
hepatocellular carcinomas HepG2 5 cells were demonstrated (Wang et al., 2015).
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Figure 1 Changes in a) pH kinetic plot, b) DPPH Antiradical activity plot and c) FRAP chelating activity plot in the extracts
during in vitro colonic fermentation from (–) blank, (··♦··) raffinose, and indigestible fraction isolated from breakfast menus: (-
■-) Modified Mexican Breakfast, MM-B, (-▲-) Traditional Mexican Breakfast, TM-B, and (-●-) Alternative Mexican
Breakfast, AM-B at different at different fermentation times, Values are means ± SEM (n=3). *Significant difference using
Two-way ANOVA/Fisher's LSD test (Samples ×Time interaction, p<0.05). *For DPPH and FRAP , the blank was subtracted of
the samples.
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3.4 Production of short chain fatty acids (SCFA) during in vitro fermentation
The SCFA production at 12, 24, 48 and 72 h of in vitro fermentation of IF isolated from breakfast
menus are showed in Table 2. At 24 h, AM-B produced the greatest acetate concentration (47.23
mmol/L), and was statistically different (p < 0.05) from the other breakfast menus. Propionic acid
concentrations were very similar (p > 0.05) between MM-B and TM-B (28.5 and 28.9 mmol/L
respectively) at 24 h. and AM-B showed the highest concentration with 35.7 mmol/L. Regarding
butyric acid, no significant differences (p>0.05) were found between fermented IF from breakfast
menus and the assay blank. Low production of SCFA may be related to the proportions of
macronutrients present in IF from the breakfast menus. A recent study suggested that the
magnitude of SCFA production during in vitro fermentation is dependent on the diet of the fecal
donor (Yang et al., 2016).
Interestingly, the substrate with lowest carbohydrate concentration (AM-B menu) showed the
highest production of SCFA. It seems also contradictory that TM-B showed a larger pH decrease
and lower SCFA production. Hypothetically, it can be thus proposed that other metabolites may be
involved in the pH decrease observed during fermentation of IF from TM-B menu. In this sense,
the volatile organic compounds produced during the fermentation are shown in Supplementary
Table SI. A greater relative concentration of hexanoic acid (ID: 31), heptanoic acid (ID: 34) and
octanoic acid (ID: 41) was recorded in the IF fermentation medium from TM-B compared to the
other breakfast menus, particularly after 48 and 72 h. Therefore, reduced pH values may be
attributed to the production of non-SCFA organic acids (Sagar et al., 2015).
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Table 2. Short-chain fatty acids (SCFAs, mmol L-1) production at 12, 24, 48 and 72 h of in vitro fermentation of blank,
raffinose and indigestible fraction isolated from breakfast menus (MM-B; Modified Mexican Breakfast, TM-B; Traditional
Mexican Breakfast, AM-B; Alternative Mexican Breakfast) 1.
SCFA/ fermentation
time
Blank Raffinose IF- Breakfast Menus
MM-B TM-B AM-B
Acetic acid 12 h 6.05 ± 0.75a 205.05 ± 25b 2.39 ± 0.08a 12.93 ± 0.69a 6.41 ± 0.87a 24 h ND 273.92 ± 33.41c 19.88 ± 2.27a 7.45 ± 0.07a 47.23 ± 0.30b 48 h 11.28 ± 6.22a 572.54 ± 24.63b 19.89 ± 2.13a 5.89 ± 0.36a 11.63 ± 1.07a 72 h ND 254.75 ± 12.48b 20.32 ± 8.69a ND 17.40 ± 6.22a Propionic acid 12 h 4.18 ± 0.62a 44.17 ± 0.89b ND ND ND 24 h ND 89.01 ± 2.61c 28.50 ± 3.15a 28.91 ± 1.13a 35.76 ± 2.30b 48 h ND 134.33 ± 8.10c 27.13 ± 1.73b ND 18.38 ± 0.45a 72 h ND 103.88 ± 2.46 ND ND ND Butyric acid
12 h 0.78 ± 0.17a 25.97 ± 5.63b 0.75 ± 0.13a 1.52 ± 0.11a 0.67 ± 0.12a 24 h 0.44 ± 0.09a 36.94 ± 7.92b 3.41 ± 1.00a 4.79 ± 0.24a 4.57 ± 0.30a 48 h 0.78 ± 0.18a 63.55 ± 7.57b 3.49 ± 0.29a 1.85 ± 0.24a 3.86 ± 0.76a 72 h ND 34.92 ± 1.32 ND ND ND
*The values are reported in mmol/L produced per 100 mg substrate as mean ± SEM of three replicate; Different lowercase
letters indicate significant differences in rows among substrates for a time (p < 0.05). No Detected (ND).
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3.5 Volatile compounds during the in vitro colonic fermentation
A total of 55 volatile compounds were detected in fermentation extracts of blank samples,
raffinose standard and IFs isolated from the different breakfast menus (See Supporting
Information Table SI). PCA was performed to determine microbial metabolic pattern of
fermentation in the samples at different fermentation times. Three principal components (PC) were
obtained (Eigenvalues > 1) that explained 71.31% of the total variance. Loading scatter plot for the
three principal components are shown in Figure 2. PC 1, 2, and 3 explained 44.75, 15.30, and 11.2
% respectively, of all variance from the gut metabolites production. PC1 on the positive axis
(Figure 2a) was highly influenced by different microbial metabolites, mainly SCFA (See
Supporting Information Table SI); butyric acid, Benzaldehyde, 4-methyl-, acetic acid, propionic
acid, butanoic acid, 3-methyl-, and ethanol (ID: 20, 23, 12, 16, 24 and 2 respectively). Increased
concentrations of these compounds were registered during in vitro colonic fermentation. On the
other hand, PC1 negative axis comprises compounds that were produced at relatively low levels
during the fermentation process, mainly medium to long chain fatty acid esters (e.g. nonanoic acid,
methyl ester, ID:19; nonanoic acid, ethyl ester, ID:21; dodecanoic acid, ethyl ester ID:36;
tridecanoic acid, methyl ester, ID:39; decanoic acid, methyl ester ID:25; decanoic acid, ethyl ester,
ID:26; pentadecanoic acid, methyl ester, ID:46), indole (ID:49), and disulfide, dimethyl (ID:5).
Regarding PC2 (Figure2a), some fatty acid esters with less than eight carbon atoms: butanoic
acid, methyl ester, ID:4; hexanoic acid, methyl ester (ID: 8); hexanoic acid, ethyl ester (ID:9);
heptanoic acid, methyl ester (ID:10); heptanoic acid, ethyl ester (ID:14) and volatile organic acids
(e.g. hexanoic acid “ID:31”, heptanoic acid “ID:34” and octanoic acid “ID:34”) were located in
the positive axis (high production). However, the volatile compounds on the negative axis (low
production) were trimethylamine, dimethyl trisulfide and indole (ID: 1, 11 and 49 respectively). In
PC 3 (Figure 2b), increased production is noticed for propionic acid (ID:16), butyric acid, (ID:20),
and butanoic acid, 3-methyl- (ID:24) and a low production observed for trimethylamine (ID:1),
trichloromethane (ID:3), pyridine, 2,4,6-trimethyl- (ID:13), phenol (ID:38), and phenol, 4-methyl-
(ID:40).
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Figure 2 Identification of Microbial metabolic pattern between the in vitro colonic fermentation extracts of blank, raffinose and
indigestible fraction isolated from breakfast menus at different fermentation times using principal component analysis (PCA):
Loading scatter plot for a) PC1 vs. PC2; b) PC3 vs. PC4 and PC5 vs. PC6 (Detailed of identification number for each volatile
compound are outlined in the Supporting Information Table SI).
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3.6 Correlation analysis between microbial metabolic pattern, pH and antioxidant capacity
Multiple regression analysis was conducted to test the relationship between microbial metabolic
pattern (component scores) pH, DPPH, and FRAP in fermentation extracts of IF isolated from
breakfast menus at different times (Figure 3). The linear regression model was significant
(p<0.05) for data with high values in the coefficients determined, indicating a proper fit of the data
with the model (Multiple R2 values= 0.91, 0.88, and 0.62 for pH, DPPH, and FRAP, respectively).
The results showed that the three components were significantly (p < 0.05) associated with the
decrease in pH values. The largest predicted decrease in pH was observed with the fermentative
metabolic pattern characterized by a high production of fatty acid esters and volatile organic acids,
with a low production of trimethylamine, dimethyl, trisulfide and indole (PC 2). Also, three PC
scores were associated with increased antioxidant capacity. Increase in predicted DPPH was
observed with increasing production of PC 2 members (Coefficient = 27.02), followed by
production of metabolites included in PC1 (high SCFA production and low fatty acid esters
production, Coefficient = 23.87) and PC3 (high propionic and butyric production and low
production of protein metabolites, Coefficient = 22.88). Finally, for chelating activity, increased
FRAP values were observed with increased scores of PC2 and PC3., while PC1 coefficient was
not significant (p>0.05). These results suggest that overall metabolite composition of the luminal
medium may affect pH and antioxidant status of the colon. They also highlight the importance of
assessing the microbial metabolic pattern instead of individual metabolites identified during IF
fermentation isolated from complete food. Actually, volatile compounds (in particular SCFAs as
pentanoic and hexanoic acids) have been proposed as potential noninvasive biomarkers, which
may reflect gut dysbiosis, and thus capable of differentiating subjects in health and disease
(Covington et al., 2013). Additionally, some compounds identified in this work have been
previously reported as harmful to health. For instance, experimental and clinical studies have
shown trimethylamine-N-oxide (formed in the liver from trimethylamine), phenol and p-cresol as
strong predictors of coronary artery disease and Crohn's disease (Trøseid et al., 2015; Walton et
al., 2013).
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Figure 3 Multiple regression analysis models exploring the association of microbial metabolic pattern (Component scores)
with: a) pH Values, b) DPPH antiradical activity and c) FRAP chelating activity. *Model fit was adjusted to: Y = β0 + β1*X1 +
β2*X2 + β3*X3, where, Y is response variable (pH, DPPH and FRAP), β0 is the intercept, β1, β2, β3, are the coefficients and X1, X2,
X3 are the Component Score (CS1, CS2 and CS3).
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3.7 Hierarchical cluster analysis (HCA)
Dendrogram of component scores (metabolic profile) from different fermentation times of IF
isolated from breakfast menus are presented in Figure 4. The HCA led to three clusters, indicating
that the production of volatile compounds by gut microbiota was different between the formed
clusters. The analysis clearly group raffinose (Cluster I) and blank samples (Cluster III) at all
times. In addition, Cluster I presented component positive scores for PC 1 (dark color), indicating
that the cluster follows a metabolic profile characterized by high SCFA production and low fatty
acid esters (medium to long chain) production. Cluster II was formed by fermentation extracts of
IF isolated from all breakfast menus. However, it is noted that TM-M (at 48 and 72 h) showed the
highest values (dark color) of the scores for PC2 (high production of C<8 fatty acid esters and
volatile organic acids). PC2 was associated with decreased pH and increased AOX. Additionally,
the remaining samples were characterized by light colors, indicating that they are not associated
with health benefits. HCA results indicated that in vitro colonic fermentation results in
considerable differences in the metabolic pattern depending on the IF samples and fermentation
time.
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Figure 4 Dendrogram of hierarchical cluster analysis based on the microbial metabolic profiles (component score) in colonic
fermentation extracts of blank, raffinose, and indigestible fraction isolated from breakfast menus (MM-B; Modified Mexican
Breakfast, TM-B; Traditional Mexican Breakfast, AM-B; Alternative Mexican Breakfast) at different fermentation times: 12 h
(T12), 24 h (T24), 48 h (T48) and 72 h (T72).
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4. Conclusions
This study shows that IF isolated from three breakfast menus consumed by Mexican
schoolchildren have different chemical composition. TM-B had non-extractable polyphenols, and
indigestible carbohydrates (DF) as main bioactive compounds. It is being increasingly shown that
the fermentative metabolism of ingested food creates a microbial metabolome that has marked
impact on host health. In this work, PCA allowed the identification of metabolic patterns that are
associated with beneficial health effects (decreased colonic pH and increased antioxidant capacity
values). Compounds associated with different diseases were also identified and associated with
harmful effects (increased pH and decreased AOX). Hierarchical cluster analysis showed that
fermentation extracts of IF isolated from a traditional Mexican breakfast presents a fermentation
time-dependent beneficial metabolic pattern. Further work evaluating the colonic fermentative
patterns of foods combined in complete realistic regimens are needed to understand the impact of
diet on intestinal and general health status.
Acknowledgments: Zamora-Gasga, VM; acknowledge the fellowship to CONACYT- Registration
number: 253795) and Sáyago-Ayerdi, SG; would also like to acknowledge the financial support to
PROMEP –ITTEP-PTC-003.
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Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of blank, rafinose and indigestible fraction isolated from breakfast menus (MM-D; Modified Mexican diet, TM-D;
Traditional Mexican Diet, AM-D; Alternative Mexican diet) analyzed by SPME–GC/MS.
ID RT Volatil Name Chemical Group
T12 T24 T48 T72 VC (%) Blank Raffinos
e MM-D TM-D AM-D Blank Raffinose MM-D TM-D AM-D Blank Raffinos
e MM-D TM-D AM-D Blank Raffinose MM-D TM-D AM-D
1 6.044 Trimethylamine Amines 37.20 0.00 24.23 16.08 21.81 24.96 0.00 26.32 12.88 49.53 33.73 0.00 12.83 8.26 27.33 61.47 0.00 18.51 18.56 28.48 77.79
2 8.475 Ethanol Alcohols and polyols 57.50 73.02 8.19 67.40 3.77 29.88 159.40 94.63 51.17 116.34 0.00 177.80 16.00 49.58 20.82 110.80 122.17 34.39 78.81 0.00 83.20
3 10.985 Trichloromethane Alkyl halides 21.18 12.36 9.37 8.56 9.41 14.18 15.55 10.50 19.30 17.46 0.00 0.00 0.00 0.00 0.00 41.05 11.72 13.80 17.86 14.31 82.71
4 12.265 Butanoic acid, methyl ester Fatty Acyls 6.87 12.15 43.68 42.09 21.33 0.00 8.26 159.80 150.40 33.66 6.69 10.52 177.65 255.49 69.02 13.14 16.81 154.62 350.23 127.92 117.57
5 14.404 Disulfide, dimethyl Organic disulfides 0.00 0.00 11.06 38.50 26.21 0.00 0.00 29.38 23.27 30.45 4.71 0.00 3.50 4.29 15.48 0.00 0.00 7.78 26.72 3.72 114.80
6 16.547 Pentanoic acid, methyl ester Fatty Acyls 6.97 0.00 71.48 102.68 78.62 4.23 3.80 327.28 311.20 171.65 13.16 4.52 312.07 414.73 196.23 17.02 4.32 406.83 637.63 395.29 109.26
7 19.273 Pentanoic acid, ethyl ester Fatty Acyls 0.00 0.00 6.81 7.80 12.07 0.00 0.00 33.23 25.32 12.75 4.16 0.00 24.54 30.05 13.75 0.00 0.00 19.06 49.35 17.15 108.82
8 20.744 Hexanoic acid, methyl ester Fatty Acyls 11.87 4.32 23.72 36.38 16.56 10.76 0.00 183.41 149.71 31.89 63.35 5.42 250.85 1720.24 576.83 57.49 13.03 1351.8
4 2292.4
5 864.02 172.62
9 23.233 Hexanoic acid, ethyl ester Fatty Acyls 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10.90 6.83 0.00 4.60 0.00 9.27 69.40 14.81 0.00 0.00 43.55 89.11 60.85 175.92
10 24.813 Heptanoic acid, methyl ester Fatty Acyls 0.00 0.00 0.00 64.33 0.00 34.70 0.00 376.35 362.08 0.00 297.16 0.00 759.18 4367.54
3657.04 250.51 0.00 4653.8
8 7933.9
4 5154.6
8 168.99
11 26.242 Dimethyl trisulfide Organic trisulfides 0.00 0.00 117.05 442.44 441.73 0.00 0.00 291.65 179.48 212.46 15.39 0.00 0.00 10.44 115.96 0.00 0.00 0.00 50.80 0.00 155.94
12 26.247 Acetic Acid Organic acid 6.05 176.44 2.39 15.30 8.52 0.00 381.06 30.83 10.11 40.38 7.52 451.15 16.50 18.37 19.00 0.00 393.48 13.55 4.09 11.60 182.91
13 26.399 Pyridine, 2,4,6-trimethyl- Pyridines and derivatives 66.24 0.00 0.00 36.24 25.00 60.69 0.00 11.81 12.60 7.41 61.69 0.00 28.83 24.83 41.47 207.55 0.00 28.07 42.56 50.65 131.15
14 26.980 Heptanoic acid, ethyl ester Fatty Acyls 0.00 0.00 4.94 8.25 9.52 0.00 0.00 40.30 25.72 15.06 23.28 0.00 34.14 238.07 96.39 12.34 0.00 189.39 368.41 272.10 163.27
15 28.571 Octanoic acid, methyl ester Fatty Acyls 31.77 7.97 566.71 920.55 593.61 49.08 7.51 1522.92
1821.15 734.62 76.73 8.80 1179.3
3 1943.9
3 1642.2
3 85.87 11.65 2063.50
3163.46
1979.41 102.20
16 29.758 Propionic Acid Organic acid 4.19 59.68 0.00 6.88 0.00 0.00 89.01 39.63 33.19 41.67 1.61 117.83 23.64 2.11 22.05 0.00 138.52 0.00 0.00 0.00 144.27
17 30.670 Octanoic acid, ethyl ester Fatty Acyls 4.18 0.00 19.21 30.30 35.46 4.68 0.00 170.39 83.29 40.55 6.90 0.00 48.70 86.91 38.52 9.40 0.00 65.73 137.07 63.42 112.90
18 31.626 Benzene, 1,3-bis(1,1-dimethylethyl)- Benzene and substituted derivatives 29.61 62.27 31.49 48.19 48.37 23.56 68.51 76.31 75.19 81.71 25.94 64.67 60.74 87.12 62.61 46.70 70.31 73.53 112.68 104.23 39.01
19 32.327 Nonanoic acid, methyl ester Fatty Acyls 13.34 0.00 116.39 155.26 183.59 12.96 0.00 203.80 239.01 189.41 12.22 0.00 141.92 194.69 321.22 20.20 0.00 303.48 280.34 202.30 87.54
20 33.352 Butiric Acid Organic acid 0.78 25.71 0.75 1.51 0.67 0.44 36.59 3.38 4.74 4.53 0.77 52.34 3.45 1.83 3.81 0.00 60.85 0.00 0.24 1.41 180.89
21 34.563 Nonanoic acid, ethyl ester Fatty Acyls 0.00 0.00 10.91 15.18 30.26 0.00 0.00 26.52 19.07 20.41 0.00 0.00 13.10 18.95 16.19 0.00 0.00 17.84 26.25 0.00 101.25
22 35.387 Acetophenone Benzene and substituted derivatives 33.95 0.00 85.37 34.17 98.99 21.14 0.00 108.65 0.00 102.57 32.22 0.00 136.65 0.00 127.21 44.36 0.00 139.74 42.49 116.66 92.89
23 35.572 Benzaldehyde, 4-methyl- Benzene and substituted derivatives 0.00 42.67 0.00 0.00 0.00 0.00 57.85 0.00 0.00 0.00 0.00 88.71 0.00 0.00 0.00 0.00 39.16 0.00 0.00 0.00 219.73
24 35.924 Butanoic acid, 3-methyl- Fatty Acyls 0.00 88.26 0.00 0.00 0.00 0.00 159.96 90.99 82.31 56.95 9.25 135.71 18.34 0.00 57.56 0.00 224.96 0.00 0.00 53.32 132.33
25 37.004 Decanoic acid, methyl ester Fatty Acyls 47.39 30.68 5726.77
5042.27
5764.08 45.75 47.56 6795.2
3 6546.7
5 4911.7
0 45.16 48.88 4594.18
4488.24
8579.37 97.90 58.02 9226.7
4 4938.2
0 5803.1
5 88.51
26 39.951 Decanoic acid, ethyl ester Fatty Acyls 0.00 0.00 332.79 307.44 344.83 0.00 0.00 626.55 338.67 441.59 0.00 0.00 340.84 318.17 343.73 0.00 0.00 399.27 430.54 113.88 93.33
27 40.452 Pentanoic acid Lipids and lipid-like molecules 0.00 0.02 0.00 6.38 0.03 0.00 0.00 0.00 5.59 0.00 0.04 0.00 0.41 6.92 0.00 0.00 0.00 0.06 8.43 0.00 203.12
28 40.987 Methyl 8-methyl-decanoate Fatty Acyls 0.00 0.00 11.81 15.64 25.66 0.00 0.00 19.34 20.84 17.26 0.00 0.00 16.57 23.77 0.00 0.00 0.00 25.69 0.00 24.42 107.67
29 43.242 Undecanoic acid, methyl ester Fatty Acyls 0.00 0.00 161.03 124.58 261.70 0.00 0.00 176.46 153.70 227.89 0.00 0.00 125.06 113.15 415.44 0.00 0.00 253.69 162.73 183.11 99.94
30 45.763 Decanoic acid, propyl ester Fatty Acyls 0.00 0.00 0.00 27.30 13.00 0.00 0.00 33.81 0.00 29.10 0.00 0.00 31.62 33.52 49.66 0.00 0.00 75.48 77.18 0.00 137.16
31 46.644 Hexanoic acid Fatty Acyls 20.53 150.65 0.00 0.00 0.00 25.80 179.41 0.00 0.00 0.00 180.54 206.85 12.97 124.38 279.26 15.82 441.89 35.87 253.72 0.00 130.45
32 46.897 Benzenepropanoic acid, methyl ester Fatty Acyls 0.00 0.00 27.66 25.72 27.00 0.00 0.00 76.60 75.74 47.32 0.00 0.00 117.21 148.41 99.45 33.71 0.00 148.44 171.44 110.48 105.39
33 48.546 Dodecanoic acid, methyl ester Fatty Acyls 114.10 26.53 3150.45
2591.42
3732.87 83.11 26.86 3350.5
5 2475.3
7 3611.2
9 65.66 24.89 2496.96
2137.17
3555.92 135.18 22.67 4175.4
9 2915.9
0 2550.8
8 84.96
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34 50.311 Heptanoic acid Fatty Acyls 0.00 99.11 0.00 0.00 0.00 0.00 23.70 0.00 0.00 17.56 223.48 85.92 0.00 309.19 221.85 80.82 163.19 0.00 748.87 218.46 164.04
35 50.449 Benzenemethanol, 4-methyl- Benzene and substituted derivatives 18.61 26.82 11.01 10.53 19.30 19.45 0.00 17.91 0.00 21.52 14.88 32.50 40.12 38.61 25.68 57.36 0.00 15.87 36.43 0.00 75.47
36 50.589 Dodecanoic acid, ethyl ester Fatty Acyls 11.01 0.00 114.55 106.14 111.59 7.76 0.00 182.77 109.46 145.00 0.00 0.00 96.40 134.26 133.24 8.55 0.00 115.48 158.58 122.06 83.76
37 51.104 Methyl 10-methyl-dodecanoate Fatty Acyls 0.00 0.00 13.35 10.94 20.95 5.12 0.00 18.72 12.89 20.72 0.00 0.00 15.78 15.72 28.45 12.15 0.00 33.37 0.00 23.34 91.70
38 51.372 Phenol Benzene and substituted derivatives
1139.60 314.26 673.60 707.71 813.83 922.3
5 609.44 973.59 884.62 1180.02
1407.16 404.40 1124.6
9 865.91 1289.40
1998.63 1171.46 1348.8
3 1412.7
5 1683.2
8 39.84
39 52.143 Tridecanoic acid, methyl ester Fatty Acyls 8.52 0.00 61.01 53.97 70.22 6.28 0.00 63.93 67.69 62.55 10.92 0.00 55.88 49.30 104.26 9.45 0.00 84.25 64.38 54.01 80.22
40 53.496 Phenol, 4-methyl- Benzene and substituted derivatives 98.09 73.24 86.32 68.48 78.07 103.0
6 66.73 103.76 93.14 103.01 294.09 77.57 136.25 158.60 169.34 422.56 89.16 225.63 453.71 345.76 74.66
41 53.717 Octanoic Acid Fatty Acyls 15.72 34.27 0.00 13.52 7.65 17.19 42.81 21.93 15.73 18.94 42.32 48.39 33.72 25.24 0.00 0.00 66.99 68.98 40.99 40.42 73.76
42 55.739 Methyl tetradecanoate Fatty Acyls 136.93 20.07 927.09 902.18 683.39 96.66 22.51 1015.69
1236.79 883.32 74.96 30.40 1123.4
8 1005.3
5 1483.7
7 141.46 28.80 1287.30
1060.94 886.56 79.18
43 55.989 Methyl myristoleate Fatty Acyls 38.87 5.32 225.70 193.58 324.91 26.14 6.71 217.00 206.21 270.32 17.40 0.00 147.48 96.05 296.95 28.24 0.00 251.75 128.26 123.92 85.41
44 56.328 Nonanoic acid Fatty Acyls 0.00 19.79 0.00 0.00 0.00 0.00 13.81 0.00 0.00 0.00 0.00 22.75 0.00 0.00 18.12 15.61 21.33 20.82 0.00 0.00 142.48
45 58.068 Methyl 13-methyltetradecanoate Fatty Acyls 8.69 0.00 67.33 55.68 0.00 12.27 0.00 67.26 83.30 65.38 10.45 0.00 0.00 60.90 82.14 16.88 0.00 122.52 85.41 37.74 99.07
46 58.489 Pentadecanoic acid, methyl ester Fatty Acyls 13.05 0.00 24.79 24.77 21.90 14.22 0.00 28.54 33.22 72.07 11.88 0.00 42.01 37.73 45.06 14.03 0.00 35.62 33.91 45.13 76.03
47 58.836 n-Decanoic acid Fatty Acyls 0.00 22.88 0.00 0.00 15.29 0.00 28.85 0.00 0.00 0.00 0.00 29.40 42.41 25.29 0.00 0.00 22.53 34.05 0.00 65.96 132.00
48 59.506 Phenol, 2,4-bis(1,1-dimethylethyl) Benzene and substituted derivatives 19.88 19.62 12.77 0.00 14.61 22.92 22.86 19.34 14.05 24.13 19.96 32.69 20.02 30.23 22.25 50.43 20.72 0.00 20.14 21.49 52.10
49 60.928 Indole Indoles and derivatives 573.25 136.14 684.85 563.83 659.64 415.94 113.85 635.20 538.83 664.10 366.17 97.76 483.55 425.13 640.96 446.87 130.70 543.16 471.55 571.74 42.54
50 62.524 9-Hexadecenoic acid, methyl ester, (Z)- Fatty Acyls 43.82 14.74 189.49 25.81 28.16 4.45 0.00 0.00 82.13 40.06 33.83 0.00 0.00 22.14 58.69 44.66 0.00 0.00 36.16 36.69 131.31
51 62.715 Methyl hexadec-9-enoate Fatty Acyls 0.00 0.00 0.00 144.68 181.80 34.73 12.88 101.23 249.57 240.02 0.00 0.00 143.58 100.90 292.95 48.04 24.07 0.00 122.98 142.12 103.18
52 63.095 Hexadecanoic acid, methyl ester Fatty Acyls 41.48 16.76 0.00 90.16 73.66 33.50 16.21 174.68 179.37 135.53 41.36 24.34 239.29 235.56 265.63 48.85 37.08 0.00 194.92 207.15 88.45
53 64.859 Hexadecanoic acid, ethyl ester Fatty Acyls 0.00 0.00 0.00 12.83 39.24 0.00 0.00 45.36 40.64 36.12 0.00 0.00 49.92 54.66 40.84 0.00 0.00 45.75 35.07 46.10 99.33
54 67.496 9-Octadecenoic acid, methyl ester, (E)- Fatty Acyls 366.22 0.00 560.31 320.40 399.65 184.0
6 161.29 552.22 706.60 717.44 290.24 0.00 782.84 480.17 952.85 219.72 185.38 578.32 470.42 590.14 61.02
55 68.442 Ethyl Oleate Fatty Acyls 0.00 0.00 102.09 0.00 98.02 0.00 0.00 131.61 169.89 105.33 0.00 0.00 135.80 0.00 159.67 0.00 0.00 0.00 253.77 205.64 123.89
ID; Identification, RT; retentiton time, VC; Variation coefficient
Capítulo 7. RESULTADOS Y DISCUSIÓN
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7.3 Fermentación colónica humana in vitro de la fracción indigestible aislada de menús
consumidos durante la comida: Impacto en el perfil de los metabolitos intestinales
Resumen
El tracto intestinal humano es el hogar de un ecosistema diverso y esencial de bacterias, conocidas
como micrbiota intestinal. Hoy en día, las interacciones huésped-microbiota se consideran un
componente fundamental subyacente a la salud y la enfermedad [1]. Así, la microbiota intestinal
genera una amplia gama de compuestos bioactivos únicos que son el resultado de la
transformación bacteriana de componentes dietéticos indigestibles. Algunos metabolitos
bacterianos (ácidos grasos de cadena corta, AGCC) desempeñan un papel vital en el
mantenimiento de la inmunidad del huésped y la homeostasis metabólica [2]. Sin embargo, los
metabolitos asociados a la disbiosis (trimetilamina, TMA), se consideran factores causales en las
enfermedades cardiovasculares [3]. La espectrometría de masas se ha convertido en una poderosa
herramienta para estudios de perfil metabólico debido a su amplio rango dinámico, capacidades
cuantitativas reproducibles y su capacidad para analizar muestras de complejidad molecular
significativa [4]. La formación de metabolitos microbianos está fuertemente influenciada por la
composición intestinal y la ingesta dietética, en particular de los carbohidratos, proteínas y grasas
dietéticas no digeribles [6]. Las enfermedades inflamatorias relacionadas con la dieta se han
convertido en serios problemas de salud pública para la sociedad occidental, incluyendo a México.
Las alteraciones en la dieta han dado lugar a un cambio en la microbiota del intestino que se ha
demostrado desempeña un papel importante en trastornos complejos como las enfermedades
inflamatorias intestinales [9]. Hipoteticamente pensamos que los metabolitos de bajo peso
molecular producidos por la microbiota intestinal influyen en las actividades biológicas en el colón
y que a su vez dependen de los componentes no digeribles de los alimentos consumidos durante
las comidas. Por lo tanto, en el presente capítulo se investigó el papel del perfil de metabolitos
microbianos sobre la actividad antioxidante y los valores de pH de durante la fermentación in vitro
de la fracción indigestible aislada de alimentos consumidos por escolares durante la comida
Capítulo 7. RESULTADOS Y DISCUSIÓN
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In vitro human colonic fermentation of indigestible fraction isolated from lunch menus:
Impact on the gut metabolites and antioxidant capacity
Abstract
Scope:
We tested the hypothesis that metabolites produced by intestinal microbiota are influenced by
indigestible fraction (IF) isolated from three lunch menus: Modified Mexican Lunch (MM-L),
Traditional Mexican Lunch (TM-L) and Alternative Mexican Lunch (AM-L).
Methods and results:
IF-Lunch menus were isolated after withstanding in vitro gastrointestinal digestion and total
soluble polyphenols (TSP), condensed tannins (CT) hydrolysable tannins and antioxidant capacity
(AOX: DPPH and FRAP) were evaluated. AOX, pH and bacterial metabolites profile changes
were monitored during in vitro colonic fermentation. Differences between lunch menus were
found for IF, TSP, CT and FRAP values (p<0.05). TM-L had the highest TSP and CT content
(0.84 and 1.89 g/100g db). TM-L and AM-L showed the highest FRAP activity. Changes on pH
and AOX during fermentation were time and substrate dependent (p<0.05). Butyric acid levels
showed no significant differences between lunch menus and blank (p>0.05). Besides, 57 volatile
compounds were detected by SPME-GC-MS. Trimethylamine, phenol and indole were associated
with increased pH and decreased AOX.
Conclusion:
Our study represents a new contribution on the potential effects of food habits on bacterial
metabolites production. Only through food complex matrix study will we improve our
understanding of the effects of diet on colon health promotion.
Capítulo 7. RESULTADOS Y DISCUSIÓN
93
Keywords:
Lunch Menus, Indigestible fraction, Antioxidant capacity, Gut metabolites.
Graphical abstract
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12-Blank
12-Raffinose
12-MM-L
12-TM-L
12-AM-L
24-Blank
24-Raffinose
24-MM-L
24-TM-L
24-AM-L
48-Blank
48-Raffinose
48-MM-L
48-TM-L
48-AM-L
72-Blank
72-Raffinose
72-MM-L
72-TM-L
72-AM-L
-10 -8 -6 -4 -2 0 2 4 6 8 10Metabolic profile A
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12-Blank
12-Raffinose
12-MM-L
12-TM-L
12-AM-L
24-Blank
24-Raffinose
24-MM-L
24-TM-L
24-AM-L
48-Blank
48-Raffinose
48-MM-L
48-TM-L
48-AM-L
72-Blank
72-Raffinose
72-MM-L
72-TM-L
72-AM-L
Dietary Lunch indigestible fraction - Gut microbiota
Bacterial metbolites profile and AOX
Capítulo 7. RESULTADOS Y DISCUSIÓN
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1 Introduction
The human intestinal tract is home to a diverse and essential ecosystem of microbes. Commensal
microorganisms establish the intestinal metabolic system by expressing genes that outnumber ours
by more than 100-fold [1] Thus, gut microbiota generates a wide range of unique bioactive
compounds which are the result of bacterial transformation of indigestible dietary components.
Some bacterial metabolites (short-chain fatty acids, SCFA) play a vital role in maintenance of host
immunity and metabolic homeostasis [2]. However, dysbiosis-associated metabolites (e.g.
trimethylamine), are considered causative factors in cardiovascular diseases [3]. The presence of
these compounds, retained in the non-solid phase of faeces that has led to the use of faecal water to
determine the potentially toxic environment to which the lumen is exposed [4, 5]. Mass
spectrometry has become a powerful tool for metabolic profile studies due to its wide dynamic
range, reproducible quantitative capabilities and its ability to analyze samples of significant
molecular complexity [6]. Data analysis involves the mining of high-density spectral data
generated from biofluids such as blood plasma or fecal water measured against a set of reference
biochemical signatures using computer pattern recognition algorithms [7]. Bacterial metabolite
formation is strongly influenced by gut composition and dietary intake [8]. Diet-related
inflammatory conditions such as obesity, type 2 diabetes, cardiovascular disease, chronic kidney
disease and autoimmune diabetes have become in serious public health problems for Western
society, including Mexico [9]. The modern western diet is one environmental factor that has
changed with increased overall caloric intake, and changes in the relative amounts of dietary
components [10]. These dietary alterations have resulted in a shift in the composite gut microbiota,
from both a structure and function perspective, that have been shown to play a role in complex
disorders such as inflammatory bowel diseases [11]. We tested the hypothesis that low molecular
weight metabolites produced by intestinal microbiota is influenced by the indigestible components
of whole foods consumed at Mexican lunch. Additionally, we investigated the role of microbial
metabolites profile on antioxidant activity and pH values of extracts at different fermentation
times.
2. Material and methods
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2.1 Lunch food preparation
Data sources on dietary patterns and food frequency consumption at lunch were obtained from a
nutritional survey carried out on eleven public schools in a Western city of Mexico (Tepic, Nayarit
State). Frequently consumed foods in each DP were used to create three lunch menus. Lunch
menus were named and comprised by the following foods; Modified Mexican Lunch (MM-L):
Soda (350 mL), three corn tortillas (90 g), refried beans (60 g) and roast beef (96 g). Traditional
Mexican Lunch (TM-L): three corn tortillas (90 g), refried beans (86 g), water (350 mL), roast
beef (96 g) and chambray onion (18 g). Alternative Mexican Lunch (AM-L): three fish ceviche
tostadas and soda (300 mL). Individual foods were purchased in a local supermarket and were
prepared in the laboratory kitchen according to traditional customs of the region. After prepared,
each menu, it was homogenized in a food processor (NB-101B, Nutribullet, China), frozen (-80
°C), freeze-dried (FreeZone 6, Labconco, USA), grounded, sieved with a mesh size of 500 µm,
and stored at -20 °C until analysis. Each menu preparation was performed by triplicate. Moisture
content of complete lunch menus, was analyzed according to AOAC [12] 925.10 methods.
2.2 Quantification and isolation of indigestible fraction (IF) in menus
A digestion procedure that mimicking the physiological situation in the upper tract (stomach and
small intestine) was utilized for indigestible fraction (IF) quantification [13]. In order to increase
the amount of isolated IF the follow amendments were realized. Briefly, 9 g of freeze-dried
samples was incubated with: a) pepsin (0.6 mL of a 300 mg/mL solution in 0.2 M HCl-KCl buffer,
pH 1.5, 40 °C, 1 h, P-7000, Sigma Aldrich, USA), b) pancreatin (3 mL of a 5 mg/ mL solution in
0.1 M phosphate buffer; pH 7.5, 37 °C, 6 h, P-1750, Sigma Aldrich, USA), c) a-amylase (3 mL of
a 120 mg/mL solution in 0.1 M Tris-maleate buffer, pH 6.9, 37 °C, 16 h, A-3176, Sigma Aldrich)
and amyloglucosidase 300 µL (pH 4.5, 60 °C 45 min, A-9913, Sigma Aldrich). Sample were
transferred into dialysis tubes (D9527 -30.48 m avg. Flat width 43 mm, 14000 Da, Sigma Aldrich)
and dialyzed against water for 48 h at 25 °C to remove digested compounds [14]. Total IF
(Dialysis retained) was collected, freeze-dried, milled (IKA M20, USA) and sieved using a 500-
micron mesh, and placed in bags with seal, for storage at -20 ° C until subsequent analyses.
2.2.3 Antioxidant compounds analysis in the total indigestible fraction
Capítulo 7. RESULTADOS Y DISCUSIÓN
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Samples (250 mg) of total IF isolated from lunch menus were extracted by aqueous-organic
solution [15]. Total soluble polyphenols (TSP) in extractable fraction were determined with the
Folin–Ciocalteu’s reagent [16] using a 96-well microplate reader (Biotek, Synergy HT, Winooski
VT, USA) with Gen5 software, and the results were expressed in g of gallic acid equivalents (g
GAE/ 100g IF). The residues of the extraction were treated for non-extractable polyphenols
quantification (Condensed tannins and hydrolyzable polyphenols). Condensed tannins (CT) were
quantified and results were expressed as CT equivalents/ 100 g IF, using a carob pod (Ceratonia
siliqua) proanthocyanidin standard [17]. Hydrolyzable polyphenols (HP) were evaluated by a
previously reported methodology [18] and results were expressed as g GAE/100 g IF.
2.2.4 Antioxidant capacity (AOX) analyses
AOX was evaluated on the extractable fraction obtained before, and methodologies were slightly
modified to a microplate reader (Biotek, Synergy HT, Winooski VT, USA) with Gen5 software.
1,1-Diphenyl-2-picryl hydrazyl (DPPH) antiradical activity assay was evaluated [19] and results
were expressed as Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic) equivalent (TE;
mmol /g IF db). Ferric reducing antioxidant power (FRAP) assay was performed [20]. Results are
expressed as mmol of Trolox equivalents (TE) /g IF db.
2.3 In vitro colonic fermentation by human microflora
Total IF isolated from three lunch menus were fermented in a batch culture system with pre-
conditioned nutritive medium and under strict anaerobic conditions at 37 °C [21]. In parallel, two
different controls were conducted under the applied conditions: a) Raffinose was incubated in
medium with faeces inoculum as a fermentable sugar reference, and b) the faecal suspension was
incubated without substrate as a negative control. All incubations were performed in triplicate and
samples and controls were collected at 12, 24, 48 and 72 h. The tubes obtained at each time of
fermentation were centrifuged (Hermle Z 323 K; Wehingen, Germany) (3500 × g ,15 min, 4 °C).
Supernatants were divided into two parts; metabolite profile analysis and antioxidant capacity
assays and were immediately stored at −80 °C until analysis.
2.4 Metabolites characterization by HS-SPME- GC/MS
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Supernatants (500 mg) were placed into a 20 mL vial sealed with a magnetic cap with a poly-tetra-
fluoro-ethylene (PTFE)/silicon septum. HS-SPME method was used for the extraction and
concentration of volatile compounds [22]; incubation with polydimethylsiloxane-divinylbenzene-
carboxen “PDMS/DVB/CAR” fiber for 120 min, 250 rpm at 45 °C; the fiber was inserted into the
injection port of the GC system for thermal desorption (240 °C for 10 min) for GC/MS analysis.
The extractions were performed in triplicate. Five runs with twelve vials / run were performed and
the tests order was completely randomized. The volatile constituents were analyzed with an
Agilent 5975C VL mass selective detector coupled to an Agilent 7890A gas chromatograph
(Agilent Technologies, Inc., Santa Clara, CA), equipped with a DB-5MS capillary column (60 m
X 250 µm X 0.25 µm; Agilent). Sample quantification was obtained by means of acetic, propionic
and butyric acid standard curves. Tentative identification of the volatile components was done
comparing the mass spectra of the samples with the data system library MSD ChemStation
software (Agilent G1701EAversion E.02.00.493). Relative concentration of all fermentation
metabolites versus acetic acid as internal standard was calculated and the results were expressed in
mmol L-1.
Antioxidant capacity (AOS) during in vitro colonic fermentation
Changes in the AOX of the sample extracts during in vitro colonic fermentation at 12, 24 and 48 h
were measured by the methods described above.
2.5 Statistical analysis
Results are expressed as mean ± SEM (n=3). Univariate testing of differences for lunch menus
was processed by ANOVA/Fisher's Least Significant Difference test. Besides, Multivariate
analysis was applied for metabolites profile interpretation. Principal component analysis (PCA)
was used to determine patterns in relative metabolites production. Finally, Pearson´s correlation
coefficients were used to evaluate the relationship between individual metabolites and changes in
pH and AOX during in vitro colonic fermentation. All analyzes were performed using
STATISTICA software, version 10.0 (StatSoft. Inc. 1984–2007, Tulsa, OK, USA). A significance
level of α = 0.05 was used in data analysis.
Capítulo 7. RESULTADOS Y DISCUSIÓN
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3. Results
3.1 Content and nutritional composition of indigestible fraction (IF) in lunch menus
The results shown in Table 1 indicate that the moisture content in TM-L and AM-L were higher
than MM-L (p<0.05), which follows that in these food mixtures are less solid components
available for gastrointestinal digestion. The increase in moisture content in TM-L could be
attributed to solubilisation of components by water addition and consequently, as a dilution effect
[23]. Meanwhile, AM-L was prepared with raw fish and raw vegetables that could increase the
amount of water and decrease the amount of soluble solids by the same dilution effect. On the
other hand, total IF content is the fraction resistant (comprising dietary fiber, resistant starch,
resistant protein, resistant lipids, certain polyphenols, and other associated compounds) to
gastrointestinal digestion and serves as substrate for the gut microbiota [13]. In this parameters,
significant differences (p<0.05) between in MM-L (5.35 g/100g wb) and TM-L (5.46 g/100g wb)
vs AM-L (2.39 g/100g wb) were found (Table 1). The main sources of dietary fiber are cereals,
legumes such as beans and lentils, vegetables, and fruits that are considered to form an important
part of a balanced healthy diet [24]. Insoluble IF was the main fraction of total IF, since it
represented from 89% in MM-L to 92% in TM-L (Table 1). Disruption of the natural matrix or
types of microstructural changes in lunch menus imparted by processing and during food ingestion
could be relevant in matrix-nutrient interactions, and their effect on composition and content of
indigestible fraction [25]. The soluble IF constituted 42% in AM-L (0.99g/100g wb) and these
values are the highest found for the three lunch menus (p<0.05). Soluble IF is associated with
viscous polysaccharides such as pectin and gums, which decrease assimilation of nutrients in
particular has been observed that these components have a postprandial glucose-lowering effect
and promote changes in the gut microbiota composition [26, 27].
3.2 Antioxidant compounds in the indigestible fraction
At present, there is scarce literature on phenolic compounds associated with indigestible fraction.
Because of the role of phenolic compounds in food complex matrix, total soluble polyphenols
(TSP), hydrolysable tannins (HT), and condensed tannins (CT) were studied in IF of lunch menus
Capítulo 7. RESULTADOS Y DISCUSIÓN
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and the results are shown in Table 1. The HT were the most abundant phenolic compounds in all
samples followed by CT and TSP.
Capítulo 7. RESULTADOS Y DISCUSIÓN
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Table 1 Chemical composition and antioxidant compounds content, and antioxidant capacity in
the indigestible fraction (IF) isolated from lunch menus1
Parameter Menus2
MM-L TM-L AM-L
Moisture content (g/100g menu )
IF (g/100g menu wb) 74.30 ± 0.39a 81.74 ± 0.13b 82.90 ± 0.14c
Insoluble IF 4.79 ± 0.29a 5.07 ± 0.38a 1.39 ± 0.12b
Soluble IF 0.56 ± 0.03a 0.39 ± 0.04a 0.99 ± 0.09b
Total IF 3 5.35 ± 0.32a 5.46 ± 0.42a 2.39 ± 0.20b
IF Antioxidant Compounds (g/ 100g IF db)
Total soluble polyphenols 0.75 ± 0.01a 0.84 ± 0.01b 0.68 ± 0.01c
Condensed Tannins 1.41 ± 0.06a 1.89 ± 0.05b 0.70 ± 0.02c
Hydrolyzable polyphenols 2.24 ± 0.13a 1.99 ± 0.05a 2.18 ± 0.00a
IF Antioxidant Capacity (mmol TE/g IF db)
DPPH ND ND ND
FRAP 8.05 ± 0.25a 10.83 ± 0.24b 10.28 ± 0.12b 1Values are mean ± standard error (n = 3); Wet basis (wb); Dry basis (db); No Detected (ND).
Means in rows marked with different letters indicate significant difference (p <0.05). 2 Menus;
MM-L: Modified Mexican Lunch, TM-L: Traditional Mexican Lunch, AM-L: Alternative
Mexican Lunch. 3 Total IF = Sum of soluble IF + insoluble IF.
The TSP, expressed as gallic acid equivalent, ranged from 0.68 to 0.84 g/100g sample db, with
statistically significant differences (p<0.05) between all the samples. TM-L showed the highest
TPC followed by MM-L and AM-L. The amount of beans in TM-L is 43% higher than in MM-L,
so this legume could be responsible for the observed differences. Hydroxybenzoic compounds
(protocatechuic acid, p-Hydroxybenzoic aldehyde and p-Hydroxybenzoic acid) were the most
abundant phenolic class detected (approximately 90% of TSP) in dietary fiber of cooked beans
Capítulo 7. RESULTADOS Y DISCUSIÓN
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[28]. With reference to CT, the highest (p<0.05) content was found in TM-L (1.89g/100g db),
followed by MM-L (1.41 g/100g db). In AM-L, CT (proanthocyanidins) were not detected. Fruits
have been found to be major sources of proanthocyanidins in the diet, besides of legumes and
cereals such as sorghum and barley, but they are not detectable in corn [29] and Mexican
vegetables[30]. Hydrolyzable polyphenols were a quantitatively important fraction of polyphenols
in all samples (approximately 50% for MM-L and TM-L and up to 75% in the case of the AM-L).
Quercetin has been reported as the individual constituent of HP identified in vegetables as onion
and cucumber consumed in Europe [31] and which were also used in AM-L preparation. In the
colon, the proanthocyanidins are catabolized by the gut microflora into a series of beneficial
metabolites such as phenyl valerolactone, phenylacetic and phenylpropionic acids [32].
Meanwhile, HT are metabolized to gallic acid, pyrogallol, phloroglucinol and finally to acetate and
butyrate via several bacterial enzymes [33] TSP linked to the IF were released in the aqueous-
organic extraction and it showed FRAP activity but DPPH activity was not observed (Table 1).
FRAP activity was higher in IF extracts isolated for TM-L and AM-L (10.83 and 10.28 mmol
TE/g IF db) and lower in MM (8.05 mmol TE/g IF db). These results suggest that antioxidant
mechanism in extracts can be as chelating agent and not as a free radical neutralizer [34]. In this
sense, polyphenols present in a mixture can interact with indigestible components (proteins, lipids
and carbohydrates), and these interactions can affect the total antioxidant capacity of extracts [35].
3.3 Changes in pH and antioxidant capacity from in vitro colonic fermentation extracts
The progress of the fermentation was followed by measuring pH changes in each sample for 72 h
(Figure 1). Raffinose control showed a significant drop (p < 0.05) of at least 3 units in their pH
values after 72 h, which is most probably caused by the fermentation end products (mainly SCFA).
After fermentation, TM-L showed a decrease in pH value (p<0.05), but the magnitude of the
reduction in pH value was markedly different depending on fermentation time. The highest pH
difference (pHto – pHt) was 0.29 unit found at 48 h. A significant increase in pH values during
MM-L and AM-L fermentation (similar to control without substrate) was observed. Differential in
pH between 0 and 72 h of fermentation was -0.11 for MM-L and -0,05 for AM-L menu. The pH of
the colon is potentially a key factor in determining the species composition and metabolic outputs
of the human intestinal.
Capítulo 7. RESULTADOS Y DISCUSIÓN
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Figure 1 Changes in a) pH kinetic plot, b) DPPH Antiradical activity plot and c) FRAP chelating activity plot in the extracts
during in vitro colonic fermentation from (–) blank, (··♦··) raffinose, and indigestible fraction isolated from lunch menus: (-■-)
Modified Mexican Lunch, MM-L, (-▲-) Traditional Mexican Lunch, TM-L, and (-●-) Alternative Mexican Lunch, AM-L at
different at different fermentation times, Values are means ± SEM (n=3). *Significant difference using Two-way
ANOVA/Fisher's LSD test (Samples ×Time interaction, p<0.05). For DPPH and FRAP, the blank was subtracted of the
samples.
Fermentation Time (h)
0 12 24 48 72
DP
PH
Ant
ioxi
dant
Act
ivity
(mM
TE
/g s
ubst
rate
db)
0
50
100
150
200
Fermentation time (h)
0 12 24 48 72
FR
AP
Ant
ioxi
dant
Act
ivity
(m
M T
E /g
sub
stra
te d
b)
0
5
10
15
20
25
Fermentation time (h)
0 12 24 48 72
pH
3.0
4.0
5.0
6.0
7.0
8.0**** **** ****
Capítulo 7. RESULTADOS Y DISCUSIÓN
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Bacteroides spp. are generally less able than many dominant Firmicutes to tolerate the presence of
short chain fatty acids at pH 5.5 limiting propionate formation and enhancing butyrate production
by the community at pH values around 5.5 compared with 6.5-6.8[36]. For bacteria that use the
butyryl-CoA:acetate-CoA-transferase route (Roseburia and Faecalibacterium prausnitzii), acetate
consumption and butyrate production are reported to increase at mildly acidic pH (5.5) compared
with near neutral pH (6.7) [37]. On the other hand, AOX evaluated by DPPH and FRAP assays
(Figure 1b and c), showed time dependent and significant differences (p <0.05) between samples.
It is noteworthy that DPPH activity (less than 35 mmol TE/g IF) was detected in fermentation
extracts at 72 h but not DPPH activity was found in aqueous-organic extracts (Table 1). In FRAP,
AM-L showed higher values (7.47 mmol TE/g IF DW) respect to MM-L (4.68 mmol TE/g IF db),
TM-L (2.43 mmol TE/g IF DW) and even that positive control (4.25 mmol TE/g IF DW) at 72 h.
Conversely, TM presented the lowest values for this AOX activity during all fermentation times.
Differences between the AOX values found by the two methods evaluated suggest that the
antioxidant mechanism of the gut metabolites is mainly as free radical neutralizers and secondarily
as chelating agents. This antioxidant mechanism was also observed in natural powdered
seasonings extracts after gastrointestinal and colonic digestion. The authors concluded that gut
metabolites present greater high free radical scavenging ability but not with significant reducing
properties [38]. . Simple soluble compounds as phenylacetic, phenylpropionic, benzoic acid
derivatives and branched-chain fatty acids (isobutyrate, 2-methylbutyrate and isovalerate) with
potential antioxidant properties can thereby be generated, mainly by amino acids and phenolic gut
metabolism [34, 39]. The combination of ascorbic acid (dietary antioxidant) with the colonic
microbiota metabolite 3,4-dihydroxyphenylacetic acid showed a potent synergic antioxidant effect
[40]. Also, 10-hydroxy-cis-12-octadecenoic acid (gut microbial metabolite of dietary linoleic acid
with immunomodulatory activity) has been associated with improved antioxidant/detoxifying
effects in a murine intestinal epithelial cell line (MODE-K)[41]
3.4 Production of SCFA
The concentrations of SCFA at 12, 24 48 and 72 h of fermentation are present in Table 2. Acetic
propionic and butyric acid concentrations in lunch menus were significantly lower than raffinose
(p<0.05). Acetate was the most abundant SCFA produced by all samples.
7. RESULTADOS Y DISCUSIÓN: CAPÍTULO 4
104
Table 2. Short-chain fatty acids (SCFAs, mmol L-1) production at 12, 24, 48 and 72 h of in vitro fermentation of blank,
raffinose and indigestible fraction isolated from lunch menus (MM-L; Modified Mexican Lunch, TM-L; Traditional Mexican
Lunch, AM-L; Alternative Mexican Lunch) 1.
SCFA/ fermentation time
Blank Raffinose
IF- Lunch Menus MM-M TM-M AM-M
Acetic acid
12 h 3.88 ± 0.93a 198.28 ± 10.05b 16.96 ± 1.28a 32.25 ± 1.54a 8.55 ± 0.26a 24 h ND 224.34 ± 3.94b 10.17 ± 0.52a 33.12 ± 2.59a 22.14 ± 3.60a 48 h 3.40 ± 0.02a 909.71 ± 81.27b 36.95 ± 1.93a 13.95 ± 1.41a 39.42 ± 8.46a
72 h 7.67 ± 1.94a 237.79 ± 8.35c 68.12 ± 8.85b 16.28 ±
2.92ab 20.84 ± 1.00ab
Propionic acid 12 h ND 30.02 ± 0.19c 3.75 ± 0.49a 7.35 ± 0.58b 2.38 ± 0.25a 24 h ND 23.78 ± 1.33c 5.18 ± 0.22a 17.44 ± 0.16b 16.30 ± 0.57b 48 h 1.85 ± 0.12a 82.95 ± 1.86d 19.72 ± 2.11c 6.82 ± 0.72b 17.35 ± 0.77c 72 h ND 28.53 ± 2.33c 12.91 ± 1.24b 4.74 ± 1.22a 5.29 ± 1.19a Butyric acid
12 h 0.60 ± 0.08a 36.22 ± 8.44b 1.08 ± 0.16a 1.97 ± 0.28a 1.32 ± 0.26a 24 h 0.39 ± 0.04a 17.80 ± 1.74b 2.81 ± 0.32a 5.14 ± 0.87a 1.99 ± 0.34a 48 h 0.92 ± 0.19a 47.99 ± 4.45b 2.45 ± 0.44a 1.35 ± 0.20a 3.42 ± 0.33a 72 h 1.13 ± 0.23a 19.36 ± 1.34b 1.82 ± 0.16a 1.46 ± 0.19a 1.41 ± 0.23a
*The values are reported in mmol/L produced per 100 mg substrate as mean ± SEM of three replicate; Different lowercase
letters indicate significant differences in rows among substrates for a time (p < 0.05).
7. RESULTADOS Y DISCUSIÓN: CAPÍTULO 4
105
IF lunch menus vs blank were not significantly different (p > 0.05) throughout the fermentation
assay at 12, 24 and 48 h. At 72 h, MM-L produced the greatest acetic acid concentration (68.12
mmol/L), and was statistically different (p < 0.05) from blank (Without substrate). Propionic acid
concentrations were fermentation time and substrate dependent (p > 0.05). TM-L produced the
highest propionate concentration after 12 h of fermentation and was statistically different (p <
0.05) from the other samples MM-L and AM-L. Propionate production remained low for MM-L
between 6-12 h bu an incresead at 48 and 72 h was observed (19.72 and 12.91 mmol/L
respectively). For AM-L the times with greater production of propionic acid were at 24 and 48 h
but it was not significantly different (p>0.05) from TM-L and AMD respectively. In Butyric acid
levels showed no significant differences between lunch menus and values were similar to blank.
The low production of AGCC in lunch menus (Mainly made with food of animal origin, meat and
fish.) suggests that the isolated fractions could have a low indigestible carbohydrate content and a
high indigestible protein and lipids content. This could have negative implications on the health of
individuals. The fermentation of proteinaceous substrates have been considered potentially
detrimental for host health and have been implicated in development of noncommunicable
diseases [42, 43] and the pathogenesis of large intestinal diseases [44]. Based on greater intake
levels of fiber through fruit and vegetables, vegan and vegetarian diets were associated with higher
production levels of SCFA, whereas the animal-based diet supported much lower levels [45]. Our
results highlight the importance of low levels of dietary fiber in lunch menus frequently consumed
by schoolchildren Mexican population. A recent study show that fiber intake is low and added
sugar and saturated fat intakes are higher than recommended for >50% of the Mexican population
aged ≥1 year [46].
3.5 Production of volatile gut metabolites
The kinetic analysis by GC-MS/SPME of the volatile metabolites produced by fecal microbiota at
the time points 12, 24, 48 and 72 h showed different metabolic profiles in relation to the different
substrates utilized (See Supporting Information Table SI). A total of 57 different metabolites
belonging to the families of amines, alcohol and polyols, alkyl halides, fatty acid ester, benzene
and substituted derivatives, organic disulfides, carbonyl compounds, organic trisulfides, pyridines
and derivatives, organic acid, alkanes, naphthalenes, benzothiazoles, indoles and derivatives and
7. RESULTADOS Y DISCUSIÓN: CAPÍTULO 4
106
SCFA were detected (Figure 2). No changes were observed in the metabolites profile of raffinose
at 12 and 72 h, which indicates that fermentation would occur over a longer portion of the colon.
Organic acids and SCFA were the most abundant compounds found in this sample. Nevertheless,
is noticeable, a decrease of organic trisulfides, indoles and derivatives and an increase in the
production of fatty acid esters and organic acid for lunch menus at 72 h. Until now, few reports on
medium chain fatty acids (MCFAs) related to colonic health have been published. Interestingly,
the MCFAs (pentanoic, hexanoic, heptanoic, octanoic and nonanoic acid were identified as the
most discriminatory metabolites between healthy controls and patients with inflammatory bowel
disease [47]. MCFAs were shown to activate the peroxisome proliferator activated receptor
(PPAR)-γ protein that regulates fatty acid storage and glucose metabolism [48]. Fatty acid esters
were the principal metabolites during in vitro colonic fermentation of the samples. These
compounds were identified in patients who presented with chronic diseases of the gastrointestinal
tract, especially in Crohn's disease [49].
Figure 2 Cumulative concentration (%) of volatile gut metabolites by chemical groups between
the in vitro colonic fermentation extracts of blank, rafinose and indigestible fraction isolated from
Modified Mexican Lunch (MM-L), Traditional Mexican Lunch (TM-L), and Alternative Mexican
Lunch (AM-L) at 12 and 72 h. Detailed of identification number for each volatile compound are
outlined in the Supporting Information Table SI.
12 hours of fermentation
Blank Raffinose MM-D TM-D AM-D
Cum
ula
tive
conce
ntr
atio
n (
%)
0
20
40
60
80
100
72 hours of fermentation
Blank Raffinose MM-D TM-D AM-D
Cum
ulat
ive
conc
entr
atio
n (%
)
0
20
40
60
80
100
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Amines Alcohols and polyols Alkyl halides Fatty acid ester Benzene and substituted derivatives Organic disulfides Carbonyl compounds Organic trisulfides Pyridines and derivatives Organic acid Alkanes Naphthalenes Benzothiazoles Indoles and derivatives SCFA
Capítulo 7. RESULTADOS Y DISCUSIÓN
107
Secondly, a statistical descriptive approach (PCA) showed that the fermentation of different corn
products for 12, 24, 48 and 72 h clearly affects the fecal metabolic fingerprint (Figure 3). The first
two principal components (PC) explained above 50% of the variation in the data (Figure 3a).
These components separate the volatiles with potentially healthy effects presenting positive
Eigenvectors in PC1 and in PC2 (eg, acetic, propionic, butyric acid, organic acids and) from those
volatiles with potentially Harmful effects presenting negative Eigenvectors on PC1 and on PC2
(e.g. phenol, phenol, 4-methyl-, trimethylamine, dimethyl disulfide, indole and fatty acid esters).
In fact, a good separation was observed for raffinose and blank at all fermentation times (Figure
3b). In lunch menus, it was notable that short fermentation times (12 and 24 h) promote a
metabolic profile similar to blank. However, long fermentation times (48 and 72 h) the samples
followed the metabolic profile B (with potentially healthy effects), specifically in MM-L and TM-
L at 72 h. For its part, raffinose was the substrate with the healthiest metabolites profile
particularly at 48 h of fermentation. In this work, the importance of improving diets with high
indigestible carbohydrates content was manifested in gut metabolic profile produced during in
vitro colonic fermentation.
Figure 3 Principal components plots (a) metabolites production “PC Loadings” (b) and sample
classification “PC scores” (%) during in vitro fermentation from indigestible fraction isolated
lunch menus (MM-L; Modified Mexican Lunch, TM-L; Traditional Mexican Lunch, AM-L;
1
23
4
5
67
8
910
11
121314
15
16
1718
19
2021
22
23
2425 26
27
28
29
30
31
32
3334
35
36
3738
39
40
4142
43
44
45
4647
48
49
50
51
52
5354
555657
-1.0 -0.5 0.0 0.5 1.0Metabolic profile A
Explained Variance: 34.81%
-1.0
-0.5
0.0
0.5
1.0
Met
abol
ic p
rofil
e B
Expl
aine
d V
aria
nce:
17.
18%
1
23
4
5
67
8
910
11
121314
15
16
1718
19
2021
22
23
2425 26
27
28
29
30
31
32
3334
35
36
3738
39
40
4142
43
44
45
4647
48
49
50
51
52
5354
555657
12-Blank
12-Raffinose
12-MM-L
12-TM-L
12-AM-L
24-Blank
24-Raffinose
24-MM-L
24-TM-L
24-AM-L
48-Blank
48-Raffinose
48-MM-L
48-TM-L
48-AM-L
72-Blank
72-Raffinose
72-MM-L
72-TM-L
72-AM-L
-10 -8 -6 -4 -2 0 2 4 6 8 10Metabolic profile A
-4
-2
0
2
4
6
Met
abol
ic p
rofil
e B
12-Blank
12-Raffinose
12-MM-L
12-TM-L
12-AM-L
24-Blank
24-Raffinose
24-MM-L
24-TM-L
24-AM-L
48-Blank
48-Raffinose
48-MM-L
48-TM-L
48-AM-L
72-Blank
72-Raffinose
72-MM-L
72-TM-L
72-AM-L
Capítulo 7. RESULTADOS Y DISCUSIÓN
108
Alternative Mexican Lunch). Detailed of identification number for each volatile compound are
outlined in the Table SI
Capítulo 7. RESULTADOS Y DISCUSIÓN
109
3.6 Correlation between gut metabolites, pH and antioxidant activity
To the best of our knowledge, this is first time to report the relationship between metabolic profile
and biological activity (pH and AOX) during the in vitro colonic fermentation of indigestible
fraction isolated from lunch menus frequently consumed by a schoolchildren Mexican population.
The pH values during in vitro colonic fermentation was positive correlated (p<0.05) with
trimethylamine, pyridine 2,4,6-trimethyl-, benzene 1,2-dichloro-, acetophenone, dodecanoic acid
ethyl ester, phenol, phenol 4-methyl-, indole and 1H-Indole 3-methyl- and negative correlated for
acetic acid, nonanal, propionic acid, benzene, 1,3-bis(1,1-dimethylethyl)-, butyric acid, butanoic
acid 3-methyl-, pentanoic acid, hexanoic acid and benzothiazole (Figure 4). A significant positive
correlations were found between DPPH antiradical activity and SCFA, butanoic acid 3-methyl-,
pentanoic acid, acetophenone, hexanoic acid and negative correlation was found for
trimethylamine, dimethyl trisulfide, dodecanoic acid ethyl ester, phenol, phenol 4-methyl-,
pentadecanoic acid methyl ester, indole and 1H-Indole 3-methyl-. Interestingly, SCFA, nonanal,
butanoic acid 3-methyl- and organic acid carried significant positive correlations with FRAP
chelating activity and fatty acid esters were negatively correlated with this activity. Some
metabolites with negative effects in pH and AOX found in this work (e.g. trimethylamine as
precursor of trimethylamine N-oxide and indole as precursor of indoxyl sulfate) have been
associated with the development of atherosclerosis and chronic kidney disease [50, 51].
Capítulo 7. RESULTADOS Y DISCUSIÓN
110
Figure 4. Pearson’s R correlations between gut microbial metabolites, pH values and antioxidant
capacity (DPPH antiradical activity and FRAP chelating activity) in extracts obtained during in
Capítulo 7. RESULTADOS Y DISCUSIÓN
111
vitro colonic fermentation of indigestible fraction isolated from lunch menus (▲Increase,
▼decrease and ▬ non-correlation, p<0.05).
5. Conclusions
Our study represents a new contribution on the in vitro potential effects of food habits on bacterial
metabolites production. IF-Lunch menus had extractable and non-extractable polyphenols, as main
bioactive compounds, but they do not contribute to an anti-radical AOX. Although the differences
in IF content, the magnitude of SCFA production in lunch menus was similar to blank (without
substrate). That could be related to the low contribution of dietary fiber in Mexican diet. PCA
allowed the identification of metabolic patterns that were associated with beneficial health effects
(Low pH and high AOX). Bacterial metabolites as trimethylamine, fatty acid esters and indole
were associated with increased pH and decreased AOX in the fermentation extracts. Bacterial
metabolites profile was dependent on fermentation time and substrate. In addition, the approach
presented here, could be used as predictive model to easily assess the indigestible components
effects coming from the food intake. Finally, we are convinced that only through food complex
matrix study will we improve our understanding of the effects of diet on colon health promotion.
Author contributions
SA-SG designed the study, supported the experimental work, and contributed to the drafting of the
manuscript and its revisions. ZG-VM conducted most of the experimental work and contributed to
the drafting of the manuscript. CC-AP contributed to the experimental work and helped revise the
manuscript; VL-PA helped design the study and supported the experimental work. T-J and LP-
MGF contributed to the revision of the manuscript and contributed to the final version
Acknowledgements The authors thank the schoolchildren who donated stool voluntarily for
participating in this study. Zamora-Gasga, VM; acknowledge the fellowship to CONACYT-
Registration number: 253795) and Sáyago-Ayerdi, SG; would also like to acknowledge the
financial support to PROMEP –ITTEP-PTC-003.
Capítulo 7. RESULTADOS Y DISCUSIÓN
112
Conflict of interest statement
The authors have declared no conflict of interest.
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Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of blank, rafinose and indigestible fraction isolated from Lunch menus (MM-L; Modified Mexican diet, TM-L; Traditional Mexican Diet, AM-
L; Alternative Mexican diet) analyzed by SPME–GC/MS (mmol/L).
ID RT Volatile Name Chemical Group
T12 T24 T48 T72
VC (%) Blank Raffinose MM-L TM-L AM-L Blank Raffinose MM-L TM-L AM-L Blank Raffinose MM-L TM-L AM-L Blank Raffinose MM-L TM-L AM-L
1 6.001 Trimethylamine Amines 18.90 0.00 24.78 8.08 23.34 21.39 0.00 19.44 22.15 28.16 36.34 0.00 10.83 5.94 16.82 24.64 0.00 8.09 9.51 9.79 74.03
2 8.359 Ethanol Alcohols and polyols 23.29 29.76 0.00 0.00 25.46 27.54 0.00 36.26 105.87 0.00 0.00 140.27 16.91 17.49 0.00 0.00 4.80 12.92 0.00 0.00 168.28
3 9.229 Methyl propionate Carboxylic acids and derivatives 0.00 4.16 4.80 0.00 5.40 0.00 0.00 6.18 5.24 5.56 0.00 6.58 0.00 5.08 4.94 0.00 0.00 8.24 7.04 4.05 88.19
4 10.754 Trichloromethane Alkyl halides 6.66 12.35 13.95 7.71 8.92 0.00 0.00 0.00 0.00 0.00 13.52 10.33 4.55 3.70 8.63 18.15 5.50 8.31 4.89 4.60 80.50
5 12.074 Butanoic acid, methyl ester Fatty Acyls 9.12 10.08 15.14 20.82 13.64 0.00 11.39 37.23 64.07 37.46 7.65 19.00 25.37 54.57 48.62 14.02 13.21 107.67 97.49 38.26 91.69
6 13.918 Toluene Benzene and substituted derivatives 0.00 9.29 0.00 0.00 0.00 13.19 9.39 12.97 0.00 0.00 0.00 0.00 69.14 0.00 0.00 32.04 20.80 0.00 0.00 0.00 201.46
7 14.254 Disulfide, dimethyl Organic disulfides 16.38 0.00 42.66 20.99 27.81 22.34 0.00 41.91 75.68 77.19 6.67 0.00 5.58 3.83 105.29 0.00 0.00 25.81 5.30 4.55 126.00
8 15.244 1-Butanol Alcohols and polyols 0.00 0.00 0.00 3.46 6.55 0.00 0.00 0.00 51.66 10.27 0.00 44.85 13.50 0.00 14.84 0.00 13.44 34.64 0.00 0.00 163.81
9 16.364 Pentanoic acid, methyl ester Fatty Acyls 0.00 3.96 29.82 27.60 9.55 3.83 0.00 109.60 113.49 124.28 9.09 3.42 89.91 107.20 248.38 9.91 0.00 225.73 145.57 147.00 111.99
10 19.121 Pentanoic acid, ethyl ester Fatty Acyls 0.00 0.00 0.00 0.00 0.00 0.00 0.00 7.42 7.02 8.50 0.00 0.00 5.76 6.02 14.03 0.00 0.00 11.08 8.83 0.00 134.95
11 20.057 Heptanal Carbonyl compounds 0.00 0.00 19.23 5.24 13.41 4.53 0.00 13.32 8.18 16.04 7.47 9.70 6.05 0.00 0.00 5.08 0.00 0.00 0.00 0.00 114.19
12 20.561 Hexanoic acid, methyl ester Fatty Acyls 0.00 3.68 54.95 28.92 15.28 5.16 0.00 177.68 150.64 76.85 6.80 0.00 212.63 373.80 615.36 10.67 3.64 666.64 541.71 364.33 135.25
13 23.045 Hexanoic acid, ethyl ester Fatty Acyls 0.00 0.00 0.00 0.00 0.00 0.00 0.00 7.89 6.26 4.68 0.00 0.00 9.08 13.47 22.69 0.00 0.00 24.23 22.00 0.00 153.67
14 24.537 Heptanoic acid, methyl ester Fatty Acyls 4.37 0.00 61.80 21.44 11.30 8.51 3.62 200.64 128.16 54.79 9.53 0.00 347.03 1042.80 934.45 10.30 6.09 1218.92 1224.46 824.62 149.03
15 25.883 Dimethyl trisulfide Organic trisulfides 392.74 0.00 674.84 283.24 461.07 176.20 0.00 133.60 183.95 157.68 37.76 0.00 39.85 28.03 25.67 34.12 0.00 11.86 13.03 17.21 139.52
16 26.235 Pyridine, 2,4,6-trimethyl- Pyridines and derivatives 0.00 0.00 14.85 0.00 17.69 10.86 0.00 19.99 14.48 6.11 23.21 0.00 13.57 9.48 0.00 20.28 0.00 16.01 12.43 14.73 84.55
17 26.247 Acetic Acid Organic acid 3.88 198.28 16.96 32.25 8.55 0.00 224.34 10.17 33.12 22.14 3.40 909.71 36.95 13.95 39.42 7.67 237.79 68.12 16.28 20.84 216.12
18 26.806 Heptanoic acid, ethyl ester Fatty Acyls 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10.68 5.48 0.00 0.00 0.00 21.05 36.51 39.91 0.00 0.00 55.38 45.89 0.00 170.50
19 28.059 Benzene, 1,2-dichloro- Benzene and substituted derivatives 4.04 0.00 0.00 0.00 5.41 7.82 0.00 0.00 0.00 4.27 3.47 0.00 0.00 0.00 3.31 13.68 0.00 0.00 0.00 4.61 154.95
20 28.156 Nonanal Carbonyl compounds 0.00 14.41 0.00 0.00 8.69 0.00 5.84 8.97 6.09 8.82 5.31 7.78 10.59 0.00 5.51 0.00 6.05 0.00 0.00 0.00 103.27
21 28.347 Octanoic acid, methyl ester Fatty Acyls 13.72 0.00 86.80 27.27 23.45 16.32 0.00 317.15 44.12 22.14 13.70 0.00 191.20 256.09 115.67 9.78 9.95 509.08 231.47 192.11 132.63
22 29.758 Propionic Acid Organic acid 0.00 30.02 3.75 7.35 2.38 0.00 23.78 5.18 17.44 16.30 1.85 82.95 19.72 6.82 17.35 0.00 28.53 12.91 4.74 5.29 131.04
23 29.837 Benzaldehyde Benzene and substituted derivatives 24.76 14.25 15.26 14.39 14.91 27.57 17.30 19.43 21.93 26.25 12.57 9.45 25.37 13.23 17.64 20.57 16.34 18.96 19.78 12.97 27.86
24 31.456 Benzene, 1,3-bis(1,1-dimethylethyl)- Benzene and substituted derivatives 12.35 45.89 38.32 19.93 30.68 16.59 44.10 42.50 23.73 29.89 22.69 26.36 23.09 13.29 33.12 15.85 27.98 26.96 13.37 32.85 38.30
25 32.114 Nonanoic acid, methyl ester Fatty Acyls 6.46 0.00 8.61 15.18 10.86 5.67 0.00 43.00 20.89 9.46 12.05 0.00 23.64 49.15 10.09 7.40 0.00 57.56 40.39 20.20 101.70
26 32.534 1,3-Benzenediol, 4-ethyl- Benzene and substituted derivatives 8.36 10.28 0.00 0.00 7.21 6.27 7.25 11.58 9.71 7.95 9.34 5.75 8.21 0.00 4.98 5.39 5.50 9.60 7.14 6.16 50.88
27 33.352 Butiric Acid Organic acid 0.60 36.22 1.08 1.97 1.32 0.39 17.80 2.81 5.14 1.99 0.92 47.99 2.45 1.35 3.42 1.13 19.36 1.82 1.46 1.41 173.57
28 34.275 Tetradecane Alkanes 4.34 0.00 0.00 8.83 0.00 0.00 0.00 7.71 7.24 0.00 5.33 0.00 5.94 0.00 0.00 0.00 4.59 0.00 3.90 0.00 133.47
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29 34.948 Acetophenone Benzene and substituted derivatives 14.44 0.00 24.87 18.28 32.43 0.00 0.00 30.85 19.45 25.63 13.18 0.00 16.60 13.33 19.66 11.10 0.00 16.52 17.08 23.32 69.88
30 35.264 Butanoic acid, 3-methyl- Fatty Acyls 0.01 108.85 0.01 37.86 25.28 7.33 146.97 29.92 101.24 28.09 10.67 220.10 74.63 0.01 68.61 11.02 121.66 23.40 25.10 33.03 109.68
31 36.508 Decanoic acid, methyl ester Fatty Acyls 21.91 13.54 38.02 163.08 48.06 25.11 11.00 421.92 201.01 44.65 11.92 5.58 333.60 734.07 65.29 22.04 12.90 347.78 101.49 58.63 141.36
32 39.159 Pentanoic acid Fatty Acyls 10.36 248.72 0.00 27.58 10.31 7.25 181.39 86.76 99.01 55.33 95.71 346.57 53.30 3.78 64.68 32.60 181.55 88.95 33.25 31.94 110.25
33 41.611 Benzeneacetic acid, methyl ester Fatty Acyls 0.00 0.00 0.00 0.00 12.16 0.00 0.00 0.00 6.49 8.42 0.00 0.00 6.44 0.00 8.83 0.00 0.00 14.93 15.62 10.65 135.84
34 42.901 Undecanoic acid, methyl ester Fatty Acyls 0.00 0.00 0.00 8.26 4.67 0.00 0.00 14.20 9.62 3.33 0.00 0.00 11.35 21.15 3.38 0.00 0.00 9.93 6.66 0.00 130.95
35 45.850 Hexanoic acid Fatty Acyls 9.34 267.33 0.00 33.11 49.17 17.47 151.41 88.20 79.45 64.43 115.46 278.26 76.85 40.89 91.40 22.30 149.34 122.69 79.07 64.47 84.27
36 46.504 Benzenepropanoic acid, methyl ester Fatty Acyls 7.82 0.00 17.69 34.91 10.66 8.50 8.87 48.55 57.92 36.94 5.32 0.00 51.38 154.43 171.88 0.00 8.86 114.92 174.03 67.93 119.38
37 47.379 Naphthalene, 1,2,3,4-tetrahydro-2,5,8-trimethyl- Naphthalenes 4.53 8.28 0.00 0.00 6.88 0.00 10.58 3.75 0.00 5.17 4.28 4.35 0.00 0.00 5.44 0.00 0.00 0.00 0.00 0.00 126.09
38 49.117 Naphthalene, 1,2,3,4-tetrahydro-1,5,7-trimethyl- Naphthalenes 0.00 9.27 0.00 6.52 5.17 0.00 11.66 0.00 8.08 6.57 0.00 0.00 0.00 0.00 4.00 0.00 0.00 0.00 4.89 0.00 137.15
39 49.715 Benzothiazole Benzothiazoles 6.41 12.42 0.00 6.32 11.19 5.83 6.43 0.00 14.61 8.75 6.58 13.95 0.00 0.00 9.23 4.21 12.31 0.00 7.88 11.63 71.96
40 50.056 Heptanoic acid Fatty Acyls 0.00 52.66 0.00 8.72 0.00 0.00 31.80 30.95 26.83 15.32 81.41 41.66 32.56 37.72 67.08 17.57 42.28 71.10 109.68 76.48 83.87
41 50.392 Dodecanoic acid, methyl ester Fatty Acyls 80.39 17.86 69.69 168.18 82.87 71.49 19.05 363.19 178.19 32.92 87.81 16.32 177.25 520.52 65.12 38.65 12.62 264.47 164.95 75.24 103.84
42 50.402 Dodecanoic acid, ethyl ester Fatty Acyls 9.32 0.00 7.71 11.94 8.00 8.00 0.00 13.76 13.24 0.00 7.35 0.00 17.01 22.88 7.12 0.00 0.00 20.82 13.06 0.00 91.45
43 50.604 Butylated Hydroxytoluene Benzene and substituted derivatives 6.62 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 31.62 0.00 34.04 23.07 26.29 23.62 40.23 32.33 34.94 124.10
44 51.061 Phenol Benzene and substituted derivatives 368.57 96.38 196.23 129.69 171.32 244.05 48.00 209.99 156.85 280.20 259.99 81.19 127.33 97.34 119.77 326.98 102.56 144.93 130.66 287.92 49.76
45 52.001 Tridecanoic acid, methyl ester Fatty Acyls 5.26 0.00 0.00 7.89 0.00 4.42 0.00 13.51 7.24 0.00 5.14 0.00 6.09 21.40 0.00 0.00 0.00 11.54 11.57 4.64 120.07
46 53.245 Phenol, 4-methyl- Benzene and substituted derivatives 436.02 132.04 683.53 469.78 286.25 680.36 97.15 862.39 613.76 551.39 703.92 124.38 805.90 687.64 926.73 441.14 91.65 1045.23 883.43 701.78 52.27
47 54.056 Tridecanoic acid, 12-methyl-, methyl ester Fatty Acyls 6.83 0.00 6.99 16.03 6.54 5.13 0.00 28.42 19.18 0.00 7.19 0.00 11.38 32.56 0.00 6.25 0.00 22.18 15.70 4.83 104.29
48 55.570 Methyl tetradecanoate Fatty Acyls 120.96 9.04 0.00 162.39 76.18 100.26 12.86 283.31 157.88 17.06 156.96 11.75 114.39 425.00 41.76 50.71 16.86 275.64 235.61 57.77 98.59
49 55.776 Methyl myristoleate Fatty Acyls 23.88 0.00 25.15 66.23 19.89 19.43 0.00 136.85 47.39 6.09 42.43 0.00 21.84 90.73 9.30 9.20 0.00 51.18 26.96 10.85 114.73
50 57.892 Pentadecanoic acid, methyl ester Fatty Acyls 0.00 0.00 12.21 9.55 7.20 6.17 0.00 29.14 9.81 0.00 15.04 0.00 14.46 23.60 7.78 6.51 0.00 25.79 15.39 9.06 93.22
51 58.738 Phenol, 2,4-bis(1,1-dimethylethyl) Benzene and substituted derivatives 9.12 11.13 6.24 7.54 9.05 13.55 8.68 13.84 10.53 10.48 14.70 8.70 11.42 13.24 10.03 16.32 10.22 0.00 13.23 13.20 33.89
52 60.703 Indole Indoles and derivatives 517.58 15.37 710.36 574.97 748.05 418.15 9.87 515.45 506.84 428.95 498.38 15.56 323.85 282.89 312.23 201.33 14.63 272.80 233.68 341.79 64.48
53 61.599 1H-Indole, 3-methyl- Indoles and derivatives 8.47 0.00 5.41 12.20 11.08 14.02 0.00 13.13 11.22 7.26 20.24 0.00 6.08 24.34 10.41 6.56 0.00 32.16 0.00 6.99 90.46
54 62.324 11-Hexadecenoic acid, methyl ester Fatty Acyls 19.63 0.00 0.00 9.81 7.04 9.62 0.00 21.55 9.49 0.00 10.57 4.21 7.21 0.00 8.63 0.00 0.00 0.00 10.87 0.00 112.67
55 62.516 Methyl hexadec-9-enoate Fatty Acyls 14.07 0.00 0.00 58.88 24.96 17.17 3.62 75.55 59.05 7.88 34.94 6.34 20.30 86.88 22.56 13.80 0.00 46.69 34.83 29.11 92.24
56 62.902 Hexadecanoic acid, methyl ester Fatty Acyls 16.37 4.86 16.04 33.05 26.94 23.63 8.00 56.34 35.49 9.75 38.82 0.00 22.03 109.29 24.37 21.24 8.48 50.21 83.48 30.98 87.15
57 67.380 9-Octadecenoic acid, methyl ester, (E)- Fatty Acyls 115.70 0.00 19.81 186.68 150.24 116.63 51.58 340.93 233.64 92.30 275.45 62.53 114.41 271.03 157.61 0.00 75.49 169.70 284.33 209.37 68.08 ID; Identification, RT; retentiton time, VC; Variation coefficient
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7.4 Efectos de los alimentos frecuentemente consumidos por escolares mexicanos en la cena
sobre el metabolismo microbiano y la capacidad antioxidante durante la fermentación
colónica in vitro
Resumen
El interés en el papel de la microbiota colónica humana en la salud o la enfermedad ha aumentado
constantemente. A través del proceso de fermentación, las bacterias del colon producen una amplia
gama de compuestos que pueden tener importantes implicaciones en los procesos fisiológicos de
colon (Nicholson y cols., 2012; Russell y cols., 2013b). Debido a la inaccesibilidad del colon
humano, los estudios in situ son difíciles y es necesaria más información para comprender los
procesos metabólicos de la microbiota. La mayoría de los estudios que presentan este enfoque han
evaluado sólo un número limitado de compuestos orgánicos volátiles. Sin embargo, poco a poco se
comienza a creer que cada metabolito tiene una importancia en la actividad biológica de todo el
extracto de fermentación. Es así que Beyer-Sehlmeyer y cols. (2003) y Campos-Vega y cols.
(2012), proponen que los efectos quimioprotectores beneficiosos en cultivos de células de cáncer
de colon podrían atribuirse a las diferencias en la magnitud de las actividades biológicas de
diferentes compuestos presentes en los extractos completos. De este modo, proponemos la micro
extracción en fase solida (MEFS) junto con la cromatografía de gases acoplada a espectrometría de
masas (GC-MS) para proveer un perfil completo de metabolitos microbianos volátiles. Hasta la
fecha, sólo unos pocos estudios han tratado de generar una visión más completa del perfil
metabólico de la microbiota utilizando heces fecales humanas como principal objeto de estudio
(De Preter y cols., 2009; Garner y cols., 2009). De acuerdo con nuestro conocimiento, ha habido
pocos intentos para determinar la composición volátil, producto del metabolismo microbiano de la
fracción indigestible de los alimentos consumidos en la cena. Por lo tanto, en este capítulo se
porpone la diferenciación de los compuestos volátiles obtenidos por MEFS/CG-EM/ACP de
diferentes tiempos de fermentación de los componentes indigestibles de mezclas de alimentos
complejos que nos permitirá idetntificar los metabolitos responsables de los cambios en el pH y
actividad antioxidante durante la fermentación colonica in vitro de la fracción indigestible aislada
de alimentos frecuentemente consumidos por escolares durante la cena.
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Effects of foods frequently eaten by Mexican schoolchildren at dinner on microbial
metabolism and antioxidant capacity during in vitro colonic fermentation
1 Introduction
Diet is one of the most influential lifestyle factors contributing to rise of inflammatory diseases
and autoimmunity in both developed and developing countries (Richards, Yap, McLeod, Mackay,
& Mariño, 2016). In particular, Western diet, including high-fat and cholesterol, high-protein,
high-sugar, and excess salt intake, promote obesity, metabolic syndrome, and cardiovascular
disease (Manzel et al., 2014). Actually, Mexican population follow these trends, wherein the
dietary fiber intake is low and added sugar and saturated fat intakes are higher than recommended
for >50% of the population aged ≥1 year (López-Olmedo et al., 2016). However, it has been
suggested that dietary polyphenol intake in a rural Mexican Diet is similar to those determined in a
healthy diet reference “Spanish Mediterranean diet” (Hervert-Hernández, García, Rosado, & Goñi,
2011). Recently evidence shown that traditional Mexican diet (based on corn, beans, and chili pep)
modestly improved insulin sensitivity under conditions of weight stability in healthy women of
Mexican descent (Santiago-Torres et al., 2016) and decreases the glucose intolerance and
biochemical abnormalities caused by a sucrose-enriched high-fat diet in rats (Avila�Nava et al.,
2017). Several studies show that long-term and shorter term dietary variation influences gut
microbiota composition and determines its metabolic profile (Kaakoush et al., 2017; Rajilić�
Stojanović, Heilig, Tims, Zoetendal, & Vos, 2013). Since many microbial products have been
shown to influence the development of diseases and the maintenance of health status of the colon
(Levy, Blacher, & Elinav, 2017; Richards et al., 2016; Yasmin et al., 2015); In this sense,
interindividual variation in microbial profiles of the intestine in humans can lead to differences in
disease risk (Conlon & Bird, 2014). Given the importance of the diet on gut metabolism-host
health, our understanding of how different sources of indigestible components from western diet
could be allow the microbial profiles to be modulated (Flint, Duncan, Scott, & Louis, 2015).
Previously, we investigated dietary patterns in Mexican schoolchildren from Tepic, State Nayarit
Mexico but the effects of foods in this dietary pattern on the gut metabolism are unknown.
Therefore, the aims were to quantify and isolate the indigestible fraction (IF) in three dinner
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menus consumed by Mexican schoolchildren and to assess how the gut metabolite profile could be
affected by indigestible components of this menus in an in vitro colonic fermentation model.
Relationships between the abundance of microbial metabolites and changes in pH, antioxidant
capacity (AOX) values were also examined.
2. Material and methods
2.1 Dinner food preparation
Data sources on dietary patterns (DP) and food frequency consumption at dinner were obtained
from a nutritional survey carried out on eleven public schools in a Western city of Mexico (Tepic,
Nayarit State). Frequently consumed foods in each DP were used to create three dinner menus.
Dinner menus were named and comprised by the following foods; Modified Mexican Dinner
(MM-D): Soda (357 mL) and three roasted meat tacos with cabbage and green sauce. Traditional
Mexican Dinner (TM-D): Whole milk (250 mL) and three bean and cheese tacos. Alternative
Mexican Dinner (AM-D): Chocolate milk (whole milk 300 mL, cane sugar 8 g, chocolate powder
16 g) and a piece of sweet bread. Individual foods were purchased in a local supermarket and were
prepared in the laboratory kitchen according to traditional customs of the region. After prepared,
each menu, it was homogenized in a food processor (NB-101B, Nutribullet, China), frozen (-80
°C), freeze-dried (FreeZone 6, Labconco, USA), grounded, sieved with a mesh size of 500 µm,
and stored at -20 °C until analysis. Each menu preparation was performed by triplicate. Moisture
content of complete dinner menus, was analyzed according to AOAC (1990) 925.10 methods.
2.2 Quantification and isolation of indigestible fraction (IF) in menus
A digestion procedure that mimicking the physiological situation in the upper tract (stomach and
small intestine) was utilized for indigestible fraction (IF) quantification (Saura-Calixto, García-
Alonso, Goni, & Bravo, 2000). In order to increase the amount of isolated IF the follow
amendments were realized (Tabernero, Venema, Maathuis, & Saura-Calixto, 2011). Insoluble IF
was considered as the digestion residues pelleted by centrifugation, while those retained by
dialysis represented soluble IF; the sum of both fractions equals total IF. Total, IF was collected,
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freeze-dried, milled (IKA M20, USA), sieved (500-micron mesh), and stored in seal bags at -20
°C.
2.2.3 Antioxidant compounds analysis in the total indigestible fraction
Samples (250 mg) of total IF isolated from dinner menus were extracted by aqueous-organic
solution (Pérez-Jiménez, Arranz, & Saura-Calixto, 2009). Total soluble polyphenols (TSP) in
extractable fraction were determined with the Folin–Ciocalteu’s reagent (Montreau, 1972) using a
96-well microplate reader (Biotek, Synergy HT, Winooski VT, USA) with Gen5 software, and the
results were expressed in g of gallic acid equivalents (g GAE/ 100g IF). The residues of the
extraction were treated for non-extractable polyphenols quantification (Condensed tannins and
hydrolyzable polyphenols). Condensed tannins (CT) were quantified and results were expressed as
CT equivalents/ 100 g IF, using a carob pod (Ceratonia siliqua) proanthocyanidin standard (Reed,
McDowell, Van Soest, & Horvath, 1982). Hydrolyzable polyphenols (HP) were evaluated by a
previously reported methodology (Hartzfeld, Forkner, Hunter, & Hagerman, 2002) and results
were expressed as g GAE/100 g IF.
2.2.4 Antioxidant capacity (AOX) analyses
AOX was evaluated on the extractable fraction obtained before, and methodologies were slightly
modified to a microplate reader (Biotek, Synergy HT, Winooski VT, USA) with Gen5 software.
1,1-Diphenyl-2-picryl hydrazyl (DPPH) antiradical activity assay was evaluated (Prior, Wu, &
Schaich, 2005). Ferric reducing antioxidant power (FRAP) assay was performed (Benzie & Strain,
1996). Results AOX were expressed as Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic) equivalent (TE; mmol /g IF db).
2.3 In vitro colonic fermentation by human microflora
Total IF isolated from three dinner menus were fermented in a batch culture system with pre-
conditioned nutritive medium and under strict anaerobic conditions at 37 °C (Campos�Vega et
al., 2009). In parallel, two different controls were conducted under the applied conditions: a)
Raffinose was incubated in medium with faeces inoculum as a fermentable sugar reference, and b)
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the faecal suspension was incubated without substrate as a negative control. All incubations were
performed in triplicate and samples and controls were collected at 12, 24, 48 and 72 h. The tubes
obtained at each time of fermentation were centrifuged (Hermle Z 323 K; Wehingen, Germany)
(3500 × g ,15 min, 4 °C). Supernatants were divided into two parts; metabolite profile analysis and
antioxidant capacity assays and were immediately stored at −80 °C until analysis.
2.4 Metabolites characterization by HS-SPME- GC/MS
Supernatants (500 mg) were placed into a 20 mL vial sealed with a magnetic cap with a poly-tetra-
fluoro-ethylene (PTFE)/silicon septum. HS-SPME method was used for the extraction and
concentration of volatile compounds (Zamora-Gasga et al., 2015); incubation with
polydimethylsiloxane-divinylbenzene-carboxen “PDMS/DVB/CAR” fiber for 120 min, 250 rpm at
45 °C; the fiber was inserted into the injection port of the GC system for thermal desorption (240
°C for 10 min) for GC/MS analysis. The extractions were performed in triplicate. Five runs with
twelve vials / run were performed and the tests order was completely randomized. The volatile
constituents were analyzed with an Agilent 5975C VL mass selective detector coupled to an
Agilent 7890A gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA), equipped with a
DB-5MS capillary column (60 m X 250 µm X 0.25 µm; Agilent). Sample quantification was
obtained by means of acetic, propionic and butyric acid standard curves. Tentative identification of
the volatile components was done comparing the mass spectra of the samples with the data system
library MSD ChemStation software (Agilent G1701EAversion E.02.00.493). Relative
concentration of all fermentation metabolites versus acetic acid as internal standard was calculated
and the results were expressed in mmol L-1.
Antioxidant capacity (AOS) during in vitro colonic fermentation
Changes in the AOX of the sample extracts during in vitro colonic fermentation at 12, 24 and 72h
h were measured by the methods described above (paragraph 2.2.4)
2.5 Statistical analysis
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125
Results are expressed as mean ± SEM (n=3). Univariate testing of differences for dinner menus
was processed by ANOVA/Fisher's Least Significant Difference test. Besides, Multivariate
analysis was applied for metabolites profile interpretation. Principal component analysis (PCA)
was used to determine patterns in relative metabolites production. Finally, Pearson´s correlation
coefficients were used to evaluate the relationship between individual metabolites and changes in
pH and AOX during in vitro colonic fermentation. All analyzes were performed using
STATISTICA software, version 10.0 (StatSoft. Inc. 1984–2007, Tulsa, OK, USA). A significance
level of α = 0.05 was used in data analysis.
3. Results
3.1 Content and nutritional composition of indigestible fraction (IF) in dinner menus
The results shown in Table 1 indicate that the moisture content in MM-D (80.03 g/100g wb) was
about five and ten percentage points higher than TMD and AM-D, respectively (p<0.05). which
follows that in this dinner menu are less solid components available for gastrointestinal digestion.
Complete digestion time depends on food structure including bolus liquid consistency; liquids are
digested faster than semisolids or cellular structures (Sensoy, 2014). The increase in moisture
content in TM-D could be attributed to soda addition and consequently as a dilution effect. On the
other hand, colonic fermentation of indigestible might act on the gut to cause symptoms have been
associated with irritable bowel syndrome (Gibson, Varney, Malakar, & Muir, 2015). In total IF
content, significant differences (p<0.05) between in MM-D (2.69 g/100g wb), AM-D (4.17 g/100g
wb) and TM-D (6.95 g/100g wb) were found (Table 1). TM-D presented the highest total IF
content that can be attributed to the presence of beans in the menu. Inclusion of black beans with a
typical Western-style meal attenuates postprandial insulin in adults with metabolic síndrome and
this effect was partly explained by the fiber content (Reverri et al., 2015). Insoluble IF was the
main fraction of total IF, since it represented from 73.2% in MM-D to 90.9% in TM-D. AM-D
showed the highest. Soluble IF content soluble indigestible fraction of the three menus (p<0.05).
Soluble IF constitute s the 5.57, 9.06 and 26.8 % of Total IF in MM-D, TM-D and AM-D
respectively. Some of the important properties of soluble IF components (e.g. β-glucan, pectins,
guar gum, arabinoxylans, and inulins) are water solubility, ability to form viscous solutions, high
Capítulo 7. RESULTADOS Y DISCUSIÓN
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fermentability in colon and has a greater hypolipidemic effect compared with Insoluble IF
(Surampudi, Enkhmaa, Anuurad, & Berglund, 2016). Interactions among food matrix may
generate different physical properties in dinner menus and thus modifying IF content. In
accordance with Zhang, Maladen, Campanella, and Hamaker (2010) electrostatic interactions
among carboxyl groups, possessing negative charge, of the free fatty acids and the poly-ionic
protein are the basis for ternary complex with starch (e.g. TM-D dinner: Corn tortilla and bean
starch, Cheese protein and vegetable oil interactions); this complex could decrease the
macronutrients digestibility and finally increase the indigestible component ratio (Parada &
Santos, 2016).
3.2 Antioxidant compounds in the indigestible fraction
Total, soluble polyphenols (TSP), hydrolysable tannins (HT), and condensed tannins (CT) were
studied in IF of dinner menus and the results are shown in Table 1. The TSP content of MM-D
(0.83 g/100g IF db) did not show significant differences (p<0.05) with AM-D (0.78 g/100g IF db)
and TM-D (0.85 g/100g IF db). High molecular weight compounds such as condensed tannins
(proanthocyanidins) and hydrolyzable polyphenols are covalently attached to dietary fiber or
proteins and can be released into the colon by gut fermentation (Bohn, 2014). In CT, the highest
(p<0.05) content was found in TM-D (2.22 g/100g IF db), followed by AM-D (1.00 g/100g db).
The presence of beans in TM-L and cocoa in AM-D could be responsible for the observed
differences. After ingestion, a small amount of flavan-3-ols or proanthocyanidins oligomers are
absorbed in the small intestine. Cocoa and common beans procyanidins (Possibly present in TM-D
and AM-D) have shown effects for preventing loss of gut barrier function and epithelial
inflammation, which are critical steps in the pathogenesis of metabolic endotoxemia, inflammatory
bowel disease and colon cancer (Bitzer et al., 2015; Moreno-Jiménez et al., 2015). The role of gut
microflora in the catabolism of proanthocyanidins has been evaluated. The microbial derived
phenylvalerolactone (5-(3´,4´-dihydroxyphenyl)-ϒ-valerolactone) and phenolic acids (flavan-3-ol
monomers) were the predominant metabolites of cocoa procyanidins in blood and urine (Ottaviani,
Kwik-Uribe, Keen, & Schroeter, 2012). Hydrolyzable tannins (HT) values ranged from 1.58 to
2.24 g/100g sample db, with statistically significant differences (p<0.05) between all the samples.
HT were a quantitatively important fraction of polyphenols in all samples (approximately 43% for
Capítulo 7. RESULTADOS Y DISCUSIÓN
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AM-D and TM-D and up to 60% in the case of the MM-D). In the colon, HT are metabolized to
gallic acid, pyrogallol, phloroglucinol and finally to acetate and butyrate via several bacterial
enzymes (Marín, Miguélez, Villar, & Lombó, 2015). TSP linked to the IF were released in the
aqueous-organic extraction and it showed FRAP activity but DPPH activity was observed only for
TM-D (2.17 g/100g IF db). FRAP activity was higher in IF extracts isolated for TM-D and AM-D
(11.29 and 12.25 mmol TE/g IF db) and lower in MM (9.66 mmol TE/g IF db). These results
suggest that antioxidant mechanism in extracts can be as chelating agent and not as a free radical
neutralizer (Valdés et al., 2015).
Table 1 Chemical composition and antioxidant compounds content, and antioxidant capacity in
the indigestible fraction (IF) isolated from dinner menus1
Parameter Menus2
MM-D TM-D AM-D
Moisture content (g/100g menu ) 80.03 ± 0.24c 75.62 ± 0.56b 71.57 ± 0.09a
IF (g/100g menu wb)
Insoluble IF 2.54 ± 0.10a 6.32 ± 0.14b 3.04 ± 0.08a
Soluble IF 0.15 ± 0.04a 0.63 0.03b 1.12 ± 0.07c
Total IF 3 2.69 ± 0.14a 6.95 ± 0.16c 4.17 ± 0.03b
IF Antioxidant Compounds (g/ 100g IF db)
Total soluble polyphenols 8.36 ± 0.27 ab 7.81 ± 0.17 a 8.57 ± 0.21 b
Condensed Tannins 0.48 ± 0.02a 2.22 ± 0.02c 1.00 ± 0.05b
Hydrolyzable polyphenols 2.08 ± 0.03a 2.24 ± 0.02 b 1.58 ± 0.05c
IF Antioxidant Capacity (mmol TE/g IF db)
DPPH ND 2.17 ± 0.44 a ND
FRAP 9.66 ± 0.27 a 11.29 ± 0.43 b 12.25 ± 0.33b 1Values are mean ± standard error (n = 3); Wet basis (wb); Dry basis (db); No Detected (ND).
Means in rows marked with different letters indicate significant difference (p <0.05). 2 Menus;
MM-D: Modified Mexican Dinner, TM-D: Traditional Mexican Dinner, AM-D: Alternative
Mexican Dinner. 3 Total IF = Sum of soluble IF + insoluble IF.
Capítulo 7. RESULTADOS Y DISCUSIÓN
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3.3 Changes in pH and antioxidant capacity from in vitro colonic fermentation extracts
The pH data were collected for 72 h during in vitro colonic fermentation of IF isolated from three
dinner menus (Figure 1). After fermentation, TM-D showed greatest decrease in pH value
(p<0.05) of dinner menus, but the magnitude of the reduction in pH value was markedly different
depending on fermentation time (pH ∆0h – 72h = 0.45 units). Aversely, AM-D presented a
significant increase in the pH value at 72 (0.35 units). Mean of pH levels were significantly lower
(p<0.05) in Raffinose control (pH ∆0h – 72h = 2.55 units) compared with dinner menus at 72 h
(p<0.05) which is most probably caused by the fermentation end products (mainly SCFA). In a
clinical trial, colonic pH levels were correlated positively with irritable bowel syndrome symptoms
severity, but higher levels of SCFAs and lower pH values are associated with decreased colon
transit time and positively with stool frequency (Ringel-Kulka et al., 2015). Colonic pH can
influence the formation of the gut metabolites. In vitro incubation was observed that the formation
of lactate is highest under slightly acidic conditions (pH 5·9), but, low pH (<5·2) lactate utilization
was strongly inhibited, resulting in lactate accumulation (Belenguer et al., 2007). Beside, low pH
(5.5) tends to increase acetate uptake and butyrate production while near neutral pH (6.7) has the
opposite effect (Kettle, Louis, Holtrop, Duncan, & Flint, 2015). In this in vitro study, determined
values demonstrated a varied antioxidant capacity (DPPH and FRAP), which differs from
substrate and fermentation time (Figure 1B and C). Figure 1B shows that the scavenging effects
of raffinose on DPPH radical were proportional to fermentation time and the values were higher
(p<0.05) of the menus from the 24 h to 72 h (28.35 to 94.31 mmol TE/gIF, respectively). Dinner
menus were more active at 72 h and there were no significant differences (p>0.05) between their
values (mean of 28 mmol TE/gIF). TM-D has the highest activity DPPH at 12 and a dramatic
decrease was observed from 48 h (From 20.33 to 3.41 mmol TE/g IF, respectively). Besides, in
FRAP, the AOX of AM-D at 12 h was similar to raffinose control (p>0.05) but significantly
greater than MM-D and TM-D (p<0.05). At 72 h, TM-D did not show FRAP activity but AM-D
and MM-D showed similar activity (4.13 and 4.69 mmol TE/g IF, respectively). Differences
between the AOX values found by the two methods evaluated suggest that the antioxidant
mechanism of the gut metabolites is mainly as free radical neutralizers and secondarily as
Capítulo 7. RESULTADOS Y DISCUSIÓN
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chelating agents. Typically, dietary polyphenols are compounds known for their antioxidant
capacity.
Capítulo 7. RESULTADOS Y DISCUSIÓN
130
Figure 1 Changes in a) pH kinetic, b) DPPH Antiradical activity and c) FRAP chelating activity in the extracts during in vitro
colonic fermentation from (–) blank, (··♦··) raffinose, and indigestible fraction isolated from dinner menus: (-■-) Modified
Mexican Dinner, MM-D, (-▲-) Traditional Mexican Dinner, TM-D, and (-●-) Alternative Mexican Dinner, AM-D at different
at different fermentation times, Values are means ± SEM (n=3). *Significant difference using Two-way ANOVA/Fisher's LSD
test (Samples ×Time interaction, p<0.05). For DPPH and FRAP, the blank was subtracted of the samples.
Fermentation Time (h)
0 12 24 48 72
DPP
H A
ntio
xidan
t Act
ivity
(mM
TE/
g su
bstra
te d
b)
0
20
40
60
80
100
120* * * *
Fermentation time (h)
0 12 24 48 72
FRAP
Anti
oxida
nt Ac
tivity
(m
M T
E /g
subs
trate
db)
0
2
4
6
8
10
12
14
16
18 * * * *
Fermentation time (h)
0 12 24 48 72
pH
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0 * * * *
Capítulo 7. RESULTADOS Y DISCUSIÓN
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In this sense, corn tortillas (important element in MM-D and TM-D menus) have a high content of
ferulic acid, carotenoids and anthocyanin’s that might lie in the colon and exert a direct antioxidant
effect (Bello-Perez, Osorio-Díaz, Agama-Acevedo, & Gonzalez-Soto, 2016). In addition,
microbial metabolites of fermentation may exert an antioxidant effect. Phenylacetic,
phenylpropionic, benzoic acid derivatives and branched-chain fatty acids (isobutyrate, 2-
methylbutyrate and isovalerate) with potential antioxidant properties can thereby be generated,
mainly by amino acids and phenolic gut metabolism (Davila et al., 2013; Valdés et al., 2015). For
its part, ellagic acid (Constituent of hydrolysable tannins) is largely metabolized by the colon
microbiota, giving rise to urolithin A (3,8-dihydroxy-6H-dibenzopyran-6-one) and its
monohydroxylated analog known as urolithin B (González-Barrio, Edwards, & Crozier, 2011)
3.4 Production of SCFA
The concentrations of SCFA at 12, 24 48 and 72 h of fermentation are present in Table 2. Acetic
propionic and butyric acid concentrations in dinner menus were significantly lower than raffinose
(p<0.05). Acetate was the most abundant SCFA produced by dinner menus at 72 h (From 20.13 in
TM-D to 48.17 mmol/L in AM-D). In propionic and butyric acid concentrations, IF dinner menus
vs blank were not significantly different (p > 0.05) throughout the all fermentation times except in
AM-D at 72 h (28.75 for propionic and 3.15 3.15 mmol/L for butyric acid). The low production of
AGCC in dinner menus (Mainly made with food of animal origin) suggests that the isolated
fractions could have a low indigestible carbohydrate content and a high indigestible protein and
lipids content. This could have negative implications on the health of individuals. An animal-based
diet increased the abundance of bile-tolerant microorganisms, including Alistipes, Bilophila, and
Bacteroides species and decreased the abundance of species specialized in the utilization of
polysaccharides (Roseburia, Eubacterium rectale, and Ruminococcus bromii) (David et al., 2014).
In contrast to the above, a higher stimulation of SCFA production in AM-D could be explained by
the presence of resistant starch in bread and consecutively cross-feeding between butyric acid-
producing bacteria and Bifidobacterium species (Rios-Covian, Gueimonde, Duncan, Flint, &
Clara, 2015).
Capítulo 7. RESULTADOS Y DISCUSIÓN
132
Table 2. Short-chain fatty acids (SCFAs, mmol L-1) production at 12, 24, 48 and 72 h of in vitro fermentation of blank,
raffinose and indigestible fraction isolated from dinner menus (MM-D; Modified Mexican Dinner, TM-D; Traditional Mexican
Dinner, AM-D; Alternative Mexican Dinner) 1.
SCFA/ fermentation time
Blank Raffinose IF- Dinner Menus
MM-D TM-D AM-D Acetic acid 12 h ND 184.71 ± 19.14 ND ND ND 24 h ND 240.20 ± 0.67b ND 5.44 ± 0.50a 5.02 ± 0.51a 48 h ND 193.79 ± 5.83b 5.97 ± 1.07a 2.96 ± 0.81a 10.98 ± 0.54a 72 h 1.68 ± 0.38a 405.97 ± 6.24d 29.57 ± 2.91b 20.13 ± 1.07b 48.17 ± 1.55c Propionic acid 12 h ND 82.28 ± 10.42 ND ND ND 24 h ND 102.06 ± 6.39b ND 17.02 ± 1.30a ND 48 h ND 93.68 ± 11.53b 2.05 ± 0.35a 3.50 ± 0.79a 8.32 ± 1.09a 72 h ND 92.31 ± 0.71c 5.08 ± 1.67a 2.76 ± 0.44a 28.75 ± 1.73b Butyric acid
12 h 1.20 ± 0.13a 18.10 ± 2.09b ND 0.83 ± 0.05a 0.70 ± 0.06a 24 h 1.23 ± 0.37a 21.09 ± 2.23b ND 1.33 ± 0.16a 0.31 ± 0.03a 48 h 0.54 ± 0.04a 15.30 ± 2.76b 0.73 ± 0.03a 0.97 ± 0.20a 1.41 ± 0.12a 72 h 0.42 ± 0.08a 9.46 ± 0.69c 0.60 ± 0.04ab 0.22 ± 0.05a 3.15 ± 0.11b
*The values are reported in mmol/L produced per 100 mg substrate as mean ± SEM of three replicate; Different lowercase
letters indicate significant differences in rows among substrates for a time (p < 0.05).
Capítulo 7. RESULTADOS Y DISCUSIÓN
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In a clinical trial, the intake of whole-grain and fiber-rich rye bread versus refined wheat bread
does not differentiate intestinal microbiota composition and main sugar-converting butyrate
producers were similarly associated (Lappi et al., 2013). However, studies on chemical
composition in IF are needed to identify foods with prebiotic potential in the Mexican diet.
3.5 Production of volatile gut metabolites
The kinetic analysis by GC-MS/SPME of the volatile metabolites produced by fecal microbiota at
the time points 12, 24, 48 and 72 h showed different metabolic profiles in relation to the different
substrates utilized (See Supporting Information Table SI). A total of 54 different metabolites
belonging to the families of amines, alcohol and polyols, alkanes, alkyl halides, benzene and
substituted derivatives, bensothiazoles, varbonyl compounds, fatty acid esters, indoles and
derivatives, organic acid, organic disulfides, organic phosphoric acids, organic trisulfides and
SCFA were detected (Figure 2). At 12 h, organic trisulfides was the most abundant compounds
found in MM-D. The fermentation of proteinaceous substrates have been considered potentially
detrimental for host health and have been implicated in development of noncommunicable
diseases (Boulangé, Neves, Chilloux, Nicholson, & Dumas, 2016; Organ et al., 2016) and the
pathogenesis of large intestinal diseases (Louis, Hold, & Flint, 2014). Notably, in MM-D, Organic
acid production was increased at 72 h. The formation of organic acid as hexanoic acid could
explain the decrease in SCFA in this sample. Hexanoic acid formation occurs through a carboxylic
acid chain elongation process, which uses reverse β-oxidation of acetic and/or n-butyric acid, and
ethanol or lactic acid as an electron donor (de Araújo Cavalcante, Leitão, Gehring, Angenent, &
Santaella, 2016). Besides, important changes were observed in the metabolites profile of dinner
menus at 12 and 72 h, which indicates that presence of fatty acid esters would occur over a longer
portion of the colon, especially in TM-D and AM-D. These compounds were identified in patients
who presented with chronic diseases of the gastrointestinal tract, especially in Crohn's disease
(Walton et al., 2013).
Capítulo 7. RESULTADOS Y DISCUSIÓN
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Figure 2 Cumulative concentration (%) of volatile gut metabolites by chemical groups between
the in vitro colonic fermentation extracts of blank, rafinose and indigestible fraction isolated from
Modified Mexican Dinner (MM-D), Traditional Mexican Dinner (TM-D), and Alternative
Mexican Dinner (AM-D) at 12 and 72 h. Detailed of identification number for each volatile
compound are outlined in the Supporting Information Table SI†.
Secondly, a statistical descriptive approach (PCA) showed that the fermentation of different corn
products for 12, 24, 48 and 72 h clearly affects the fecal metabolic fingerprint (Figure 3). The first
two principal components (PC) explained above 50% of the variation in the data (Figure 3A).
These components separate the volatiles with potentially healthy effects presenting positive
Eigenvectors in PC1 (eg, acetic, propionic and butyric acid,) from those volatiles with potentially
harmful effects presenting positive Eigenvectors on PC2 (e.g. dimethyl disulfide, dimethyl
trisulfide, indole and fatty acid esters). In fact, a good separation was observed for raffinose and
blank at all fermentation times (Figure 3b). In dinner menus, it was notable that MM-D promote a
harmful metabolic profile During the first 48 h of fermentation. TM-D and AM-D in intermediate
and long fermentation times (24, 48 and 72 h) the samples do not follow identified patterns. In this
work, the importance of improving diets with high indigestible carbohydrates content was
manifested in gut metabolic profile produced during in vitro colonic fermentation.
12 h of fermentation
Blank Raffinose MM-D TM-D AM-D
Cum
ulat
ive
Con
cent
ratio
n (%
)
0
20
40
60
80
100
72h of fermentation
Blank Raffinose MM-D TM-D AM-D
Cum
ulat
ive
Con
cent
ratio
n (%
)
0
20
40
60
80
100
Amines Alcohols and polyolsAlkanesAlkyl halidesBenzene and substituted derivativesBenzothiazolesCarbonyl compoundsFatty acid estersIndoles and derivativesOrganic acidOrganic disulfidesOrganic phosphoric acidsOrganic trisulfidesSCFA
Capítulo 7. RESULTADOS Y DISCUSIÓN
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Figure 3 Principal components plots (a) metabolites production “PC Loadings” (b) and sample
classification “PC scores” (%) during in vitro fermentation from indigestible fraction isolated
dinner menus (MM-D; Modified Mexican Dinner, TM-D; Traditional Mexican Dinner, AM-D;
Alternative Mexican Dinner). Detailed of identification number for each volatile compound are
outlined in the Table SI†.
3.6 Correlation between gut metabolites, pH and antioxidant activity
The pH values during in vitro colonic fermentation was positive correlated (p<0.05) with
trimethylamine, phenol, phenol 4-methyl-, pentadecanoic acid, ethyl esther, phenol, 2,4-bis(1,1-
dimethylethyl) and indole negative correlated for SCFA and pentanoic acid, (Figure 4). A
significant positive correlations were found between DPPH and FRAP AOX and SCFA and
pentanoic acid and negative correlation with was found mainly for phenol and phenol 4-methyl.
Some metabolites with negative effects in pH and AOX found in this work (e.g. trimethylamine as
precursor of trimethylamine N-oxide and indole as precursor of indoxyl sulfate) have been
associated with the development of atherosclerosis and chronic kidney disease (Bäckhed, 2013;
Poesen et al., 2014).
1
2
3
45
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28 29
30
31
32
33
34
35
36
37
38
39
40
41
42
4344
45 46
47
48
49
50
51
52
53
54
-1.0 -0.5 0.0 0.5 1.0PC 1
Variance Explained: 30.27%
-1.0
-0.5
0.0
0.5
1.0PC
2V
aria
nce
Expl
aine
d: 1
8.66
% 1
2
3
45
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
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28 29
30
31
32
33
34
35
36
37
38
39
40
41
42
4344
45 46
47
48
49
50
51
52
53
54
12-Blank
12-Raffinose
12-MM-D
12-TM-D12-AM-D
24-Blank24-Raffinose
24-MM-D
24-TM-D
24-AM-D
48-Blank
48-Raffinose
48-MM-D
48-TM-D
48-AM-D 72-Blank
72-Raffinose72-MM-D
72-TM-D
72-AM-D
-10 -8 -6 -4 -2 0 2 4 6 8PC 1
-6
-4
-2
0
2
4
6
8
PC 2
12-Blank
12-Raffinose
12-MM-D
12-TM-D12-AM-D
24-Blank24-Raffinose
24-MM-D
24-TM-D
24-AM-D
48-Blank
48-Raffinose
48-MM-D
48-TM-D
48-AM-D 72-Blank
72-Raffinose72-MM-D
72-TM-D
72-AM-D
A) B)
Capítulo 7. RESULTADOS Y DISCUSIÓN
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Figure 4. Pearson’s R correlations between gut microbial metabolites, pH values and antioxidant
capacity (DPPH antiradical activity and FRAP chelating activity) in extracts obtained during in
vitro colonic fermentation of indigestible fraction isolated from dinner menus (▲Increase,
▼decrease and ▬ non-correlation, p<0.05).
pH DPPH FRAPTrimethylamineAcetic acid, methyl esterEthanolMethyl propionateTrichloromethaneButanoic acid, methyl esterDisulfide, dimethylButanoic acid, ethyl esterPentanoic acid, methyl esterBenzene, 1,3-dimethyl-Pentanoic acid, ethyl esterHexanoic acid, methyl ester2-Heptanone, 4-methyl-Hexanoic acid, ethyl esterHeptanoic acid, methyl esterDimethyl trisulfideAcetic AcidHeptanoic acid, ethyl esterDodecaneBenzene, 1,3-dichloro-Octanoic acid, methyl esterPropanoic acidBenzaldehydeOctanoic acid, ethyl esterNonanoic acid, methyl ester1,3-Benzenediol, 4-ethyl-Butanoic acidNonanoic acid, ethyl esterAcetophenoneDecanoic acid, methyl esterPentanoic acidDecanoic acid, ethyl esterUndecanoic acid, methyl esterHexanoic acidDodecanoic acid, methyl esterBenzothiazoleHeptanoic acidDodecanoic acid, ethyl esterButylated HydroxytoluenePhenolTridecanoic acid, methyl esterPhenol, 4-methyl-Octanoic AcidMethyl tetradecanoateMethyl myristoleateTributyl phosphatePentadecanoic acid, methyl esterPhenol, 2,4-bis(1,1-dimethylethyl)IndoleDodecanoic acidMethyl hexadec-9-enoateHexadecanoic acid, methyl esterHexadecanoic acid, ethyl esterEthyl Oleate
Capítulo 7. RESULTADOS Y DISCUSIÓN
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5. Conclusions
This study represents a new contribution on the in vitro potential effects of food habits on bacterial
metabolites production. IF-Dinner menus had extractable and non-extractable polyphenols, as
main bioactive compounds, but they do not contribute to an anti-radical AOX. Although the
differences in IF content, the magnitude of SCFA production in dinner menus was similar to blank
(without substrate) except for the AM-D menú which may contain resistant starch in IF
composition. That could be related to the low contribution of dietary fiber in Mexican diet. PCA
allowed the identification of metabolic patterns that were associated with beneficial health effects
(Low pH and high AOX). MM-D microbial metaboitos may cause adverse effects on the health of
the population. Bacterial metabolites as phenol and phenol 4-methyl were associated with
increased pH and decreased AOX in the fermentation extracts. More studies are needed to confirm
our results.
Acknowledgements The authors thank the schoolchildren who donated stool voluntarily for
participating in this study. Zamora-Gasga, VM; acknowledge the fellowship to CONACYT-
Registration number: 253795).
Conflict of interest statement
The authors have declared no conflict of interest.
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Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of blank, rafinose and indigestible fraction isolated from dinner menus (MM-D; Modified Mexican diet, TM-D; Traditional Mexican Diet, AM-D;
Alternative Mexican diet) analyzed by SPME–GC/MS.
ID RT Volatil Name Chemical Group T12 T24 T48 T72 VC (%) Blank Raffinose MM-D TM-D AM-D Blank Raffinose MM-D TM-D AM-D Blank Raffinose MM-D TM-D AM-D Blank Raffinose MM-D TM-D AM-D
1 6.02 Amines Trimethylamine 17.91 0.00 28.77 14.38 29.97 11.25 0.00 12.80 8.24 28.33 19.65 0.00 18.49 4.92 24.00 45.05 0.00 10.11 4.02 11.43 84.45
2 7.41 Carboxylic acids and derivatives Acetic acid, methyl ester 0.00 0.00 0.00 19.78 23.72 0.00 0.00 0.00 22.35 34.89 0.00 0.00 0.00 19.71 42.97 4.03 0.00 34.29 14.50 7.27 127.59
3 8.36 Alcohols and polyols Ethanol 0.00 35.05 93.49 0.00 117.13 0.00 28.46 71.25 56.29 117.25 48.85 98.12 50.32 0.00 122.75 95.10 93.99 71.29 0.00 70.44 74.02
4 9.30 Carboxylic acids and derivatives Methyl propionate 3.80 3.70 0.00 4.79 5.16 0.00 4.76 0.00 12.39 9.87 4.24 4.51 0.00 5.58 12.78 0.00 10.42 9.25 3.69 0.00 89.27
5 10.92 Alkyl halides Trichloromethane 17.55 11.06 0.00 8.20 0.00 10.10 10.78 0.00 14.12 17.62 15.41 18.80 0.00 13.21 15.40 28.89 20.99 10.55 4.80 0.00 75.26
6 12.21 Fatty Acyls Butanoic acid, methyl ester 13.17 7.17 5.50 32.19 41.35 12.77 38.46 6.21 82.69 63.04 10.04 11.13 0.00 26.25 73.49 15.44 9.65 24.31 17.46 48.43 88.78
7 14.23 Organic disulfides Disulfide, dimethyl 135.14 0.00 199.66 173.63 148.02 0.00 0.00 106.90 133.76 163.97 63.17 0.00 15.80 6.50 41.54 32.00 0.00 19.01 17.09 22.86 109.28
8 14.94 Fatty Acyls Butanoic acid, ethyl ester 0.00 0.00 96.60 49.38 44.78 0.00 0.00 101.63 7.70 7.01 0.00 0.00 40.29 5.01 11.18 0.00 0.00 3.43 0.00 69.12 153.41
9 16.40 Fatty Acyls Pentanoic acid, methyl ester 12.24 3.68 15.04 86.12 32.18 8.76 0.00 30.25 139.41 167.17 17.55 0.00 12.63 132.47 302.25 32.69 4.98 114.83 205.02 99.51 117.86
10 18.60 Benzene and substituted derivatives Benzene, 1,3-dimethyl- 5.83 0.00 6.35 3.96 5.85 4.08 4.08 5.36 5.62 4.39 0.00 3.39 0.00 4.57 7.45 8.99 9.69 8.88 0.00 3.87 64.19
11 19.15 Fatty Acyls Pentanoic acid, ethyl ester 0.00 0.00 0.00 149.88 107.01 0.00 0.00 302.09 10.26 14.81 0.00 0.00 260.79 11.52 43.64 0.00 0.00 16.05 29.93 793.46 216.72
12 20.56 Fatty Acyls Hexanoic acid, methyl ester 14.17 0.00 8.42 52.57 21.64 49.25 0.00 27.25 273.36 42.32 164.77 6.59 50.62 1085.23 78.63 211.31 0.00 940.18 1065.90 21.68 176.91
13 21.30 Carbonyl compounds 2-Heptanone, 4-methyl- 3.72 0.00 10.09 5.70 8.68 0.00 4.81 8.80 0.00 5.01 4.67 4.24 3.94 0.00 4.99 7.81 7.89 0.00 0.00 4.30 79.09
14 23.08 Fatty Acyls Hexanoic acid, ethyl ester 0.00 0.00 76.74 87.38 34.88 0.00 0.00 324.84 13.50 8.28 9.35 0.00 629.83 45.45 37.24 13.93 0.00 70.70 100.33 573.19 183.93
15 24.59 Fatty Acyls Heptanoic acid, methyl ester 15.21 0.00 7.89 76.37 43.20 114.80 0.00 51.18 902.36 146.15 510.74 0.00 183.23 7048.82 169.21 634.37 13.46 8426.79 9204.20 76.37 215.83
16 26.00 Organic trisulfides Dimethyl trisulfide 502.99 0.00 1350.18 955.66 590.14 0.00 0.00 555.46 477.72 603.53 65.98 0.00 42.11 8.90 193.20 64.85 0.00 29.04 27.88 51.02 138.32
17 26.38 SCFA Acetic Acid 0.00 184.72 0.00 0.00 0.00 0.00 240.20 0.00 5.44 5.02 0.00 193.79 5.97 2.96 10.98 1.68 405.97 29.57 20.13 48.17 191.14
18 26.86 Fatty Acyls Heptanoic acid, ethyl ester 0.00 0.00 103.97 34.82 36.13 5.11 0.00 595.93 32.52 8.05 31.02 0.00 1883.68 293.14 21.98 44.96 0.00 710.49 756.77 764.87 177.75
19 27.45 Alkanes Dodecane 0.00 0.00 5.77 4.60 4.98 0.00 0.00 4.89 3.29 4.14 0.00 4.70 4.39 0.00 3.96 0.00 3.90 0.00 0.00 3.89 94.98
20 28.03 Benzene and substituted derivatives Benzene, 1,3-dichloro- 0.00 0.00 12.00 13.37 0.00 0.00 0.00 7.66 10.09 14.97 0.00 0.00 5.82 0.00 10.74 0.00 0.00 6.86 6.73 5.65 113.58
21 28.51 Fatty Acyls Octanoic acid, methyl ester 31.33 10.33 0.00 674.68 501.52 114.96 0.00 7.80 1031.37 1016.15 299.41 0.00 23.14 1957.45 1459.89 439.78 5.43 2625.29 2202.15 263.12 129.91
22 29.49 SCFA Propanoic acid 0.00 82.29 0.00 0.00 0.00 0.00 102.06 0.00 17.02 0.00 0.00 93.68 2.05 3.50 8.32 0.00 92.31 5.08 2.76 28.76 169.42
23 30.02 Benzene and substituted derivatives Benzaldehyde 26.98 18.36 24.41 26.29 26.40 23.32 21.91 14.22 19.57 0.00 22.19 31.97 14.09 11.81 16.55 55.60 0.00 0.00 0.00 21.36 70.28
24 30.55 Fatty Acyls Octanoic acid, ethyl ester 0.00 0.00 138.90 578.83 364.07 5.42 0.00 66.18 0.00 47.41 15.24 0.00 164.57 0.00 121.96 33.02 0.00 214.37 127.10 348.92 141.75
25 32.20 Fatty Acyls Nonanoic acid, methyl ester 11.79 0.00 0.00 117.22 136.02 0.00 0.00 0.00 218.52 301.83 44.85 0.00 5.15 224.52 465.74 53.64 0.00 193.85 113.38 63.71 130.53
26 32.70 Benzene and substituted derivatives 1,3-Benzenediol, 4-ethyl- 13.25 3.90 0.00 24.90 0.00 4.86 6.29 15.72 23.86 25.92 6.30 9.29 6.68 12.13 10.11 4.45 8.96 10.20 4.83 6.66 76.30
27 33.39 SCFA Butanoic acid 1.20 18.10 0.00 83.00 0.70 1.23 21.09 0.00 1.33 0.31 0.54 15.30 0.73 0.97 1.41 0.43 9.46 0.61 0.22 3.15 235.52
28 34.41 Fatty Acyls Nonanoic acid, ethyl ester 0.00 0.00 50.56 38.95 51.21 0.00 0.00 26.23 17.03 21.40 0.00 0.00 24.29 14.69 46.22 10.61 0.00 21.30 11.31 45.26 98.58
29 35.14 Benzene and substituted derivatives Acetophenone 12.47 0.00 197.71 46.25 43.02 0.00 0.00 145.34 49.95 44.22 15.02 0.00 96.48 32.74 33.28 36.99 0.00 91.68 0.00 35.20 119.74
30 36.69 Fatty Acyls Decanoic acid, methyl ester 40.71 39.16 15.01 3159.65 4109.32 32.96 4.54 14.64 3172.16 4468.53 52.76 4.76 24.62 6513.80 9647.04 85.27 24.86 491.03 1879.75 2228.69 148.77
31 39.24 Fatty Acyls Pentanoic acid 20.48 185.54 7.94 16.85 0.00 23.04 160.60 9.93 33.14 0.00 29.85 246.82 7.56 38.28 30.80 20.30 180.94 45.16 0.00 22.37 137.33
32 39.80 Fatty Acyls Decanoic acid, ethyl ester 0.00 0.00 287.55 1155.40 1382.79 0.00 0.00 123.20 210.62 372.26 0.00 0.00 123.46 217.17 852.44 0.00 0.00 65.67 137.04 1687.66 155.09
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33 43.04 Fatty Acyls Undecanoic acid, methyl ester 0.00 0.00 0.00 59.60 116.55 0.00 0.00 0.00 98.97 211.90 0.00 0.00 0.00 131.07 310.56 6.82 0.00 19.78 53.70 60.85 157.74
34 45.97 Fatty Acyls Hexanoic acid 33.52 32.22 0.00 0.00 0.00 46.51 34.24 11.07 0.00 0.00 112.52 71.02 20.20 255.38 0.00 152.78 38.91 310.87 155.66 0.00 140.96
35 48.45 Fatty Acyls Dodecanoic acid, methyl ester 110.98 34.31 0.00 1578.77 4941.14 111.89 8.76 0.00 2675.44 7864.62 145.25 15.33 0.00 3410.43 9299.80 180.01 16.24 348.87 1634.76 2865.39 155.70
36 49.92 Benzothiazoles Benzothiazole 6.92 5.33 21.42 25.30 0.00 5.83 5.74 18.21 7.04 0.00 7.70 0.00 0.00 7.18 0.00 10.78 17.27 12.06 16.98 20.14 85.40
37 50.22 Fatty Acyls Heptanoic acid 18.24 15.57 0.00 7.95 0.00 74.56 22.46 33.82 34.20 0.00 268.61 35.67 0.00 686.81 0.00 341.28 51.79 784.48 598.19 0.00 169.34
38 50.45 Fatty Acyls Dodecanoic acid, ethyl ester 17.72 0.00 127.86 379.57 801.69 11.76 0.00 64.52 93.90 278.78 19.52 0.00 54.63 107.89 473.15 47.88 0.00 49.14 110.18 866.25 148.50
39 50.70 Benzene and substituted derivatives Butylated Hydroxytoluene 41.27 28.58 9.21 70.91 47.37 32.81 17.56 9.23 39.89 0.00 44.14 22.15 19.80 44.63 35.12 57.76 29.84 60.33 36.83 0.00 60.38
40 51.14 Benzene and substituted derivatives Phenol 177.85 20.91 168.73 82.06 64.76 176.89 32.34 134.11 76.30 111.24 157.18 41.68 268.82 84.17 218.92 207.88 65.83 156.51 59.69 119.36 56.59
41 52.04 Fatty Acyls Tridecanoic acid, methyl ester 8.61 0.00 0.00 27.21 43.65 6.35 0.00 0.00 51.30 65.23 19.21 0.00 0.00 68.06 63.36 10.74 0.00 12.40 36.37 22.88 112.03
42 53.30 Benzene and substituted derivatives Phenol, 4-methyl- 398.84 86.17 384.39 305.84 262.02 370.28 115.45 434.07 337.66 400.50 583.56 115.41 684.37 468.19 321.18 903.69 165.89 834.26 377.03 254.97 56.90
43 53.58 Fatty Acyls Octanoic Acid 0.00 12.94 0.00 0.00 9.89 0.00 0.00 0.00 0.00 10.84 0.00 0.00 0.00 0.00 15.00 28.70 33.98 19.65 35.76 12.36 135.53
44 55.62 Fatty Acyls Methyl tetradecanoate 106.13 7.54 23.83 517.15 698.56 101.85 7.09 39.23 951.70 921.04 216.28 12.85 41.00 1260.18 975.61 198.24 16.21 274.45 776.63 357.54 108.58
45 55.86 Fatty Acyls Methyl myristoleate 18.99 4.22 13.64 183.92 83.29 19.69 0.00 21.25 190.76 278.51 36.13 0.00 18.88 200.10 389.79 37.70 0.00 75.13 85.17 0.00 131.85
46 56.22 Organic phosphoric acids and derivatives Tributyl phosphate 3.58 4.06 0.00 8.22 7.68 4.16 0.00 0.00 0.00 0.00 4.70 0.00 0.00 0.00 0.00 11.06 17.35 13.26 0.00 5.44 130.05
47 58.12 Fatty Acyls Pentadecanoic acid, methyl ester 15.84 0.00 0.00 19.53 24.53 8.45 0.00 7.43 31.07 38.79 21.84 0.00 0.00 32.65 28.08 13.43 0.00 23.28 25.81 11.88 84.60
48 58.76 Benzene and substituted derivatives Phenol, 2,4-bis(1,1-dimethylethyl) 16.70 0.00 0.00 12.00 9.94 0.00 0.00 0.00 10.00 11.15 16.83 0.00 0.00 19.23 15.33 19.13 0.00 0.00 13.58 12.53 98.08
49 60.75 Indoles and derivatives Indole 483.61 27.41 867.43 926.11 869.79 253.76 21.94 583.60 560.05 691.16 304.05 18.17 532.96 517.63 631.22 624.72 49.76 557.18 308.95 401.66 61.69
50 62.23 Fatty Acyls Dodecanoic acid 0.00 0.00 0.00 11.75 16.24 0.00 0.00 0.00 34.97 31.10 10.00 16.36 0.00 47.13 58.41 12.85 83.09 28.40 55.39 25.45 111.06
51 62.53 Fatty Acyls Methyl hexadec-9-enoate 0.00 0.00 14.22 104.63 0.00 0.00 0.00 14.43 78.49 152.16 0.00 0.00 14.51 105.05 146.72 0.00 0.00 0.00 71.38 55.14 139.52
52 62.94 Fatty Acyls Hexadecanoic acid, methyl ester 29.88 0.00 11.34 26.49 67.59 21.08 0.00 12.11 83.42 115.46 28.71 0.00 14.58 132.46 137.12 41.57 0.00 117.26 101.51 52.96 96.64
53 64.73 Fatty Acyls Hexadecanoic acid, ethyl ester 0.00 0.00 25.64 29.75 41.68 0.00 0.00 26.00 0.00 27.05 0.00 0.00 23.46 22.10 40.75 0.00 0.00 21.69 19.42 26.03 99.16
54 68.36 Fatty Acyls Ethyl Oleate 84.55 0.00 98.53 72.52 217.24 0.00 0.00 90.83 0.00 203.76 81.61 0.00 100.84 0.00 252.13 209.45 0.00 201.05 142.85 145.54 91.81
ID;Identification,RT;retentitontimeVC;Variationcoefficient
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7.5 Los metabolitos intestinales se asocian al pH y cambios en la actividad antioxidante
durante la fermentación colónica in vitro de productos procesados mexicanos de maíz.
Resumen
La principal fuente de carbohidratos en la mayoría de las dietas es el almidón, que es el
carbohidrato de almacenamiento de legumbres, hortalizas de raíz y cereales como maíz.1 México
tiene el mayor consumo per cápita de maíz en el mundo, que se consume principalmente como
tortilla y otros productos de maíz nixtamalizados.2 Existe una creciente evidencia que muestra
asociaciones entre patrones dietéticos tradicionales que incluían productos de maíz y la
disminución en el riesgo de enfermedades crónicas no transmisibles en la población mexicana.3,4
La presencia de polifenoles, almidón resistente (AR) y polisacáridos no amiláceos (PNA) en los
productos de maíz hacen que estos alimentos puedan ser considerados como nutracéuticos.5
Recientes investigaciones han reportado una relación inversa entre el consumo de fibra
(incluyendo RS), los polifenoles dietéticos y el riesgo de obesidad y diabetes asociados con
cambios en la microbiota intestinal.6 La microbiota intestinal puede producir metabolitos dañinos
asociados con enfermedades o compuestos beneficiosos que protegen contra la enfermedad. La
microbiota intestinal ejerce un papel primordial promoviendo la fermentación de fibra soluble y
RS para la producción de ácidos grasos de cadena corta (AGCC), principalmente ácido acético,
propiónico y butírico.7 Además, los compuestos nitrogenados que tienen el potencial de promover
el cáncer.8 Claramente, la dieta influye en el perfil de los metabolitos intestinales y estos juegan un
papel importante en la salud del huésped. Sin embargo, no existen datos sobre la fermentación
colónica de componentes indigestibles aislados de productos de maíz mexicanos. En este sentido,
se han utilizado modelos de fermentación fecal in vitro para evaluar la producción de metabolitos
intestinales debido a que estos resultados son difíciles de medir in vivo.9 La microextracción en
fase sólida (MEFS) seguida de cromatografía de gases-espectrometría de masas (CG- EM) se han
utilizado para analizar el perfil de los metabolitos intestinales.10 Por lo anterior, el objetivo de este
capítulo fue determinar las correlaciones entre los cambios en el pH, los valores de la actividad
antioxidante y la concentración de metabolitos intestinales formados durante la fermentación
colónica in vitro de la fracción indigestible aislada de tres productos de maíz: Totopos del istmo,
tortilla de maíz horneadas y una tortilla de maíz tradicional.
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Gut metabolites are associated to pH and antioxidant activity changes during in vitro colonic
fermentation of Mexican processed corn products.
Abstract
Diet is a major factor driving the composition and metabolism of the colonic microbiota. In
Mexico, nixtamalized corn products are widely consumed. However, there are no reports
addressing the production of metabolites during the colonic fermentation of their indigestible
fraction (IF). The aim of this work was to determine changes in antioxidant capacity (AOX: DPPH
and FRAP), pH values, short chain fatty acid (SCFA) concentration and relative metabolite
production during in vitro colonic fermentation of IF isolated from three corn products: Istmo
Totopos (IT), baked corn tortilla (BCT), and traditional corn tortilla (TCT). IFs were isolated after
withstanding a simulated in vitro gastrointestinal digestion, characterized and used as substrate for
in vitro colonic fermentation using a human fecal batch culture model. Fermentation extracts were
characterized by gas chromatography–mass spectrometry analysis using a headspace solid-phase
micro extraction technique (SPME-GC-MS.) Significant differences (p<0.05) were found on pH
values and AOX of the samples and a time dependence was observed. TCT and BCT presented the
highest DPPH values after 24 h of fermentation (60.21 and 53.21 mmol TE/g IF, respectively).
Butyric acid production was higher (p<0.05) from TCT (331.96 mmol·L-1 at 48h) than from a
fermentable reference substrate (raffinose: 144.93 mmol·L-1). Besides, 46 volatile compounds
were detected by SPME-GC-MS and two principal components were identified. Changes in
bacterial metabolites profile were found after 12 and 48 h of fermentation, except for IT.
Trimethylamine, phenol and phenol 4-methyl- were associated with increased pH and decreased
AOX. Corn products may have potential beneficial effects on gut health.
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Graphical Abstract
1 Introduction
Our diet is composed of a variety of dietary macronutrients- carbohydrates, proteins, fats and
fibers. Starch is the main is storage carbohydrate in dried legumes, root vegetables and cereals,
such as corn, and represents the major source of carbohydrates in most diets.1 Mexico has the
highest per capita consumption of corn, which is mainly eaten as corn tortilla (325 g of tortilla per
person/ per day) and others nixtamalized corn products.2 Growing evidence shows associations
between traditional dietary patterns that include corn products and decreased risk of chronic non-
communicable diseases in the Mexican population.3, 4 The presence of polyphenols (PP), resistant
starch (RS) and non-starch polysaccharides (NSP) in corn products make these foods good
candidates to qualify as nutraceuticals or functional foods, promoting health benefits.5 Recent
research has revealed an inverse relationship between consumption of dietary fiber (including RS)
and dietary PP and the risk of obesity and diabetes, a relationship that seems associated with
changes in the gut microbiota.6 The gut microbiota can either produce harmful metabolites
associated with certain pathologies or beneficial compounds that protect against disease. The
microbiota exerts a primordial role in the fermentation of soluble dietary fiber and RS that leads to
the production of short-chain fatty acids (SCFA), mainly acetic, propionic, and butyric acids.7 In
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addition, nitrogenous compounds (fermentation products of resistant proteins), particularly N-
nitrous, are potential cancer promoters and exert carcinogenic effects via DNA alkylation, which
can cause mutations.8 Clearly, diet has marked influence on gut metabolites profile and this has an
important impact on the host health. It is noteworthy, however, that despite the relative abundance
of corn products in the Mexican diet, no data are available on the colonic fermentation of their
indigestible components. In this sense, in vitro fecal fermentation models have been used to
evaluate the production of gut metabolites, since these outcomes are difficult to measure in vivo.9
Headspace solid-phase microextraction (HS-SPME) followed by Gas Chromatography–Mass
Spectrometry (GC-MS) have been used to analyze gut metabolites profile, given its better matrix
clean-up features, relatively inexpensiveness, as well as its fats operation and easy automation
possibilities.10 In view of all the above, in the present study, an in vitro gastrointestinal digestion
model was used to quantify and isolate the indigestible fraction (IF) in three nixtamalized corn
products of ample consumption in Mexico: Istmo Totopo (IT), baked corn tortilla (BCT) and
traditional corn tortilla (TCT). The main objective was to submit the isolated IF to in vitro colonic
fermentation, to determine the correlations between changes in pH, antioxidant capacity (AOX)
values and gut metabolites concentration.
2 Materials and methods
2.1 Raw material
Three corn products consumed popularly in Mexico were utilized in this study: Istmo Totopos
(IT), baked corn tortilla (BCT), and traditional corn tortilla (TCT). IT were supplied by the
National Center for Genetic Resources (Jalisco, México) and were traditionally prepared with a
corn breed, ‘Zapalote Chico’, cultivated in Oaxaca, Mexico. BCT (Saníssimo, Corporativo Bimbo,
S.A. de C.V., Mexico DF, Mexico) and TCT (Maseca®, Gruma, S.A.B. de C.V., Nuevo Leon,
Mexico) were purchased from the local market. Each sample was homogenized in a food
processor (NB-101B, Nutribullet, China), frozen (-80 °C), freeze-dried (FreeZone 6, Labconco,
USA), milled, sieved with a mesh size of 500 µm and stored at -20 °C until analysis.
2.2 Chemical composition of corn products
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Moisture, ash, protein and fat contents were analyzed according to AOAC (1990)11 methods
925.10, 923.03, 920.87 and 920.39, respectively. Additionally, resistant starch (RS) was assessed
following a multienzymatic digestion protocol12 using pepsin (P-7000, Sigma Aldrich, St Louis
Missouri, USA), α-amylase (A-3176, Sigma Aldrich), and amyloglucosidase (A-9913, Sigma
Aldrich).
2.3 Simulated in vitro gastrointestinal digestion
2.3.1 Quantification and isolation of indigestible fraction
A digestion procedure that mimics the physiological situation in the upper digestive tract (stomach
and small intestine) was utilized for the quantification of indigestible fraction (IF).13 In order to
increase the yield of isolated IF, certain preparative adjustments were included. Briefly, 9 g of
freeze-dried samples were sequentially incubated with: a) pepsin (0.6 mL of a 300 mg/mL solution
in 0.2 M HCl-KCl buffer, pH 1.5, 40 °C, 1 h, P-7000, Sigma Aldrich, USA), b) pancreatin (3 mL
of a 5 mg/mL solution in 0.1 M phosphate buffer; pH 7.5, 37 °C, 6 h, P-1750, Sigma Aldrich,
USA), c) alpha-amylase (3 mL of a 120 mg/mL solution in 0.1 M Tris-maleate buffer, pH 6.9, 37
°C, 16 h, A-3176, Sigma Aldrich). Samples were transferred into dialysis tubes (D9527 -30.48 m
avg. Flat width 43 mm, 14000 Da, Sigma Aldrich) and dialyzed against tap water for 48 h at 25 °C
to remove the digestion-released compounds.14 Total IF (dialysis retentate) was collected, freeze-
dried, milled (IKA M20, USA) and sieved using a 500 µm mesh, and placed in bags with seal,
which were stored at -20 ° C until subsequent analyses.
2.4 In vitro colonic fermentation by human microflora
Total IF isolated from the corn products was fermented in a batch culture system with pre-
conditioned nutritive medium and under strict anaerobic conditions, at 37 °C.15 Fresh faecal
samples were collected (maximum 2 h after defecation) from four healthy adults (25–35 years)
who declared no gastrointestinal diseases and no intake of antibiotics at least 3 months before the
beginning of the study. The medium was adjusted to pH 7 using HCl and reduced in an anaerobic
chamber for 12 h prior to the fermentation. A 1:10 (w/v) dilution of the faecal samples with 0.1
mol/L, pH 7 phosphate buffer was prepared and homogenized in a digital high-speed homogenizer
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system (IKA-Ultra-Turrax, T18, USA; 1 min, 6000 rpm). The resulting faecal suspension (1 mL)
was distributed in disposable tubes (containing 9 mL nutritive medium) and 0.1 g of the isolated
total IF from each corn product were added. In parallel, two different controls were incubated
under the same conditions: a) rafinosse was incubated in medium with faeces inoculum as a
fermentable indigestible sugar reference, and b) the faecal suspension was incubated without any
additional substrate, and used as a negative control. All incubations were performed in triplicate
and both samples and controls were analyzed after 12, 24, and 48 h of fermentation. Changes in
pH values were measured at each time point. The material obtained at each time of fermentation
was centrifuged (Hermle Z 323 K; Wehingen, Germany) at 3500 × g for 15 min at 4 °C.
Supernatants were divided into two parts: one was used for metabolite profile analysis and the
other was for AOX assays. Samples were always kept at −80 °C until analysis.
2.4.1 Metabolite profile analysis
Headspace solid-phase microextraction (HS-SPME) conditions
Fermentation supernatants (500 mg) were placed into a 20 mL vial sealed with a magnetic cap
with a poly-tetra-fluoro-ethylene (PTFE)/silicon septum. HS-SPME method was used for the
extraction and concentration of volatile compounds.16 Briefly, a 2 cm polydimethylsiloxane-
divinylbenzene-carboxen (PDMS/DVB/CAR) solid phase microextraction (SPME) fiber was used.
Sample vials were placed into a Gerstel MPS2Autosampler. The samples were stirred (250 rpm) at
45 °C for 5 min. The DVB/CAR/PDMS fiber was then exposed to the headspace, and the samples
were stirred for 120 min (250 rpm at 45 °C). The fiber was then inserted into the injection port of
the GC system for thermal desorption (240 °C for 10 min) for GC/MS analysis. The extractions
were performed in triplicate. Five runs with twelve vials / run were performed and the tests order
was completely randomized.
Gas chromatography–mass spectrometry (GC–MS) analysis
The volatile constituents were analyzed with an Agilent 5975C VL mass selective detector
coupled to an Agilent 7890A gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA),
equipped with a DB-5MS capillary column (60 m X 250 µm X 0.25 µm; Agilent). Helium (flow
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rate, 1 mL/min) was used as the carrier gas. The injector temperature was 250 °C. The oven
temperature program was 40°C for 5 min, increased by 5°C /min to 200 °C and was maintained for
2 min, then increased by 20°C/min to 230°C and held for 15 min. Mass spectra were recorded at
70 eV in electron impact (EI) ionization mode. The temperature of the quadrupole mass detector
and ion source were 150 and 230°C, respectively. The injector was used in the splitless mode.
SCFA were quantified using standard curves of acetic, propionic and butyric acids. Tentative
identification of the volatile components was done by comparing the mass spectra of the samples
with the data system library MSD ChemStation software (Agilent G1701EAversion E.02.00.493).
Relative concentration of all fermentation metabolites versus acetic acid as internal standard was
calculated and the results were expressed in mmol/L extract.
2.4.2 Antioxidant capacity (AOX) during in vitro colonic fermentation
Changes in the AOX of the sample extracts during in vitro colonic fermentation at 12, 24 and 48 h
were measured by two methods: a) DPPH• radical scavenging activity: 1,1-Diphenyl-2-picryl
hydrazyl (DPPH) radical scavenging activity assay was determinate in aqueous-organic extracts.
Extract or Trolox standard (30 µL) were reacted with 200 µL of DPPH solution (190 µM) and
absorbance readings at 517 nm were taken after 10 min reaction using a multi-detection microplate
reader (Biotek, Synergy HT,Winooski VT, USA) with Gen5 software. Trolox (37.5 - 600 µM) was
used as the standard for the calibration curve, and the DDPH radical-scavenging activities were
expressed as Trolox equivalent (TE; mmol/g dry basis).17 b) Ferric-ion reducing antioxidant
power: The stock solutions included 0.3 M acetate buffer (0.3 M acetic acid and 0.3 M sodium
acetate), pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40 mM HCl and 20 mM
ferric chloride (FeCl3.6H2O) solution. The fresh working solution was prepared in a 10:1:1 ratio
prior to use and heated to 37oC in water bath before use. Briefly, 24 µL sample extracts or Trolox
standard were added to each well of a 96-well microplate and was dispensed 180 µL of FRAP
solution using multi-channel dispenser. Absorbance at 595 nm was taken after 30 min of reaction
using microplate reader (Biotek, Synergy HT, Winooski VT, USA) with Gen5 software. The
standard curve was used between 8.125x10-3 - 0.13 mmol Trolox and results are expressed in
Trolox equivalent (TE; mmol/g dry matter).18
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2.5 Statistical analysis
Results are expressed as mean ± SEM (n=3). Univariate testing of differences for corn products
was processed by one-way ANOVA/Fisher's Least Significant Difference test. Besides,
Multivariate analysis was applied for metabolites profile interpretation. Principal component
analysis (PCA) was used to determine patterns in relative metabolites production. Finally,
Pearson´s correlation coefficients were used to evaluate the relationship between individual
metabolites and changes in pH and AOX during in vitro colonic fermentation. All analyzes were
performed using STATISTICA software, version 10.0 (StatSoft. Inc. 1984–2007, Tulsa, OK,
USA). A significance level of α = 0.05 was used in data analysis.
3 Results and Discussion
3.1 Chemical composition
Table 1 shows the chemical composition of the different nixtamalized corn products. Differences
were found among the products (p<0.05). BCT exhibited the highest ash content of the three corn
samples, followed by IT and TCT (3.39, 2.40 and 1.35 g/100g db, respectively). The type of corn
(agronomic varieties) and calcium salts used as additives in the formulation of BCT could
influence the high value recorded for this sample.19 Regarding fat content, BCT and IT did not
exhibit appreciable difference (p>0.05), but TCT had a significantly lower content. The maize
cultivar employed can play an important role in these results; ‘Zapalote chico’ race is
characterized by high oil content and flotation index, and a low pericarp content.20 Fat content in
corn tortilla prepared with different nixtamalization procedures (3.41 to 4.60 g/100g db) have been
previously shown to have higher fat values than those reported here.21 On the other hand, the three
samples exhibited different protein contents (p<0.05); with TCT showing the highest level (8.91
g/100g db). Those differences in protein content may be attributed to temperature differences
during the hydrothermal treatment (nixtamalization); moisture content and time of processing
greatly influence the salt-soluble protein content of corn kernels.22
3.1.1 Resistant starch (RS) and indigestible fraction (IF) in corn products
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Starch is another important component in corn products. Starches can be classified into three types
according to their digestibility: rapidly digestible, slowly digestible, and resistant starch (RS).23 RS
is considered a bioactive compound forming part of the indigestible fraction. RS, insoluble and
soluble IF contents in corn products are presented in Table 1. RS content in TCT was the highest
(5.2 g/100 g) although not significantly different (p>0.05) from the level in IT (4.34 g/100g db).
These values were similar to those reported in corn tortilla made with kernels of different
endosperm (floury, intermediate, and vitreous) stored for 96 h at 4 °C (4.9, 4.4 and 5.6 g/100 g
respectively).24 The variations in RS contents may be related to starch structural changes
(intermolecular associations between amylose and amylopectin, amylose and lipids or amylose-
protein complexes) occurring during processing. In addition, storage of corn products potentiates
the formation of retrograded RS fractions .25
Table 1 Chemical composition and indigestible fraction content from three Mexican corn
products: Istmo Totopos (IT), Baked corn tortilla (BCT) and traditional corn tortilla (TCT (g/100g
db) 1
Parameter Mexican Corn products
IT BCT TCT
Chemical composition
Moisture 2 4.67 ± 0.13a 4.93 ± 0.06a 49.58 ± 0.72b
Ash 2.40 ± 0.02b 3.39 ± 0.04c 1.35 ± 0.05a
Fat 2.36 ± 0.12a 2.44 ± 0.03a 0.7 ± 0.03b
Protein 3 7.20 ± 0.13a 8.26 ± 0.29b 8.91 ± 0.04c
Available Starch 74.64 ± 2.03a 77.05 ± 0.28ab 78.02 ± 0.89b
Resistant Starch 4.34 ± 0.78a 3.2 ± 0.03b 5.2 ± 0.1a
Indigestible Fraction (IF) content
Total IF 4 27.22 ± 0.11a 25.54 ± 3.51a 25.39 ± 0.28a
Soluble IF 2.89 ± 0.60a 2.05 ± 0.16a 2.13 ± 0.48a
Insoluble IF 24.34 ± 0.50a 24.49 ± 3.41a 23.262 ± 0.78a
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1Values are mean ± standard error (n = 3); Dry basis (db). Means in rows marked with different
letters indicate significant difference (p <0.05). 2Expressed in wet basis. 3Conversion factor: % N x
6.25. 4 Total IF = Sum of soluble IF + insoluble IF.
Considering a 30 g serving, IT would supply 1.24 g RS/portion), which is almost twice RS supply
than in TCT (0.78 g/portion), a consequence of dilution effect by differences in moisture content.
(4.67 g/100 g wb for IT and 49.58 g/100 g wb for TCT). RS reaches the colon were it is fermented
producing short-chain fatty acids (SCFA), especially butyric acid), limiting glucose absorption in
the small intestine, causing a decrease in the rate of starch hydrolysis and reducing the total caloric
intake.5 IF content consists in food components that being unavailable for digestion in the small
intestine, pass into the colon where are also used as a substrate for fermentation by the gut
microbiota. IF is classified as: soluble IF, that comprises monosaccharides, disaccharides, and
oligosaccharides, and insoluble IF, which includes RS, indigestible protein, indigestible fat,
polyphenols, and non-starch polysaccharides (NSP: cellulose, hemicelluloses and lignin).13 The
studied corn products did not show differences in their soluble, insoluble and total IF contents
(p>0.05). Total IF content may be modified during the nixtamalization process; overcooking
caused by high temperatures allows softening and removal of the pericarp in corn kernels,
resulting in decreased indigestible carbohydrates contents.26 Besides, soluble to insoluble IF ratio
in corn products can be also affected by the temperature during processing. High cooking
temperatures produce pirodextrinization of starch and NSP, increasing the content of indigestible
soluble carbohydrates with concomitant decrease in the insoluble indigestible carbohydrate
fractions.21 Despite the lack of differences in IF content, present results support the idea of that
corn products as appreciable sources of dietary fiber (DF) in Mexican diets. DF exerts various
physiological functions associated whit human health, as it contributes to normal postprandial
glycemic/insulinemic responses, reduce blood cholesterol levels and may provide protective
effects against colon cancer.27, 28
3.3 pH and antioxidant capacity (AOX) change during in vitro colonic fermentation
3.3.1 pH Change
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Figure 1 A shows the pH values of the fermentation cultures at the beginning and end of the
incubation period. Changes in pH provide an overview of the process of fermentation. Starting pH
values ranged approximately between 7.05 and 7.3 before fermentation, and decreased in the IF-
containing samples already after 12 h fermentation, a change that is related to the production of
SCFA (Table 2). Predominant SCFA (acetic, propionic and butyric acids) have pKa values around
4.8 and their production upon carbohydrate fermentation decreases the intestinal lumen pH.29 The
pH values in corn products extracts were higher compared with the control sample (raffinose) at
all-time points (p < 0.05), indicating a relatively lower fermentability of IF. The pH significantly
declined from 7.05 to 4.83 during the fermentation of TCT (0 to 12 h), and then showed a slight
increase at 24, 48 and 72 h. This behavior was similar in the IT and BCT preparations. The
approximately 2 pH units decrease recorded during fermentation of the corn samples is indicative
of potential beneficial action of these products on the host health. The pH lowering effect has been
used as a marker of colonic cancer preventive power, impact on gut microbiota composition and
ability to prevent overgrowth of pH-sensitive pathogenic bacteria such as Enterobacteriaceae and
Clostridia.30, 31
3.3.2 DPPH radical scavenging activity and Ferrous ion chelating activity
Oxidative stress is involved in both inflammation, the process of initiation and progression of
different pathologies, such as colorectal cancer.32 In this in vitro study, AOX values showed
differences, depending on the substrate and time of fermentation (Figure 1B and C). Figure 1B
shows that the scavenging effects of raffinose on DPPH radical were proportional to fermentation
time. TCT and BCT preparations were more active after 24 h (60.1 and 53.2 mmol TE/g IF,
respectively), followed by a decline of about 50% at 48 h. IT exerted the highest DPPH
antioxidant activity at 12 h followed by a marked decrease from 24 h on (from 48.47 to 22.78
mmol TE/g IF, respectively). According to the FRAP antioxidant activity assay the various corn
products showed similar AOX after being fermented for 12 h, but their values were significantly
greater than those recorded for the raffinose reference (p<0.05). After 24 h of fermentation, FRAP
values of BCT preparation increased significantly (p<0.05), reaching the maximum value of the
three corn products, followed by a reduction up at the end of the experiment (48 h), maintaining a
value of 10.93 mmol TE/g IF. FRAP values of IT decreased by 82.1% in the 12 to 48 h
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fermentation period. Typically, the in vitro antioxidant action of foods is limited to liberation of
antioxidant components during the gastrointestinal digestion. Corn tortillas have a high content of
ferulic acid, carotenoids and anthocyanins that might reach the colon and exert a direct antioxidant
effect assigned to radical scavenging or metal ion chelation.5, 33
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Figure 1 Changes during in vitro colonic fermentation of (-○-) blank, (-●-) raffinose (positive control), and indigestible fraction
isolated from (-�-) Istmo Totopo, (-◊-) Baked corn tortilla and (-□-) Traditional corn tortilla: a) pH values plot b) DPPH
Antiradical activity plot and b) FRAP chelating activity plot. Values are means ± SEM (n=3). For DPPH and FRAP, the values
are reported after subtraction of blank sample (no added substrate) at the same time point. *Significant difference using Two-
Way ANOVA/Fisher's LSD test (Samples ×Time interaction, p<0.05)
Fermentation time (h)
0 12 24 48
pH
3.0
4.0
5.0
6.0
7.0
8.0 * * *
Fermentation Time (h)
0 12 24 48
DPP
H A
ntio
xidan
t Act
ivity
(mM
TE/
g su
bstra
te d
b)
10
20
30
40
60
70
0
50
* * *
Fermentation time (h)
0 12 24 48
FRA
P A
ntio
xidan
t Act
ivity
(m
M T
E /g
subs
trate
db)
0
5
10
15
20
25* * *
*
A) B) C)
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Also, RS can stimulate the fermentative production of butyric acid and this metabolite may also
exert in vitro and in vivo antioxidant effect.34 Pre-clinical study has shown that oxidative stress in
the colonic mucosa can be beneficially modulated by butyric acid, increasing glutathione (GSH)
production and decreasing the formation of reactive oxygen species.35 Therefore, the effect that
fermentable indigestible components exert on the growth of various colonic microbial strains and
the production of gut metabolites with AOX activity stress the impact that the type of ingested
food has on human health.
3.4 Short Chain Fatty Acids (SCFA): Profile During Fermentation
The concentrations of SCFA measured at 12, 24 and 48 h of fermentation are shown in Table 2.
Acetic and propionic acid concentrations in the corn products preparations were significantly
lower than those registered using raffinose as substrate (p<0.05). However, butyric acid levels
after 24 and 48 h were higher in TCT than in the raffinose control (p<0.05). The concentrations of
acetic, propionic, and butyric acids in TCT were significantly higher than IT and BCT (p<0.05) at
48 h. The molar proportions of the different SCFA varied throughout the fermentation of the corn
products. At 12 h, raffinose exhibited a 54: 34: 13 molar ratio for acetic, propionic and butyric
acids, respectively; this profile resembled that found at 48 h (55: 32: 13).
But, for the IT substrate, a 41: 34: 25 molar ratio was found after 12 h, and a 34: 23: 43 proportion
was detected at the end of the 48 h period. These results indicate an increase in the production of
butyric acid and a decrease of acetic and propionic acid as the fermentation proceeds. The same
behavior was observed for the BCT and TCT substrates. This can be considered beneficial, given
that butyric acid stimulate apoptosis of colorectal carcinoma cells.36 The stimulation of butyric
acid production by IF from corn products may be explained by the cross-feeding between butyric
acid-producing bacteria and Bifidobacterium species. The ability of Faecalibacterium prausnitzii,
for instance, to increase butyric acid levels in the fermentation medium is explained by its higher
affinity for the substrate and it´s ability to the partially consume the acetic acid produced by
Bifidobacterium strains.37 Bifidobacterium longum, Bifidobacterium catenulatum,
Bifidobacterium bifidum, Lactobacillus gasseri and Lactobacillus salivarius were the main species
involved in SCFA production (mainly acetic acid).38
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Table 2. Production of short-chain fatty acids (SCFAs, mmol L-1) at 12, 24 and 48 h of in vitro fermentation of blank, raffinose
and indigestible fraction isolated from Istmo Totopo (IT), baked corn tortilla (BCT) and traditional corn tortilla (TCT) *.
Metabolite / fermentation time Substrate
Blank Raffinose IT BCT TCT Acetic acid
12 h 27.54 ± 1.55aA 367.51 ± 2.53cA 145.10 ± 1.11bA 199.77 ± 47.51bA 333.90 ± 49.4cA 24 h 35.86 ± 2.64aA 729.15 ± 28.80eB 230.37 ± 19.77bB 308.48 ± 13.91cB 468.55 ± 12.33dB 48 h 19.09 ± 9.62aA 611.85 ± 4.17dC 188.58 ± 5.10bAB 171.73 ± 18.83bA 498.00 ± 18.60cB
Propionic acid 12 h 9.03 ± 4.60aA 232.04 ± 1.56cA 120.45 ± 0.51bA 150.07 ± 30.64bA 176.33 ± 15.01bcA
24 h ND 421.44 ± 38.43dB 135.29 ± 12.71bA 160.72 ± 25.04bA 284.98 ± 10.40cB 48 h 4.12 ± 4.12aA 362.17 ± 58.37dB 123.93 ± 9.13bA 135.72 ± 9.64bA 293.75 ± 6.15cB
Butyric acid 12 h 3.46 ± 1.84aA 86.19 ± 0.14bA 89.90 ± 4.51bA 145.88 ± 1.96bA 141.44 ± 59bA
24 h 7.51 ± 7.51aA 109.61 ± 22.81bA 174.53 ± 20.79bB 178.53 ± 29.19bAB 363.01 ± 13.18cB 48 h 3.88 ± 1.95aA 144.93 ± 27.30bA 235.21 ± 47.6bB 236.58 ± 30.03bB 331.96 ± 9.34dB
*The values are reported in mmoL· L-1 produced per 100 mg substrate as mean ± SEM of three replicates; Different lowercase letters indicate significant differences in rows among substrates for a time. Different capital letters in columns indicate significant differences between times for a same substrate (Two-way ANOVA and Fisher LSD Test, p< 0.05). ND: Not detected.
Capítulo 7. RESULTADOS Y DISCUSIÓN
161
The composition of the indigestible fractions, particularly their content of RS, could be involved in
the SCFA profile produced by fermentation of the corn product substrates. Recently the
manipulation of the gut microbiota and SCFA production using RS was related with protection
against colitis-associated colorectal cancer in rats.39
3.5 Production of volatile gut metabolites
The kinetic analysis by GC-MS/SPME of the volatile metabolites produced by fecal microbiota at
the time points 12, 24, and 48 h showed different metabolic profiles in relation to the different
substrates utilized (See Supporting Information Table SI). A total of 46 different metabolites
belonging to the families of alcohols and polyols, alkanes, amines, benzene and substituted
derivatives, carbonyl compounds, ethers, fatty acid esters, indoles and derivatives, olefins, organic
acids, organic disulfides, organic trisulfides and SCFA were detected (Figure 2). No changes were
observed in the relative concentrations of organic acids, SCFA and indole during the fermentation
of IT, indicating that this substrate is steadily fermented, which suggests that fermentation of this
substrate would occur over a longer portion of the colon. Structural differences in the indigestible
carbohydrates present in the samples could favor the maintenance of the bacteria and their
metabolites.40
Nevertheless, it is noticeable that as the fermentation process the fermentation of TCT and BCT
went on the concentration of organic acids decreased and the production of metabolites from the
indole group increased. Indole and derivatives are products of the metabolism of aminoacids by
bacteria including Bacteroides thetaiotaomicron, B. eggerthii, B. ovatus, B. fragilis,
Parabacteroides distasonis, Clostridium bartlettii, and Eubacterium hallii.41 The products of the
fermentation of amino acids, such as phenol and p-cresol, are considered toxic to the intestine.42
However, indole (metabolite synthesized from tryptophan) is able to modulate the hormone GLP-1
secretion by L-enteroendocrine cells43 and could be involved in development and neural
function.44
Capítulo 7. RESULTADOS Y DISCUSIÓN
162
Figure 2 Cumulative concentration (%) of volatile gut metabolites by chemical groups during in vitro colonic fermentation
extracts of blank, raffinose and indigestible fraction isolated from Istmo Totopo (IT), Baked corn tortilla (BCT) and traditional
corn tortilla (TCT) at 12 and 48 h. Detailed of identification number for each volatile compound are outlined in the Table SI†.
12 hours of fermentation
Blank Raffinose Istmo Totopo Baked Tortilla Corn Tortilla
Cum
ulativ
e co
ncen
tratio
n (%
)
0
20
40
60
80
100
48 hours of fermentation
Blank Raffinose Istmo Totopo Baked Tortilla Corn Tortilla
Cum
ulativ
e co
ncen
tratio
n (%
)
0
20
40
60
80
100
Alcohols and polyolsAlkanes Amines Benzene and substituted derivatives Carbonyl compounds EthersFatty acid ester Indoles and derivatives Olefins Organic acid Organic disulfides*0 Organic trisulfidesSCFA
IT BCT TCT IT BCT TCT
Capítulo 7. RESULTADOS Y DISCUSIÓN
163
Using statistical descriptive approach (PCA), here it is shown that the fermentation of different
corn products for 12, 24, and 48 h clearly affects the fecal metabolic fingerprint (Figure 3). The
first two principal components (PC) explained nearly 45% of the variation in the data (Figure
3A).These components separate the volatiles that are generally obtained from the fermentative
metabolism of carbohydrates with positive Eigenvectors in PC1 and negative Eigenvectors in PC2
(e.g., butyric acid, ethanol, organic acids and fatty acid esters) from those volatiles that are most
prevalent as products from protein metabolism with low Eigenvectors on PC1 and positive
Eigenvectors on PC2 (e.g. phenol, phenol, 4-methyl-, trimethylamine and dimethyl trisulfide). In
fact, a good separation of the 12 h time point from the other fermentation points was observed for
all samples, most evidently in the case of TCT (Figure 3B). Notably, the increase of butyric acid
represented the greatest variation registered during fermentation of the corn products. No other
SCFA was found to be significantly modified, suggesting that butyric acid was the main final
product from the microbial degradation of the IF isolated from corn products, especially in TCT.
Figure 3 Principal components plots A) metabolites production “PC Loadings” and B) sample
classification “PC scores” (%) during in vitro fermentation from indigestible fraction isolated from
Istmo Totopo (IT), Baked corn tortilla (BCT) and Traditional Corn Tortilla (TCT).
Trim ethy lam ine
Ethy l ether
Hexane
Ethanol
Butanoic acid, m ethy l ester
Disulfide, dim ethy l
Toluene
2,4-Dim ethy l-1-heptene
Pentanoic acid, m ethy l ester
Heptanal
Acetic Acid
Dim ethy l trisulfide
Propionic Acid
Benzene, 1,4-dichloro-
Benzaldehy de
Butiric Acid
Nonanal
Butanoic acid, 3-m ethy l-
Octanoic acid, m ethy l ester
Pentanoic acid Acetophenone
Benzaldehy de, 4-m ethy l-
2-Nonenal, (E)-
DecanalNonanoic acid, m ethy l ester
Pentanoic acid, 4-m ethy l-
Benzene, 1,3-bis(1,1-dim ethy lethy l)-
Hexanoic acidDecanoic acid, m ethy l ester
Heptanoic acid
Phenol
Benzenem ethanol, 4-m ethy l-
Phenol, 4-m ethy l-
Octanoic Acid
Dodecanoic acid, m ethy l ester
Nonanoic acid
Buty lated Hy droxy toluene
n-Decanoic acid
2-Propenoic acid, tridecy l ester
Indole
Diethy l Phthalate
Tetradecanal
9-Hexadecenoic acid, m ethy l ester, (Z)-
Hexadecanoic acid, m ethy l ester
Dibuty l phthalate
9,12-Octadecadienoic acid (Z,Z)-, m ethy l ester
-1.0 -0.5 0.0 0.5 1.0PC 1 (25.55%)
-1.0
-0.5
0.0
0.5
1.0
PC 2
(17.
88%
)
Trim ethy lam ine
Ethy l ether
Hexane
Ethanol
Butanoic acid, m ethy l ester
Disulfide, dim ethy l
Toluene
2,4-Dim ethy l-1-heptene
Pentanoic acid, m ethy l ester
Heptanal
Acetic Acid
Dim ethy l trisulfide
Propionic Acid
Benzene, 1,4-dichloro-
Benzaldehy de
Butiric Acid
Nonanal
Butanoic acid, 3-m ethy l-
Octanoic acid, m ethy l ester
Pentanoic acid Acetophenone
Benzaldehy de, 4-m ethy l-
2-Nonenal, (E)-
DecanalNonanoic acid, m ethy l ester
Pentanoic acid, 4-m ethy l-
Benzene, 1,3-bis(1,1-dim ethy lethy l)-
Hexanoic acidDecanoic acid, m ethy l ester
Heptanoic acid
Phenol
Benzenem ethanol, 4-m ethy l-
Phenol, 4-m ethy l-
Octanoic Acid
Dodecanoic acid, m ethy l ester
Nonanoic acid
Buty lated Hy droxy toluene
n-Decanoic acid
2-Propenoic acid, tridecy l ester
Indole
Diethy l Phthalate
Tetradecanal
9-Hexadecenoic acid, m ethy l ester, (Z)-
Hexadecanoic acid, m ethy l ester
Dibuty l phthalate
9,12-Octadecadienoic acid (Z,Z)-, m ethy l ester 12-B
12-R
12-IT
12-BCT
12-TCT
24-B
24-R
24-IT
24-BCT 24-TCT
48-B
48-R
48-IT
48-BCT
48-TCT
-6 -4 -2 0 2 4 6 8PC1 (25.55%)
-6
-4
-2
0
2
4
6PC
2 (1
7.88
%)
12-B
12-R
12-IT
12-BCT
12-TCT
24-B
24-R
24-IT
24-BCT 24-TCT
48-B
48-R
48-IT
48-BCT
48-TCT
A) B)
Capítulo 7. RESULTADOS Y DISCUSIÓN
164
3.6 Correlation between gut metabolites, pH and antioxidant activity
To the best of our knowledge, this is first study reporting the relationship between metabolic
profile and biological activity (pH and AOX) during the in vitro colonic fermentation of IF
isolated on processed corn products. pH values during in vitro colonic fermentation were
positively correlated (p<0.05) with trimethylamine, dimethyl trisulfide, benzaldehyde, phenol and
4-methyl-phenol and inversely correlated with ethanol, 2,4-dimethyl-1-heptene, propionic acid,
butyric acid, acetophenone, benzene 1,3-bis(1,1-dimethylethyl)- and nonanoic acid (Figure 4).
Significant positive correlations were found between DPPH antiradical activity and ethanol, 2,4-
Dimethyl-1-heptene, acetic acid, propionic acid, butyric acid, nonanal, acetophenone, and 1,3-
bis(1,1-dimethylethyl)-benzene, while it correlated negatively with trimethylamine, dimethyl
trisulfide, phenol and 4-methyl-phenol production. Interestingly, acetic acid, propionic acid,
ethanol, and nonanal, exhibited significant positive correlations with FRAP chelating activity,
whereas trimethylamine, phenol, and indole were negatively correlated with this activity. In this
work, some metabolites showed negative relationship with pH and AOX values (e.g.
trimethylamine and 4-methyl-phenol), and they have been recently associated with the
development of cardiovascular diseases and genotoxic effects on colonic epithelial cells.45, 46
Capítulo 7. RESULTADOS Y DISCUSIÓN
165
Figure 4. Pearson’s R correlations between gut microbial metabolites and pH values and
antioxidant capacity (DPPH antiradical activity and FRAP chelating activity) in extracts obtained
during in vitro colonic fermentation of blank, raffinose and indigestible fraction isolated from corn
products
In vitro Gut metabolite pH DPPH FRAPTrimethylamineEthyl etherHexaneEthanolButanoic acid, methyl esterDisulfide, dimethylToluene2,4-Dimethyl-1-heptenePentanoic acid, methyl esterHeptanalAcetic AcidDimethyl trisulfidePropionic AcidBenzene, 1,4-dichloro-BenzaldehydeButiric AcidNonanalButanoic acid, 3-methyl-Octanoic acid, methyl esterPentanoic acidAcetophenoneBenzaldehyde, 4-methyl-2-Nonenal, (E)-DecanalNonanoic acid, methyl esterPentanoic acid, 4-methyl-Benzene, 1,3-bis(1,1-dimethylethyl)-Hexanoic acidDecanoic acid, methyl esterHeptanoic acidPhenolBenzenemethanol, 4-methyl-Phenol, 4-methyl-Octanoic AcidDodecanoic acid, methyl esterNonanoic acidButylated Hydroxytoluenen-Decanoic acid2-Propenoic acid, tridecyl esterIndoleDiethyl PhthalateTetradecanal9-Hexadecenoic acid, methyl ester, (Z)-Hexadecanoic acid, methyl esterDibutyl phthalate9,12-Octadecadienoic acid (Z,Z)-, methyl ester
p Va
lue
-1-0.8-0.6-0.4-0.200.20.40.60.81Pe
arso
n's c
orre
latio
n co
effic
ient
<0.001<0.01<0.05
<0.001<0.01<0.05>0.05
>0.05
>0.05
Capítulo 7. RESULTADOS Y DISCUSIÓN
166
4. Conclusion
This contribution shows the changes in of bacterial metabolites, pH and AOX values during in
vitro colonic fermentation of IF isolated from three corn products. Chemical composition and RS
content was different among maize products. The IF content was the same but the magnitude of
SCFA production, particularly butyric acid, during in vitro fermentation was dependent on
fermentation time and substrate. Bacterial metabolites as trimethylamine, phenol and 4-methyl-
phenol were associated with increased pH and decreased AOX in the fermentation extracts. In
general terms, TCT and IT could be perceived as foods with potential health effects on colon
potential. In addition, the approach followed here, may be used as predictive model to assess the
metabolic implications of food substrates present in the traditional Mexican diet. Only through the
study of foods frequently consumed by a population will we improve our understanding of the
effects of diet on colon health promotion.
Acknowledgments: Zamora-Gasga, VM; acknowledge the fellowship to Consejo Nacional de
Ciencia y Tecnología (CONACYT- Registration number: 253795) and Sáyago-Ayerdi, SG; would
also like to acknowledge the financial support to PROMEP –ITTEP-PTC-003.
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Table SI. Volatile compounds characterization of in vitro colonic fermentation extracts of blank, rafinose and indigestible fraction isolated from Istmo Totopo (IF-IT), Baked corn tortilla (IF-BCT) and traditional corn tortilla (IF-
TCT) analyzed by SPME–GC/MS (mmol/L)
ID RT 12 24 48
VC (%) Volatile Name Chemical Group Blank Raffinose IF-IT IF-BCT IF-TCT Blank Raffinose IF-IT IF-BCT IF-TCT Blank Raffinose IF-IT IF-BCT IF-TCT
1 5.960 Trimethylamine Amines 20.33 0.00 0.00 0.00 0.00 21.29 0.00 0.00 0.00 0.00 66.31 0.00 0.00 0.00 0.00 248.81
2 6.252 Ethyl ether Ethers 38.96 88.01 77.03 68.81 59.68 0.00 0.00 30.35 24.37 49.55 0.00 0.00 0.00 0.00 0.00 111.46
3 7.082 Hexane Alkanes 0.00 0.00 0.00 0.00 0.00 0.00 41.46 0.00 39.80 73.02 33.21 0.00 0.00 0.00 45.29 155.73
4 7.906 Ethanol Alcohols and polyols 0.00 86.33 0.00 168.88 220.16 0.00 187.16 227.31 222.99 301.79 0.00 187.65 0.00 168.50 191.25 80.57
5 11.280 Butanoic acid, methyl ester Fatty Acyls 0.00 0.00 34.67 29.07 82.99 0.00 0.00 36.08 23.92 64.55 0.00 0.00 41.45 0.00 48.96 112.87
6 12.885 Disulfide, dimethyl Organic disulfides 37.13 0.00 0.00 0.00 0.00 38.34 0.00 0.00 0.00 0.00 0.00 0.00 50.46 0.00 81.26 184.95
7 13.083 Toluene Benzene and substituted derivatives 0.00 0.00 0.00 0.00 473.50 295.15 251.42 407.64 449.17 226.80 246.74 0.00 273.54 0.00 281.99 92.15
8 14.499 2,4-Dimethyl-1-heptene Olefins 0.00 0.00 32.64 32.86 30.57 0.00 0.00 19.84 30.90 31.38 0.00 21.39 22.11 28.00 32.81 76.41
9 15.022 Pentanoic acid, methyl ester Fatty acid ester 0.00 0.00 0.00 0.00 19.70 0.00 0.00 30.43 0.00 23.47 0.00 0.00 42.56 0.00 27.25 154.32
10 18.079 Heptanal Carbonyl compounds 0.00 0.00 23.92 0.00 20.79 0.00 0.00 25.11 0.00 37.00 0.00 19.56 0.00 0.00 28.10 131.94
11 20.028 Acetic Acid SCFA 27.54 367.51 145.10 199.77 333.90 35.86 729.15 230.37 308.48 468.55 19.09 611.85 188.58 171.73 498.00 74.19
12 21.978 Dimethyl trisulfide Organic trisulfides 115.60 0.00 0.00 0.00 0.00 752.69 0.00 0.00 0.00 0.00 46.50 0.00 26.15 29.55 153.32 257.96
13 22.223 Propionic Acid SCFA 9.03 232.04 120.45 150.07 176.33 0.00 421.44 135.29 160.72 284.98 4.12 362.17 123.93 135.72 293.75 72.39
14 23.182 Benzene, 1,4-dichloro- Benzene and substituted derivatives 0.00 24.80 24.38 26.66 33.52 30.65 30.20 29.23 29.59 33.14 29.55 18.85 26.46 23.13 26.74 31.47
15 23.624 Benzaldehyde Benzene and substituted derivatives 54.76 0.00 0.00 0.00 0.00 35.03 0.00 0.00 0.00 0.00 0.00 42.84 0.00 0.00 0.00 211.33
16 24.351 Butiric Acid SCFA 3.46 86.19 89.90 145.88 141.44 7.51 109.61 174.53 178.53 363.01 3.88 144.93 235.21 236.58 331.96 72.77
17 25.180 Nonanal Carbonyl compounds 34.57 19.24 34.73 0.00 28.75 0.00 25.31 0.00 20.85 41.89 0.00 33.80 20.79 31.82 38.56 68.96
18 25.410 Butanoic acid, 3-methyl- Organic acid 0.00 44.41 0.00 30.15 0.00 0.00 53.89 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 213.63
19 25.521 Octanoic acid, methyl ester Fatty acid ester 0.00 0.00 0.00 0.00 43.01 0.00 0.00 35.06 26.84 60.28 50.52 0.00 0.00 0.00 29.39 135.32
20 26.948 Pentanoic acid Organic acid 0.00 0.00 0.00 112.19 202.69 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 276.19
21 27.127 Acetophenone Benzene and substituted derivatives 0.00 0.00 34.46 33.07 30.83 0.00 0.00 0.00 35.96 32.09 0.00 0.00 0.00 44.77 36.00 112.15
22 27.425 Benzaldehyde, 4-methyl- Benzene and substituted derivatives 35.67 29.57 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 44.01 26.67 24.78 21.14 133.22
23 27.554 2-Nonenal, (E)- Carbonyl compounds 19.78 0.00 54.96 75.03 27.84 28.11 0.00 51.13 26.51 36.31 0.00 0.00 31.78 43.13 45.03 77.68
24 28.251 Decanal Carbonyl compounds 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 33.22 22.77 0.00 74.81 0.00 0.00 238.94
25 28.506 Nonanoic acid, methyl ester Fatty acid ester 0.00 0.00 54.63 37.81 76.24 0.00 0.00 135.26 60.92 117.97 0.00 0.00 152.58 0.00 114.26 113.46
26 28.544 Pentanoic acid, 4-methyl- Organic acid 0.00 0.00 0.00 0.00 0.00 0.00 20.52 0.00 0.00 0.00 0.00 24.34 180.50 0.00 0.00 309.18
27 28.960 Benzene, 1,3-bis(1,1-dimethylethyl)- Benzene and substituted derivatives 48.12 62.17 103.04 106.87 91.25 34.60 86.78 88.07 120.09 126.03 36.10 106.08 106.85 175.75 148.27 41.22
28 29.461 Hexanoic acid Organic acid 0.00 34.12 25.18 26.34 0.00 0.00 31.24 28.23 0.00 26.67 45.68 39.29 43.29 33.83 166.13 120.42
29 31.270 Decanoic acid, methyl ester Fatty acid ester 0.00 0.00 0.00 0.00 26.02 0.00 0.00 0.00 0.00 22.46 0.00 0.00 0.00 0.00 21.45 207.93
30 31.876 Heptanoic acid Organic acid 0.00 22.05 22.30 0.00 0.00 0.00 0.00 0.00 0.00 29.72 67.19 46.06 0.00 0.00 0.00 168.02
31 32.368 Phenol Benzene and substituted derivatives 100.74 0.00 0.00 0.00 0.00 573.21 0.00 0.00 0.00 0.00 574.45 0.00 0.00 0.00 0.00 241.34
Capítulo 7. RESULTADOS Y DISCUSIÓN
174
32 32.569 Benzenemethanol, 4-methyl- Benzene and substituted derivatives 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 39.14 61.28 50.75 0.00 211.14
33 34.080 Phenol, 4-methyl- Benzene and substituted derivatives 0.00 0.00 0.00 0.00 0.00 33.92 0.00 0.00 0.00 0.00 165.71 0.00 0.00 0.00 0.00 323.52
34 34.197 Octanoic Acid Organic acid 38.81 78.71 70.52 78.21 61.30 23.13 75.23 61.76 85.55 0.00 0.00 49.47 0.00 42.30 0.00 72.92
35 36.240 Dodecanoic acid, methyl ester Fatty acid ester 22.49 0.00 24.43 22.31 45.09 25.46 0.00 48.22 45.57 54.50 32.02 0.00 81.30 0.00 76.78 83.59
36 36.432 Nonanoic acid Organic acid 221.55 334.35 372.36 394.35 320.70 118.46 257.73 275.88 291.24 189.57 81.55 396.79 506.23 238.43 163.01 41.32
37 36.594 Butylated Hydroxytoluene Benzene and substituted derivatives 28.03 32.62 23.63 24.98 20.76 30.82 35.83 21.87 20.79 24.78 0.00 35.05 0.00 0.00 20.06 56.98
38 38.697 n-Decanoic acid Organic acid 33.28 67.65 44.38 53.88 43.05 0.00 33.26 34.41 39.92 38.36 0.00 92.07 86.22 0.00 31.27 69.81
39 40.245 2-Propenoic acid, tridecyl ester Fatty acid ester 108.18 242.24 179.61 201.88 174.85 203.06 361.73 247.22 254.43 290.62 205.51 518.10 350.42 461.24 332.36 40.60
40 40.705 Indole Indoles and derivatives 2043.97 292.58 1032.86 752.14 890.08 2200.81 418.91 1714.30 1184.04 1769.78 2161.86 410.78 1913.45 2101.52 1571.39 50.59
41 41.408 Diethyl Phthalate Benzene and substituted derivatives 41.95 60.31 24.19 50.62 44.89 23.19 34.07 28.78 42.89 40.58 46.51 43.83 46.48 54.60 36.83 25.48
42 42.649 Tetradecanal Carbonyl compounds 0.00 0.00 0.00 0.00 0.00 0.00 29.60 0.00 0.00 0.00 0.00 47.18 24.20 23.00 0.00 182.69
43 44.168 9-Hexadecenoic acid, methyl ester, (Z)- Fatty acid ester 0.00 0.00 22.01 20.52 35.32 20.01 0.00 44.48 25.41 39.96 27.86 0.00 50.19 0.00 49.06 84.69
44 44.321 Hexadecanoic acid, methyl ester Fatty acid ester 19.71 0.00 31.11 27.19 32.21 0.00 0.00 30.52 26.04 28.02 28.42 0.00 34.36 31.87 35.72 64.75
45 47.872 Dibutyl phthalate Benzene and substituted derivatives 70.82 0.00 59.48 58.92 57.16 67.19 121.81 82.80 78.74 0.00 80.06 66.93 75.02 74.54 75.69 46.95
46 48.073 9,12-Octadecadienoic acid (Z,Z)-, methyl ester Fatty acid ester 0.00 0.00 25.80 28.42 53.26 0.00 0.00 47.00 55.91 84.21 0.00 0.00 49.22 22.04 78.27 101.41
ID; Identification, RT; retentiton time, VC; Variation coefficient
Capítulo 8. CONCLUSIONES
175
Capítulo 8. CONCLUSIONES
En este trabajo se evaluó el impacto de los componentes indigestibles de alimentos frecuentemente
consumidos y sus posibles efectos en el mantenimiento de la salud del colon en una población de
escolares que viven en Tepic, Nayarit, México.
El uso de una combinación de análisis de conglomerados (AC) y el análisis de componentes
principales (ACP) determinó siete factores dietéticos y tres dietas diferentes: Dieta Tradicional
Mexicana, Dieta Modificada Mexicana y Dieta Alternativa Mexicana.
Un patrón dietético caracterizado por una alta ingesta de leguminosas, bocadillos y bajas ingesta
bebidas se asoció negativamente con el peso y el IMC, lo que sugiere que el patrón dietético
tradicional mexicano podría conducir a la reducción de la prevalencia de sobrepeso y obesidad
entre los escolares.
No se encontró una relación clara entre el estatus de sobrepeso-obesidad y los tipos de dieta.
El perfil de ingesta de macronutrientes de los escolares depende del tipo de dieta consumida. El
consumo de energía de azúcares, dulces, pasteles y bebidas endulzadas aparece en todos los
patrones dietéticos.
Se deben considerar nuevas políticas para el combate de la prevalencia de sobrepeso y obesidad en
escolares mexicanos, incluyendo el diseño de un programa de intervención nutricional
personalizado tomando en cuenta los patrones dietéticos de cada zona geográfica.
En cuanto a la digestión gastrointestinal y fermentación colínica in vitro de los alimentos
frecuentemente consumidos por los escolares en las dietas previamente identificadas, se obtuvo
que las fracciones indigestibles (FI) aisladas de los tres menús del desayuno presentaron una
composición química variada.
TM-B mostró el mayor contenido polifenoles no extraíbles, y carbohidratos no digeribles (DF)
como principales compuestos bioactivos. Estos compuestos podrían influir en el metabolismo
Capítulo 8. CONCLUSIONES
176
fermentativo al ingerir estos alimentos modificando el perfil de metabolitos microbianos que tiene
un impacto marcado en la salud del huésped.
En este trabajo, ACP permitió la identificación de patrones metabólicos que se asocian con efectos
beneficiosos para la salud (disminución del pH del colon y aumento de los valores de la capacidad
antioxidante). También se identificaron compuestos asociados con diferentes enfermedades que
además se relacionaron con potenciales efectos nocivos (aumento del pH y disminución de
capacidad antioxidante “AOX”) durante la fermentación colónica in vitro.
El análisis de agrupamiento jerárquico mostró que los extractos de fermentación de las FI´s
aisladas de un desayuno mexicano tradicional presentan un patrón metabólico beneficioso
dependiente del tiempo de fermentación.
Para entender el impacto de la dieta sobre el estado de salud intestinal, se necesitan trabajos
adicionales que evalúen los patrones fermentativos colónicos de los alimentos combinados en
regímenes realistas completos.
Nuestro estudio representa una nueva contribución sobre los efectos potenciales in vitro de los
hábitos alimentarios en la producción de metabolitos bacterianos. Las FI´s aisladas de menús de la
comida presentaron una apreciable cantidad de polifenoles extraíbles y no extraíbles, como
principales compuestos bioactivos, pero no contribuyen a un AOX anti-radical.
Aunque existieron diferencias en el contenido de FI, la magnitud de la producción de ácidos
grasos de cadena corta (AGCC) en los menús de de las comidas fueron similares al blanco (sin
sustrato). Eso puede estar relacionado con la baja contribución de la fibra dietética en la dieta
mexicana. ACP permitió la identificación de patrones metabólicos que se asociaron con efectos
beneficiosos para la salud (pH bajo y alto AOX).
Los metabolitos bacterianos como trimetilamina, ésteres de ácidos grasos e indol se asociaron con
el aumento del pH y la disminución de AOX en los extractos de fermentación. El perfil de los
metabolitos bacterianos fue dependiente del tiempo de fermentación y del sustrato. El enfoque
Capítulo 8. CONCLUSIONES
177
presentado aquí, podría ser utilizado como modelo predictivo para evaluar fácilmente los efectos
de los componentes indigestibles provienente de la ingesta de alimentos comunes en la dieta.
Los menús de las cenas mostraron diferencias importantes y similitudes con los resultados
obtenidos durante el desayuno y la comida. La fracción indigestible de la dieta tradicional presentó
el mayor contenido de polifenoles extraíbles y no extraíbles, como principales compuestos
bioactivos, con una ligera contribución en la AOX anti-radical. A pesar de las diferencias en el
contenido de FI, la magnitud de la producción de AGCC en los menús de cena fueron bajos, y
similares a los encontrados en los otros tiempos de ingesta evaluados. El menú de la dieta
alternativa puede contener almidón resistente en la composición de FI lo que favorecíó la
producción de AGCC. Fue apreciable la relación entre el bajo contenido de AGCC y la baja
contribución de la fibra dietética en la todas las dietas mexicanas.
El ACP permitió la identificación de patrones metabólicos que se asociaron con efectos
beneficiosos para la salud (pH bajo y alto AOX). Siendo los AGCC los que se relacionaron con
efectos benéficos en los extractos de fermentación. Sin embargo, los metaboitos microbianos del
menú de la Dieta Modificada Mexicana fueron mayormente relacionados a la fermentación de
proteínas (principalmente trisulfidos) lo que sugiere posibles efectos adversos en la salud de la
población.
Los metabolitos bacterianos como el fenol y el fenol 4-metilo se asociaron con el aumento del pH
y la disminución de AOX en los extractos de fermentación, estos metabolitos estuvieron presentes
durante las distintas femrentaciones de los alimentos consumidos durante el desayuno, comida y la
cena. Sin embargo, son necesarios estudios in vivo para relacionar nuestros resultados con los
posibles efectos de la dieta sobre la salud intestinal
Por último, la fermentación de alimentos procesadores de maíz, mostraron una relación directa
entre los metabolitos bacterianos, el pH y los valores de AOX durante la fermentación colónica in
vitro. La composición química y el contenido de AR fueron diferentes entre los productos de maíz.
A diferencias de los menús, y a pesar de que el contenido de FI fue el mismo, la magnitud de la
producción de AGCC, particularmente de ácido butírico, durante la fermentación in vitro,
dependió del tiempo de fermentación y del sustrato. Los metabolitos bacterianos como la
Capítulo 8. CONCLUSIONES
178
trimetilamina, el fenol y el 4-metilfenol se asociaron con el aumento del pH y la disminución de
AOX en los extractos de fermentación, siendo estos compuestos relacionados negativamente
efectos saludables en todas las muestras analizadas durante el desarrollo de este trabajo.
En términos generales, la tortilla tradicional de maíz y el totopo del istmo de Tehuantepec podrían
percibirse como alimentos con efectos potenciales para la salud intestinal debido a la alta
producción de ácido butírico lo que sugiere una relación con el contenido de AR.
Finalmente, este estudio demuestra que sólo a través del análisis de los alimentos consumidos con
frecuencia por una población, se logrará mejorar nuestra comprensión de los efectos de la dieta en
la promoción de la salud del colon.
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