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Efecto de la pectina sobre la actividad de algunas enzimas
digestivas y la digestión de lípidos
Mauricio Espinal Ruiz
Universidad Nacional de Colombia
Facultad de Ciencias
Departamento de Química
Bogotá DC, Colombia
2016
Efecto de la pectina sobre la actividad de algunas enzimas
digestivas y la digestión de lípidos
Mauricio Espinal Ruiz
Tesis presentada como requisito para optar al título de:
Doctor en Ciencias Química
Director:
Carlos Eduardo Narváez Cuenca, PhD
Grupo de Investigación:
Estudio de los cambios químicos y bioquímicos de alimentos frescos y procesados
Universidad Nacional de Colombia
Facultad de Ciencias
Departamento de Química
Bogotá DC, Colombia
2016
Effect of pectin on the activity of some digestive enzymes
and the digestion of lipids
Mauricio Espinal Ruiz
Universidad Nacional de Colombia
Facultad de Ciencias
Departamento de Química
Bogotá DC, Colombia
2016
Effect of pectin on the activity of some digestive enzymes
and the digestion of lipids
Mauricio Espinal Ruiz
Thesis submitted in fulfillment of the requirements for the degree of:
Doctor of Science - Chemistry
Director:
Carlos Eduardo Narváez Cuenca, PhD
Research Group:
Study of the chemical and biochemical changes of fresh and processed foods
Universidad Nacional de Colombia
Facultad de Ciencias
Departamento de Química
Bogotá DC, Colombia
2016
Resumen
Aunque se ha demostrado que la pectina tiene varias funciones fisiológicas benéficas para la
salud humana, la información disponible sobre los mecanismos mediante los cuales la pectina es
capaz de ejercer dichas funciones es limitada. En esta tesis se estudiaron los mecanismos
mediante los cuales la pectina es capaz de ejercer sus funciones fisiológicas. Se evaluó el efecto
de la pectina sobre la actividad de las principales enzimas digestivas [lipasa pancreática, -
amilasa, fosfatasa alcalina y proteasa (quimotripsina)]. Entre las enzimas estudiadas, la lipasa
pancreática fue la enzima más susceptible de ser inhibida por la pectina. Se hizo especial énfasis
en el efecto de la pectina sobre el destino gastrointestinal de los lípidos, haciendo uso de una
emulsión como modelo experimental. Se utilizó un modelo de digestión in vitro que consistió en
la simulación de las fases oral, gástrica e intestinal con el objetivo de evaluar el efecto de la
pectina sobre la velocidad y la magnitud del proceso de digestión de lípidos. Las digestiones in
vitro mostraron que la velocidad y la magnitud del proceso de digestión fueron inhibidos cuando
se aumentó la concentración de pectina. Además, se encontró que la pectina de alto grado de
metoxilación (HMP) tuvo mayor capacidad de inhibir el proceso de digestión que la pectina de
bajo grado de metoxilación (LMP). Esto permitió sugerir que los mecanismos fisicoquímicos que
pueden explicar la influencia de la pectina sobre la digestión de lípidos son el incremento de la
viscosidad de los fluidos gastrointestinales, la floculación de lípidos y las interacciones existentes
entre la pectina y los lípidos, así como con los componentes gastrointestinales que participan en
el proceso de digestión (lipasa pancreática, sales biliares, CaCl2, y NaCl). Finalmente, se
encontró que el aumento de la viscosidad de los fluidos gastrointestinales por efecto de la pectina
y las interacciones electrostáticas (atractivas y repulsivas) inhibió la velocidad y la magnitud del
proceso de transferencia de masa de los compuestos nutricionales más importantes
(monosacáridos, aminoácidos y lípidos emulsificados). Los resultados obtenidos en esta tesis
podrían conllevar a la comprensión de la funcionalidad fisiológica de la pectina y cómo esta
funcionalidad puede verse afectada por las características estructurales de la pectina.
Palabras clave: Pectina, grado de metoxilación, enzimas digestivas, emulsión, digestión in vitro.
Abstract
Although it has been demonstrated that pectin has several physiological functions beneficial to
human health, limited information is available concerning the mechanisms by which pectin is
able to exert such functions. In this thesis, the mechanisms by which pectin is able to exert its
physiological functions were studied. The effect of pectin on the activities of the major digestive
enzymes [pancreatic lipase, -amylase, alkaline phosphatase and protease (chymotrypsin)] was
evaluated. Among the studied enzymes, pancreatic lipase was the most likely to be inhibited by
pectin. Special emphasis was made on the effect of pectin on the gastrointestinal fate of lipids
(and pancreatic lipase) by using a corn oil-in-water emulsion as the experimental model. A
simulated in vitro digestion model consisting of oral, gastric, and small intestinal phases was used
to elucidate the impact of pectin on the rate and extent of the digestion process of emulsified
lipids. The simulated in vitro digestions revealed that both the rate and extent of the digestion
process of emulsified lipids were inhibited with increasing concentration of pectin. In addition,
high methoxylated pectin (HMP) was found to be more effective in inhibiting the digestion
process of emulsified lipids as compared to low methoxylated pectin (LMP). We suggest that the
physicochemical mechanisms that may account for the influence of pectin on the digestion of
emulsified lipids are the modification of the viscosity of the gastrointestinal (GI) fluids, the
flocculation of emulsified lipids, and the interactions between pectin and both the emulsified
lipids and the GI components involved in the lipid digestion process (e.g., pancreatic lipase, bile
salts, CaCl2, and NaCl). Finally, the increase of the GI fluids viscosity by means of pectin and
electrostatic interactions (repulsive and attractive) were found to affect the rate and extent of the
mass transfer process of the most important nutritional compounds (monosaccharides, amino
acids, and emulsified lipids). The results obtained in this thesis might lead to the comprehension
of the physiological functionality of pectin and how this functionality can be affected by the
structural characteristics of pectin.
Keywords: Pectin, methoxylation degree, digestive enzymes, emulsion, in vitro digestion.
Objetivos
Objetivo General
Estudiar los efectos que ejerce la pectina sobre la actividad de algunas enzimas digestivas y sobre
la digestión de lípidos en un sistema gastrointestinal simulado.
Objetivos específicos
1. Evaluar el efecto de la pectina sobre la actividad de las principales enzimas digestivas (lipasa,
proteasa y -amilasa) en soluciones modelo.
2. Evaluar el efecto de la pectina sobre la transferencia de masa de nutrientes (azúcares, lípidos
y aminoácidos) en un sistema de difusión controlada.
3. Evaluar el efecto de pectinas de alto y bajo grado de metoxilación sobre la digestión de
lípidos en un sistema gastrointestinal simulado y compararlo con diferentes fuentes de fibra
dietaria (quitosano y metil celulosa).
4. Evaluar las posibles interacciones moleculares de la pectina con cada uno de los principales
componentes del sistema gastrointestinal (lipasa pancreática, sales biliares y electrolitos).
Objectives
General objective
Study the effects exerted by pectin on both the activity of some digestive enzymes and lipid
digestion in a simulated gastrointestinal system.
Specific objectives
1. Evaluate the effect of pectin on the activity of the main digestive enzymes (lipase, protease,
and -amylase) in model solutions.
2. Evaluate the effect of pectin on the mass transfer of nutrients (monosaccharides, lipids, and
amino acids) in a diffusion controlled system.
3. Evaluate the effect of both low and high methoxylated pectins on lipid digestion in a
simulated gastrointestinal system, and compare them with different sources of dietary fiber
(chitosan and methyl cellulose).
4. Evaluate the possible molecular interactions of pectin with each of the main components of
the gastrointestinal system (pancreatic lipase, bile salts, and electrolytes).
Table of contents
Resumen – Abstract
Objetivos – Objectives
Chapter 1 General introduction 1
Chapter 2
Optimization of the reaction conditions affecting the activity of
digestive enzymes
34
Chapter 3 Inhibition of digestive enzyme activities by pectins in model solutions 56
Chapter 4
Impact of dietary fibers [methyl cellulose, chitosan, and pectin] on
digestion of lipids under simulated gastrointestinal conditions
86
Chapter 5
Impact of pectin properties on lipid digestion under simulated
gastrointestinal conditions: Comparison of citrus and banana passion
fruit (Passiflora tripartita var. mollisima) pectins
117
Chapter 6
Interaction of a dietary fiber (pectin) with gastrointestinal components
(bile salts, calcium, and lipase): A calorimetry, electrophoresis, and
turbidity study
154
Chapter 7
Effect of pectin on the mass transfer kinetics of monosaccharides,
amino acids, and a corn oil-in-water emulsion in a Franz diffusion cell
187
Chapter 8 General discussion 214
Concluding remarks 233
Academic production 234
Acknowledgements 236
Chapter 1
General introduction
Chapter 1
2
1.1. Background
In recent years, an increase in the incidence of cardiovascular disease (CVD) and coronary heart
disease (CHD) has increased among some European countries, United States, and also in
Colombia (Threapleton, Greenwood, Evans, Cleghorn, Nykjaer, Woodhead, et al., 2013). CVD
and CHD account for almost half (48 and 52%) of all deaths in Europe and Colombia,
respectively, and a third (33%) of all deaths in the United States (Bolívar-Mejía & Vesga-
Angarita, 2013). Epidemiologic studies have shown that consumption of grains, cereals, fruits,
and vegetables may lower the risk of suffering both CVD and CHD (Brownlee, 2011). Dietary
fiber is one of the components that may be responsible for the beneficial effect of these foods
(Hollmann, Themeier, Neese, & Lindhauer, 2013). The protective connection between the
consumption of dietary fiber and both CVD and CHD was proposed in the 1970s (Phillips & Cui,
2011). Many experimental studies have examined the relationship between different sources of
dietary fiber and controlling both CVD and CHD risk factors. Results from experiments have
shown that dietary fiber may lower the concentrations of glucose and cholesterol in blood
(Threapleton, et al., 2013), reduce blood pressure (Streppel, Arends, & van’tVeer, 2005),
promote body-weight loss (Howarth, Saltzman, & Roberts, 2001), and improve insulin sensitivity
(de Leeuw, Jongbloed, & Verstegen, 2004), thereby reducing the risk of CVD and CHD
mortalities. Studies regarding the effect of dietary fiber on the CVD and CHD risk factors have
been focused on the major representative sources of soluble dietary fiber: legumes, fruits, seeds,
and grains (Galisteo, Duarte, & Zarzuelo, 2008). Nevertheless, information of purified soluble
dietary fiber (e.g., pectin, chitosan, and methyl cellulose) is scarce. While information on the
physiological effects of soluble dietary fiber has been provided, no information on the molecular
mechanisms by which soluble dietary fiber may act on CVD and CHD risk factors, is available.
In this chapter, we present the theoretical background required to address this thesis. First, we
make a brief description of the definition, classification, and functional properties of dietary fiber.
Then, we focus on the structural characteristics and classification of pectin. Next, we make a
description of the structure and properties of emulsions. Finally, we emphasize on the use of an in
vitro digestion model for evaluating the gastrointestinal fate of emulsified lipids.
Chapter 1
3
1.2. Dietary fiber
1.2.1. Definition of dietary fiber
Currently, the most accepted definition of dietary fiber is that of the European Commission of
Nutrition and Health, which has defined dietary fiber as the carbohydrate polymers with three or
more monomeric units, which are neither digested nor absorbed in the upper gastrointestinal tract
(GIT) of humans (mouth, stomach, and small intestine), and belong to the following categories: i)
edible carbohydrate polymers which have been obtained from food raw material by physical,
enzymatic or chemical means and that have a beneficial physiological effect demonstrated by
accepted scientific evidence; and ii) edible synthetic carbohydrate polymers which also have a
beneficial physiological effect demonstrated by accepted scientific evidence (Cummings, Mann,
Nishida, & Vorster, 2009; DeVries, 2003; Harris & Pijls, 2013). Although dietary fiber is defined
as carbohydrate polymers, it has been stated that lignin and other compounds can be classified as
dietary fiber, as long as they are closely associated with the carbohydrate polymers in plant cell
walls and behave chemically as dietary fiber (Mann & Cummings, 2009; Phillips & Cui, 2011;
Turner & Lupton, 2011).
1.2.2. Classification of dietary fiber
Dietary fiber includes a large variety of carbohydrates with various constituent monosaccharide
compositions, molecular weights, physical properties, and physiological effects. Dietary fiber is
usually classified based on their physical properties, especially based on their solubility in water,
viscosity, and fermentability. Dietary fiber can be classified as soluble (viscous and fermentable)
and insoluble (non-viscous and slowly fermentable) (Ramulu & Udayasekhara Rao, 2003).
Solubility has proven to be a very convenient parameter in the understanding of the
physicochemical properties of dietary fiber, allowing a simple division into those that principally
have effects on glucose and lipid absorption from the small intestine (soluble), and those which
are slowly and incompletely fermented and have pronounced effects in the large intestine
(insoluble) (Cummings & Stephen, 2007).
Chapter 1
4
Figure 1.1. Classification of dietary fiber based on their digestibility in the upper gastrointestinal tract
(GIT) and their molecular weight. Grey box indicates the carbohydrates that can be classified as dietary
fiber (Cummings & Stephen, 2007). FOS, fructo-oligosaccharides; GOS, galacto-oligosaccharides.
It has been mentioned, nevertheless, that the classification of dietary fiber based on the water
solubility is less convenient, because solubility of dietary fiber can also be influenced by several
environmental factors, such as pH, ionic strength, and temperature (Cummings & Stephen, 2007).
Therefore, it was suggested to classify dietary fiber based either on their chemical structure (e.g.,
molecular weight) or on recognized physiological properties (e.g., digestibility in the upper GIT)
(Cummings & Stephen, 2007).
Figure 1.1 illustrates the classification of dietary fiber based on their digestibility in the upper
GIT (mouth, stomach, and small intestine) and their molecular weight. Figure 1.1 shows that
dietary fiber can be classified as non-digestible oligosaccharides (NDOs), resistant starch (RS),
and non-starch polysaccharides (NSPs).
Digestible
starch
Resistant
starch (RS) Glucans
Chitosan
Mannans
Fructans
Xylans
Pectins
Maltodextrins
Polysaccharides
Non-starch polysaccharides (NSPs)
Non-digestible
oligosaccharides
(NDOs)
FOS and GOS
Oligosaccharides
Non-digestible polysaccharides
Dietary fiber
Digestible Non-digestible
Monosaccharides
Disaccharides
Non-digestible
disaccharides
Lactulose
Digestibility in the upper GIT
Mo
lecu
lar
wei
gh
t
Cellulose
Methyl cellulose
Hemicellulose
Chapter 1
5
1.2.2.1. Non-digestible oligosaccharides (NDOs)
Oligosaccharides (from 3 to 10 monomeric units) which are not digested in the upper GIT can be
included as a dietary fiber source according to the definition of the European Commission of
Nutrition and Health (Harris & Pijls, 2013). Considering the extent of the definition, NDOs are
very diverse, including degradation products of NSPs and some synthetized oligosaccharides,
excluding mono and disaccharides. Therefore, non-digestible disaccharides such as lactulose
cannot considered as dietary fiber (Phillips & Cui, 2011). The most recognized NDOs are fructo-
oligosaccharides (FOS) and galacto-oligosaccharides (GOS), which have been demonstrated to
present prebiotic activity by promoting the growth of beneficial bacteria in the colon (Mussatto &
Mancilha, 2007). In addition, NDOs are suitable ingredients to be used in the fabrication of low-
caloric foods for diabetic patients (Mussatto & Mancilha, 2007).
1.2.2.2. Resistant starch (RS)
Chemically, all starch molecules are similar among them, since they have glucose residues linked
by -(1,4) and -(1,6) linkages (amylose and amylopectin fractions). Although human -amylase
can hydrolyze the -(1,4) linkages, some fractions of starch are not available to be digested in the
upper GIT. The portion of starch that is not digested in the upper GIT is known as RS. RS can be
divided into four groups (Fuentes-Zaragoza, Riquelme-Navarrete, Sánchez-Zapata, & Pérez-
Álvarez, 2010): i) RS1, which is physically inaccessible because of entrapment in a non-
digestible matrix; ii) RS2, which is physically inaccessible because its compacted structure; iii)
RS3, retrograded starch (crystalline structures of amylopectin); and iv) RS4, modified starches
obtained by chemical treatments, e.g., di-starch phosphate ester. Sources and properties of RS
have been reviewed in detail elsewhere (Fuentes-Zaragoza, Riquelme-Navarrete, Sánchez-
Zapata, & Pérez-Álvarez, 2010; Haralampu, 2000; Raigond, Ezekiel, & Raigond, 2015; Sajilata,
Singhal, & Kulkarni, 2006). Among the four types of RS, it is important to stress that RS3 is of
particular interest, because of its thermal stability. This allows RS3 to be stable in normal
processing conditions, and allows its use as a functional ingredient in a wide variety of food
systems (Haralampu, 2000).
Chapter 1
6
1.2.2.3. Non-starch polysaccharides (NSPs)
All non-digestible polysaccharides besides RS belong to this group. NSPs, therefore, include a
wide diversity of polysaccharides with several constituent monosaccharide composition and
linkage types, as described in Table 1.1. The majority of dietary fibers are NSPs. The diversity in
the chemical structures of NSPs leads to diverse physical and chemical properties, as well as
diversity in their physiological effects (Kumar, Sinha, Makkar, de Boeck, & Becker, 2012). NSPs
are usually categorized based on their water solubilities (Sasaki, Kohyama, & Yasui, 2004).
However, physical properties of NSPs depend on their chemical structures (e.g., molecular
weight and monosaccharide composition) as well as environmental factors, including how the
NSPs can be isolated and processed (Izydorczyk, Macri, & MacGregor, 1998; You, Xie, Liu, &
Gu, 2010). Some of the most representative NSPs are insoluble NSPs such as cellulose,
hemicellulose, and chitin that can be found in cereals, legumes, and crustaceans, respectively; and
soluble NSPs such as pectin, chitosan, and alginate that can be found in fruits, crustaceans, and
algae, respectively (Table 1.1).
1.2.3. Health effects of dietary fiber
Dietary fiber is recognized for having a positive role in regulating body weight (Slavin, 2005),
alleviating diabetes (Post, Mainous, King, & Simpson, 2012), preventing cardiovascular diseases
(Threapleton, et al., 2013), maintaining colon health (Brownlee, 2011), and preventing various
types of cancer (Kritchevsky & Story, 1982). In the description that follows, the mechanisms by
which dietary fiber can influence the health of the consumers at different sites in the GIT are
described.
1.2.3.1. Dietary fiber in the upper GIT
The presence of dietary fiber in food potentially reduce energy uptake (Brownlee, 2011). Dietary
fiber provides bulk volume with less energy as compared to other nutrients, thus reducing the
energy density of foods (Howarth, Saltzman, & Roberts, 2001; Slavin, 2005).
Chapter 1
7
Table 1.1. Examples of the diversity of non-starch polysaccharides (NSPs) (Cummings & Stephen, 2007).
Dietary Fiber Fiber Variability Main Constituents Sources
Cellulose Cellulose
Methyl Cellulose
Glucose
Methyl-glucose
Plants
Synthetic
-Glucans -(1,3)-(1,4)-glucan
-(1,3)-(1,6)-glucan Glucose Cereals
Hemicelluloses
Arabinoxylans
Xyloglucans
Glucomannan
Galactoglucomannan
Arabinose, xylose
Glucose, xylose
Mannose,glucose
Mannose, glucose, galactose
Cereals
Dicots and conifers
Amorphophallus konjac
Gymnospermae
Pectins
Homogalacturonan
Rhamnogalacturonan I
Rhamnogalacturonan II
Galacturonic acid
Galacturonic acid, rhamnose,
arabinose, galactose
Galacturonic acid, rhamnose,
arabinose, galactose
Fruits and vegetables
Plant exudate gums
Arabic gum
Ghatti gum
Karaya gum
Tragacanth gum
Arabinose, galactose,
rhamnose, glucuronic acid
Arabinose, galactose,
mannose, xylose, rhamnose
Galactose, rhamnose,
galacturonic acid
Arabinose, galactose, xylose,
rhamnose, fucose
Acacia senegal
Anodeissus latifolia
Sterculia spp.
Astragalus spp.
Mucilages Ispaghula husk
Xylose, arabinose, rhamnose,
galacturonic acid
Plantago ovate
Galactomannans Guar gum
Locust bean gum
Mannose, galactose
Mannose, galactose
Guar plant
Carob tree
Seaweed polysaccharides
Alginate
Carrageenan
Agar
Guluronic acid,
mannuronic acid
Galactose, anhydro galactose
Galactose, anhydro galactose
Brown seaweed
Red seaweed
Redseaweed
Chitin and chitosan Chitin
Chitosan
N-acetyl glucosamine
Glucosamine Crustaceans, insects
Microbial polysaccharides
Xanthan gum
Reuteran
Gellan gum
Glucose, mannose
Glucose, rhamnose
Glucose, rhamnose
Xanthomonas campestris
Lactobacillus reuteri
Pseudomonas elodea
Chapter 1
8
The effects of dietary fiber after consumption of foods start in the oral phase. Foods that are rich
in dietary fiber are usually recognized as less palatable (Yuan, Smeele, Harington, van Loon,
Wanders, & Venn, 2014), and they usually require a lot of mastication. The effects of dietary
fiber in the gastric and small intestinal phases are usually related to their physicochemical
features, mainly by increasing the viscosity of digesta (Wanders, Jonathan, van den Borne, Mars,
Schols, Feskens, et al., 2013). It has been shown that a high viscosity digesta may delay gastric
emptying and induce satiety (Burton-Freeman, 2000). Gastric emptying is one of the many
factors that have a role in satiation and regulation of food intake (Janssen, Vanden Berghe,
Verschueren, Lehmann, Depoortere, & Tack, 2011). In the small intestine, increased viscosity of
the digesta upon consumption of dietary fiber has been related to a decreased rate of nutrient
digestion and absorption (Torsdottir, Alpsten, Holm, Sandberg, & Tölli, 1991). Besides having a
role in the regulation of food intake, the reduction of the glycemic index can prevent the
incidence of type II diabetes (Post, Mainous, King, & Simpson, 2012). The effects of viscous
dietary fiber on glycemic response (Yuan, Smeele, Harington, van Loon, Wanders, & Venn,
2014), blood lipids (Jenkins, Wolever, Rao, Hegele, Mitchell, Ransom, et al., 1993), intestinal
enzymatic activity (Dunaif & Schneeman, 1981), and nutrient digestibility (Zhang, Li, Liu, Zang,
Duan, Yang, et al., 2013) have been documented. The effects of dietary fiber aforementioned
might be due to the increased viscosity of the GIT fluids. Dietary fiber without viscous properties
(such as insoluble dietary fiber), therefore, do not have considerable effect in the upper GIT
(Dikeman & Fahey, 2006; Wanders, et al., 2013). In addition, it has been demonstrated that
soluble dietary fiber with a high water holding capacity (e.g., chitosan, pectin, and methyl
cellulose) can reduce appreciably the free water content and increase the viscosity of the digesta
(Dikeman & Fahey, 2006).
1.2.3.2. Dietary fiber in the large intestine
After passing the upper GIT tract, dietary fiber reaches the large intestine. In the large intestine
there is a large community of microorganisms that utilizes the dietary fiber as energy source for
their growth (prebiotic activity). It has been proposed that the fermentation of dietary fiber by
these microorganisms is one of the main mechanism by which dietary fiber can influence health
Chapter 1
9
(Hamaker & Tuncil, 2014). Dietary fiber that can be fermented by these microorganisms are
capable to modify the composition and diversity of the microbiota (Hamaker & Tuncil, 2014).
For example, FOS and GOS are reported to stimulate the growth of Bifidobacteria and
Lactobacilli (Sims, Ryan, & Kim, 2014), which are considered to be beneficial for health
(Guarner & Malagelada, 2003). The final products of dietary fiber fermentation in the large
intestine are gasses (e.g., H2 and CH4) and short chain fatty acids (SCFAs). The SCFAs are
mainly acetic, propionic, and butyric acids. An increased amount of SCFAs in the digesta is
associated with a lower pH, which contributes to the inhibition of the growth of pathogenic
bacteria (Sun & O’Riordan, 2013). As mentioned above, dietary fiber has an important number of
physiological functions. However, the molecular mechanisms by which the dietary fiber is able to
exert their physiological functions remain unknown for most of the dietary fiber sources
available. Among the different sources of dietary fiber, pectin stands out because of its
recognized functional properties and its versatility to be used in several industrial applications.
Below, we provide an overview of the structural characteristics of pectin.
1.3. Pectin
1.3.1. Structure of pectin
Pectin is a polysaccharide with several applications in the pharmaceutical, food, and
biotechnology industries (Munarin, Tanzi, & Petrini, 2012). It has been used successfully for
many years in the food and beverages industries as a thickening and gelling agent, as well as a
colloidal stabilizer (Yapo, 2011). Pectin is a complex mixture of polysaccharides that comprises
about the third part of the cell wall of higher plants. The highest concentration of pectin is found
in the middle lamella of cell walls, with a gradual decrease toward the plasma membrane (Fruk,
Cmelik, Jemric, Hribar, & Vidrih, 2014). Different types of pectin fractions can be isolated from
the cell wall, including homogalacturonan (HG), rhamnogalacturonan I (RG-I), and
rhamnogalacturonan II (RG-II). Typically, HG is the most abundant polysaccharide, constituting
about 65% (mol/mol) of the pectin, whereas RG-I and RG-II constitute about 25 and 10%
(mol/mol), respectively (Ridley, O'Neill, & Mohnen, 2001).
Chapter 1
10
1.3.1.1. Homogalacturonan (HG)
HG is a linear chain of 1,4-linked -D-galactopyranosyl uronic acid (GalA) units in which some
of the carboxyl groups can be methyl esterified (methoxylated) in the C6 position, as shown in
Figure 1.2. HG can be partially O-acetylated at C2 and C3 positions, depending on the plant
source (Ridley, O'Neill, & Mohnen, 2001). The methoxylation degree (MD) is defined as the
percentage of carboxyl groups which have been methoxylated. If more than 50% of the carboxyl
groups are methoxylated, the pectin is called high methoxylated pectin (HMP), and less than that
methoxylation degree is called low methoxylated pectin (LMP). The MD is variable and affects
the overall physicochemical properties of pectin, especially, its electrical properties and capacity
to form calcium-mediated interactions between HG chains (Mohnen, 2008). In addition, the
physicochemical properties of HG are significantly affected by the spontaneous ionization of the
free carboxyl groups in water (–COOH + H2O –COO⊝ + H3O⊕) and by the binding capacity
of metal ions (divalent cations such as Ca2⊕ and Fe
2⊕), especially in LMP (Yapo, 2011).
Figure 1.2. Structure of homogalacturonan (HG). The pKa value of the carboxyl group is 3.5. In high-
acidic conditions (pH<3.5) the carboxyl groups will be protonated (neutral), whereas in low-acidic,
neutral, or alkaline conditions (pH>3.5), the carboxyl groups will be dissociated, forming the carboxylate
group (anionic). Each number represents a carbon atom of the ring (Ridley, O´Neill, & Mohnen 2001).
-(1,4) linkage
O-acetylation
O-acetylation
Methoxylation
CarboxylatepH>3.5
CarboxylpH<3.5
23
6
Chapter 1
11
The experimental pKa value of the free carboxyl group of pectin is 3.5 (Caffall & Mohnen, 2009).
Thus, in high-acidic conditions (pH<3.5) a large fraction of the carboxyl groups will be
protonated and unavailable for ionic cross-linking by cations, whereas in low-acidic, neutral, or
alkaline conditions (pH>3.5), the carboxyl groups of pectin will be partially or completely
ionized (depending on the pH), and available for ionic cross-linking by cations, especially
divalent cations such as Ca2⊕, forming the egg-box structures as shown in Figure 1.3 (Pérez,
Mazeau, & Hervé du Penhoat, 2000; Ridley, O'Neill, & Mohnen, 2001).
It has been suggested that HG are relatively rigid chains (Pérez, Mazeau, & Hervé du Penhoat,
2000). The binding of calcium ions by two HG chains facilitates their alignment, which
consequently allows the binding of another calcium ion, and so on along the HG chains, as
observed in the Figure 1.3. Subsequent additions of calcium ions promotes the formation of
trimers, tetramers and hexamers of HG chains, promoting the growing of the HG chains and the
formation of complex three-dimensional structures that are able to trap water and compounds of
nutritional interest (Munarin, Tanzi, & Petrini, 2012; Yang, Zhang, Hong, Gu, & Fang, 2013;
Yapo, 2011).
Figure 1.3. The egg-box structures in homogalacturonan. Adapted from http://genialab.de.
Egg-box structures
(LMP chain)
Rhamnosyl fold
HMP
chain
Ca2+
Chapter 1
12
1.3.1.2. Rhamnogalacturonan I (RG-I)
RG-I is a branched polymer with a backbone of the repeated disaccharide [4)--D-GalA-
(12)--L-Rha-(1]n, where Rha corresponds to rhamnose (Silva, Jers, Meyer, & Mikkelsen,
2016). The Rha units in the backbone can be substituted with -(1,4) galactan, branched
arabinan, and arabinogalactan branches (Figure 1.4). The predominant branches of RG-I contain
both linear and branched -L-arabinofuranosyl, and -D-galactopyranosyl residues (Figure 1.4).
In addition, the glycosyl residues -L-fucosyl, -D-glucuronosyl, and 4-O-methyl--D-
glucuronosyl may also be present (Khodaei & Karboune, 2013), as may be polyphenolics such as
ferulic and coumaric acids (Oosterveld, Pol, Beldman, & Voragen, 2001). As the structure of
RG-I is very heterogeneous, there is no knowledge of how its structure relates to functionality
(Silva, Jers, Meyer, & Mikkelsen, 2016). It has been suggested, nevertheless, that RG-I is
responsible for preventing the linear backbone of HG to form multivalent associations (Pérez,
Rodríguez-Carvajal, & Doco, 2003). In addition, RG-I may control the interaction of HG with
other cell wall components such as proteins and cellulose (Khodaei & Karboune, 2013).
Figure 1.4. Schematic structure of pectin. Pectin consists of three different types of polysaccharides,
including homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II).
Kdo, 3-deoxy-D-manno-2-octulosonic acid; D-Dha, 3-deoxy-D-lyxo-2-heptulosaric acid. Adapted from
http://plantphysiol.org (Plant Physiology. 153 (2010): 384-395).
RG-II RG-IHG
D-Galacturonic acid
L-Rhamnose
D-Glucoronic acid
Kdo
L-Arabinose
D-Galactose
L-Aceric acid
D-Dha
L-Apiose
L-Fucose
D-Xylose
L-Galactose
O-Acetyl
O-Methyl
Borate
Chapter 1
13
1.3.1.3. Rhamnogalacturonan II (RG-II)
RG-II is present in the primary cell wall of higher plants (Pérez, Rodríguez-Carvajal, & Doco,
2003). RG-II is quite different to RG-I since its backbone is composed of 1,4-linked -D-GalA
units rather than the repeated disaccharide [4)--D-GalA-(12)--L-Rha-(1]n (Ishii &
Matsunaga, 2001). Complex branches are attached to C2 and C3 positions in the backbone to
form RG-II (Figure 1.4). These branches are composed of up to 12 types of glycosyl units,
linked together by at least 22 different glycosidic bonds (Ridley, O'Neill, & Mohnen, 2001).
Some of the monosaccharide units and glycosidic linkages found in RG-II branches are
considered unique in plant polysaccharides, such as L-aceric acid, L-fucose, L-apiose, 3-deoxy-
D-manno-2-octulosonic acid, and 3-deoxy-D-lyxo-2-heptulosaric acid (Pérez, Rodríguez-
Carvajal, & Doco, 2003). Frequently, RG-II forms a dimer mediated by borate ion (BO33⊝)
attached to L-apiose units (Whitcombe, O'Neill, Steffan, Albersheim, & Darvill, 1995). RG-II
dimerization is a very important process for ensuring the integrity of the cell wall (Kaneko, Ishii,
& Matsunaga, 1997). Therefore, although RG-II is a minor component of pectin, it has an
essential role in the pectin structure stability (Whitcombe, O'Neill, Steffan, Albersheim, &
Darvill, 1995).
1.3.2. Classification of pectin
During the early work with pectins, different names were reported in literature, leading to
confusion. Because of this, a committee was organized by the American Chemical Society (ACS,
1944), giving a rigid classification of pectin. Pectins are mainly classified as function of both
molecular weight and MD since they are the main parameters influencing their physicochemical
and functional properties (Yapo, 2011) (Figure 1.5). Pectins may be grouped into three
categories: protopectins, pectinic acids, and pectic acids (Worth, 1967). Protopectin can be found
in immature fruits, whereas pectinic and pectic acids can be found in ripe and overripe fruits,
respectively. In addition, pectinic and pectic acids can be obtained by enzymatic hydrolysis of
protopectin and they represent the wide variety of naturally processed pectins (Worth, 1967).
Chapter 1
14
1.3.2.1. Protopectin
Protopectin is a water-insoluble pectic substance which upon hydrolysis (either enzymatic or
chemical), yields pectic and pectinic acids. Protopectins are characterized by their high molecular
weights (MW; n>2000, where n is the number of GalA units per molecule of pectin) as well as
their high MD (100% mol/mol).
1.3.2.2. Pectic acids
Pectic acids are pectic substances characterized by their intermediate MW (n ranging from 50 to
2000) and for having all of the carboxyl groups completely free [0% (mol/mol) MD, LMP]. The
salts of pectic acids are referred as pectates.
1.3.3.3. Pectinic acids
Pectinic acids are pectic substances characterized by their intermediate MW (n ranging from 50
to 2000) and for having variable MD. Pectinic acids can be classified as LMP and HMP
depending on their MD [LMP if MD<50% (mol/mol); and HMP if MD>50% (mol/mol)]. The
salts of pectinic acids are referred as pectinates.
As aforementioned, pectins are a group of polysaccharides with complex three-dimensional
structures which are responsible for their functional properties. It has been postulated that the
presence of pectins in the upper GIT will result in a decreased rate of intestinal digestion of a
range of nutrients, including carbohydrates, lipids and proteins (Brownlee, 2011). Furthermore,
the molecular mechanisms involved in the interaction of pectin with gastrointestinal components
as well as the control of lipid digestion by means of pectin are complex and remain under
investigation. As part of the experimental chapters we use emulsified lipids as a model for
evaluating the effect of the physicochemical properties of pectins on the rate and extent of the
lipid digestion process. Below, we provide an overview of the emulsion characteristics,
preparation, and the main factors affecting their long-term stability.
Chapter 1
15
Figure 1.5. Classification of pectic substances according to the American Chemical Society (ACS, 1944). MD, methoxylation degree; MW,
molecular weight; n, number of galacturonic acid units per molecules of pectin; LMP, low methoxylated pectin; HMP, high methoxylated pectin.
Chapter 1
16
1.4. Emulsions
1.4.1. General characteristics of emulsions
An emulsion is formed by two immiscible liquids (usually oil and water), with one of the liquids
dispersed as small spherical droplets in the other one (Figure 1.6). Emulsions can be classified
according to the spatial distribution of the oil and aqueous phases (Rodríguez-Abreu & Lazzari,
2008). An emulsion consisting of oil droplets dispersed into an aqueous phase is called an oil-in-
water emulsion (o/w emulsion), whereas an emulsion consisting of water droplets dispersed into
an oil phase is called water-in-oil emulsion (w/o emulsion). The compound forming the droplets
in an emulsion is referred to as the dispersed phase, whereas the compound forming the bulk
liquid is referred to as the continuous phase (Leal-Calderon, Thivilliers, & Schmitt, 2007). The
process of converting two separate bulk immiscible liquids into an emulsion and of reducing the
size of the droplets in a coarse emulsion is known as homogenization (Yang, Marshall-Breton,
Leser, Sher, & McClements, 2012). This process is often performed by using mechanical devices
known as homogenizers, which subject the bulk liquids to strong mechanical mixing (Kizling,
Kronberg, & Eriksson, 2006).
Figure 1.6. An example of an emulsion, consisting of oil droplets dispersed in an aqueous phase (o/w
emulsion). Picture obtained from McClements (2010).
Chapter 1
17
The production of an emulsion directly from two separate bulk liquid phases can be defined as
primary homogenization, whereas the reduction in size of the droplets in an coarse emulsion can
be defined as secondary homogenization (Figure 1.7) (Elwell, Roberts, & Coupland, 2004). It is
feasible to form an emulsion by homogenizing oil and water together. However, the two phases
rapidly separate into an oil layer on top and a water layer at the bottom. The driving force of this
separation process is the fact that the contact between the oil and water phases is
thermodynamically unfavorable (Kabalnov, 1998). However, it is possible to produce long-term
kinetically stable (metastable) emulsions by adding substances known as emulsifiers (Dickinson,
2015). An emulsifier is a compound (e.g., proteins, polysaccharides, surfactants, and
phospholipids) that can be used to improve the stability of an emulsion (Dickinson, 2009).
Emulsifiers are surface-active compounds that absorb onto the surface of the lipid droplets during
the homogenization process, forming a layer which prevents them to aggregate (Ozturk &
McClements, 2016). All in all, the chemical nature of the ingredients composing the emulsion
(oil phase, aqueous phase, and emulsifier), as well as the homogenization process used for
fabricating the emulsion, are determining factors defining the physicochemical properties of the
emulsion and its further stability (Dickinson, 2009; Kabalnov, 1998; Leal-Calderon, Thivilliers,
& Schmitt, 2007).
Figure 1.7. Homogenization can be conveniently classified into two categories: primary and secondary
homogenization. Primary homogenization is the conversion of two bulk liquids into a coarse emulsion,
whereas secondary homogenization is the reduction of the droplet size in an existing coarse emulsion to
form a fine emulsion. Picture obtained from McClements (2010).
Water
Oil
Primary
homogenization
Secondary
homogenization
Bulk
liquids
Coarse
emulsion
Fine
emulsion
Chapter 1
18
1.4.2. Physicochemical properties of emulsions
1.4.2.1. Particle size distribution
The most important properties of emulsions such as shelf life, appearance, texture, and flavor are
influenced by the size of the lipid droplets (Huang, Kakuda, & Cui, 2001). For example, the
stability of an emulsion decreases as the droplet size increases because larger droplets are highly
susceptible to aggregation (Saiki, Horn, & Prestidge, 2008). Lipid droplets are formed by
homogenization and its particle size depends on the chemical nature of the components of the
emulsion, the environmental conditions, and the homogenization method (Guzey & McClements,
2006). If the lipid droplets of an emulsion are of the same particle size, it is referred to as a
monodisperse emulsion, whereas if there is a range of lipid droplet sizes in the emulsion, it is
referred to as a polydisperse emulsion (Figure 1.8). The droplet size of a monodisperse emulsion
can be characterized by a single number (e.g., droplet diameter or radius). Monodisperse
emulsions are often produced for fundamental studies since the interpretation of the experimental
measurements is much easier than for polydisperse emulsions. Emulsions in food systems,
nevertheless, contain a wide distribution of lipid droplet sizes, and therefore, the characterization
of their droplet sizes is more complicated than that for monodisperse emulsions. Therefore, the
whole particle size distribution in polydisperse emulsions should be reported (Guzey &
McClements, 2006).
Figure 1.8. Schematic representation of monodisperse and polydisperse emulsions (McClements, 2010).
Monodisperse
emulsion
Polydisperse
emulsion
Chapter 1
19
1.4.2.2. Surface electrical charge
The physicochemical and sensory properties of emulsions are influenced by both the magnitude
and sign of the electrical charge on the lipid droplets (Guzey & McClements, 2006). The origin
of this charge is usually the absorption of emulsifier molecules possessing an electrical charge
(Ichikawa, Dohda, & Nakajima, 2006). Emulsifiers often have ionizable groups that may be
neutral, positively charged, or negatively charged, depending on the pH. Therefore, an emulsion
droplet may have an electrical charge depending on the surfactant type and the pH of the aqueous
phase. The electrical charge of a droplet can be characterized by several ways (Snarski & Dunn,
1991), including the surface charge density (), the electrical surface potential (0), and the zeta-
potential (). The surface charge density is the electrical charge per unit surface area, whereas the
electrical surface potential is the free energy required to increase the surface charge density from
0 to . The zeta-potential is the net surface potential of a droplet and it takes into account the
electrical charge of the species in the surrounding medium, as well as the environmental
conditions such as the ionic strength and the pH (McClements, 2004). Zeta-Potential influences
the interaction between emulsion droplets and other charged species such as biopolymers,
surfactants, vitamins, antioxidant, flavors, and minerals (Leunissen, van Blaaderen,
Hollingsworth, Sullivan, & Chaikin, 2007). These interactions usually have significant
implications for the overall stability and quality of an emulsion. For example, the volatility of a
flavor can be reduced if the flavor is electrostatically attracted to the surface of the lipid droplet,
altering the flavor profile of the emulsion (Given, 2009), or the susceptibility of lipid droplets to
oxidation depends on the attraction ability of the metal ion to the lipid droplet surface (Coupland
& McClements, 1996).
1.4.3. Stability of emulsions
The term emulsion stability is widely used to describe the capacity of an emulsion to resist
changes in its properties over time (Kabalnov, 1998; Kizling, Kronberg, & Eriksson, 2006). The
more stable the emulsion, the more slowly its properties change. Nevertheless, there are a wide
variety of physicochemical mechanisms that may account for the alteration in emulsion
Chapter 1
20
physicochemical properties. The most common physical mechanisms that are responsible for the
instability of emulsions are shown schematically in Figure 1.9. A brief description of the main
physical mechanisms of emulsion instability is given below: Gravitational separation, droplet
aggregation, and phase inversion.
1.4.3.1. Gravitational separation
Lipid droplets in an emulsion possess a different density to that of the continuous phase
surrounding them, and therefore a gravitational and a buoyant force acts on them (Robins, 2000).
Creaming and sedimentation are mechanisms of gravitational separation. Creaming implies the
upward movement of the lipid droplets because of their lower density as compared to the
continuous phase (Robins, 2000), whereas sedimentation implies the downward movement of the
lipid droplets because of the higher density as compared to the continuous phase (Yan &
Masliyah, 1993). The densities of the most of edible oils are lower than that of water. Therefore,
there is a trend for lipid droplets to accumulate at the top of an emulsion and water at the bottom.
Thus, the lipid droplets in an o/w emulsion tend to cream, whereas those in a w/o emulsion tend
to sediment (Basaran, Demetriades, & McClements, 1998). Gravitational separation is often
considered as having an adverse effect on the quality of emulsions. The separation of an emulsion
into an opaque cream layer on top and a clear aqueous layer at the bottom is undesirable (Robins,
2000). In addition, the textural attributes of an emulsion are also negatively affected by
gravitational separation, since the cream layer on top tends to be more viscous than the aqueous
layer on bottom (Robins, Watson, & Wilde, 2002).
1.4.3.2. Droplet aggregation
The lipid droplets in an emulsion are in continual movements because of the effects of the
thermal energy, gravity, or mechanical forces (e.g., agitation), and therefore, they can collide to
each other. After a collision takes place, the emulsion droplets may either move away or
aggregate, depending on the magnitude of both the attractive and repulsive interactions between
them (Saiki, Horn, & Prestidge, 2008). Flocculation and coalescence are both types of droplet
Chapter 1
21
aggregation. Flocculation takes place when two or more lipid droplets collide and form an
aggregate in which lipid droplets maintain their individual integrity (Starov & Zhdanov, 2003).
Flocculation increases the rate of the gravitational separation in o/w emulsions, which is often
undesirable since flocculation reduces their shelf life (Dickinson, 2010). Flocculation also
induces a pronounced increase of the emulsion viscosity, which may lead to the formation of a
gel network structure (Starov & Zhdanov, 2003).
Coalescence is the process by which two or more droplets collide to form a single large droplet
(Tcholakova, Denkov, Ivanov, & Campbell, 2006). Coalescence is the main mechanism by which
an emulsion evolves toward its most thermodynamically stable state since coalescence involves
an appreciable decrease in the contact area between the oil and water phases (Krebs, Schroën, &
Boom, 2013). Extensive lipid droplet coalescence eventually leads to the phases separation,
forming a layer of oil on top, which is known as the oiling-off process (Degner, Chung, Schlegel,
Hutkins, & McClements, 2014).
Figure 1.9. Emulsions may become unstable through a variety of physical mechanisms, including
creaming, sedimentation, flocculation, coalescence, and phase inversion (McClements, 2010).
Creaming Sedementation Flocculation Coalescence
Phase
inversionKinetically stable
emulsion
Chapter 1
22
1.4.3.3. Phase inversion
Phase inversion is the process by which an o/w emulsion is converted into a w/o emulsion or vice
versa (Perazzo, Preziosi, & Guido, 2015). Phase inversion is usually produced by alterations in
the composition or environmental conditions of an emulsion, for example, modification of the
disperse phase volume fraction (emulsion concentration), emulsifier type and concentration,
solvent presence, temperature, or mechanical agitation (Rao & McClements, 2010). Phase
inversion is an important step in the production of a wide number of food systems, including
butter and margarine. However, in other food systems, phase inversion is undesirable because it
has a negative effect on their appearance, texture, stability, and taste.
Emulsions are often used as a model to evaluate the stability and the gastrointestinal fate of lipids
upon digestion (Hur, Lim, Decker, & McClements, 2011). In vitro digestion models are therefore
needed to test the efficacy of different approaches of controlling lipid digestion (e.g., by addition
of pectins or other sources of dietary fiber) under conditions that simulate the human GIT. In the
next section, we provide an overview of the major physicochemical and physiological events that
occur in each region of the human GIT for simulating the in vitro digestion of emulsified lipids.
Emphasis will be placed in the composition, structure, and dynamics of the different regions of
the GIT and their further influence on the gastrointestinal fate of emulsified lipids. This approach
utilizes a number of sequential steps (oral, gastric, and small intestine phases) to more accurately
mimic the entire digestion.
1.5. In vitro digestion model of emulsified lipids
After ingestion, emulsified lipids are subjected to a complex series of chemical and physical
changes as they pass through the mouth, stomach, small intestine, and large intestine phases,
which affect their ability to be digested and absorbed. A schematic diagram of the
physicochemical conditions in the different regions of the human GIT is shown in Figure 1.10
(Hur, Lim, Decker, & McClements, 2011; McClements & Li, 2010).
Chapter 1
23
Figure 1.10. Schematic diagram of the physicochemical conditions in the different regions of the human
gastrointestinal tract. These conditions are used for simulating the in vitro digestion of emulsified lipids
and were obtained from previous reports (Hur, Lim, Decker, & McClements, 2011; McClements & Li,
2010).
By using this GIT model, the emulsified lipids are prepared and then subjected to three sequential
steps designed to mimic particular regions of the human GIT (e.g., mouth, stomach, and small
intestine). It is important to clarify that although the colonic phase will be mentioned, this phase
was not simulated in this thesis because there is evidence suggesting that emulsified lipids are
fully digested and adsorbed within the small intestine, therefore this step can be omitted (Hur,
Lim, Decker, & McClements, 2011; Singh, Ye, & Horne, 2009).
1.5.1. Oral phase
The main function of the mouth is to ingest the foods and to convert them into a form appropriate
for swallowing. The composition, structure, and properties of lipid droplets change appreciably
Oral phase:
• pH 5 - 7
• -Amylase
• Salts
• Biopolymers (mucin)
• 5 - 60 s
Gastric phase:
• pH 1 - 3
• Pepsin
• Salts
• Biopolymers (mucin)
• Agitation
• 30 min – 4 h
Intestinal phase:
• pH 6 - 8
• Pancreatin
• Bile salts
• Agitation
• 1 - 2 h
Colonic phase:
• pH 5 - 7
• Bacterial enzymes
• Anaerobic conditions
• Agitation
• 12 – 24 h
Chapter 1
24
during mastication due to the complex physicochemical and physiological processes that occur
within the mouth (Singh, Ye, & Ferrua, 2015). An ingested food is subjected to a wide number of
physicochemical processes: mixing with saliva; changing the pH, ionic strength, and temperature;
attacking by various digestive enzymes (e.g., -amylase); interacting with biopolymers from
saliva (e.g., mucin); and breaking down after mastication (Singh, Ye, & Horne, 2009;
Vingerhoeds, Blijdenstein, Zoet, & van Aken, 2005). The most important factor affecting the
behavior of emulsified lipids in the mouth is the interaction with saliva (Vingerhoeds,
Blijdenstein, Zoet, & van Aken, 2005). Human saliva is usually around pH 5.5 to 6.0 during
fasting and around 6.5 to 7.0 after food ingestion (Brandão, Soares, Mateus, & de Freitas, 2014).
Saliva contains water (99%), minerals (<1%), and proteins (0.1 to 0.5%). The protein fraction is
complex and contains digestive enzymes (e.g., -amylase), immunoglobulins, and glycosylated
proteins (e.g., mucins) (Brandão, Soares, Mateus, & de Freitas, 2014). The mucins are proteins
capable of inducing flocculation of the lipid droplets (Vingerhoeds, Blijdenstein, Zoet, & van
Aken, 2005). Therefore, limited lipid digestion may occur during the mastication process because
of the flocculation of lipid droplets and to the lack of lingual lipases secreted within the mouth in
adults (Neyraud, Palicki, Schwartz, Nicklaus, & Feron, 2012). The material that is swallowed
after mastication is referred to as bolus.
1.5.2. Gastric phase
After the bolus is swallowed it rapidly passes through the esophagus and reaches the stomach,
where it is mixed with the gastric fluids containing gastric enzymes (e.g., gastric lipase and
pepsin), minerals, and surface-active materials, and is also subjected to mechanical forces due to
stomach motility (Pal, Abrahamsson, Schwizer, Hebbard, & Brasseur, 2003). The pH of the
human stomach has been reported to be between 1.0 to 3.0 during fasting and around 2.0 to 2.7
after food ingestion (Singh, Ye, & Horne, 2009). The high acidity of stomach plays a wide
variety of physiological roles, including activating enzymes (e.g., zymogens), hydrolysis of food
components (both enzymatic and chemical hydrolysis), and the inactivation of microorganisms
(Guerra, Etienne-Mesmin, Livrelli, Denis, Blanquet-Diot, & Alric, 2012). The digestion of
emulsified lipids begins in the stomach because of the presence of gastric lipase. Gastric lipase
Chapter 1
25
adsorbs onto the lipid droplet surfaces, where it is able to convert triacylglycerols (TAG) into
diacylglycerols (DAG), monoacylglycerols (MAG), and free fatty acids (FFA). It has been
established, however, that the gastric digestion of emulsified lipids is negligible as compared to
that occurring in the small intestine, because gastric lipases are considerably less active than
intestinal lipases (Canaan, Roussel, Verger, & Cambillau, 1999; Reis, Holmberg, Miller, Leser,
Raab, & Watzke, 2009; Reis, Holmberg, Watzke, Leser, & Miller, 2009). The FFA released in
the stomach after gastric digestion of TAG, nevertheless, may play an important role in the
subsequent digestion and absorption process of nutrients since FFA are capable to i) promote
lipid digestion by enhancing droplet disruption, ii) increase solubilization of lipid digestion
products, iii) stimulate hormone release, iv) increase the binding capacity of colipase protein, and
v) increase the activity of pancreatic lipase in the small intestine (Singh, Ye, & Ferrua, 2015;
Singh, Ye, & Horne, 2009). The partially digested and disrupted food that leaves the stomach and
reaches the small intestine is usually known as chyme.
1.5.3. Small intestine phase
The small intestine is the region in the GIT where most of the lipid digestion and absorption
processes normally occur. After reaching the small intestine phase, the chyme is mixed with
bicarbonate (HCO3⊝), bile salts, phospholipids, and enzymes secreted by the liver and the
pancreas (McClements & Li, 2010). The bicarbonate secreted into the small intestine increases
the pH from highly acidic (around pH 1.0 to 3.0) to neutral (around pH 5.8 to 6.5), where the
pancreatic enzymes work more efficiently (Wilde & Chu, 2011). Lipid hydrolysis is carried out
in the small intestine by the enzymatic action of lipases secreted by the pancreas (Singh, Ye, &
Ferrua, 2015). Pancreatic lipase plays an important role in the lipid digestion process because it is
the digestive enzyme responsible for hydrolyzing TAGs into FFAs, DAGs, and MAGs. To
catalyze this reaction, pancreatic lipase needs to adsorb onto the lipid droplet surfaces to be in
proximity to TAG (Reis, Holmberg, Miller, Leser, Raab, & Watzke, 2009). Pancreatic lipase
often does this as part of a complex with a protein known as colipase, and also with bile salts
(Erlanson-Albertsson, 1983). Usually, colipase binds to the C-terminal domain of pancreatic
lipase, increasing its hydrophobicity and facilitating its adsorption onto the lipid droplet surfaces
Chapter 1
26
(Erlanson-Albertsson, 1983). Therefore, both pancreatic lipase and colipase come together in
commercial enzymatic preparations so then, further addition of colipase is not necessary (Singh,
Ye, & Horne, 2009).
Bile salts are capable of stabilizing lipid droplets against aggregation, and forming micelles that
solubilize and transport hydrophobic molecules such as TAGs and the lipid digestion products
(Maldonado-Valderrama, Wilde, Macierzanka, & Mackie, 2011). Once the digestion process of
emulsified lipids has been carried out by pancreatic lipase, the undigested lipids, lipid digestion
products, and organic-soluble compounds (e.g., FFAs, DAGs, MAGs, cholesterol, phospholipids,
and fat-soluble vitamins) are solubilized within micelles formed by bile salts and phospholipids,
and then, they are transported to the epithelium cells for further absorption (Golding & Wooster,
2010; Singh, Ye, & Horne, 2009; Wilde & Chu, 2011).
1.5.4. Large intestine phase
The material that is not digested and absorbed within the small intestine reaches the large
intestine. The main physiological function of the large intestine is the absorption of water and
electrolytes, the fermentation of polysaccharides and proteins, the re-absorption of bile salts, and
the formation, storage, and elimination of fecal matter (Emmanuel & Butt, 2015). Any material
that is undigested in the upper GIT eventually reaches the colon. Usually, lipids are fully digested
in the stomach and small intestine. However, undigested lipids may pass through the GIT and
reach the colon (Guerra, Etienne-Mesmin, Livrelli, Denis, Blanquet-Diot, & Alric, 2012;
McClements & Li, 2010; Singh, Ye, & Horne, 2009). For example, if lipid droplets are
surrounded by an indigestible layer or trapped within an indigestible matrix (e.g., a dietary fiber
matrix), then they are not capable to be fully digested in the upper GIT (McClements, 2010). The
large intestine contains different kinds of anaerobic microorganisms, which are capable to
ferment some food components that were not digested in the upper GIT (Gibson, Probert, Loo,
Rastall, & Roberfroid, 2004). Therefore, lipids trapped within dietary fiber matrices may only be
released after they reach the large intestine by means of bacterial fermentation where they cannot
longer be digested or absorbed (McClements, 2010, 2015; Yao, Xiao, & McClements, 2014).
Chapter 1
27
1.6. Aim and outline of the thesis
In recent years, the effect of pectin consumption on a number of physiological responses such as
the regulation of body-weight and blood pressure, as well as the control of the glucose and lipid
levels in blood have been identified. However, the mechanisms by which pectin is able to exert
its physiological functions have not yet been studied. For example, little information is available
about the inhibitory capacity of pectin on the activity of the major digestive enzymes (pancreatic
lipase, -amylase, alkaline phosphatase, and protease), or about the inhibitory capacity of pectin
on the rate and extent of the mass transfer process of nutrients (e.g., monosaccharides, amino
acids, and lipids). Furthermore, few attempts on the evaluation of the effect of pectin properties
(e.g., molecular weight and methoxylation degree) on the gastrointestinal fate of emulsified lipids
have been done. Finally, little is known about the molecular interactions between pectin and the
major gastrointestinal components governing the digestion process of emulsified lipids.
Therefore, with the objective to evaluate the mechanisms by which pectin is able to exert its
physiological functions, the aim of this thesis is to obtain fundamental understanding of the
influence of pectin properties (methoxylation degree) on the activity of the major digestive
enzymes (pancreatic lipase, -amylase, alkaline phosphatase, and protease), and the rate and
extent of the mass transfer process of nutrients (monosaccharides, amino acids, and lipids). This
thesis is also aimed at evaluating the incidence of pectin properties on the gastrointestinal fate of
emulsified lipids by using a simulated in vitro GIT model, and to evaluate both the nature and
magnitude of the molecular interactions between pectin and the compounds related to the
digestion of emulsified lipids.
In chapter 2, the evaluation of the factors affecting the activity of the main digestive enzymes
[pancreatic lipase, -amylase, alkaline phosphatase, and protease (chymotrypsin)] is described.
The results obtained in this chapter are used in chapter 3, where the influence of pectin
properties (methoxylation degree) on the enzymatic activity of these enzymes is evaluated.
Because pancreatic lipase was found to be the enzyme affected the most by pectin, subsequent
chapters are focused on evaluating the effect of pectin on the gastrointestinal fate of lipids. In
Chapter 1
28
chapter 4, the inhibitory effect of pectin on the rate and extent of the digestion of emulsified
lipids is evaluated by using a simulated in vitro GIT model, and the results are compared with
those obtained with other sources of dietary fiber (chitosan, and methyl cellulose). In addition,
chapter 5 describes the influence of pectin properties (molecular weight and methoxylation
degree) on the gastrointestinal fate of emulsified lipids by using a simulated in vitro GIT model.
To give a deeper insight on the mechanism governing the effect of pectin on the digestion of
emulsified lipids, chapter 6 describes both the nature and magnitude of the molecular
interactions between pectin and the major components of the GIT. Considering that pectin is
known for increasing the viscosity of the digesta and it is able to provide a restriction of the
nutrient mobility, chapter 7 describes the effect of pectin properties (methoxylation degree) on
the rate and extent of the diffusion process of the most important nutrients (monosaccharides,
amino acids, and an emulsion). Finally, in chapter 8, a general discussion is presented and
implications of the findings are elaborated.
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Chapter 2
Optimization of the reaction conditions affecting
the activity of digestive enzymes
Chapter 2
35
Abstract
Enzymatic activities were evaluated spectrophotometrically by using the artificial chromogenic
substrates p-nitrophenyl palmitate, 2-chloro-p-nitrophenyl--D-maltotrioside, p-nitrophenyl
phosphate, and p-nitrophenyl acetate for pancreatic lipase, -amylase, alkaline phosphatase, and
protease, respectively. Artificial chromogenic substrates were used because they allow obtaining
fast, sensitive, and reproducible results. Both temperature (37 °C) and pH (7.0) of each of the
reaction mixtures were fixed to ensure the optimum performance of digestive enzymes. Buffer
and cofactor types and buffer, cofactor, and enzyme concentrations were evaluated as the reaction
conditions affecting the activity of the digestive enzymes. Pancreatic lipase and alkaline
phosphatase were more active in Tris-HCl buffer (at concentrations of 50 and 20 mM,
respectively) as compared to phosphate buffer. Conversely, -amylase and protease were more
active in phosphate buffer (at concentrations of 20 and 50 mM, respectively) as compared to Tris-
HCl buffer. -Amylase, alkaline phosphatase, and protease required the presence of NaCl as
cofactor (at concentrations of 10, 15, and 20 mM, respectively), whereas pancreatic lipase
required the presence of both NaCl and CaCl2 (at concentrations of 150 and 1.0 mM,
respectively). The optimum enzyme concentrations were found to be 900, 10, 0.8, and 3 U mL-1
for pancreatic lipase, -amylase, alkaline phosphatase, and protease, respectively. Finally, the
optimization process of the reaction conditions allowed improving the activities of all of the
digestive enzymes during each step. However, the optimization of the reaction conditions had no
a significant effect on the reproducibility of the measurements.
Keywords: Digestive enzymes, buffer, cofactor, specific activity, physiological conditions.
Chapter 2
36
2.1. Introduction
The chemical reactions occurring in living organisms are controlled by enzymes
(Martínez Cuesta, Rahman, Furnham, & Thornton, 2015). Enzymes are globular proteins with
complex three-dimensional structures, responsible for their unique catalytic functions (Jacobson,
Kalyanaraman, Zhao, & Tian, 2014). Regarding the complex structures of enzymes, it is
reasonable to expect that many environmental parameters are capable to affect their three-
dimensional conformation and their subsequent catalytic activities (Daniel & Danson, 2013). The
activity of an enzyme can be largely affected by several environmental conditions such as
temperature, pH, types of buffer and cofactor, and concentrations of buffer, cofactor, substrate,
and enzyme (Iyer & Ananthanarayan, 2008). Enzymes display their highest activities at their
optimum conditions; deviations from these conditions may cause a reduction of the enzymatic
activity, depending on the degree of the deviation (Vendruscolo, 2002). Moderate deviations
produce small activity decrease. However, severe deviations may lead to a complete loss of the
enzymatic activity. Consequently, it is important to establish the optimum parameters for
measuring the activity of enzymes to ensure their optimal performance.
The enzymatic reactions can be carried out by using either native or artificial substrates. The use
of native substrates is preferred because of the biological relevance of the results. However, the
reproducibility obtained with native substrates is sometimes unacceptable, especially, when the
substrates have a high structural complexity (Svendsen, Blombäck, Blombäck, & Olsson, 1972).
Instead, it is possible to use artificial substrates to obtain faster and reliable results as compared
to native substrates (Bisswanger, 2014). A wide variety of artificial chromogenic substrates are
available for the rapid and reliable in vitro assaying of the activity of several enzymes (Goddard
& Reymond, 2004). Artificial substrates are structurally similar to their analogous native
substrates, but they are conjugated to a chromogenic leaving group. After enzymatic reaction
takes place, the chromogenic leaving group is liberated and it can be conveniently monitored by
changes in absorbance.
Chapter 2
37
It has been established that enzymes are less specific (ability of the enzyme to select the
substrate) but more selective (ability of the substrate to bind to the enzyme) to artificial substrates
(Svendsen, Blombäck, Blombäck, & Olsson, 1972). Therefore, the use of artificial substrates has
some advantages and disadvantages. In the one hand, the high selectivity existing between
enzymes and artificial substrates allows the enzymatic assay to be more sensitive, faster, and
reproducible as compared to native substrates. In the other hand, the low specificity of enzymes
by artificial substrates may lead to collateral enzymatic reactions if other enzymes are present
(Wenger, Sattler, Clark, & Wharton, 1976). However, because the enzymes used in this study are
commercial and relatively pure, interferences can be considered as negligible. Furthermore, extra
development efforts for adjusting the experimental conditions when using artificial substrates are
required because the experimental conditions reported in literature for native substrates may
differ considerably to those required for artificial substrates (Nagaki, Kimura, Kimura, Maki,
Goto, Nishino, et al., 2001).
In this chapter the reaction conditions affecting the activity of some hydrolytic digestive enzymes
were studied. Pancreatic lipase, -amylase, and protease were selected because they are the main
enzymes involved in the digestion of lipids, carbohydrates, and proteins, respectively. Although
alkaline phosphatase is an enzyme not directly involved in the digestive processes, it was also
selected because it participates in the dephosphorylation of nutrients, which is a necessary
process for their further digestion (Lallès, 2014). Enzymatic activities were evaluated
spectrophotometrically by using the artificial chromogenic substrates p-nitrophenyl palmitate
(pNPPA), 2-chloro-p-nitrophenyl--D-maltotrioside (G3CNP), p-nitrophenyl phosphate (pNPP),
and p-nitrophenyl acetate (pNPA) for pancreatic lipase, -amylase, alkaline phosphatase, and
protease, respectively (Figure 2.1). Buffer and cofactor types and buffer, cofactor, and enzyme
concentrations were evaluated as the reaction conditions affecting the activities of the
aforementioned digestive enzymes. Because of the human physiological conditions were taken
into account, both temperature (37 °C) and pH (7.0) were fixed to ensure the optimum
performance of digestive enzymes. The best reaction conditions obtained in this study for the
measurement of the activity of digestive enzymes will be used in further studies (Chapter 3) to
evaluate the effect of pectin on the activity of these enzymes.
Chapter 2
38
Figure 2.1. Chemical structures of the artificial chromogenic substrates 2-chloro-p-nitrophenyl--D-
maltotrioside (G3CNP), p-nitrophenyl acetate (pNPA), p-nitrophenyl palmitate (pNPPA), and p-
nitrophenyl phosphate (pNPP) for -amylase, protease, pancreatic lipase, and alkaline phosphatase,
respectively. Arrows indicate the sites of bond cleavage by the enzymes. Chromogenic leaving groups are
represented in red: 2-chloro-p-nitrophenol (CNP) and p-nitrophenol (pNP).
2.2. Materials and methods
2.2.1. Chemicals
The hydrolytic enzymes porcine pancreatic -amylase, from Sus scrofa (16 U mg-1
type VI-B,
E.C. 3.2.1.1); porcine pancreatic lipase, from Sus scrofa (100 U mg-1
type II, E.C. 3.1.1.3);
bovine pancreatic protease (chymotrypsin), from Bos taurus (5 U mg-1
type I, E.C. 3.4.21.1); and
bovine intestinal mucosa alkaline phosphatase, from Bos taurus (10 U mg-1
type I, E.C. 3.1.3.1);
the artificial substrates G3CNP, pNPPA, pNPA, and pNPP; the reaction products p-nitrophenol
(pNP) and 2-chloro-p-nitrophenol (CNP); and the protein determination reagents brilliant blue G-
250 (Coomassie Blue) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich
Chemical Company (St. Louis, MO, USA). Ox bile extract with cholic acid content higher than
55% (w/w) was purchased from MP Biomedicals (Solon, OH, USA). Other chemicals were
purchased from Merck KGaA (Darmstadt, Germany).
( )14
G3CNP pNPA pNPPA pNPP
NO2
O
O
CH3
NO2
O
O
CH3
NO2
O
P
O
OH OH
O
OO
OH
OH
OH
OH
OO
OH
OH
OH
OH
O
OH
OH
OH
OH
NO2
Cl
NO2
O
O
CH3
NO2
O
O
CH3
NO2
O
P
O
OH OH
O
OO
OH
OH
OH
OH
OO
OH
OH
OH
OH
O
OH
OH
OH
OH
NO2
Cl
pNPCNP
O
OOH
OH
OH
OH
O
OOH
OH
OH
OH
O
OOH
OH
OH
OH
NO2
Cl
NO2
O
O
CH3
pNP pNP
Chapter 2
39
2.2.2. Reaction conditions affecting the activity of digestive enzymes
The reaction conditions affecting the enzymatic activities for each of the tested enzymes were
adjusted by using a stepwise design: the best result obtained for one parameter was fixed and then
used to evaluate the next one. Because of the variability in the reaction conditions that can be
found for each enzyme, depending on the nature of the substrate and the source of the enzyme, a
range of experimental conditions typically found in literature were selected to be evaluated for
each enzyme. The reaction mixtures for all of the tested enzymes were prepared in the proper
buffer solution at pH 7.0 (depending on the buffer that was evaluated). All buffer solutions
evaluated for each of the tested enzymes contained 3.5 mg mL-1
bile acid extract (equivalent to
5.0 mM cholic acid in the total mixture). It is known that pancreatic lipase is the only enzyme
which is dependent on bile salts (Reis, Holmberg, Watzke, Leser, & Miller, 2009); however, bile
salts were also added to the reaction mixtures of -amylase, alkaline phosphatase, and protease
because bile salts are capable to modify the chemical environment of these enzymes and affect
their further stabilities (Robic, Linscott, Aseem, Humphreys, & McCartha, 2011). All reaction
mixtures for each of the tested enzymes were incubated at 37 °C and the absorbance of the
product was measured every 5 s throughout 120 s. The product obtained for pancreatic lipase,
alkaline phosphatase, and protease was pNP and the absorbance was recorded at 415 nm, whereas
the product obtained for -amylase was CNP and the absorbance was recorded at 405 nm
(Figure 2.1).
2.2.2.1. Pancreatic lipase
The conditions for measuring the activity of pancreatic lipase are based on those reported by
(Tsujita, Takaichi, Takaku, Sawai, Yoshida, & Hiraki, 2007). Buffer type [Tris-HCl and
phosphate buffer solution (PBS)], buffer concentration (Tris-HCl at concentrations ranging from
20 to 100 mM), cofactor type (NaCl, CaCl2, and a mixture of both NaCl and CaCl2), cofactor
concentration (NaCl at concentrations ranging from 30 to 270 mM, and CaCl2 at concentrations
ranging from 0.3 to 2.7 mM), and enzyme concentration (pancreatic lipase at concentrations
ranging from 300 to 1500 U mL-1
) were evaluated in the mentioned order. For each of the
Chapter 2
40
enzymatic assays, the substrate was kept constant at a concentration of 4.0 mM pNPPA. All
reaction mixtures contained 100 g mL-1
Tween-20 to improve the solubility of the substrate.
2.2.2.2. -Amylase
The conditions for measuring the activity of -amylase are based on those reported by
(Morishita, Iinuma, Nakashima, Majima, Mizuguchi, & Kawamura, 2000). Buffer type (Tris-HCl
and PBS), buffer concentration (PBS at concentrations ranging from 5 to 45 mM), cofactor type
(NaCl, CaCl2, and a mixture of both NaCl and CaCl2), cofactor concentration (NaCl at
concentrations ranging from 3 to 23 mM), and enzyme concentration (-amylase at
concentrations ranging from 3 to 15 U mL-1
) were evaluated in the mentioned order. For each of
the enzymatic assays, the substrate was kept constant at a concentration of 0.4 mM G3CNP. All
reaction mixtures contained 15% (v/v) glycerol required to improve the stability of the enzyme.
2.2.2.3. Alkaline phosphatase
The conditions for measuring the activity of alkaline phosphatase are based on those reported by
(Chaudhuri, Chatterjee, Venu-Babu, Ramasamy, & Thilagaraj, 2013). Buffer type (Tris-HCl and
PBS), buffer concentration (Tris-HCl at concentrations ranging from 5 to 45 mM), cofactor type
(NaCl, CaCl2, and a mixture of both NaCl and CaCl2), cofactor concentration (NaCl at
concentrations ranging from 3 to 23 mM), and enzyme concentration (alkaline phosphatase at
concentrations ranging from 0.3 to 1.3 U mL-1
) were evaluated in the mentioned order. For each
of the enzymatic assays, the substrate was kept constant at a concentration of 0.4 mM pNPP.
2.2.2.4. Protease
The conditions for measuring the activity of protease are based on those reported by (Verma &
Ghosh, 2010). Buffer type (Tris-HCl and PBS), buffer concentration (PBS at concentrations
ranging from 10 to 60 mM), cofactor type (NaCl, CaCl2, and a mixture of both NaCl and CaCl2),
cofactor concentration (NaCl at concentrations ranging from 5 to 30 mM), and enzyme
Chapter 2
41
concentration (protease at concentrations ranging from 1 to 5 U mL-1
) were evaluated in the
mentioned order. For each of the enzymatic assays, the substrate was kept constant at a
concentration of 1.0 mM pNPA.
2.2.2.5. Determination of the specific enzymatic activities
To determine the specific enzymatic activities, the absorbance obtained from the enzymatic
activity experiments was plotted versus time. From the straight lines, the slope (expressed as
absorbance units min-1
) was calculated and then interpolated in the calibration lines for each
reaction product to express it as μmol product min-1
. The calibration lines were obtained using
pNP at concentrations ranging from 5 to 50 M (7 data points, r2=0.999) for lipase, alkaline
phosphatase, and protease; and using CNP at concentrations ranging from 5 to 60 M (7 data
points, r2=0.998) for -amylase. The protein concentration of each working enzyme solution was
determined by using the modified Bradford method (Zor & Selinger, 1996), with BSA as the
standard. The specific enzymatic activities of pancreatic lipase, alkaline phosphatase, and
protease were then expressed as mol pNP min-1
mg-1
protein, while the specific enzyme activity
of -amylase was expressed as mol CNP min-1
mg-1
protein.
2.2.3. Data analysis
All measurements were conducted with three analytical and three technical (instrumental)
replicates, for a total of nine replicates (n=9). The mean values and their standard deviations were
reported. Statistical descriptive analyses were performed by using STATGRAPHICS Centurion
XVI version 16.1.11 for Windows (Statpoint Technologies Inc., Warrenton, VA, United States).
Chapter 2
42
2.3. Results and discussion
2.3.1. Effect of the buffer type and concentration
Figure 2.2 presents the specific enzymatic activities of pancreatic lipase, -amylase, alkaline
phosphatase, and protease in the presence of either Tris-HCl or PBS (pH 7.0). The specific
enzymatic activities of pancreatic lipase (Figure 2.2a) and alkaline phosphatase (Figure 2.2c) in
the presence of Tris-HCl were found to be 2.4 and 6.1 times higher, respectively, than those
obtained in the presence of PBS. Conversely, the specific enzymatic activities of -amylase
(Figure 2.2b) and protease (Figure 2.2d) in the presence of PBS were found to be 4.7 and 1.6
times higher, respectively, than those obtained in the presence of Tris-HCl. After defining the
appropriate buffer type (Tris-HCl for pancreatic lipase and alkaline phosphatase, and PBS for -
amylase and protease), the effect of buffer concentrations was then evaluated. The buffer
concentration dependence on the activity of each of the tested digestive enzymes is shown in
Figure 2.3. The specific enzymatic activities increased from the low buffer concentration region
up to a maximum value (optimum buffer concentration), and then decreased to the high buffer
concentration region. For pancreatic lipase, the maximum specific activity was reached at a
concentration of 50 mM Tris-HCl (Figure 2.3a). This value was higher than that determined for
alkaline phosphatase, in which the optimum Tris-HCl concentration was about 20 mM (Figure
2.3c). For -amylase the maximum specific activity was reached at a concentration of 20 mM
PBS (Figure 2.3b). This value was lower than that determined for protease, in which the
optimum PBS concentration was about 50 mM (Figure 2.3d). The effect of buffer on the activity
of the digestive enzymes can be addressed by considering two criteria: i) buffer capacity and ii)
buffer concentration (Bisswanger, 2014).
i) Buffer serves to adjust and stabilize the pH during the enzymatic reaction. The pKa value
indicates the pH where the buffer possesses its highest buffer capacity (Chiriac & Balea, 1997). It
is accepted that the maximum capacity of a buffer comprises a range from one pH unit below to
one pH unit above the pKa value (pH=pKa 1) (Chiriac & Balea, 1997).
Chapter 2
43
Figure 2.2. Effect of the buffer type on the enzymatic activity of pancreatic lipase (a), -amylase (b),
alkaline phosphatase (c), and protease (d). The reaction conditions are presented below. Pancreatic lipase:
Either Tris-HCl or phosphate (PBS) buffer solutions (50 mM, pH 7.0), 0.6 mM CaCl2, 600 U mL-1
enzyme concentration; and 4.0 mM pNPPA. -Amylase: Either Tris-HCl or phosphate (PBS) buffer
solutions (25 mM, pH 7.0), 15 mM NaCl, 5 U mL-1
enzyme concentration, and 0.4 mM G3CNP. Alkaline
phosphatase: Either Tris-HCl or phosphate (PBS) buffer solutions (15 mM, pH 7.0), 10 mM NaCl, 0.5 U
mL-1
enzyme concentration, and 0.4 mM pNPP. Protease: Either Tris-HCl or phosphate (PBS) buffer
solution (20 mM, pH 7.0), 15 mM NaCl, 2 U mL-1
enzyme concentration, and 1.0 mM pNPA. The
temperature of each of the enzymatic reactions was fixed at 37 °C.
0.0
0.3
0.6
0.9
1.2
Tris-HCl PBS
Lip
ase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
Buffer (50 mM pH 7.0)
a.
0
2
4
6
8
Tris-HCl PBS
-Am
yla
se A
ctiv
ity
(m
ol
CN
P m
in-1
mg
-1p
rote
in)
Buffer (25 mM pH 7.0)
b.
0
10
20
30
Tris-HCl PBS
Ph
osp
ha
tase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
Buffer (15 mM pH 7.0)
c.
0
4
8
12
Tris-HCl PBS
Pro
tease
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
Buffer (20 mM pH 7.0)
d.
Chapter 2
44
Figure 2.3. Effect of the buffer concentration on the enzymatic activity of pancreatic lipase (a), -amylase
(b), alkaline phosphatase (c), and protease (d). The reaction conditions are presented below. Pancreatic
lipase: Tris-HCl buffer solution (pH 7.0) at concentrations ranging from 20 to 100 mM, 0.6 mM CaCl2,
600 U mL-1
enzyme concentration; and 4.0 mM pNPPA. -Amylase: Phosphate buffer solution (PBS; pH
7.0) at concentrations ranging from 5 to 45 mM, 15 mM NaCl, 5 U mL-1
enzyme concentration, and 0.4
mM G3CNP. Alkaline phosphatase: Tris-HCl buffer solution (pH 7.0) at concentrations ranging from 5 to
45 mM, 10 mM NaCl, 0.5 U mL-1
enzyme concentration, and 0.4 mM pNPP. Protease: Phosphate buffer
solution (PBS, pH 7.0) at concentrations ranging from 10 to 60 mM, 15 mM NaCl, 2 U mL-1
enzyme
concentration, and 1.0 mM pNPA. The temperature of each of the enzymatic reactions was fixed at 37 °C.
0
3
6
9
0 10 20 30 40 50
-Am
yla
se A
ctiv
ity
(m
ol
CN
P m
in-1
mg
-1p
rote
in)
PBS (mM)
b.
0
5
10
15
20
25
0 10 20 30 40 50
Ph
osp
ha
tase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
Tris-HCl (mM)
c.
0
3
6
9
12
0 20 40 60 80
Pro
tea
se A
ctiv
ity
(m
ol p
NP
min
-1m
g-1
pro
tein
)
PBS (mM)
d.
0.0
0.3
0.6
0.9
1.2
0 30 60 90 120
Lip
ase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
Tris-HCl (mM)
a.
Chapter 2
45
The pKa values have been reported to be 7.20 and 8.06 for PBS and Tris-HCl, respectively
(Good, Winget, Winter, Connolly, Izawa, & Singh, 1966). Therefore, PBS is expected to have a
higher buffer capacity than Tris-HCl at pH 7.0 because the pKa of PBS is closer to the pH of the
reaction than that of Tris-HCl. Therefore, the highest buffer capacity of PBS as compared to that
of Tris-HCl may be responsible for the higher enzymatic activity exhibited by -amylase (Figure
2.2b) and protease (Figure 2.2d), when they were prepared in PBS at pH 7.0.
ii) It has been established that the more concentrated a buffer system, the higher its capacity to
stabilize the pH (Iyer & Ananthanarayan, 2008). However, most enzymes accept only moderate
concentrations of buffer, commonly between 5 and 200 mM. At low concentrations of buffer, the
enzymatic activities can be inhibited by pH shifts during the reaction (Ballou & Luck, 1941). It is
important to consider that some enzymatic reaction themselves can cause pH shifts and
consequently, a continuous decrease of the enzymatic activity, e.g., if an acid or alkaline
component becomes released during a hydrolysis reaction, such as the liberation of fatty acids by
pancreatic lipase or the liberation of amino acids by protease (Li & McClements, 2010). In such
cases, the pH should be kept constant during the reaction by using a concentrated buffer solution.
Therefore, the high buffer concentrations obtained here for pancreatic lipase (Figure 2.3a) and
protease (Figure 2.3d) necessary for reaching their maximum activities may be required to
compensate the continuous acidification of the reaction medium by the release of acid products.
Conversely, high concentrations of buffer can destabilize the intermolecular interactions which
maintain the enzymes folded, leading to denaturation (Bauduin, Nohmie, Touraud, Neueder,
Kunz, & Ninham, 2006). For example, the destabilizing effect of Tris-HCl on the tertiary
structure of -amylase (Ballou & Luck, 1941), and the inhibitory effect of PBS on the activity of
alkaline phosphatase (Coburn, Mahuren, Jain, Zubovic, & Wortsman, 1998) have been reported.
The inhibition of the activity due to the extreme concentrations of buffer was observed for all of
the tested enzymes (Figure 2.3). Therefore, the low enzymatic activities exhibited by -amylase
(Figure 2.2b) and alkaline phosphatase (Figure 2.2c) can be attributed to the destabilizing
influence of Tris-HCl and the inhibitory effect of PBS, respectively.
Chapter 2
46
2.3.2. Effect of the cofactor type and concentration
After defining the appropriate type and concentration of buffer for each of the tested enzymes,
the effect of both cofactor type and its concentration was evaluated. Figure 2.4 shows the effect
of the cofactor type on the specific enzymatic activities of pancreatic lipase, -amylase, alkaline
phosphatase, and protease. For each of the tested enzymes, enzymatic activities in the absence of
cofactor (control) were negligible. However, the addition of a cofactor significantly increased the
enzymatic activities. NaCl, CaCl2, and a mixture of both NaCl and CaCl2 were evaluated as
cofactors of the digestive enzymes. For pancreatic lipase (Figure 2.4a) addition of NaCl or CaCl2
had little effect on the enzymatic activity. However, when a mixture of both NaCl and CaCl2 was
added, the enzymatic activity was significantly increased. The results obtained for -amylase
(Figure 2.4b), alkaline phosphatase (Figure 2.4c), and protease (Figure 2.4d) were quite
different to those obtained for pancreatic lipase. The individual addition of CaCl2 had little effect
on the enzymatic activities, whereas the individual addition of NaCl significantly increased the
activities of these enzymes. In addition, when a mixture of both NaCl and CaCl2 was added, the
enzymatic activities were fairly similar to those obtained with only NaCl. This result suggests
that NaCl is the only cofactor affecting the activity of -amylase, alkaline phosphatase, and
protease, whereas CaCl2 has no significant contribution to the overall activity of these enzymes.
The cofactor concentration dependence towards the activity of each of the tested digestive
enzymes resembles in some respect the buffer concentration dependence (Figure 2.5): increasing
when raising the cofactor concentration, reaching a maximum value, followed by a decrease. For
pancreatic lipase (Figure 2.5a) the maximum specific activity was reached at concentrations of
150 and 1.0 mM for NaCl and CaCl2, respectively; whereas for -amylase (Figure 2.5b),
alkaline phosphatase (Figure 2.5c), and protease (Figure 2.5d) the maximum specific activities
were reached at concentrations of 10, 15, and 20 mM NaCl, respectively. It has been reported that
pancreatic lipase is highly dependent on calcium concentration, whereas -amylase, alkaline
phosphatase and protease do not require a cofactor for its normal operation (Schonheyder &
Volqvartz, 1945). However, the enzymatic activities of all the digestive enzymes studied here
were considerably enhanced upon addition of cofactors (Figures 2.4 and 2.5).
Chapter 2
47
Figure 2.4. Effect of the cofactor type on the enzymatic activity of pancreatic lipase (a), -amylase (b),
alkaline phosphatase (c), and protease (d). The reaction conditions are presented below. Pancreatic lipase:
50 mM Tris-HCl buffer solution (pH 7.0), 0.6 mM NaCl, 0.6 mM CaCl2, or a mixture of NaCl and CaCl2
(each component at a concentration of 0.6 mM), 600 U mL-1
enzyme concentration, and 4.0 mM pNPPA.
-Amylase: 20 mM Phosphate buffer solution (PBS, pH 7.0), 15 mM NaCl, 15 mM CaCl2, or a mixture of
NaCl and CaCl2 (each component at a concentration of 15 mM), 5 U mL-1
enzyme concentration, and 0.4
mM G3CNP. Alkaline phosphatase: 20 mM Tris-HCl buffer solution (pH 7.0), 10 mM NaCl, 10 mM
CaCl2, or a mixture of NaCl and CaCl2 (each component at a concentration of 10 mM), 0.5 U mL-1
enzyme concentration, and 0.4 mM pNPP. Protease: 50 mM Phosphate buffer solution (PBS, pH 7.0), 15
mM NaCl, 15 mM CaCl2, or a mixture of NaCl and CaCl2 (each component at a concentration of 15 mM),
2 U mL-1
enzyme concentration, and 1.0 mM pNPA. The temperature of each of the enzymatic reactions
was fixed at 37 °C. Control corresponds to the sample with no addition of cofactor.
0.0
0.3
0.6
0.9
1.2
Control NaCl CaCl2 NaCl+CaCl2
Lip
ase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
Cofactor (0.6 mM)
a.
0
3
6
9
Control NaCl CaCl2 NaCl+CaCl2
-A
my
lase
Act
ivit
y(
mo
l C
NP
min
-1m
g-1
pro
tein
)
Cofactor (15 mM)
b.
0
10
20
30
40
Control NaCl CaCl2 NaCl+CaCl2
Ph
osp
ha
tase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
Cofactor (10 mM)
c.
0
2
4
6
8
10
Control NaCl CaCl2 NaCl+CaCl2
Pro
tea
se A
ctiv
ity
(m
ol p
NP
min
-1m
g-1
pro
tein
)
Cofactor (15 mM)
d.
Chapter 2
48
Figure 2.5. Effect of the cofactor concentration on the enzymatic activity of pancreatic lipase (a), -
amylase (b), alkaline phosphatase (c), and protease (d). The reaction conditions are presented below.
Pancreatic lipase: 50 mM Tris-HCl buffer solution (pH 7.0), NaCl at concentrations ranging from 30 to
270 mM, and CaCl2 at concentrations ranging from 0.3 to 2.7 mM, 600 U mL-1
enzyme concentration, and
4.0 mM pNPPA. -Amylase: 20 mM Phosphate buffer solution (PBS, pH 7.0), NaCl at concentrations
ranging from 3 to 23 mM, 5 U mL-1
enzyme concentration, and 0.4 mM G3CNP. Alkaline phosphatase: 20
mM Tris-HCl (pH 7.0), NaCl at concentrations ranging from 3 to 23 mM, 0.5 U mL-1
enzyme
concentration, and 0.4 mM pNPP. Protease: 50 mM Phosphate buffer solution (PBS, pH 7.0), NaCl at
concentrations ranging from 5 to 30 mM, 2 U mL-1
enzyme concentration, and 1.0 mM pNPA. The
temperature of each of the enzymatic reactions was fixed at 37 °C.
0 1 2 3
0.0
0.3
0.6
0.9
1.2
0 100 200 300
CaCl2 (mM)
Lip
ase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
NaCl (mM)
NaCl
CaCl2
0
2
4
6
0 5 10 15 20 25
-Am
yla
se A
ctiv
ity
(m
ol
CN
P m
in-1
mg
-1p
rote
in)
NaCl (mM)
b.a.
0
10
20
30
0 5 10 15 20 25
Ph
osp
hata
se A
ctiv
ity
(m
ol p
NP
min
-1m
g-1
pro
tein
)
NaCl (mM)
c.
0
2
4
6
8
10
0 5 10 15 20 25 30 35
Pro
tease
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
NaCl (mM)
d.
Chapter 2
49
Interestingly, pancreatic lipase required both NaCl and CaCl2 to exhibit an appreciable catalytic
activity (Figures 2.4a and 2.5a). This result can be explained by considering two different
situations: i) There might be a synergistic effect between NaCl and CaCl2 probably due to the fact
that the two metal ions can act as cofactors of pancreatic lipase (Kapoor & Gupta, 2012), and ii)
it has been reported that NaCl can operate as a cofactor of pancreatic lipase by binding to its
catalytic site, whereas CaCl2 is capable to act as both a cofactor and an allosteric activator of the
catalytic reaction (Kimura, Futami, Tarui, & Shinomiya, 1982).
There are other divalent ions such as Fe2+
, Cu2+
, and Zn2+
that may behave as cofactors.
However, Ca2+
is very important for its biological prevalence on the activity of digestive
enzymes. Unlike Fe2+
and Cu2+
, Ca2+
has a single oxidation state (+2) under physiological
conditions, therefore, Ca2+
does not participate in redox reactions catalyzed by oxidoreductases,
being a selective cofactor for hydrolases (Kimura, Futami, Tarui, & Shinomiya, 1982). Similar to
Ca2+
, Zn2+
also has a single oxidation state (+2), however, Zn2+
is a cofactor often associated to
oxidoreductases because of its capacity to form coordination bonds (Broderick, 2001). It is also
important to stress that although digestive enzymes do not require NaCl as a cofactor, the activity
of all of them was stimulated upon addition of NaCl (Figures 2.4 and 2.5). Although Na+ is not a
very common ion acting as cofactor, it has been reported that NaCl is capable to add functionality
to enzymes by providing strongly electrophilic centers (Broderick, 2001). Therefore, NaCl is a
very versatile cofactor that can be used either to increase the activity of digestive enzymes or to
replace other cofactors if required.
The effect of the cofactor concentration on the activity of the digestive enzymes can be addressed
by considering similar criteria to those considered for the buffer concentration. Enzymes are less
active at low cofactor concentrations because the substrates cannot adequately orientate in the
active site (Broderick, 2001). Conversely, high concentrations of cofactor can destabilize the
intermolecular interactions which maintain the enzymes folded, leading to denaturation (Bauduin,
Nohmie, Touraud, Neueder, Kunz, & Ninham, 2006). The inhibition of the activity due to the
extreme concentrations of cofactor was observed for all of the tested enzymes (Figure 2.5).
Chapter 2
50
2.3.3. Effect of the enzyme concentration
It has been reported that enzymatic activity is directly proportional to the enzyme concentration,
showing a linear dependence at low concentrations of enzyme (Al-Zuhair, Ramachandran, &
Hasan, 2004; Bisswanger, 2014; Miranda, Ferreira, Cordeiro, & Freire, 2006; Rami Tzafriri &
Edelman, 2007). This behavior was observed for all of the tested digestive enzymes (Figure 2.6).
The linear dependence was maintained for all of the tested enzymes until the optimum enzyme
concentration was reached. From this optimum, the enzymatic activity decreased as the enzyme
concentration increased, indicating an inhibition of the enzymatic activities by either substrate
depletion or inhibition by product (Bisswanger, 2014; Cao & De La Cruz, 2013; Goddard &
Reymond, 2004). For pancreatic lipase (Figure 2.6a), -amylase (Figure 2.6b), alkaline
phosphatase (Figure 2.6c), and protease (Figure 2.6d) the maximum specific activities were
reached at enzyme concentrations of 1000, 10, 0.8, and 3 U mL-1
, respectively.
The concentration of all components directly involved in the enzymatic reaction should be
present at saturating concentrations, excepting the enzyme, i.e., the reaction rate must be
independent of the substrate, buffer, and cofactor concentrations (Bisswanger, 2014). Unlike the
other components involved in the enzymatic reaction, the concentration of the enzyme should be
as low as possible because only catalytic amounts of enzyme are required. This is because the
Michaelis-Menten model is derived on the assumption of minor, even negligible amounts of
enzyme to ensure the enzyme saturation with the substrate (Johnson & Goody, 2011). The
evaluation of the substrate concentrations, however, was postponed for further studies (Chapter
3). Finally, the best conditions for measuring the activities of the digestive enzymes evaluated in
this study are summarized in Table 2.1.
2.3.4. Overall effect of the optimization process
The optimization of the reaction conditions allowed to improve the activities of all of the
digestive enzymes studied during each step of the optimization process.
Chapter 2
51
Figure 2.6. Effect of the enzyme concentration on the enzymatic activity of pancreatic lipase (a), -
amylase (b), alkaline phosphatase (c), and protease (d). The reaction conditions are presented below.
Pancreatic lipase: 50 mM Tris-HCl buffer solution (pH 7.0); 150 mM NaCl and 1.0 mM CaCl2; enzyme
concentration ranging from 300 to 1500 U mL-1
; and 4.0 mM pNPPA. -Amylase: 20 mM Phosphate
buffer solution (PBS; pH 7.0); 10 mM NaCl; enzyme concentration ranging from 3 to 15 U mL-1
; and 0.4
mM G3CNP. Alkaline phosphatase: 20 mM Tris-HCl buffer solution (pH 7.0); 15 mM NaCl; enzyme
concentration ranging from 0.3 to 1.3 U mL-1
; and 0.4 mM pNPP. Protease: 50 mM Phosphate buffer
solution (PBS; pH 7.0); 20 mM NaCl; enzyme concentration ranging from 1 to 5 U mL-1
; and 1.0 mM
pNPA. The temperature of each of the enzymatic reactions was fixed at 37 °C.
0.00
0.05
0.10
0.15
0.20
0 6 12 18
-Am
yla
se A
ctiv
ity
(m
ol
CN
P m
in-1
)
-Amylase (U mL-1)
b.
0.00
0.02
0.04
0.06
0.08
0.0 0.5 1.0 1.5
Ph
osp
hata
se A
ctiv
ity
(m
ol p
NP
min
-1)
Phosphatase (U mL-1)
c.
0.00
0.05
0.10
0.15
0.20
0.25
0 2 4 6
Pro
tease
Act
ivit
y(
mo
l p
NP
min
-1)
Protease (U mL-1)
d.
0.0
0.5
1.0
1.5
2.0
0 600 1200 1800
Lip
ase
Act
ivit
y(
mo
l p
NP
min
-1)
Lipase (U mL-1)
a.
Chapter 2
52
Table 2.1. Optimum conditions for the measurement of the enzymatic activities of pancreatic lipase, -
amylase, alkaline phosphatase, and protease.
Enzyme Buffer (pH 7.0) Cofactor concentration Enzyme concentration
(U mL-1
)
Pancreatic lipase 50 mM Tris-HCl 150 mM NaCl; 1 mM CaCl2 1000
-Amylase 20 mM PBS1 10 mM NaCl 10
Alkaline phosphatase 20 mM Tris-HCl 15 mM NaCl 0.8
Protease 50 mM PBS 20 mM NaCl 3
1 Phosphate buffer solution (PBS).
The use of Tris-HCl allowed increasing the activity of pancreatic lipase and alkaline phosphatase
by 2.4 and 6.1 times, respectively, as compared to PBS, whereas PBS allowed to increase the
activity of amylase and protease 2.4 and 6.1 times, respectively, as compared to Tris-HCl.
Increasing the Tris-HCl concentration from 20 to 50 mM increased 1.6 times the activity of
pancreatic lipase, whereas increasing the Tris-HCl concentration from 5 to 20 mM increased 3.9
times the activity of alkaline phosphatase. In addition, increasing the PBS concentration from 5 to
20 mM increased 2.6 times the activity of -amylase, whereas increasing the PBS concentration
from 10 to 50 mM increased 2.1 times the activity of protease.
The cofactor optimization also presented a considerable effect on the activity of all of the tested
digestive enzymes. The enzymatic activities in presence of the optimum cofactor at the optimum
concentration (NaCl and CaCl2 for pancreatic lipase, and NaCl for -amylase, alkaline
phosphatase, and protease) were 8.1, 21.1, 28.3, and 9.3 times higher for pancreatic lipase, -
amylase, alkaline phosphatase, and protease, respectively, than those obtained when the cofactor
was suppressed. Increasing the enzyme concentration from 300 to 1000 U mL-1
increased 2.9
times the activity of pancreatic lipase, whereas increasing the enzyme concentration from 3 to 10
U mL-1
increased 2.8 times the activity of -amylase. In addition, increasing the enzyme
concentration from 0.3 to 0.8 U mL-1
increased 2.6 times the activity of alkaline phosphatase,
whereas increasing the enzyme concentration from 1 to 3 U mL-1
increased 2.9 times the activity
of protease.
Chapter 2
53
Finally, the optimization of the reaction conditions had no a significant effect on both the overall
maximum enzymatic activity and the reproducibility of the measurements. The average
maximum enzymatic activities during the optimization process of pancreatic lipase, alkaline
phosphatase, and protease were 0.88 0.05, 23.25 3.72, and 7.66 0.99 μmol pNP min-1
mg-1
protein, respectively, whereas the average maximum enzymatic activity during the optimization
process of -amylase was 6.17 0.96 μmol CNP min-1
mg-1
protein. In addition, the variation
coefficients of the enzymatic activities during the optimization process were 5.7, 15.6, 16.0, and
12.9% for pancreatic lipase, -amylase, alkaline phosphatase, and protease, respectively.
2.4. Conclusions
This is the first reported evidence on the evaluation of the reaction conditions affecting the
activity of the main digestive enzymes (pancreatic lipase, -amylase, alkaline phosphatase, and
protease) by using artificial chromogenic substrates. Artificial chromogenic substrates are known
to increase the sensitivity and reliability of enzymatic assays. However, the adjustment of the
experimental conditions affecting the activity of digestive enzymes is required since the
experimental conditions reported in literature for native substrates may differ considerably to
those required for artificial substrates. The enzymatic activities of each of the tested digestive
enzymes were enhanced by selecting appropriate buffer and cofactor type as well as the
concentration of buffer, cofactor, and enzyme. In addition, the optimization of the reaction
conditions allowed enhancing the specific activities of all of the tested digestive enzymes.
However, no significant effect was observed on the reproducibility of the measurements. The best
conditions obtained here for measuring the activities of digestive enzymes (summarized in Table
2.1) will be used in further studies to evaluate the effect of pectin on the enzymatic activities of
these enzymes (Chapter 3).
Chapter 2
54
Acknowledgements
We are grateful to Departamento Administrativo de Ciencia, Tecnología e Innovación de
Colombia (COLCIENCIAS) and Vicerrectoría Académica of Universidad Nacional de Colombia
for providing a fellowship to Mauricio Espinal-Ruiz supporting this work.
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Chapter 3
Inhibition of digestive enzyme activities by pectins in model solutions
Published as:
Espinal-Ruiz, M., Parada-Alfonso, F.; Restrepo-Sánchez, L. P., & Narváez-Cuenca, C. E.
Bioactive Carbohydrates and Dietary Fibre. 4 (2014): 27 – 38.
Chapter 3
57
Abstract
The presence of dietary fiber (e.g., pectin) in the gastrointestinal tract may decrease the caloric
uptake and reduce the risk of developing cardiovascular diseases. These phenomena are governed
by several mechanisms, such as the regulation of the rate and extent of nutrient absorption and
the alteration of the normal activity of the gastrointestinal tract enzymes. In this study, we
evaluated the effect of pectins with five methoxylation degrees (MD) on the activities of lipase,
-amylase, alkaline phosphatase, and protease. The MD of pectins ranged (in % mol/mol) from
87.4% (high methoxylated pectin, HMP) to 7.1% (low methoxylated pectin, LMP). The
enzymatic activities were evaluated in model solutions after incubation with pectins. The
Michaelis-Menten constant (Km) remained unmodified whereas the apparent maximum velocity
(Vmaxapp) decreased with increasing pectin concentrations. The Vmaxapp represented 13.3, 38.6,
41.9, and 44.4% of the Vmax (without pectins) for lipase, -amylase, alkaline phosphatase, and
protease, respectively, when they were inhibited with 100 g mL-1
HMP. Kinetic analyses
showed that all of the tested pectins behaved as non-competitive inhibitors of digestive enzymes.
Increasing both the concentration and MD of pectins, the enzymatic activities were reduced by
decreasing the non-competitive inhibition constant (Ki). In plotting Ki versus MD, a straight line
was obtained, with slopes of 1.943, 1.558, 1.344, and 1.165 g mL-1
%-1
for lipase, -amylase,
alkaline phosphatase, and protease, respectively. Among them, lipase was most likely to be
inhibited by pectins. Our results suggested that pectins might be able to suppress digestion by
inhibiting digestive enzymes.
Keywords: Pectin, methoxylation degree, digestive enzymes, hydrophobic interactions, non-
competitive inhibition.
Chapter 3
58
3.1. Introduction
The main function of the gastrointestinal tract is the absorption of nutrients derived from food
digestion. This function is controlled by a series of digestive processes that occur in different
sections of the gastrointestinal tract. These digestive processes are controlled by the secretion of
digestive enzymes and their associated cofactors and by the stability of the pH and temperature
conditions of the gastrointestinal tract (Dawson, 1993). Lipases, -amylases, alkaline
phosphatases, and proteases are the main gastric and pancreatic enzymes present in the
gastrointestinal tract (Rothman, 1977). These enzymes are responsible for the hydrolysis of the
triglycerides, carbohydrates, and proteins that are consumed in diet, which are carriers of a great
caloric content. It has been postulated that the presence of any type of dietary fiber in the
gastrointestinal tract may result in the decrease of the total caloric uptake (Amarowicz, Kmita-
Glazewska, & Kostyra, 1990). This phenomenon is governed by several mechanisms, such as the
regulation of the nutrient absorption rate, perturbations of the intestinal physiological conditions,
the encapsulation of minerals and vitamins needed for metabolic processes, and alterations of the
normal activities of the digestive enzymes (Kumar & Chauhan, 2010).
Recent epidemiological studies have shown that the consumption of dietary fiber is associated
with the reduction of the risk of developing chronic cardiovascular diseases. Consequently, it has
been recommended to increase the intake of products of plant origin that have high dietary fiber
levels, particularly soluble dietary fiber, together with other phytochemical constituents (Kris-
Etherton, et al., 2002). Pectins are a type of soluble dietary fiber that cannot be digested in the
upper gastrointestinal tract due to their resistance to the hydrolytic action of digestive enzymes
(Holloway, Tasman-Jones, & Maher, 1983). Pectins also promote bacterial fermentation in the
large intestine, improving the proliferation of intestinal microbiota that is beneficial for human
health (Louis, Scott, Duncan, & Flint, 2007). In the gastrointestinal tract, pectins form a complex
three-dimensional matrix with fibrous and amorphous characteristics. Physicochemical
properties, such as the methoxylation degree (MD), acetylation degree, molecular weight
distribution, distribution of non-methoxylated galacturonic acid residues, methoxylation and
Chapter 3
59
acetylation distribution patterns, and gel-forming capacity (Brownlee, 2011), as well as the
structure of the three-dimensional matrix, play important roles in the homeostatic and therapeutic
functionality of pectins in human nutrition (Kay, 1982). Enhancement of the gastrointestinal
viscosity, inhibition of digestion and the absorption of nutrients, control of gastrointestinal
motility and immunity, regulation of the activity of the colonic microbiota, and regulation of a
systemic stimulus associated with the feeling of satiety are among the therapeutic properties and
physiologic effects that are beneficial for human health that have been attributed to pectins
(Brownlee, 2011).
Regulation of the gastrointestinal tract enzymatic activity by dietary fiber from different plant
materials was previously reported (Dunaif & Schneeman, 1981; Isaksson, Lundquist, & Ihse,
1982a, 1982b; Ikeda & Kusano, 1983; Tsujita, Takaichi, Takaku, Sawai, Yoshida, & Hiraki,
2007). The dietary fiber materials contribute to inhibiting the activity of gastrointestinal tract
enzymes. According to the afore-cited work, the occurrence of physical interactions (such as
ionic interactions, hydrogen bonding, dispersive forces, and hydrophobic interactions) might play
an important role in the capacity of the dietary fiber to inhibit enzymatic activities. However, the
experiments conducted in those studies, did not identify a kinetic mechanism underlying the
inhibition of such enzymes. Inhibition of the activity of the enzymes by ingested pectins might
play an important role in reducing the quantity of free fatty acids, monosaccharides, and amino
acids that can be absorbed at the gastrointestinal tract level (Ikeda & Kusano, 1983). Identifying
the mechanism by which pectins act as inhibitors of digestive enzymes in model solutions might
be useful in understanding the physiological phenomena involved in the in vivo regulation that
pectins exert on nutrient absorption at the gastrointestinal tract level (Brownlee, 2011).
The aim of this study was, therefore, to evaluate in model solutions the effects of both the
concentration and MD of pectins on the activities of lipase, -amylase, alkaline phosphatase, and
protease (chymotrypsin). This study also aimed to determine a kinetic mechanism by which
pectins inhibit the activity of enzymes, as well as to evaluate theoretically, through molecular
docking calculations, the influence of structural parameters on the enzyme-pectin surface
interaction.
Chapter 3
60
3.2. Materials and methods
3.2.1. Chemicals
The enzymes porcine pancreatic -amylase, from Sus scrofa (16 U mg-1
type VI-B, E.C. 3.2.1.1)
porcine pancreatic lipase, from Sus scrofa (100 U mg-1
type II, E.C. 3.1.1.3) bovine pancreatic
protease (chymotrypsin), from Bos taurus (5 U mg-1
type I, E.C. 3.4.21.1), and bovine intestinal
mucosa alkaline phosphatase, from Bos taurus (10 U mg-1
type I, E.C. 3.1.3.1); the artificial
substrates 2-chloro-p-nitrophenyl--D-maltotrioside (G3CNP), p-nitrophenyl palmitate (pNPPA),
p-nitrophenyl acetate (pNPA), and p-nitrophenyl phosphate (pNPP); the reaction products p-
nitrophenol (pNP) and 2-chloro-p-nitrophenol (CNP); and the protein determination reagent
brilliant blue G-250 (Coomassie Blue); as well as bovine serum albumin were purchased from
Sigma-Aldrich Chemical Company (St. Louis, MO, USA). A high-methoxylated citrus pectin
(HMP) was purchased from CIMPA (Bogotá, Colombia). Ox bile extract with a cholic acid
content higher than 55% (w/w) was purchased from MP Biomedicals (Solon, OH, USA). Other
chemicals were purchased from Merck KGaA (Darmstadt, Germany).
3.2.2. Preparation and characterization of pectins with different MD levels
3.2.2.1. De-esterification of pectins
Alkaline de-esterification of the HMP was performed as described by Dongowski (1997) to
obtain pectins with different MD levels. One gram of HMP was mixed with 50 mL of 0.25 M
NaOH (pH of the mixture 10.0) and stirred for 0, 9, 15, 30, or 45 min at 25 °C. The mixture
was neutralized to pH 7.0 with 3.0 M HCl and then, 150 mL of 80% (v/v) ethanol were added to
induce pectin precipitation. The partially de-esterified pectins were filtered and washed with 100
mL of 80% (v/v) ethanol. Pectins were dried at 70 °C for 5 h and then the MD, total uronic acid
content, acetylation degree, and molecular weight distribution were evaluated.
Chapter 3
61
3.2.2.2. Characterization of pectins
3.2.2.2.1. Determination of the total uronic acid content
The total uronic acid content was determined according to van den Hoogen et al. (1998). An
aliquot of 400 L of each pectin solution (100 g mL-1
) was mixed with 2 mL of concentrated
sulfuric acid (98% w/w) containing 120 mM sodium tetraborate and incubated for 60 min at
80°C. After cooling to room temperature, the background absorbance of the samples was
measured at 540 nm. Then, 400 L of m-hydroxydiphenyl reagent (100 L of 100 mg mL-1
m-
hydroxydiphenyl in dimethyl sulfoxide, mixed with 4.9 mL of 80% (v/v) sulfuric acid) was added
and mixed with the samples. After 15 min, the absorbance of the pink-colored samples was
measured at 540 nm. A calibration line was obtained using galacturonic acid at concentrations
ranging from 0.1 to 1.0 g mL-1
(7 data points, r2=0.999). Total uronic acid content was
expressed as the moles of uronic acid residues per 100 g of pectin.
3.2.2.2.2. Determination of the methoxylation and acetylation degrees
The MDs and acetylation degrees were determined according to Voragen, Schols, and Pilnik
(1986). A model LC-20AT liquid chromatograph (Shimadzu Corporation, Kyoto, Japan)
equipped with an Aminex HPX-87H column (300 mm x 7.8 mm x 9 m; Bio-Rad Labs,
Richmond, CA, USA) was used. The column was operated at 18 °C, at a flow rate of 0.3 mL min-
1, with 5 mM sulfuric acid as the eluent. The components eluted from the column were detected
using a RID-10A refractive index detector (Shimadzu Corporation) at 40 °C. Each pectin sample
(30 mg) was suspended in 1.0 mL of 0.4 M NaOH and stirred at 18 °C for 2 h. The suspension
was then centrifuged (4,000 x g; 4 °C; 30 min) and then, 20 L of the clear supernatant was
injected into the column. The amounts of methanol and acetic acid were determined using the
external standard method. Calibration lines were obtained with methanol and acetic acid at
concentrations ranging from 5 to 100 g mL-1
(7 data points, r2=0.999 for methanol and r
2=0.997
Chapter 3
62
for acetic acid). The MD and acetylation degree were expressed as moles of methyl and acetyl
esters, respectively, per 100 mol of uronic acid.
3.2.2.2.3. Determination of the molecular weight distribution
The molecular weight distribution was determined using high performance size exclusion
chromatography (HPSEC) according to the method of Houben, Jolie, Fraeye, Van Loey, and
Hendrickx (2011), using a LC-20AT liquid chromatograph model (Shimadzu Corporation),
equipped with a mixed-bed column of TSK-gel (GMPWXL, 300 mm x 7.8 mm, pore size 100-
1000 Å, particle size 13 m; Tosoh Biosciences, Stuttgart, Germany). A 20 L injection loop was
used. Elution was performed using 50 mM NaNO3 at a flow rate of 0.7 mL min-1
for 25 min at 35
°C. A RID-10A refractive index detector (Shimadzu Corporation) at 40 °C was used to monitor
the eluents. Dextran standards with molecular weights ranging from 1.1 to 400 kDa (Sigma-
Aldrich Chemical Company, St. Louis, MO, USA) were used to estimate the molecular weight
distribution of pectins. A straight calibration curve was obtained when plotting the log of the
molecular weight versus the elution time.
3.2.3. Kinetics of the inhibition of digestive enzyme activities by pectins in model solutions
3.2.3.1. Experimental design
The kinetic inhibition profile for each enzyme by pectins was obtained according to the
methodology proposed by Kakkar, Boxenbaum, and Mayersohn (1999). Several experimental
factors were used to evaluate the activity of lipase, -amylase, alkaline phosphatase, and protease
(chymotrypsin). These experimental factors were derived by using six substrate concentration
points (ranging from 2 to 10 mM pNPPA for lipase, from 0.2 to 1.0 mM G3CNP for -amylase,
from 0.2 to 1.0 mM pNPP for alkaline phosphatase, and from 0.4 to 2.0 mM pNPA for protease),
six concentration points for each pectin (ranging from 20 to 100 g mL-1
), and five types of each
pectin, with MDs ranging (in % mol/mol) from 7.1% (low methoxylated pectin, LMP) to 87.4%
Chapter 3
63
(high methoxylated pectin, HMP). The inhibitory mechanism of each pectin sample toward the
enzyme activities was evaluated by kinetic analysis using the Lineweaver-Burk plot.
3.2.3.2. Determination of the inhibition of digestive enzyme activities by pectins
3.2.3.2.1. Determination of the activity of lipase
The enzymatic activity of lipase was determined according to Tsujita, Takaichi, Takaku, Sawai,
Yoshida, and Hiraki (2007). A reaction mixture consisting of 500 L of pNPPA at concentrations
ranging from 6 to 30 mM, 500 L of each pectin solution at concentrations ranging from 60 to
300 g mL-1
, and 500 L of a working solution of lipase at 3,000 U mL-1
(one unit is the amount
of enzyme required to convert 1 mol of pNPPA) was prepared. All of the solutions were
prepared in 50 mM Tris-HCl buffer pH 7.0 containing 150 mM NaCl, 1 mM CaCl2, 100 g mL-1
Tween-20, and 3.5 mg mL-1
bile acid extract (equivalent to 5.0 mM cholic acid in the total
mixture). The mixture was incubated at 37 °C and the absorbance of the pNP produced was
measured at 415 nm every 5 s throughout 120 s.
3.2.3.2.2. Determination of the activity of -amylase
The enzymatic activity of -amylase was determined according to Morishita, Iinuma,
Nakashima, Majima, Mizuguchi, and Kawamura (2000). A reaction mixture consisting of 500 L
of G3CNP at concentrations ranging from 0.6 to 3.0 mM; 400 L of 13.1 mg mL-1
bile acid
extract (equivalent to 18.7 mM cholic acid in the total mixture), 500 L of each pectin solution at
concentrations ranging from 60 to 300 g mL-1
, and 100 L of a working solution of -amylase
of 150 U mL-1
(one unit is the amount of enzyme required to convert 1 mol of G3CNP) was
prepared. All of the solutions were prepared in 20 mM phosphate buffer pH 7.0 containing 10
mM NaCl and 15 % (v/v) glycerol. The mixture was incubated at 37 °C and the absorbance of the
CNP produced was measured at 405 nm every 5 s throughout 60 s.
Chapter 3
64
3.2.3.2.3. Determination of the activity of alkaline phosphatase
The enzymatic activity of alkaline phosphatase was determined according to Chaudhuri,
Chatterjee, Venu-Babu, Ramasamy, and Thilagaraj (2013). A reaction mixture consisting of 500
L of pNPP at concentrations ranging from 0.6 to 3.0 mM, 400 L of 13.1 mg mL-1
bile acid
extract (equivalent to 18.7 mM cholic acid in the total mixture), 500 L of each pectin solution at
concentrations ranging from 60 to 300 g mL-1
, and 100 L of a working solution of alkaline
phosphatase of 12 U mL-1
(one unit is the amount of enzyme required to convert 1 mol of
pNPP) was prepared. All of the solutions were prepared in 20 mM Tris-HCl buffer pH 7.0
containing 15 mM NaCl. The mixture was incubated at 37 °C and the absorbance of the pNP
produced was measured at 415 nm every 5 s throughout 60 s.
3.2.3.2.4. Determination of the activity of protease
The enzymatic activity of protease (chymotrypsin) was determined according to Verma & Ghosh
(2013). A reaction mixture consisting of 500 L of pNPA at concentrations ranging from 1.2 to
6.0 mM, 400 L of 13.1 mg mL-1
bile acid extract (equivalent to 18.7 mM cholic acid in the total
mixture), 500 L of each pectin solution at concentrations ranging from 50 to 300 g mL-1
, and
100 L of a working solution of protease of 45 U mL-1
(one unit is the amount of enzyme
required to convert 1 mol of pNPA) was prepared. All of the solutions were prepared in 50 mM
phosphate buffer pH 7.0 containing 20 mM NaCl. The mixture was incubated at 37 °C and the
absorbance of the pNP produced was measured at 415 nm every 5 s throughout 60 s.
3.2.3.2.5. Determination of the specific enzymatic activities
To determine the enzymatic activities, the slope of the straight lines obtained when plotting the
absorbance versus time data obtained from the enzymatic activity experiments was interpolated
in the calibration lines for each reaction product. The calibration lines were obtained using pNP
at concentrations ranging from 5 to 50 M (7 data points, r2=0.999) for lipase, alkaline
Chapter 3
65
phosphatase, and protease and using CNP at concentrations ranging from 5 to 60 M (7 data
points, r2=0.998) for -amylase. The protein concentrations of the working enzyme solutions
were determined using the Bradford method as modified by Zor & Selinger (1996), using bovine
serum albumin (BSA) as the standard. The specific enzymatic activities of lipase, alkaline
phosphatase, and protease were expressed as mol of pNP released per minute per mg protein
(mol pNP min-1
mg-1
protein), while the specific enzyme activity of -amylase was expressed as
the mol of CNP released per minute per mg protein (mol CNP min-1
mg-1
protein).
3.2.4. Theoretical studies of digestive enzyme inhibition by pectins
3.2.4.1. Structure of the gastrointestinal enzymes
The structure of each enzyme was obtained from the Protein Data Bank
(http://www.rcsb.org/pdb, consulted on March 5th
2013). Porcine pancreatic lipase from Sus
scrofa (EC 3.1.1.3, pdb code 1ETH), porcine pancreatic -amylase from Sus scrofa (EC 3.2.1.1,
pdb code 1DHK), bovine pancreatic chymotrypsin from Bos taurus (EC 3.4.21.4, pdb code
1S0Q), and human placental alkaline phosphatase from Homo sapiens (EC 3.1.3.1, pdb code
3MK1) were used for the molecular docking calculations.
3.2.4.2. Structure of the artificial substrates
The molecular structure of the artificial substrates pNPPA, G3CNP, pNPA, and pNPP was fully
optimized using the Hartree-Fock method, as implemented in GAMESS (General Atomic and
Molecular Electronic Structure System) version May 01 of 2012 (Schmidt et al., 1993) in the 6-
311G(d,p) basis set. Solvent effects were simulated by placing the substrates in dielectric
medium simulating water, using the IEF-PCM model (Cossi, Barone, Mennucci, & Tomasi,
1998). The geometric optimization and the energy calculations were performed using this
medium.
Chapter 3
66
3.2.4.3. Structure of pectins
Pectin fragments -D-GalA-(1-4)-[-D-GalA-(1-4)]18--D-GalA-(1-1)-OH with MDs ranging
(in % mol/mol) from 0% to 100% were constructed in the biomolecular force field GLYCAM 06
for carbohydrates (Kirschner et al., 2008). Energy minimization of pectin fragments was
performed using the AMBER 9 force field, and the assigning of partial charges was conducted
using Gasteiger function partial charges. In all cases, the pdb format files that were built were
employed in the molecular docking calculations.
3.2.4.4. Molecular docking protocol
The basic docking protocol was performed using the default settings provided by AutoDock
Tools, according to Neuhaus (2010). The enzymes, artificial substrates, and pectin fragments
were converted to pdbqt format in Autodock Tools. The Lamarckian Genetic Algorithm with a
population size of 150 dockings and five million energy evaluations was used. All other
parameters, e.g., the crossover rate and mutation rate, were obtained using the default settings.
The grid size for specifying the search space was set at 21 x 21 x 21 Å3 in a centered position
with default grid-point spacing of 0.375 Å. Autodock 4.0 was launched from Autodock Tools on
Devian-Linux operating system, and the docking logs were analyzed using the graphical user
interface of Autodock Tools. The docked energy was defined as the sum of the intermolecular
and the internal energies. For a representative docking instance, the orientation or pose with the
lowest estimated free energy (G) of binding, corresponding to the docking energy and unbound
free energy of the system, was chosen in each calculation. The binding free energy ratio (BFER)
was defined as the ratio of the free energy binding of each enzyme-pectin complex and the free
energy binding of the enzyme-substrate complex.
3.2.5. Data analysis
All measurements were conducted with three analytical and three technical (instrumental)
replicates, for a total of nine replicates (n=9). The mean values and their standard deviations were
Chapter 3
67
reported. Comparisons among the mean values were performed by one-way variance analysis
(ANOVA) and using Fisher's least significant difference test (p<0.05), using the R program
(version 2.13.1, 08 July 2011).
3.3. Results and discussion
3.3.1. Characterization of pectins
Alkaline hydrolysis of HMP was performed to obtain pectins with different MD, thus pectins
obtained had MDs (% mol/mol) ranging from 87.4 5.4% (HMP) to 7.1 2.7% (LMP) (Table
3.1). In addition to the MD, the acetylation degree and molecular weight distribution of the
obtained pectins were also evaluated because these molecules are potentially susceptible the
effects of the alkaline hydrolysis conditions, and these features determine the structure, three-
dimensional conformation, and functional properties of pectins (Mohnen, 2008). The alkaline
treatment did not affect (p<0.05) the acetylation degree [average of acetylation degree was 4.9
0.4% (mol/mol)] nor the molecular weight values (HPSEC profiles of the obtained pectins did not
show significant differences; Figure 3.1). Alkaline hydrolysis was performed at low temperature
(pH 10.0; 25 °C; 45 min) because it has been established that alkaline hydrolysis
Table 3.1. Effect of the alkaline hydrolysis of high methoxylated pectin on the methoxylation and
acetylation degrees.
Time (min) Methoxylation (% mol/mol)1 Acetylation (% mol/mol)
2
0 87.4 5.4ª 4.4 0.3ª
9 64.6 5.3b 4.8 0.3
a
15 39.1 5.0c 5.3 0.1
a
30 28.4 3.8d 4.9 0.4
a
45 7.1 2.7e 5.4 0.4
a
1 Expressed as moles of methyl esters per 100 mol of uronic acid.
2 Expressed as moles of acetyl esters per 100 mol of uronic acid.
Different letters within the same column indicate significant differences as calculated using Fisher's least
aaaaaaaaaaaaaaasignificant difference test (p<0.05).
Chapter 3
68
Figure 3.1. High performance size exclusion chromatography (HPSEC) elution profiles of pectins after 0
(continuous line) and 45 min (dotted line) of de-esterification with 0.25 M NaOH at 25 °C, to obtain
pectins with methoxylation degrees of 87.4 and 7.1% (mol/mol), respectively. The scale was shifted
upwards by 2 refractive index (RI) units for the 45 min profile (dotted line).
of the -(1,4) glycosidic bond of pectins should not be performed at temperatures above 60 °C to
prevent -elimination from becoming the primary mechanism of degradation (Krall &
McFeeters, 1998). We found that the molecular weight distribution was not affected by this
process (Figure 3.1). This result is most likely because hydrolysis of the -(1,4) glycosidic bond
of pectins, which is required to decrease the molecular weight, is highly efficient under extreme
temperature and acidity conditions (pH ≤ 2.0; 100 °C; 3 h) (Ramaswamy, Kabel, Schols, &
Gruppen, 2013). It is well known that the alkaline hydrolysis conditions may affect both the MD
and acetylation degree, depending on the temperature, alkali concentration, and the initial number
of available methyl and acetyl groups present in pectin. Nevertheless, in this study, we found that
the alkaline conditions did not affect significantly the acetylation degree, most likely due to the
low number of initial acetylation sites available compared to the high number of initial
methoxylation sites present in HMP (Sundar-Raj, Rubila, Jayabalan, & Ranganathan, 2012;
Garna, Mabon, Nott, Wathelet, & Paquot, 2006). In contrast to the stability of the acetylation
degree and molecular weight during the alkaline treatment, there was a statistically significant
0
2
4
6
8
10
0 5 10 15 20
RI
Res
po
pn
se
Time (min)
Chapter 3
69
decrease (p<0.05) of the MD over time. After 45 min of alkaline treatment, almost complete de-
esterification of HMP was obtained, yielding an LMP with a MD of 7.1 2.7% (mol/mol).
The MD was the only structural parameter that differentiated pectins used in the enzyme
inhibition experiments, meaning that the observed differences in the biological functionalities of
pectins are most likely due to differences in their MD. The structural importance of MD in
pectins lies in the fact that the methyl-ester groups neutralize the negative charges present in the
free carboxyl groups, decreasing their polar nature and increasing their hydrophobicity (Mohnen,
2008). Thus, the MD is a parameter that affects the biological functionality of pectins and it can
be a factor that determines the interaction of them with other biologically relevant biomolecules,
such as proteins (Benjamin, Lassé, Silcock, & Everett, 2012).
3.3.2. Kinetics of the inhibition of the digestive enzymatic activities by pectins
The effect of the MD and of the concentration of each pectin sample on the activities of the
enzymes lipase, -amylase, alkaline phosphatase, and protease (chymotrypsin) was evaluated.
The kinetic profiles of the in vitro digestion of artificial substrates by the enzymes in the presence
of HMP are shown in Figure 3.2. Increasing the concentration of HMP decreased (p<0.05) the
activity of lipase, -amylase, alkaline phosphatase, and protease. The same trend was observed
using each of the other pectins. Because the increase in the concentration of each pectin sample in
the reaction medium caused a decrease in the activity of each enzyme, one might suppose that the
tested pectins displayed inhibitory behavior toward the enzymatic activities and that pectins
therefore might behave kinetically as a defined type of enzyme inhibitor (competitive, non-
competitive or uncompetitive). Inhibitory behavior towards enzymes was previously reported for
other types of dietary fiber, such as cellulose (Dunaif & Schneeman, 1981), agar-agar,
carboxymethyl cellulose, sodium alginate, xylan, inulin (Ikeda & Kusano, 1983) pectin,
mucilages, polyethylene glycol (Isaksson, Ihse, & Lundquist, 1982a), and dietary fiber extracted
from wheat bran, oat bran, and alfalfa (Dunaif & Schneeman, 1981). In all cases, increasing the
substrate concentration did not overcome the inhibitory effect of pectins (the maximum velocity,
Vmax, of each enzyme inhibited by pectins did not equal the Vmax of the uninhibited enzyme at
Chapter 3
70
Figure 3.2. Effect of the concentration of high methoxylated pectin (HMP, methoxylation degree of
87.4% mol/mol) on the kinetic behaviors of lipase (a), -amylase (b), alkaline phosphatase (c), and
protease (d). The concentrations of HMP were 0 (), 20 (), 40 (), 60 (), 80 (), and 100 g mL-1
(). Points correspond to experimental data and lines correspond to fitted data according to the Michaelis-
Menten equation.
any substrate concentration). In addition, lipase was the enzyme most affected by the inhibitory
effect of each pectin sample among the enzymes studied (Figure 3.2a).
That HMP utilized the kinetic mechanism of non-competitive inhibition of the activities of the
studied enzymes was confirmed using the double-reciprocal plot method of Lineweaver-Burk
0
2
4
6
0.0 0.2 0.4 0.6 0.8 1.0
-A
my
lase
Act
ivit
y(
mo
l C
NP
min
-1m
g-1
pro
tein
)
G3CNP (mM)
b.
0.0
0.4
0.8
1.2
0 2 4 6 8 10
Lip
ase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
pNPPA (mM)
a.
0
10
20
30
0.0 0.2 0.4 0.6 0.8 1.0
Ph
osp
ha
tase
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
pNPP (mM)
c.
0
3
6
9
0.0 0.5 1.0 1.5 2.0
Pro
tease
Act
ivit
y(
mo
l p
NP
min
-1m
g-1
pro
tein
)
pNPA (mM)
d.
Chapter 3
71
(Figure 3.3). All of the tested pectins behaved as non-competitive inhibitors of the enzymes. This
behavior suggested that pectins can interact with both the free enzyme and the enzyme-substrate
complex (ES) at a domain different from the catalytic site of the enzymes (Rehm & Becker,
1988). It was also possible to verify that the Vmax of the enzymes inhibited with pectins did not
equal the Vmax of the non-inhibited enzyme at any substrate concentration because the apparent
maximum velocity (Vmaxapp) decreased significantly as pectin concentrations increased (Figure
3.4; only the results from using HMP are shown). The presence of pectins in the reaction medium
did not significantly modify the ES affinity or the efficiency of ES complex formation
(kinetically represented by the Michaelis-Menten constant, Km), which are distinctive behaviors
of non-competitive enzyme inhibition.
The variance analysis (ANOVA, with p<0.05) performed for each kinetic profile (from Figure
3.4) revealed that neither the MD levels nor pectin concentration affected the Km value. The
observed kinetic mechanism of non-competitive inhibition explains the fact that this type of
inhibition cannot be overcome by increasing the substrate concentration because in this type of
inhibition pectins do not compete for the catalytic site of the enzyme. This result is important in
understanding the enzyme-pectin interaction phenomenon because it could be expected that an
interaction of enzymes with pectins could be only superficial (e.g., at an allosteric regulation site)
without any compromise of the catalytic site of the enzyme.
The decrease in the Vmaxapp (Equation 3.1) was dependent on the concentration of each pectin
sample. According to the non-competitive inhibition mechanism, this parameter corresponds to
the following expression:
[ ]
(3.1)
Where Ki is the non-competitive inhibition constant of an inhibited enzyme with each pectin
sample at a given concentration [Pectin]. This constant governs the process of non-competitive
inhibition of each enzyme. Using Equation 3.1, it was possible to find the Ki constant for each
Chapter 3
72
Figure 3.3. Lineweaver-Burk plot of lipase (a), -amylase (b), alkaline phosphatase (c), and protease (d)
inhibited with high methoxylated pectin (HMP, methoxylation degree of 87.4% mol/mol). The kinetic
profile obtained suggested that HMP behaves as a non-competitive inhibitor of digestive enzymes. The
concentrations of HMP were 0 (), 20 (), 40 (), 60 (), 80 (), and 100 g mL-1
(). Points
correspond to experimental data and lines correspond to fitted data according to the Lineweaver-Burk
equation.
pectin sample with a given MD. Figure 3.5 shows that a linear tendency, with a negative slope,
was revealed upon plotting the Ki against MD. An increase in the MD caused a significant
decrease in the Ki. In the context of non-competitive enzymatic inhibition, the Ki can be
interpreted as the inhibitor concentration required to reduce the enzymatic activity by 50%
0
2
4
6
8
10
-1.2 -0.8 -0.4 0.0 0.4 0.8
1/L
ipa
se A
ctiv
ity
1/pNPPA (mM-1)
a.
0.0
0.5
1.0
1.5
-3 0 3 6
1/
-Am
yla
se A
ctiv
ity
1/G3CNP (mM-1)
b.
0.00
0.05
0.10
0.15
0.20
-4 -2 0 2 4 6
1/P
ho
sph
ata
se A
ctiv
ity
1/pNPP (mM-1)
c.
0.0
0.1
0.2
0.3
0.4
0.5
-4 -2 0 2 4
1/P
rote
ase
Act
ivit
y
1/pNPA (mM-1)
d.
Chapter 3
73
Figure 3.4. Effect of the concentration of high methoxylated pectin (HMP, methoxylation degree of
87.4% mol/mol) on the apparent maximum velocity (Vmaxapp) and Michaelis-Menten constant (Km) of
lipase (a), -amylase (b), alkaline phosphatase (c), and protease (d). In non-competitive inhibition, the Km
remains constant whereas the Vmaxapp decreases when the concentration of HMP increases. An ANOVA
analysis (p<0.05) showed that the HMP concentration did not significantly affect the Km.
(IC50, Cheng & Prussof, 1973), namely, pectins with a high Ki (LMP) are less efficient as a non-
competitive inhibitor of enzymes than pectins with a low Ki (HMP). All of the tested enzymes
were inhibited by pectins with any MD by means of the same molecular mechanism (non-
competitive inhibition). The efficiency of the inhibition was determined from the slope (m) of the
straight line that was obtained when plotting Ki vs MD (Figure 3.5).
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
0 20 40 60 80 100
Km
(mM
)Vm
ax
Ap
p
(m
ol p
NP
min
-1m
g-1
pro
tien
)
Pectin (g mL-1)
Vmax App
Km
a.
0.0
0.2
0.4
0.6
0.8
1.0
0
2
4
6
8
10
0 20 40 60 80 100
Km
(mM
)Vm
ax
Ap
p
(m
ol
CN
P m
in-1
mg
-1p
roti
en)
Pectin (g mL-1)
Vmax App
Km
b.
0.0
0.2
0.4
0.6
0.8
1.0
0
10
20
30
40
0 20 40 60 80 100
Km
(mM
)Vm
ax
Ap
p
(m
ol p
NP
min
-1m
g-1
pro
tien
)
Pectin (g mL-1)
Vmax App
Km
c.
0.0
0.2
0.4
0.6
0.8
1.0
0
2
4
6
8
10
0 20 40 60 80 100
Km
(mM
)Vm
ax
Ap
p
(m
ol p
NP
min
-1m
g-1
pro
tien
)
Pectin (g mL-1)
Vmax App
Km
d.
Chapter 3
74
Figure 3.5. Effect of the methoxylation degree (MD) of pectins on the non-competitive inhibition constant
(Ki) of lipase (a), -amylase (b), alkaline phosphatase (c), and protease (d). The efficiency of inhibition
was measured as the slope (m) of the straight line obtained when plotting Ki vs MD. High methoxylated
pectin (HMP) has the highest ability to inhibit the activities of the digestive enzymes (r2=0.984, 0.986,
0.961, and 0.930 for lipase, -amylase, alkaline phosphatase, and protease, respectively).
Interestingly, each enzyme was inhibited with different efficiencies. Lipase (m = -1,943 g mL-1
%-1
) was more likely to be inhibited by pectins than were -amylase (m = -1,558 g mL-1
%-1
),
alkaline phosphatase (m = -1,344 g mL-1
%-1
), or protease (m = -1,165 g mL-1
%-1
). These
differences could be attributed to the interaction capacity of each enzyme and pectins, which is
0
50
100
150
200
0 20 40 60 80 100
Ki(
g m
L-1
)
MD (% mol/mol)
a.
m = -1.943 0.112
0
50
100
150
200
0 20 40 60 80 100
Ki(
g m
L-1
)
MD (% mol/mol)
b.
m = -1.558 0.109
0
50
100
150
200
0 20 40 60 80 100
Ki(
g m
L-1
)
MD (% mol/mol)
c.
m = -1.344 0.154
0
50
100
150
200
0 20 40 60 80 100
Ki(
g m
L-1
)
MD (% mol/mol)
d.
m = -1.165 0.139
Chapter 3
75
mediated by the structural characteristics of each of the molecule involved in the interaction
(Rodríguez-Patiño & Pilosof, 2011).
Ikeda & Kusano (1983) have suggested two hypotheses to explain the inhibitory effect of dietary
fiber on the activity of trypsin (a protease). The first hypothesis states that the inhibition of
enzymatic activity may result from the interaction of dietary fiber with the substrate, preventing
the enzyme-substrate interaction, and the second hypothesis states that there is a direct interaction
between dietary fiber and trypsin, making the enzyme unable to perform its catalytic function.
Regarding these hypothesis, we found that the inhibitory effect of pectins on the activities of the
enzymes appears to be due to a non-competitive enzyme-pectin interaction. The non-competitive
inhibitory mechanism of pectins on the activities of the enzymes revealed in this study (Figure
3.3) is most likely due to the large size of pectins, which generally have sizes and molecular
dimensions comparable to those of the enzymes, suggesting that the interaction of pectin with an
enzyme may be superficial (Zhao, Diao, & Zong, 2013). Nevertheless, it is important to consider
that the overall inhibitory effect of pectins toward a gastrointestinal tract activity might be caused
by several mechanisms, such as modification of the composition and structure of the interface,
substrate coating with a pectin layer or embedding of the substrates within pectin particles
(Miled, Beisson, de Caro, de Caro, Arondel, & Verger, 2001; Reis, Holmberg, Watzke, Leser, &
Miller, 2009; McClements & Li, 2010). Also, it has been previously established that the
inhibition of some digestive processes can be enhanced by the increase of viscosity of the
gastrointestinal fluids and by flocculation of lipids due to the presence of polysaccharides in the
gastrointestinal tract (McClements, 2000). At sufficiently high concentration, polysaccharides
form a three-dimensional network of interacting or entangled molecules than traps substrates and
enzymes and effectively inhibit their movements and interactions (Dickinson, 2009).
3.3.3. Nature of the enzyme-pectin interaction
The interaction of pectins with enzymes may be controlled by the structural parameters of both
pectins (MD, acetylation degree, and molecular weight distribution) and the digestive enzymes
(size, molecular weight, surface hydrophobicity, and three-dimensional structure).
Chapter 3
76
Table 3.2. Structural properties of lipase, -amylase, alkaline phosphatase, and protease. Lipase has the
greater probability of having surface interactions with hydrophobic pectins (such as high methoxylated
pectin, HMP) due to their larger size and its large exposed surface area. The high content of hydrophobic
amino acids confers upon the lipase enzyme its predominantly hydrophobic character and its high capacity
for interaction with HMP.
Enzyme MW1 pI
2 V
3 As
4 THA (%)
5 Gtr w→o
6
Lipase 99.7 (896) 6.11 (-12.0) 170,960 31,063 58.1 (521) -74.5
-Amylase 55.4 (496) 6.17 (-5.2) 111,734 16,811 56.0 (278) -40.5
Alkaline phosphatase 52.7 (484) 6.12 (-7.8) 76,503 14,611 52.5 (254) -35.7
Protease 23.3 (223) 8.34 (+6.3) 33,795 6,753 49.8 (111) -16.1
1 MW: Molecular weight (kDa). The number of total amino acids is in parentheses. Obtained from Protein Data Bank. 2 pI: Isoelectric point. The estimated charge at pH 7.0 is in parentheses. Obtained from Protein Data Bank. 3 V: Protein volume (Å3). Calculated according to Voss & Gerstein (2010). 4 As: Accessible surface area (Å2). Calculated according to Miller, Janin, Lesk & Chothia (1987). 5 THA: Total hydrophobic amino acids (sum of Phe, Trp, His, Tyr, Ala, Ile, Leu, Val, Pro, and Gly). The percent of hydrophobic
residues is given as number of hydrophobic residues per 100 residues. The number of hydrophobic residues is in parentheses.
Obtained from Protein Data Bank. 6 Gtr w→o (kcal mol-1): free energy transfer from water to the bilayer interface (surface hydrophobicity). Calculated according to
Eisenhaber (1996).
Table 3.2 shows some of the structural characteristics of the enzymes of interest in this study.
Size is a structural parameter that might be important in the interaction of each enzyme with
pectins. The size of the enzymes may be represented by several structural characteristics, such as
the molecular weight, volume and accessible surface area. Lipase, with the highest degree of
inhibition by pectins, is the enzyme with the highest molecular weight, volume, and surface
accessible area of those studied. The large size of the lipase molecule might result in an increased
probability of interaction with pectins and therefore, an increased susceptibility of being inhibited
by them. Thus, as the size (As) of the enzyme decreases (lipase, 31,063 Å2; -amylase, 16,811
Å2; alkaline phosphatase, 14,611 Å
2; and protease, 6,753 Å
2), its ability to interact with and to be
inhibited by pectins also decreases.
It was also observed that each enzyme was more susceptible to inhibition by HMP than by LMP.
This behavior could be governed by the physical nature of the non-covalent intermolecular forces
operating in the enzyme-pectin interaction (McClements, 2006). The difference in the
hydrophobicity of LMP and HMP might also explain the non-covalent interaction of pectins with
each protein. Due to the presence of negatively charged free carboxyl groups, LMP is hydrophilic
Chapter 3
77
in nature and can interact more efficiently with proteins via an electrostatic mechanism. In
contrast, due to the presence of methoxylated carboxyl (carbomethoxyl ester group) groups of
neutral nature, HMP is more hydrophobic and can interact more efficiently with proteins via a
hydrophobic mechanism. Lipase, -amylase, and alkaline phosphatase, which have isoelectric
points (pI) slightly below the pH (pH 7.0) of the model solution, would have a negative
electrostatic charge (net charge of the enzymes at pH 7.0 in all cases is very close to neutrality).
The negative electrostatic nature of these enzymes could hinder their interaction with LMP due to
negative electrostatic repulsions. Such repulsive electrostatic forces could explain the weak
interaction between the enzymes and the LMP.
The total hydrophobic amino acids (THA) and the free transfer energy of the enzyme from the
aqueous phase (w) toward the organic (lipidic) phase (o, Gtr wo) were calculated to estimate the
hydrophobicity (Eisenhaber, 1996) of each enzyme. Lipase (containing 58.1% THA) is the
enzyme with the greatest number of hydrophobic amino acids (Phe, Trp, His, Tyr, Ala, Ile, Leu,
Val, Pro, and Gly) present in its structure; whereas, as the hydrophobicity of the enzymes
decreased (-amylase, 56.0% THA; alkaline phosphatase, 52.5% THA; and protease, 49.0%
THA), their ability to interact and be inhibited by HMP also decreased. The parameter Gtr wo
(Equation 3.2) is defined as:
(
) (Eq. 3.2)
Where So corresponds to the solubility of the enzyme in the organic (lipidic) phase and Sw
corresponds to the solubility of the enzyme in the aqueous phase. In the case of hydrophobic
enzymes, for which So > Sw, the Gtr wo is negative, whereas in hydrophilic enzymes, for which
So < Sw, the Gtr wo is positive. The more negative the Gtr wo, the greater the So, as well as the
hydrophobicity of the enzyme, meaning that a protein has a strong tendency to be transferred
from within the aqueous solution to the interface.
Chapter 3
78
Examining this parameter showed that lipase (Gtr wo = -74.51 kcal mol-1
) is the enzyme with
the highest hydrophobicity, whereas -amylase, alkaline phosphatase, and protease were the
enzymes with low hydrophobicity. Interestingly, the enzymes with low Gtr wo values (-
amylase, -40.46 kcal mol-1
; alkaline phosphatase, -35.66 kcal mol-1
; and protease, -16.07 kcal
mol-1
) were those which were less inhibited by HMP than lipase. This finding might indicate that
electrostatic interactions are less important in the overall enzyme-pectin interaction as compared
to hydrophobic interactions, which are generally dominant (Miled, Beisson, de Caro, de Caro,
Arondel, & Verger, 2001; McClements, 2006), and that the mechanism that could be determinant
in the enzyme-pectin interactions might be hydrophobic in nature (McClements, 2006; Hur, Lim,
Decker, & McClements, 2011).
The interfacial phenomena that occur at the oil-water interface of the simulated gastrointestinal
fluid utilized in this study also might govern the efficiency of the enzyme-pectin interaction.
Lipase is the enzyme that is most affected by interfacial characteristics, such as the substrate, bile
salts, and lipase interfacial concentrations, as well as by its hydrophobic character and its high
susceptibility to be affected by the highly hydrophobic HMP molecule (McClements, 2006; Reis,
Holmberg, Watze, Leser, & Miller, 2009). Lipases are water-soluble enzymes with a limited
activity toward substrates in aqueous media, but they exhibit high activity when the substrate is at
a concentration high enough to form micelles in the presence of a surfactant or when the substrate
is present in an emulsified medium (Miled, Beisson, de Caro, de Caro, Arondel, & Verger, 2001).
This particular behavior occurs because lipases are enzymes that are resistant to the denaturing
conditions of interfaces, such as the high concentration of surfactant agents (Reis, Holmberg,
Watzke, Leser, & Miller, 2009). It can be suggested, therefore, that lipases are enzymes that are
particularly susceptible to being inhibited by pectins, not only due to the direct effect they have
on the activity of these enzymes, but also due to the effect they have on the stability of the
emulsion formed under the simulated gastrointestinal tract conditions. It has also been suggested
that pectins can behave as anionic surfactants that may compete with a lipase for a position in the
interface, displacing the enzyme and inhibiting its activity by decreasing its interfacial
concentration (Reis, Holmberg, Watze, Leser, & Miller, 2009).
Chapter 3
79
In this study we did not conduct any emulsion preparation for any enzyme test. For the lipase
model, the chromogenic group of the pNPPA allows this compound to remain in aqueous
solution. It was only necessary the addition of a small amount of Tween 20 to improve the
solubility of pNPPA in the buffer solution. It is known, nevertheless, that to catalyze its
enzymatic reaction, lipase must absorb to the oil-water interface so that it is in close proximity to
lipid droplets. Lipase generally does this as part of a complex with co-lipase and possibly with
bile-salts (Singh, Ye, & Horne, 2009). Competitive adsorption processes occur at the lipid-
droplet surfaces among the enzyme complex, bile salts, phospholipids, digestion products, and
other surface-active substances such as pectins, which could interfere with lipase adsorbing to the
droplet surfaces. Thus, in an emulsified system (not tested by us), lipid-droplet surfaces might be
coated with pectin layers that inhibit the direct access of the lipase/co-lipase complex to the lipid
droplets (Singh, Ye, & Horne, 2009; McClements & Li, 2010).
3.3.4. Theoretical studies of digestive enzyme inhibition by pectins
To evaluate the differences in the enzyme-pectin interaction that were experimentally observed in
this study, a theoretical model was proposed. This model allowed calculating the interaction
energy for each enzyme-pectin pair. The interaction energies were calculated using molecular
docking methodology, by searching for the site on the enzyme where the interaction with pectin
was most likely to occur. Figure 3.6 shows the structures of the enzyme-HMP interaction. In all
cases, the enzyme-pectin interaction occurred at a site different from the catalytic site of each
enzyme (where substrate is located), which is consistent with a non-competitive inhibition
mechanism. Due to the large size that pectins can have, they were not expected to have a
significant interaction with the catalytic site of an enzyme, which generally represents only a
small fraction of the total surface area of an enzyme. The sites of the enzymes for which pectins
had more affinity were protein domains with a relatively high abundance of superficial
hydrophobic amino acids. For example, in the case of lipase, it was found that HMP had high
affinity for a highly hydrophobic domain that includes Gly47, Leu136, Leu140, Trp131, Tyr142,
Gly354, Tyr373, Gly415, Trp436, Val437, and Leu443, all of which are amino acids with a
hydrophobic nature.
Chapter 3
80
Figure 3.6. Structure of the enzyme-substrate-pectin complexes formed by the substrates and lipase (a),
-amylase (b), alkaline phosphatase (c), or protease (d) when inhibited by high methoxylated pectin
(HMP) with a methoxylation degree of 100% (mol/mol). In all cases, pectin fragments were docked on the
surface of the enzymes at a site different from the active (catalytic) site.
This observation suggested that the hydrophobic interactions in the enzyme-pectin coupling are
the interactions that could mostly govern the non-competitive enzyme inhibition that was
experimentally observed. As was experimentally observed, although all of the enzymes were
inhibited by pectins with any MD by means of the same molecular mechanism, each enzyme was
inhibited with different efficiencies. Figure 3.7 shows the calculated enzyme-pectin interaction
energies expressed as the BFER. The BFER value corresponds to the relationship between the
free energy of the enzyme-pectin interaction and the free energy of the enzyme-substrate
interaction. The BFER is a measure of how many fold greater is the affinity of enzyme for pectin
than that for its respective substrate. It was observed that for any MD, all the enzymes had greater
affinity for pectins than for the substrates. The BFER increased as the MD increased.
a. b.
c. d.
Chapter 3
81
Figure 3.7. Effect of the methoxylation degree (MD, ranging from 0% to 100% mol/mol) of pectins on the
free energy of binding of the enzyme-pectin complexes. The binding free energy ratio (BFER) was
defined as the ratio of the free energy of binding of an enzyme-pectin complex and the free energy of
binding of the enzyme-substrate complex (dimensionless). Lipase (), -amylase (), alkaline
phosphatase (), and protease ().
These results suggested that an increase in the MD promotes enzyme-pectin interactions and
increases the efficiency of the non-competitive enzyme inhibition. The trend of inhibition
observed in our experiments (Figure 3.5) was theoretically validated (Figures 3.6 and 3.7). For a
pectin with a theoretical MD of 100% (mol/mol), lipase had 18.4 times greater affinity for the
inhibitor than for the substrate, whereas -amylase (12.3 times), alkaline phosphatase (8.0 times),
and protease (4.2 times) had weaker interactions with the inhibitor (Figure 3.7). Furthermore, for
a pectin with a theoretical MD of 0% (mol/mol), lipase had 6.2 times greater affinity for the
inhibitor than for the substrate, whereas -amylase (4.3 times), alkaline phosphatase (2.7 times)
and protease (1.4 times) were less inhibited (Figure 3.7). Thus, it could be suggested that the
enzyme-pectins hydrophobic interactions are critical to the efficiency with which pectins inhibit
the activity of the enzymes via a non-competitive mechanism.
0
5
10
15
20
0 40 80 120
BF
ER
MD (% mol/mol)
Chapter 3
82
Figure 3.8. Non-competitive inhibition of digestive enzyme activities upon addition of pectin. Digestive
enzyme (yellow), pectin (white), substrate (red), and product (blue).
Finally, our results allow us to suggest that the mechanism by which pectin is able to reduce the
digestive enzyme activities is the non-competitive inhibition (Figure 3.8): Pectin binds to the
digestive enzymes somewhere other than the active site, or the substrate cannot correctly
orientate on the active site of the digestive enzymes.
3.4. Conclusions
The kinetic analyses performed in this study suggested that pectins function as non-competitive
inhibitors of the gastrointestinal tract enzymes. The high degree of methoxylation of pectins and
the hydrophobicity of the enzymes significantly contribute to the enzyme-pectin surface
interactions and to the efficiency of the inhibitory effect of pectins on the in vitro activity of the
enzymes in model solutions. The order of the magnitude with which the enzymes were inhibited
by pectins was lipase>-amylase>alkaline phosphatase>protease. Based on our results, we
suggest that pectins might be able to inhibit nutrient digestion in the small intestine by inhibiting
gastrointestinal tract enzymatic activities.
Enzymatic
reaction
Inhibition of
enzymatic activity
No orientation of
the substrate
Chapter 3
83
Acknowledgments
The authors are grateful to COLCIENCIAS and the Universidad Nacional de Colombia for
providing a fellowship to Mauricio Espinal-Ruiz that supported this study.
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Chapter 4
Impact of dietary fibers [methyl cellulose, chitosan, and pectin]
on digestion of lipids under simulated gastrointestinal conditions
Published as:
Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E.,
& McClements, D. J. Food & Function. 5 (2014): 3083 – 3095.
Chapter 4
87
Abstract
A simulated in vitro digestion model was used to elucidate the impact of dietary fibers on the
digestion rate of emulsified lipids. The influence of polysaccharide type (chitosan (cationic),
methyl cellulose (non-ionic), and pectin (anionic)) and initial concentration (0.4 to 3.6% (w/w))
was examined. 2% (w/w) corn oil-in-water emulsions stabilized by 0.2% (w/w) Tween-80 were
prepared, mixed with polysaccharides, and then subjected to an in vitro digestion model (37 °C):
initial (pH 7.0); oral (pH 6.8; 10 min); gastric (pH 2.5; 120 min); and, intestinal (pH 7.0; 120
min) phases. The impact of polysaccharides on lipid digestion, -potential, particle size,
viscosity, and stability was determined. The rate and extent of lipid digestion decreased with
increasing pectin, methyl cellulose, and chitosan concentrations. The free fatty acids released
after 120 min of lipase digestion were 46, 63, and 81% (w/w) for methyl cellulose, pectin, and
chitosan, respectively (3.6% (w/w) initial polysaccharide), indicating that methyl cellulose had
the highest capacity to inhibit lipid digestion, followed by pectin and chitosan. In the absence of
polysaccharides, lipid droplets remained stable to flocculation throughout the digestion model.
Methyl cellulose and pectin promoted depletion flocculation of the lipid droplets, whereas
chitosan promoted bridging flocculation. These results have important implications for
understanding the influence of dietary fibers on lipid digestion, since they promote droplet
flocculation and therefore inhibit digestion. The control of lipid digestibility within the
gastrointestinal tract might be important for the development of reduced-calorie emulsion-based
functional food products.
Keywords: Pectin, chitosan, methyl cellulose, emulsion, lipid digestion, gastrointestinal tract,
flocculation.
Chapter 4
88
4.1. Introduction
Diets rich in fat have been associated with high incidences of obesity and elevated risks of
coronary heart disease, diabetes, and certain forms of cancer (Bray, Paeratakul, & Popkin, 2004;
van Dam & Seidell, 2007). A potential strategy for combating these chronic diseases is therefore
to reduce the total level of fat present in food products (Chung, Degner, & McClements, 2013;
Mao & Julian McClements, 2012; Wu, Degner, & McClements, 2013). However, the
development of these products is challenging because fats have a major impact on the
physicochemical, sensory, and nutritional properties of foods (Heertje, 2014; Narine &
Marangoni, 1999). For instance, fat contributes to the desirable texture of dairy products
(Rousseau, 2000), the mouthfeel and texture of bakery products (Ghotra, Dyal, & Narine, 2002),
and the creamy texture, milky appearance, desirable flavor, and satiating effects of emulsion-
based products, such as sauces, spreads, dressings, and dips (Ghosh & Rousseau, 2011). Foods
with reduced fat levels must therefore be carefully formulated to ensure that they maintain their
desirable physicochemical, sensory, and nutritional properties (e.g., appearance, flavor, texture,
shelf life, and satiety effects), otherwise they will not be acceptable to consumers (Heertje, 2014).
Rather than simply reducing the total amount of fat present within foods, it may also be possible
to improve their healthfulness using other strategies associated with controlling fat digestion. For
example, if the rate and extent of lipid digestion within the small intestine can be decreased then
the spike in blood lipid levels that normally occurs can be reduced (Michas, Micha, & Zampelas,
2014). In addition, retarded lipid digestion may also increase the feelings of satiety and satiation,
which may lead to lower overall calorie consumption (Kritchevsky, 1988; Li, Hu, &
McClements, 2011; Li & McClements, 2014). Dietary fibers are known to have an impact on the
behavior of lipids within the gastrointestinal tract and can therefore be used to modulate the
response of humans to ingested lipids (Galisteo, Duarte, & Zarzuelo, 2008; Gunness & Gidley,
2010; Kritchevsky, 1988; Slavin, 2005). Dietary fibers may influence lipid digestion through a
wide variety of different mechanisms (Gunness & Gidley, 2010): i) they may bind to species that
play a critical role in digestion such as bile salts, phospholipids, enzymes or calcium
(Dongowski, 2007); ii) they may increase the viscosity of the intestinal phase, and thereby alter
Chapter 4
89
mass transport processes (Fabek, Messerschmidt, Brulport, & Goff, 2014; Kristensen & Jensen,
2011); iii) they may form protective coatings around lipid droplets thereby inhibiting lipase
access (Li & McClements, 2014; Simo, Mao, Tokle, Decker, & McClements, 2012; Tokle,
Lesmes, & McClements, 2010); iv) they may promote lipid droplet aggregation thereby changing
the amount of lipid surface exposed to lipase (Hur, Lim, Decker, & McClements, 2011;
McClements & Li, 2010a); v) they may inactive digestive enzymes (Beysseriat, Decker, &
McClements, 2006; Brownlee, 2011; Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, &
Narváez-Cuenca, 2014); and vi) they may alter their microbial population within the large
intestine (Castillo, Martín-Orúe, Anguita, Pérez, & Gasa, 2007). The ability of dietary fibers to
impact lipid digestion through these and other mechanisms ultimately depends on their molecular
and physicochemical properties (Mudgil & Barak, 2013). At present, there is a relatively poor
understanding of the relationship between dietary fiber structure and their impact on the lipid
digestion process.
In the present study, we used a simulated static in vitro digestion model to study the influence of
a cationic (chitosan), non-ionic (methyl cellulose), and anionic (pectin) polysaccharides on the
digestion of emulsified lipid droplets. The aim of the study was to obtain a better understanding
of the role of dietary fiber characteristics on the gastrointestinal fate of ingested lipids. The
knowledge gained from this study might be useful for the fabrication of healthier functional food
products designed to promote health and wellness (Chung, Degner, & McClements, 2013; Khan,
Grigor, Winger, & Win, 2013).
4.2. Materials and methods
4.2.1. Chemicals
Corn oil was purchased from a commercial food supplier (Mazola, ACH Food Companies Inc.,
Memphis, TN, USA) and stored at 4 °C until use. The manufacturer reported that the corn oil
contained approximately 14, 29, and 57% (w/w) of saturated, monounsaturated, and
polyunsaturated fatty acids, respectively. Powdered methyl cellulose (M0262, 41 kDa molecular
Chapter 4
90
weight, 27.5-31.5% (mol/mol) methylation degree, viscosity of 2% (w/w) aqueous solution
=400 cps, and powdered chitosan (448877, medium molecular weight (190-310 kDa), 75-85%
deacetylation, viscosity of 1% (w/w) solution in 1% (w/v) acetic acid =200-800 cps, were
purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA).
Commercial powdered high methoxylated pectin (Genu Pectin (Citrus), USP/100) was kindly
donated by CP Kelco (Lille Skensved, Denmark) and was used without further purification. The
composition of this material as provided by the manufacturer was 6.9% (w/w) moisture, 89.0%
(mol/mol) galacturonic acid, and 8.6% (w/w) methoxyl groups, which corresponds to a degree of
methoxylation of approximately 62% (mol/mol). The average molecular weight was reported by
the manufacturer as 200 kDa. Fat soluble fluorescent dye Nile Red (N3013), lipase from porcine
pancreas (Type II, L3126, triacylglycerol hydrolase E.C. 3.1.1.3), bile extract (porcine, B8631),
mucin from porcine stomach (Type II, M2378, bound sialic acid ≤ 1.2% w/w), and pepsin A from
porcine gastric mucose (P7000, endopeptidase E.C. 3.4.23.1, activity ≥ 250 units mg-1
solid) were
purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). The supplier has
reported that lipase activity is 100-400 units mg-1
protein (using olive oil) and 30-90 units mg-1
protein (using triacetin) for 30 min incubation (one unit of lipase activity was defined as the
amount of enzyme required for the release of 1 eq of fatty acid from either triacetin (pH 7.4) or
olive oil (pH 7.7) in 1 h at 37 °C).
The composition of the bile extract has been reported as 49% (w/w) total bile salt (BS),
containing 10-15% glycodeoxycholic acid, 3-9% taurodeoxycholic acid, 0.5-7.0% deoxycholic
acid, 1-5% hydrodeoxycholic acid, and 0.5-2.0% cholic acid; 5% (w/w) phosphatidyl choline
(PC); Ca2+
≤0.06% (w/w); critical micelle concentration of bile extract 0.07 0.04 mM; and mole
ratio of BS to PC being around 15:1. All other chemicals were purchased from Sigma-Aldrich
Chemical Company (St Louis, MO, USA). Double distilled water was used to prepare all
solutions.
Chapter 4
91
4.2.2. Solution and emulsion preparation
4.2.2.1. Polysaccharide stock solutions preparation
Pectin, chitosan, and methyl cellulose stock solutions (4% w/w) was prepared by dispersing 10 g
of powdered pectin, chitosan, or methyl cellulose into 240 g of 5 mM phosphate buffer pH 7.0 for
pectin and methyl cellulose, and 5 mM acetate buffer pH 4.0 for chitosan. The solutions were
stirred at 800 rpm for 12 h (overnight) at room temperature to ensure complete dispersion and
dissolution. Pectin, chitosan, and methyl cellulose stock solutions were finally adjusted to pH 7.0
using 1.0 M sodium hydroxide and hydrochloric acid solutions, and were equilibrated for 10 min
before analysis.
4.2.2.2. Stock emulsion preparation
A stock emulsion was prepared by mixing together 20% (w/w) corn oil and 80% (w/w) buffered
emulsifier solution (5 mM phosphate buffer pH 7.0, containing 2.5% (w/w) Tween 80) for 5 min
using a bio-homogenizer (Speed 2, Model MW140/2009-5, Biospec Products Inc., ESGC,
Switzerland). The coarse emulsion obtained was then passed 5 times through a high-pressure
homogenizer (Microfluidizer M-110L processor, Microfluidics Inc., Newton, MA, USA)
operating at 11,000 psi (75.8 MPa).
4.2.2.3. Polysaccharide-emulsion mixtures preparation
Polysaccharide-emulsion mixtures were prepared by mixing the stock emulsion (containing
20.0% (w/w) corn oil and 2.0% (w/w) Tween 80) with buffered stock solutions of 4.0% (w/w)
chitosan (cationic), methyl cellulose (non-ionic), or pectin (anionic), to obtain systems of varying
composition: 2.0% (w/w) corn oil, 0.2% (w/w) Tween 80, and 0.2-3.6% (w/w) polysaccharide
(corresponding to mass ratio polysaccharide to corn oil ranging from 0.1 to 1.8). The emulsion-
polysaccharide mixtures were then stirred with a high-speed stirrer (Fisher Steadfast Stirrer,
Model SL 1200, Fisher Scientific, Pittsburgh, PA, USA) at 1000 rpm and stored overnight at
Chapter 4
92
room temperature. The emulsion-polysaccharide mixtures were characterized to obtain the initial
phase, prior to subjection to the static in vitro digestion model.
4.2.3. Static in vitro digestion model
Each emulsion sample (initial phase) was passed through a simulated static in vitro digestion
model that consisted of oral (Section 4.2.3.1), gastric (Section 4.2.3.2), and intestinal (Section
4.2.3.3) phases. Measurements of emulsion microstructure and stability, particle size distribution,
particle charge, and viscosity were performed after each phase (Section 4.2.4). The standardized
static in vitro digestion model used in this study was a modification of those described previously
(Li, Hu, & McClements, 2011; Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, et al.,
2014).
4.2.3.1. Oral phase
Simulated saliva fluid (SSF, pH 6.8) containing 3.0% (w/w) mucin was prepared according to the
composition shown in Table 4.1. The SSF composition was based on those reported in previous
studies (Mao & McClements, 2012). Each emulsion (initial phase) was mixed with SSF (ratio 1:1
w/w) and the resulting mixture containing 1% (w/w) corn oil and 0.1-1.8% (w/w) pectin was used
for characterization after the incubation period. The oral phase model consisted of a conical flask
containing emulsion-SSF mixture incubated at 37 °C with continuous shaking at 100 rpm for 10
min in a temperature controlled air incubator (Excella E24 Incubator Shaker, New Brunswick
Scientific, NJ, USA) to mimic the conditions in the mouth. The resulting oral phase (bolus) was
used in the gastric phase (Section 4.2.3.2).
4.2.3.2. Gastric phase
Simulated gastric fluid (SGF) was prepared by adding 2.0 g NaCl, 7.0 mL concentrated HCl
(37% w/w), and 3.2 g pepsin A (from porcine gastric mucose, 250 units mg-1
) to a flask and then
diluting with double distilled water to a volume of 1.0 L, and finally adjusting to pH 1.2
Chapter 4
93
Table 4.1. Chemical composition of simulated saliva fluid (SSF) used to simulate oral conditions.
Compound Chemical formula Concentration (g L-1
)1
Sodium chloride NaCl 1.594
Ammonium nitrate NH4NO3 0.328
Potassium dihydrogen phosphate KH2PO4 0.636
Potassium chloride KCl 0.202
Potassium citrate K3C6H5O7•H2O 0.308
Uric acid sodium salt C5H3N4O3Na 0.021
Urea H2NCONH2 0.198
Lactic acid sodium salt C3H5O3Na 0.146
Porcine gastric mucin (Type II) ---- 30
1The SSF was prepared in double distilled water and then pH 6.8 was adjusted using 0.1 M NaOH.
using 1.0 M HCl. Samples taken from the oral phase (bolus) were mixed with SGF (ratio 1:1
w/w) so that the final mixture contained 12.0 mM NaCl, 0.16% (w/w) pepsin A (corresponding to
an enzymatic activity of 400 units mL-1
), 0.5% (w/w) corn oil, and 0.05-0.90% (w/w) pectin. This
mixture was then adjusted to pH 2.5 using 1.0 M NaOH and incubated at 37 °C with continuous
shaking at 100 rpm for 2 h (this time represents the half emptying of a moderately nutritious and
semi-solid meal (Hur, Lim, Decker, & McClements, 2011)). Since lipase activity is markedly
lower in the gastric compartment compared to that in the duodenal tract, the addition of gastric
lipase in this phase can be omitted (McClements & Li, 2010a). Samples were taken for
characterization at the end of the incubation period (gastric phase). The resulting gastric phase
(chyme) was used in the intestinal phase (Section 4.2.3.3).
4.2.3.3. Intestinal phase
Samples obtained from the gastric phase (20.0 mL chyme containing 0.5% (w/w) corn oil and
0.05-0.90% (w/w) pectin) were incubated for 2 h at 37 °C in a simulated small intestine fluid
(SIF) containing 2.5 mL pancreatic lipase (24.0 mg mL-1
), 3.5 mL bile extract solution (54.0 mg
mL-1
), and 1.5 mL salt solution containing 0.25 M CaCl2 and 3.0 M NaCl, to obtain a final
Chapter 4
94
composition of the intestinal fluid in the reaction vessel of 0.36% (w/w) corn oil, 0.05-0.65%
(w/w) pectin, 2.0 mg mL-1
pancreatic lipase (corresponding to an enzymatic activity of 550 units
mL-1
), 7.0 mg mL-1
bile extract, 15.0 mM CaCl2, and 150.0 mM NaCl. The free fatty acids (FFA)
released were monitored by determining the amount of 0.10 M NaOH needed to maintain a
constant pH 7.0 within the reaction vessel using an automatic titration unit (pH stat titrator, 835
Titrando, Metrohm USA, Inc., Riverview, FL, USA). All additives were dissolved in phosphate
buffer solution (5 mM, pH 7.0) before use. Lipase addition and initialization of the titration
program were carried out only after the addition of all pre-dissolved ingredients and balancing
the pH to 7.0. Samples were taken for physicochemical and structural characterization at the end
of the digestion period (intestinal phase). The volume of 0.10 M NaOH added to the emulsion
was recorded over time and then was used to calculate the concentration of FFA generated by
lipolysis. The amount of FFA (% w/w) released was calculated using the following equation:
(
) (4.1)
Where, CNaOH is the concentration of the sodium hydroxide (0.10 M), MWLipid is the average
molecular weight of corn oil (872 g mol-1
), WLipid is the initial weight of corn oil in the intestinal
phase (0.10 g), and VNaOH is the volume of NaOH (L) titrated into the reaction vessel to
neutralize the FFA released, assuming that all triacylglycerols (TAG) are hydrolyzed in two
molecules of FFA and one molecule of monoacylglycerol (MAG). Titration blanks were
performed by inactivating lipase in boiling water for 15 min prior to initialization of the titration
program.
4.2.4. Emulsion characterization
4.2.4.1. Creaming stability measurements
Ten milliliters of emulsion was transferred into a test tube (internal diameter 15 mm, height 125
mm), tightly sealed with a plastic cap, and then stored at room temperature for 24 h, after which
Chapter 4
95
appreciable phase separation into an opaque layer at the top, a turbid layer in the middle, and a
transparent layer at the bottom was observed in some of the systems. We defined the serum layer
to be the sum of the turbid and transparent layers. The total height of the emulsion (HE) and the
height of the serum layer (HS) were measured using a laser vertical profiling system (Turbiscan
Classic MA2000, Formulaction, Wynnewood, PA, USA). The extent of creaming was then
characterized by the creaming index (CI) as follows: ⁄ . The creaming index
provided indirect information about the extent of droplet aggregation.
4.2.4.2. Emulsion microstructure
The microstructure of the emulsions was characterized by confocal microscopy. An optical
microscopy (C1 Digital Eclipse, Nikon, Tokyo, Japan) with a 60x objective lens was used to
capture images of the emulsions. Emulsions were gently stirred to form a homogeneous mixture
without introducing air bubbles. A small aliquot of the emulsions (6 L) was then transferred to a
glass microscope slide and covered with a glass cover slip. The cover slip was fixed to the slide
using nail polish to avoid evaporation. A small amount of immersion oil (Type A, Nikon,
Melville, NY, USA) was placed on the top of cover slip. Emulsions samples were stained with fat
soluble fluorescent dye Nile Red (0.1% (w/w) dissolved in ethanol) to visualize the location of
the oil phase. All confocal images were taken using an excitation (543 nm) argon laser and
emitted light was collected between 555-620 nm, and then characterized using the instrument
software (EZ CS1 version 3.8, Niko, Melville, NY, USA).
4.2.4.3. Apparent viscosity measurements
The apparent viscosity () of samples was measured using a dynamic shear rheometer (Kinexus
Rotational Rheometer, Malvern Instruments Ltd., Worcestershire, UK). A cup and bob geometry
consisting of a rotating inner cylinder (diameter 25.0 mm) and a static outer cylinder (diameter
27.5 mm) was used. The samples were loaded into the rheometer measurement cell and allowed
to equilibrate at 37 °C for 5 min before the beginning all experiments. Samples underwent a
Chapter 4
96
constant shear treatment (10 s-1
; 10 min) prior to analysis to standardize the shear rate of each
sample. The apparent viscosity was then obtained from measurements with a shear rate of 10 s-1
.
4.2.4.4. Particle size distribution measurements
The emulsions were diluted to a droplet concentration of approximately 0.005% (w/w) using
buffer solution at the appropriate pH prior to analysis to avoid multiple scatterings effects. The
particle size distribution of emulsions was then measured using a static light scattering instrument
(Mastersizer 2000, Malvern Instruments Ltd., Worcestershire, UK). A refractive index ratio of
1.47 (corn oil) was used in the calculations of the particle size distribution. Background
corrections and system alignment were performed prior to each measurement when the
measurement cell was filled with the appropriate buffer solution. Particle sizes were reported as
particle size distribution profiles (volume fraction (%) vs. particle diameter (m)) for a mass ratio
polysaccharide:corn oil of 1.8.
4.2.4.5. Particle electrical charge measurements
The surface electrical charge (-potential) of emulsions was determined using a particle
electrophoresis instrument (Zetasizer NanoSeries, Malvern Instruments Ltd., Worcestershire,
UK). The emulsions were diluted to a droplet concentration of approximately 0.005% (w/w)
using buffer solution at the appropriate pH prior to analysis. Diluted emulsions were injected into
the measurement chamber, equilibrated for 120 s and then the -potential was determined by
measuring the direction and velocity that the droplets moved in the applied electric field. Each -
potential measurement was calculated from the average of 20 continuous readings made per
sample. To determine the effect of pH on the -potential of the polysaccharides (0.5% w/w), a
titration between pH 2.0-8.0 was performed with an automatic titration unit (Multi Purpose
Titrator MPT-2, Malvern Instruments Ltd., Worcestershire, UK) and 0.25 M NaOH. The -
potential was recorded at each pH after 60 s equilibrium.
Chapter 4
97
4.2.5. Data analysis
All measurements were performed at least three times using freshly prepared samples. Averages
and standard deviations were calculated from these triplet measurements.
4.3. Results and discussion
4.3.1. Electrical properties of dietary fibers
Initially, we measured the -potential versus pH profiles of the three polysaccharides used in this
study to characterize their electrical properties (Figure 4.1). The -potential of the chitosan went
from highly positive at pH 2.0 to close to zero at pH 8.0, which can be attributed to the presence
of cationic amino groups (–NH2 + H2O –NH3⊕
+ OH⊝) with a pKa value around pH 6.5 along
the polymer backbone (Yuan, Gao, Decker, & McClements, 2013). The -potential of the methyl
cellulose was close to zero across the entire pH range studied due to the fact that it is a neutral
polymer with no charged groups. The -potential of the pectin went from close to zero at pH 2.0
to highly negative at pH 8.0, which can be attributed to the presence of anionic carboxyl groups
(–COOH + H2O –COO⊝ + H3O⊕) with a pKa value around pH 3.5 (Jones, Lesmes, Dubin, &
McClements, 2010). Visual observations of the samples indicated that they remained transparent
across the entire pH range studied, suggesting that self-association, precipitation, and
sedimentation did not occur.
4.3.2. Influence of dietary fibers on physicochemical properties of lipid droplets in
simulated gastrointestinal tract (GIT)
In this series of experiments, we examined the influence of the three polysaccharides on the
physicochemical and structural properties of lipid droplets as they passed through a simulated
GIT. Different types and amounts of dietary fiber were mixed with stock emulsion, and then the
properties of the resulting mixtures were characterized as they were passed through the simulated
Chapter 4
98
Figure 4.1. Influence of pH on the surface electrical charge (-potential) of diluted 0.5% (w/w) chitosan,
methyl cellulose, and pectin solutions.
oral, gastric, and small intestine stages. The particle size distribution, microstructure, charge, and
stability of the samples were measured after each stage of the GIT model.
4.3.2.1. Initial samples
The particle size distribution measured by static light scattering (SLS) indicated that all of the
initial emulsions contained relatively small droplets, with a monomodal distribution with a peak
around 310 nm (Figure 4.2a). Confocal microscopy images suggested that there were very large
flocs present in the emulsions containing methyl cellulose and pectin, and some small flocs in the
emulsions containing chitosan (Figure 4.3). The fact that droplet flocculation was not evident in
the light scattering data, but was in the microscopy images, can be attributed to the fact that the
emulsions were highly diluted prior to SLS measurements, which will breakdown any weakly
flocculated droplets (McClements, 2000).
-60
-40
-20
0
20
40
60
2 3 4 5 6 7 8
-P
ote
nti
al
(mV
)
pH
Chitosan
Methyl Cellulose
Pectin
Chapter 4
99
Figure 4.2. Influence of chitosan, methyl cellulose, and pectin (mass ratio polysaccharide:corn oil of 1.8)
on the particle size distribution of emulsions under simulated gastrointestinal conditions consisting of an
initial (a), oral (b), gastric (c), and intestinal (d) phases. Control corresponds to the emulsion without the
addition of polysaccharides. The scale was shifted upwards by 25, 50, and 75% for chitosan, methyl
cellulose, and pectin, respectively.
One would not expect an electrostatic attraction between anionic or neutral polymers and oil
droplets stabilized by a non-ionic surfactant. We therefore attribute the extensive droplet
flocculation observed in the emulsions containing methyl cellulose or pectin to a depletion effect
(Furusawa, Ueda, & Nashima, 1999), i.e., the generation of an osmotic attraction between the
droplets due to the exclusion of non-adsorbed polymers from the droplet
0
20
40
60
80
100
0.05 0.5 5 50 500
Volu
me
Fra
ctio
n (
%)
Particle Diameter (m)
Pectin
Methyl Cellulose
Chitosan
Control
0
20
40
60
80
100
0.05 0.5 5 50 500
Volu
me
Fra
ctio
n (
%)
Particle Diameter (m)
Pectin
Methyl Cellulose
Chitosan
Control
0
20
40
60
80
100
0.05 0.5 5 50 500
Volu
me
Fra
ctio
n (
%)
Particle Diameter (m)
Pectin
Methyl Cellulose
Chitosan
Control
c.
0
20
40
60
80
100
0.05 0.5 5 50 500
Volu
me
Fra
ctio
n (
%)
Particle Diameter (m)
Pectin
Methyl Cellulose
Chitosan
Control
d.
a. b.
Chapter 4
100
Figure 4.3. Influence of chitosan, methyl cellulose, and pectin (mass ratio polysaccharide:corn oil of 1.8)
on the microstructure of emulsions observed by confocal fluorescence microscopy under simulated
gastrointestinal conditions consisting of an initial (a), oral (b), gastric (c), and intestinal (d) phases.
Control corresponds to the emulsion without the addition of polysaccharides.
surfaces (Jenkins & Snowden, 1996; McClements, 2000). Conversely, the small amount of
flocculation observed in the emulsions containing chitosan may be attributed to either a depletion
or bridging effect (Furusawa, Ueda, & Nashima, 1999). Measurement of the -potential of the
Tween 80-stabilized oil droplets indicated that they had a slight negative charge (-6.0 mV) at
neutral pH, which may have been due to the presence of anionic impurities (such as fatty acids)
in the oil or surfactant, or due to preferential adsorption of hydroxyl ions (rather than hydronium
ions) from water by the oil droplet surfaces (Nikiforidis & Kiosseoglou, 2011). Thus, there may
have been a weak electrostatic attraction between the anionic fat droplets and cationic chitosan
molecules initially leading to some bridging flocculation in this system (Biggs, Habgood,
Jameson, & Yan, 2000). In addition, any non-adsorbed chitosan molecules may have promoted
ChitosanMethyl
CellulosePectin
a. Initial
b. Oral
c. Gastric
d. Intestinal
Control
Chapter 4
101
Figure 4.4. Influence of the concentration (mass ratio polysaccharide (P):corn oil (CO)) of chitosan,
methyl cellulose, and pectin on creaming stability of emulsions under simulated gastrointestinal conditions
consisting of an initial (a), oral (b), gastric (c), and intestinal (d) phases.
depletion flocculation (Biggs, Habgood, Jameson, & Yan, 2000; Renault, Sancey, Badot, &
Crini, 2009). However, the fact that much less flocculation occurred within the sample containing
chitosan suggests that neither depletion nor bridging effects were particularly strong (Furusawa,
Ueda, & Nashima, 1999). Bridging flocculation may have been limited due to the relatively weak
electrostatic interactions at this pH (Biggs, Habgood, Jameson, & Yan, 2000), whereas depletion
flocculation may have been limited because of the relatively low molecular weight of the
chitosan used (Li & McClements, 2013; Renault, Sancey, Badot, & Crini, 2009; Yuan, Gao,
Decker, & McClements, 2013).
ChitosanMethyl
CellulosePectin
a. Initial
b. Oral
c. Gastric
d. Intestinal
0.0 0.1 0.6 1.2 1.8 0.0 0.1 0.6 1.2 1.8 0.0 0.1 0.6 1.2 1.8Mass Ratio (P:CO)
Chapter 4
102
Measurements of the creaming stability of the initial emulsions in the presence of the different
polysaccharides also supported the observation that flocculation occurred in some of the samples
(Figure 4.4). In the absence of dietary fiber, the emulsions appeared homogenous after storage
and could therefore be considered to be stable to creaming. The initial emulsions containing
chitosan were stable to creaming at all dietary fiber concentrations studied, which suggests that
extensive droplet flocculation did not occur. The stability of the chitosan emulsions could be
attributed to an increase in aqueous phase viscosity, since the chitosan gave a larger increase than
the pectin (Figure 4.5). The emulsions containing methyl cellulose and pectin were stable to
creaming at low levels (0.4% w/w), but highly susceptible to creaming at higher levels (Figure
4.4). At low levels of these polysaccharides, the depletion attraction is not strong enough to
overcome the steric and/or electrostatic repulsion between the oil droplets and therefore
flocculation does not occur. At higher polysaccharide levels, the depletion attraction is strong
enough to promote flocculation and therefore rapid creaming occurs because of the resulting
increase in particle size (McClements, 2000). The thickness of the cream layer increases at high
polysaccharide levels because of the formation of a three-dimensional network of strongly
aggregated droplets that inhibits their movement (Mao & McClements, 2012). Viscosity
measurements of the samples containing high levels of the polysaccharides indicated that they
were relatively viscous, and could therefore inhibit droplet creaming (Kawakatsu, Trägårdh, &
Trägårdh, 2001) (Figure 4.5).
Measurements of the electrical charge in the emulsion-polysaccharide systems showed that there
was little change in the -potential when methyl cellulose or chitosan was added, but that there
was an appreciable increase in the negative charge when pectin was added (Figure 4.6). These
results suggest that methyl cellulose and chitosan did not strongly interact with the emulsion
droplets, which can be attributed to the relatively low charge of the fat droplets and
polysaccharides at this pH. The large increase in negative charge that occurred when pectin was
added can probably be attributed to the fact that the micro-electrophoresis instrument measured
the electrical characteristics of the pectin molecules rather than those of the fat droplets (Tsai,
Chen, Kuo, Lin, Wang, Hsien, et al., 2014).
Chapter 4
103
Figure 4.5. Influence of the concentration of chitosan, methyl cellulose, and pectin on the apparent
viscosity of emulsions under simulated gastrointestinal conditions consisting of an initial (a), oral (b),
gastric (c), and intestinal (d) phases. Apparent viscosity () was obtained from measurements with a shear
rate of 10 s-1
.
4.3.2.2. Oral phase
The emulsion samples were then subjected to a simulated oral phase, and their physicochemical
and structural properties were measured. The particle size distribution measured by SLS indicated
that the majority of fat droplets in all of the emulsions remained relatively small, but that there
was a population of highly aggregated droplets in all of the systems (Figure 4.2b).
0.00
0.25
0.50
0.75
1.00
1.25
1.50
0.0 0.2 0.4 0.6 0.8 1.0
Ap
pa
ren
t V
isco
sity
(P
a s
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
c.
0.000
0.005
0.010
0.015
0.020
0.025
0.0 0.2 0.4 0.6 0.8 1.0
Ap
pa
ren
t V
isco
sity
(P
a s
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
d.
0
100
200
300
400
500
600
700
0.0 1.0 2.0 3.0 4.0
Ap
pa
ren
t V
isco
sity
(P
a s
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
a.
0
10
20
30
40
50
60
70
0.0 0.5 1.0 1.5 2.0
Ap
pa
ren
t V
isco
sity
(P
a s
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
b.
Chapter 4
104
Figure 4.6. Influence of the concentration of chitosan, methyl cellulose, and pectin on the surface
electrical charge (-potential) of emulsions under simulated gastrointestinal conditions consisting of an
initial (a), oral (b), gastric (c), and intestinal (d) phases.
The confocal microscopy images confirmed that large flocs were present in all of the emulsions
containing polysaccharides, but that there were also some smaller flocs in the control emulsion
containing no dietary fiber (Figure 4.3). Visual observations indicated that all the emulsions
were highly unstable to gravitational separation: after storage they all had a thin white layer of fat
droplets at the top and a watery serum layer at the bottom (Figure 4.4). These results suggest that
the conditions in the oral phase promoted extensive droplet flocculation in all of the emulsions. In
-60
-40
-20
0
20
40
60
0.0 1.0 2.0 3.0 4.0
-P
ote
nti
al
(mV
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
a.
-60
-40
-20
0
20
40
60
0.0 0.5 1.0 1.5 2.0
-P
ote
nti
al
(mV
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
b.
-60
-40
-20
0
20
40
60
0.0 0.2 0.4 0.6 0.8 1.0
-P
ote
nti
al
(mV
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
c.
-60
-40
-20
0
20
40
60
0.0 0.2 0.4 0.6 0.8
-P
ote
nti
al
(mV
)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
d.
Chapter 4
105
the control emulsion, droplet aggregation can be attributed to depletion flocculation induced by
the presence of the mucin molecules in the simulated oral fluids (Vingerhoeds, Blijdenstein, Zoet,
& van Aken, 2005). In the other emulsions, droplet flocculation may have been a result of
depletion and bridging flocculation caused by the mucin and dietary fiber molecules
(McClements, 2000; Vingerhoeds, Blijdenstein, Zoet, & van Aken, 2005). The presence of mucin
would have increased the osmotic attraction between the fat droplets due to the presence of non-
adsorbed polysaccharides in the aqueous phase. In addition, there may have been some
electrostatic attraction between the anionic mucin and cationic chitosan in the emulsions
containing chitosan (Svensson, Thuresson, & Arnebrant, 2008).
Similar to the initial samples (Section 4.3.2.1), measurements of the electrical charge
characteristics of the emulsion-polysaccharide systems showed that there was little change in the
-potential when methyl cellulose or chitosan was added, but that there was a large increase in
negative charge when pectin was added (Figure 4.6b). Again, these results suggest that methyl
cellulose and chitosan did not strongly interact with the fat droplets under oral conditions, which
can be attributed to the relatively low charge of the fat droplets (-12.0 mV) and these two
polysaccharides (Figure 4.1) at this pH. The large increase in negative charge that was observed
when pectin was added to the emulsions can again be attributed to the fact that the micro-
electrophoresis instrument was more sensitive to the pectin molecules than the fat droplets
(Tholstrup Sejersen, Salomonsen, Ipsen, Clark, Rolin, & Balling Engelsen, 2007).
Shear viscosity measurements indicated that all of the samples containing polysaccharides were
relatively viscous after exposure to oral conditions (Figure 4.5, >1 Pa s). The increase in
viscosity in the presence of the polysaccharides depended on dietary fiber type: methyl
cellulose>chitosan>pectin. These differences can be attributed to differences in the molecular
characteristics of the dietary fibers, such as molecular weight, conformation, and molecular
interactions. In general, the apparent viscosity of a polymer solution increases with increasing
molecular weight, decreasing branching, and increasing interactions (Dunstan, Chai, Lee, &
Boger, 1995).
Chapter 4
106
4.3.2.3. Gastric phase
After passage through the oral phase, the samples were subjected to a simulated gastric phase,
and again changes in their physicochemical and structural properties were measured. Both the
light scattering and confocal microscopy measurements indicated that extensive droplet
aggregation occurred in all of the systems (Figures 4.2c and 4.3). The irregular shape of the
particles observed in the confocal microscopy images suggested that the droplets were
flocculated, rather than coalesced under gastric conditions. Visual observations indicated that all
the control and chitosan emulsions were relatively stable to gravitational separation: after storage
they had a fairly uniform cloudy appearance throughout (Figure 4.4). On the other hand, the
emulsions containing methyl cellulose or pectin had white sediments at the bottom of the test
tubes after exposure to the gastric phase (Figure 4.4). The amount of sediment present in these
samples increased as the polysaccharide concentration increased. These results suggest that the
flocs formed by these two polysaccharides in the simulated gastric fluids were large and dense
enough to rapidly sediment. Furthermore, the flocs formed in the control and chitosan emulsions
did not appear to be strongly susceptible to gravitational separation, perhaps because of their
smaller size or lower density contrast (McClements, 2000).
Electrical charge measurements of the emulsion-polysaccharide systems under gastric conditions
showed that there was little change in -potential when methyl cellulose or pectin was added, but
that there was a large increase in positive charge when chitosan was added (Figure 4.6c). These
results suggest that methyl cellulose and pectin did not strongly interact with the fat droplets
through electrostatic interactions under gastric conditions, which can be attributed to the
relatively low charge of the fat droplets (-1.0 mV) and these polysaccharides (Figure 4.1) at pH
3.0. The large increase in positive charge that occurred when chitosan was added to the emulsions
can be attributed to the fact that the chitosan molecules became strongly cationic under acidic
conditions (Figure 4.1). The measured positive charge may therefore have been indicative of
interactions between the fat droplets and chitosan (Yuan, Gao, Decker, & McClements, 2013), or
due to the fact that the micro-electrophoresis instrument was more sensitive to the chitosan
molecules than the fat droplets. The viscosity of all the emulsions was relatively low under
Chapter 4
107
simulated gastric conditions, which can be attributed to the fact that the samples were diluted at
each stage of the gastrointestinal tract model so the polymer concentration would be relatively
low, i.e., below the polymer overlap region(Simo, Mao, Tokle, Decker, & McClements, 2012).
4.3.2.4. Intestinal phase
After passage through the gastric phase, the samples were subjected to a simulated small intestine
phase, and changes in their physicochemical and structural properties were again measured. Light
scattering and confocal microscopy measurements suggested that extensive droplet aggregation
occurred in all of the systems, but that there were distinct differences between their
microstructures (Figures 4.2d and 4.3). The fat phase was fairly evenly distributed throughout
the sample in the control emulsion containing no polysaccharide (Figure 4.3) and many small
particles were detected by SLS (Figure 4.2d). Presumably, the majority of these particles were
mixed micelles formed by the lipid digestion process (Hur, Lim, Decker, & McClements, 2011;
McClements & Li, 2010a; Minekus, et al., 2014). Mixed micelles consist of small (<10 nm)
micelle structures, as well as much larger (50-5000 nm) liposome structures (McClements & Li,
2010b). They consist of phospholipids and bile salts from the intestinal fluids, as well as free
fatty acids and monoacylglycerols resulting from digestion of the triacylglycerols (Almgren,
2000; Yang & McClements, 2013).
The mixed emulsions containing chitosan contained some irregular shaped particles, but these
were appreciably smaller than those observed in the mixed emulsions containing either pectin or
methyl cellulose (Figure 4.3). The particles in these systems were probably a mixture of
undigested fat droplets and mixed micelles. Visual observations indicated that the control
emulsions and the emulsions containing chitosan had a relatively uniform yellowish brown
appearance (Figure 4.4). The emulsions containing methyl cellulose or pectin also had a
yellowish brown color but there was evidence of some sediment at the bottom of the test tubes
after exposure to the intestinal phase (Figure 4.4). The brownish yellow color can be attributed to
the presence of bile salts, since the stock solution of these digestive components had a dark
brown color.
Chapter 4
108
Electrical charge measurements indicated that the control emulsions had a relatively high
negative charge (-35.0 mV) under simulated intestinal conditions (Figure 4.6d), which can be
attributed to the presence of anionic substances at the particle surfaces, such as free fatty acids,
phospholipids, and bile salts. The -potential changed appreciably with increasing polysaccharide
concentration, with the direction of the change depending on initial polysaccharide type. The
particles became more positive when chitosan was added, more negative when pectin was added,
and changed little when methyl cellulose was added (Figure 4.6d). These results suggest that
methyl cellulose did not strongly interact with the fat droplets through electrostatic interactions
under intestinal conditions, which can be attributed to its neutral charge characteristics (Figure
4.1) at pH 7.0. On the other hand, the increase in positive charge on the particles when chitosan
was added to the emulsions may have been due to the fact that cationic chitosan molecules
interacted with the anionic lipid particles. The increase in negative charged when increasing
amounts of pectin were added may have been due to binding of pectin to the negative lipid
particles, but this is unlikely due to strong electrostatic repulsion between them (Beysseriat,
Decker, & McClements, 2006; Simo, Mao, Tokle, Decker, & McClements, 2012). Instead, the
micro-electrophoresis instrument may have been more sensitive to the pectin molecules than the
lipid particles. In addition, the viscosity of all the emulsions was relatively low under simulated
intestinal conditions (Figure 4.5d), which can be attributed to the progressive dilution that occurs
after passage through each stage of the gastrointestinal model (Hur, Lim, Decker, & McClements,
2011; McClements & Li, 2010a; Minekus, et al., 2014). Finally, we examined the influence of
polysaccharide type and concentration on the rate and extent of lipid digestion using a pH stat
method (Figure 4.7). In the absence of polysaccharide, the emulsions were rapidly and
completely digested. Indeed, the fat phase was almost fully digested within the first 5 minutes of
incubation. In the presence of polysaccharides, there was a decrease in both the rate and extent of
lipid digestion (principally the extent rather than rate), with the extent of the digestion process
depending on the polysaccharide concentration. In addition, there was a slight decrease in the
total amount of fatty acids produced after 2 hours of digestion with increasing chitosan
concentration, but a much more appreciable decrease with increasing pectin or methyl cellulose
concentration (Figure 4.8). These results suggest that both pectin and methyl cellulose were able
to appreciably inhibit lipid digestion.
Chapter 4
109
Figure 4.7. Influence of the concentration of chitosan (a), methyl cellulose (b), and pectin (c) on in vitro
hydrolysis (percentage of free fatty acids (FFA) released by the pH stat method) of lipid droplets (0.5 %
w/w) under simulated gastrointestinal conditions.
0
20
40
60
80
100
0 20 40 60 80 100 120
FF
A R
elea
sed
(%
w/w
)
Digestion Time (min)
0.00%
0.07%
0.15%
0.44%
0.65%
0
20
40
60
80
100
0 20 40 60 80 100 120
FF
A R
elea
sed
(%
w/w
)
Digestion Time (min)
0.00%
0.07%
0.15%
0.44%
0.65%
0
20
40
60
80
100
0 20 40 60 80 100 120
FF
A R
elea
sed
(%
w/w
)
Digestion Time (min)
0.00%
0.07%
0.15%
0.44%
0.65%
c.
b.
a.
Chapter 4
110
Figure 4.8. Influence of the concentration of chitosan, methyl cellulose, and pectin on free fatty acids
(FFA) released after 2 hours of digestion (intestinal phase).
Based on the confocal microscopy images (Figure 4.3), the creaming stability of the emulsions
throughout the gastrointestinal digestion (Figure 4.4), and the aggregation state of the lipid
droplets (Figure 4.2), the most likely mechanism for this phenomenon is the ability of these
polysaccharides to promote extensive droplet flocculation by depletion attraction. Presumably,
the lipid droplets are trapped within large flocs that reduce the ability of the lipase molecules to
interact with the fat droplet surfaces (by steric hindrance) and digest the lipids (Figures 4.7 and
4.8). One would expect that the inhibition of lipid digestion would increase as the floc size
increased, and as the packing of the droplets and polymers within the flocs increased.
4.3.3. Potential mechanisms
Overall, this study has shown that different polysaccharides have different effects on the extent of
lipid digestion. In particular, our results suggest that both pectin and methyl cellulose were able
to appreciably inhibit lipid digestion. In this section, we examine some potential mechanisms that
may account for the observed influence of polysaccharides on lipid digestion.
0
20
40
60
80
100
0.0 0.2 0.4 0.6 0.8
Fin
al
Dig
esti
on
(%
FF
A)
Polysaccharide (% w/w)
Chitosan
Methyl Cellulose
Pectin
Chapter 4
111
4.3.3.1. Rheology
The type and amount of polysaccharides present in the initial system influenced the rheological
properties of the fluids in the various stages of the simulated gastrointestinal tract (Figure 4.5).
Changes in the rheology of the gastrointestinal fluids may impact the rate and extent of lipid
digestion through a number of mechanisms. At the molecular level, an increase in the micro-
viscosity of a sample will slow down the movement of any molecular species involved in the
lipid digestion process, e.g., bile salts and lipase towards the droplet surfaces, or free fatty acids
and monoacylglycerols away from the droplet surfaces. Consequently, dietary fibers could
decrease the rate and extent of lipid digestion due to their ability to slow down molecular
diffusion. However, it should be stressed that polysaccharides may cause a large increase in the
macro-viscosity of a sample, but have little effect on the micro-viscosity since small molecules
can easily diffuse through the large pores in polymer networks. An increase in the macro-
viscosity associated with the presence of dietary fibers may influence the intimate mixing of the
samples with the digestive components, which could also inhibit the ability of lipase to get the
lipid droplets surfaces. In the small intestine phase, the increase in apparent viscosity due to the
presence of the different polysaccharides was relatively modest (Figure 4.5), and therefore we do
not believe that this mechanism played a major role in influencing lipid digestion.
4.3.3.2. Flocculation
The presence of polysaccharides within the gastrointestinal fluids may have promoted
flocculation of the lipid droplets due to bridging, depletion, or other flocculation mechanism. The
ability of lipase to interact with the lipid droplet surfaces and digest the encapsulated triglycerides
may be reduced if the droplets trapped within the large flocs (Figure 4.9). One would expect that
the inhibition of lipid digestion would increase as the floc size increased, and as the packing of
droplets and polymers within the flocs increased, since these factors would reduce the ability of
lipase molecules to rapidly diffuse through the entire flocs. Based on our confocal microscopy
images (Figure 4.3) and other measurements, this mechanism appears to be important in
accounting for the observed inhibition of lipid digestion, since the emulsions
Chapter 4
112
Figure 4.9. Schematic representation of the inhibition of lipid droplets digestion extent upon addition of
polysaccharides. Lipid droplets of the corn oil-in-water emulsion stabilized by Tween 80 (a), digestion of
lipid droplets by lipase (b), polysaccharides may lead a decrease on the digestion extent of lipid droplets
by embedding them into their structure (c).
containing methyl cellulose and pectin were highly flocculated (Figure 4.3) and also had reduced
digestion rates (Figure 4.8).
4.3.3.3. Electrostatic interactions
One would expect cationic chitosan molecules to interact with various anionic species involved in
the lipid digestion process, such as lipid droplets, bile salts, phospholipids, free fatty acids, and
mixed micelles. These interactions may either inhibit or promote lipid digestion depending on
their nature. For example, chitosan may bind free fatty acids produced during triglyceride
lipolysis and remove them from the lipid droplet surfaces, thereby allowing the lipase to continue
acting on the non-digested triglycerides. On the other hand, if chitosan forms a protective layer
around the lipid droplet surfaces, then it may inhibit lipid digestion by preventing the lipase from
reaching the non-digested triglycerides within the lipid droplets. One would also expect anionic
pectin molecules to interact with any cationic species involved in the lipid digestion process. For
example, anionic pectin may strongly bind calcium ions and prevent them for precipitating long-
chain fatty acids at the lipid droplet surfaces. As a result, lipid digestion may be inhibited because
a. b. c.
Chapter 4
113
the formation of a layer of free fatty acids around the lipid droplets can prevent lipase from
reaching the non-digested triglycerides. Electrostatic interactions may therefore also play an
important role in the ability of certain polysaccharides to inhibit lipid digestion. In future studies,
it would be useful to carry out a more detailed study of the interactions of dietary fibers with
specific digestive components so as to better understand the potential importance of these
interactions.
Finally, through this study it was possible to obtain a better understanding of the role of dietary
fiber characteristics (viscosity and surface electrical charge) on the gastrointestinal fate of
ingested lipids. The knowledge gained from this study might be useful for the design,
development, and fabrication of healthier functional food products specifically engineered for
controlling obesity and for promoting health and wellness of the consumers.
4.4. Conclusions
The objective of this work was to study the impact of three polysaccharides (chitosan, methyl
cellulose, and pectin) on the physicochemical characteristics and microstructure of emulsified
lipids during passage through a simulated gastrointestinal tract. Pectin and methyl cellulose
promoted depletion flocculation when present at sufficiently high concentrations, whereas
chitosan promoted bridging flocculation under acidic pH conditions. Pectin and methyl cellulose
reduced the extent of lipid digestion appreciably, whereas chitosan caused a slight decrease.
These results have important implications for understanding the influence of dietary fibers on
lipid digestion, since they promote droplet flocculation and therefore inhibit digestion. Our
results suggest that droplet flocculation may have restricted the access of lipase to the fat droplet
surfaces, thereby reducing the magnitude of the hydrolysis of the emulsified lipids (Figure 4.9).
In addition, electrostatic interactions of polysaccharides with oppositely charged species involved
in lipid digestion may also impact the digestion process. This information may be used for
designing functional foods that give healthier lipid profiles and thereby promote health and
wellness of the consumers.
Chapter 4
114
Acknowledgments
The authors are grateful to COLCIENCIAS and Universidad Nacional de Colombia for providing
a fellowship to Mauricio Espinal-Ruiz supporting this work. We also thank the United States
Department of Agriculture (NIFA Program) for supporting this research.
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Chapter 5
Impact of pectin properties on lipid digestion under simulated gastrointestinal
conditions: Comparison of citrus and banana passion fruit
(Passiflora tripartita var. mollissima) pectins
Published as:
Espinal-Ruiz, M., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E., & McClements D. J.
Food Hydrocolloids. 52 (2016): 329 – 342.
Chapter 5
118
Abstract
Medium methoxylated pectin (52% mol/mol, MMP) was isolated from banana passion fruit
(Passiflora tripartita var. mollisima) by hot acidic extraction. The impact of MMP on lipid
digestion was compared to that of commercial citrus pectins with high (71% mol/mol, HMP) and
low (30% mol/mol, LMP) methoxylation degree. A static in vitro digestion model was used to
elucidate the impact of pectin properties (methoxylation degree and molecular weight) on the
gastrointestinal fate of emulsified lipids. A 2.0% (w/w) oil-in-water emulsion stabilized with
0.2% (w/w) Tween 80 was prepared, mixed with 1.8% (w/w) pectin samples, and then subjected
to the static in vitro digestion model (37 °C): initial (pH 7.0); oral (pH 6.8, 10 min, mucin);
gastric (pH 2.5, 120 min, pepsin); and intestinal (pH 7.0, 120 min, bile salts, and pancreatic
lipase) phases. The impact of the three pectin samples on surface particle charge (-potential),
particle size distribution of lipid droplets, microstructure, rheology, and lipid digestion (free fatty
acids (FFAs) released) was determined. The rate and extent of lipid digestion decreased with
increasing simultaneously both the molecular weight and pectin methoxylation, with the FFAs
released after 120 min of intestinal digestion being 47, 70, and 91% (w/w) for HMP, MMP, and
LMP, respectively. These results have important implications for understanding the influence of
pectin on lipid digestion. The control of lipid digestibility within the gastrointestinal tract might
be important for the designing and development of novel functional foods to control bioactive
release or to modulate satiety.
Keywords: Pectin, Passiflora tripartita var. mollisima, emulsion, lipid digestion, gastrointestinal
tract, depletion flocculation.
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5.1. Introduction
Tropical fruits are a good source of bioactive agents suitable for utilization in the food,
pharmaceutical, and cosmetic industries (Schieber, Stintzing, & Carle, 2001). Certain bioactive
agents found in tropical fruits have been shown to inhibit cardiovascular diseases and some types
of cancer (Rufino, Alves, de Brito, Pérez-Jiménez, Saura-Calixto, & Mancini-Filho, 2010).
Banana passion fruit (Passiflora tripartita var. mollissima) may be a particularly good source of
bioactive agents because of its relatively high levels of phenolics, carotenoids, and dietary fibers
(Gil, Restrepo, Millán, Alzate, & Rojano, 2014), which are known to be beneficial to human
health and wellbeing (Wootton-Beard & Ryan, 2011). Previous studies have shown that dietary
fibers from fruits have a positive effect on the treatment of diseases such as hyperlipidemia,
coronary heart disease, and certain types of cancer (Kumar, Sinha, Makkar, de Boeck, & Becker,
2011). The major source of non-cellulosic dietary fiber in fruits are pectins (Voragen, Timmers,
Linssen, Schols, & Pilnik, 1983). Pectins are acidic hetero-polysaccharides composed mainly of
-(1,4) linked D-galacturonic acid (GalA) residues (Ridley, O'Neill, & Mohnen, 2001). The
carboxyl moieties of the GalA unit may be esterified with methanol, which alters the electrical
characteristics of the molecule. Overall, the degree and patterning of methoxylation, as well as
the molecular weight, are important parameters determining the functional attributes of different
pectins (Funami, Nakauma, Ishihara, Tanaka, Inoue, & Phillips, 2011).
Although is usually accepted that pectin cannot be digested by the human gastrointestinal tract
(GIT), it is possible to get some nutrients from pectins due to the presence of symbiotic bacteria
in the GIT. Some bacteria are able to produce a group of enzymes that break down pectin into
simple sugars (mainly galacturonic acid), which are then in turn fermented to create short chain
fatty acids that human cells can absorb and which can contribute as much as 10 percent of the
calories required by cells. It has been established that two species of gut bacteria in mammals
have evolved to break down some particular kinds of foods. Bacteroides ovatus and Bacteroides
thetaiotaomicron are able to break down hemicelluloses and pectins, as well as other complex
carbohydrates that human intestinal cells secrete as mucus. These bacteria are a crucial part of the
Chapter 5
120
bacterial cells that make up our gastrointestinal tract, so further understanding of the metabolism
of these gut bacteria could help to improve the influence of pectin on human nutrition (Inman,
2011).
Overconsumption of fat is a major contributing factor to obesity, cardiovascular disease, and
diabetes (Bray & Popkin, 1998). For this reason, there has been considerable interest in the
development of effective strategies to reduce the caloric content of foods, or to reduce the spike
in blood lipids that occurs after consuming a fatty meal. Several studies have suggested that
certain types of dietary fibers can inhibit the digestion and absorption of lipids (Beysseriat,
Decker, & McClements, 2006; Edashige, Murakami, & Tsujita, 2008; Tsujita, Sumiyoshi, Han,
Fujiwara, Tsujita, & Okuda, 2003; Yonekura & Nagao, 2009). Numerous physicochemical and
physiological mechanisms may contribute to this effect, including the ability of dietary fibers to
alter the rheology of the gastrointestinal fluids, to bind digestive components (such as bile salts
and digestive enzymes), to alter the aggregation state of lipid droplets, to form protective coatings
around lipid droplets, and to be fermented within the large intestine by colonic bacteria
(Grabitske & Slavin, 2009; Lattimer & Haub, 2010; McClements, Decker, & Park, 2009). In a
recent study, we showed that pectin reduced the rate and extent (principally the extent rather than
rate) of the digestion of emulsified lipids (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sanchez,
Narvaez-Cuenca, & McClements, 2014). Increased consumption of pectin may therefore prove to
be one strategy of reducing the caloric content of fatty food products or of modulating blood lipid
levels (Mesbahi, Jamalian, & Farahnaky, 2005).
The lipids in food may be consumed in a wide variety of different physical structures such as oils
(edible oils), bulk fats (margarine and butter), or emulsified fats (milk, cream, soups, and sauces).
Nevertheless, most fatty foods are broken down into oil-in-water emulsions within mouth during
mastication and within the stomach and small intestine during the digestion process (McClements
& Li, 2010). Consequently, lipid digestion within the gastrointestinal tract typically involves
digestion of emulsified fats. Lipid digestion involves several sequential steps that include various
physicochemical and biochemical events (Torcello-Gomez, Maldonado-Valderrama, Martin-
Rodriguez, & McClements, 2011).
Chapter 5
121
In the mouth, an ingested food is mixed with saliva (pH 7.0), undergoes temperature changes
(T 37 °C), and is subjected to mechanical forces that may alter the structure, physical state, and
interfacial properties of the lipid phase (Li, Kim, Park, & McClements, 2012). In the stomach, the
lipids are mixed with a highly acidic aqueous solution that contains minerals, biopolymers,
surface active compounds, and digestive enzymes (Singh, Ye, & Horne, 2009). The lipid phase
may undergo further changes in structure due to droplet disruption and coalescence processes, as
well as changes in the nature and composition of the surface active materials adsorbed at the
lipid-water interface (Singh & Sarkar, 2011). In particular, gastric lipase adsorbs to the lipid-
water interface and initiates the lipid digestion process, converting some of the triacylglycerols
(TAGs) to diacylglycerols (DAGs), monoacylglycerols (MAGs), and free fatty acids (FFAs)
(Wilde & Chu, 2011). In the small intestine, the emulsified lipids are mixed with digestive juices
that contain pancreatic lipase, colipase, bile salts, and phospholipids (Golding & Wooster, 2010).
The bile salts and phospholipids compete and displace any surface active material present at the
lipid-water interface, and the lipase-colipase complex binds to the lipid droplet surfaces (Reis,
Holmberg, Watzke, Leser, & Miller, 2009). The pancreatic lipase converts TAGs into MAGs and
FFAs, which leave the lipid droplet surfaces and are incorporated into mixed micelle structures
consisting of phospholipids and bile salts, which then transport them to the epithelial cells, where
they are adsorbed (Yao, Xiao, & McClements, 2014).
In this study, a simulated in vitro gastrointestinal model was used to evaluate the impact of
commercial high (HMP) and low (LMP) methoxylated pectins from citrus, and medium
methoxylated pectin (MMP) isolated from banana passion fruit (Passiflora tripartita var.
mollisima) on the gastrointestinal fate of emulsified lipids. These three pectin samples were
selected because of their different charge (methoxylation) and size (molecular weight)
characteristics, and because they can be used as functional ingredients in food and beverage
products (Willats, Knox, & Mikkelsen, 2006). We hypothesized that these three pectins would
have different effects on lipid digestion due to their different molecular and physicochemical
characteristics. In particular, we focused on their influence on the rheology of the gastrointestinal
fluids, the aggregation stability of lipid droplets in different stages of the gastrointestinal tract,
and the rate and extent of lipid digestion. The aim of the study was to obtain a better
Chapter 5
122
understanding of the role of pectin characteristics on the gastrointestinal fate of ingested lipids.
The knowledge obtained in this study might be useful for the design, fabrication, and
implementation of pectin-based functional foods designed to promote health and wellness by
modulating lipid digestion.
5.2. Materials and methods
5.2.1. Chemicals
Corn oil was purchased from a commercial food supplier (Mazola, ACH Food Companies Inc.,
Memphis, TN, USA) and stored at 4 °C until use. The manufacturer reported that the corn oil
contained approximately 14, 29, and 57% (w/w) of saturated, monounsaturated, and
polyunsaturated fatty acids, respectively. Commercial powdered high methoxylated pectin (HMP,
Genu Citrus Pectin USP/100) was kindly donated by CP Kelco Co. (Lille Skensved, Denmark)
and was used without further purification. The methoxylation degree of this material was 71%
(mol/mol) and the average molecular weight 181 kDa. Commercial powdered low methoxylated
pectin (LMP) was kindly donated by TIC Gums Inc. (Belcamp, MD, USA) and was also used
without further purification. The methoxylation degree of this material was 30% (mol/mol) and
the average molecular weight 130 kDa. Fat soluble fluorescent dye Nile Red (N3013), lipase
from porcine pancreas (Type II, L3126, triacylglycerol hydrolase E.C. 3.1.1.3), bile extract
(porcine, B8631), mucin from porcine stomach (Type II, M2378, bound sialic acid ≤ 1.2%), and
pepsin A from porcine gastric mucose (P7000, endopeptidase E.C. 3.4.23.1, activity ≥ 250 units
mg-1
solid) were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). One
unit of activity of pepsin A will increase A280nm to 0.001 per min at pH 2.0 and 37 °C, using
hemoglobin as substrate. The supplier reported that the lipase activity at 37 °C was 100-400 units
mg-1
protein (pH 7.7 using olive oil) and 30-90 units mg-1
protein (pH 7.4 using triacetin) for 30
min incubation (one unit of activity of lipase corresponds to the release of 1 μeq of free fatty
acids). The composition of the bile extract has been reported as 49% (w/w) total bile salt (BS),
containing 10-15% glycodeoxycholic acid, 3-9% taurodeoxycholic acid, 0.5-7% deoxycholic
Chapter 5
123
acid, 1-5% hydrodeoxycholic acid, and 0.5-2% cholic acid; 5% (w/w) phosphatidyl choline (PC);
Ca2+ ≤ 0.06% (w/w); critical micelle concentration of bile extract 0.07 0.04 mM; and mole ratio
of BS to PC being around 15:1. Dextran analytical standards (25, 150, and 410 kDa) for high
performance size exclusion chromatography (HPSEC) were purchased from Sigma-Aldrich
Chemical Company (St Louis, MO, USA). All other chemicals were purchased from Sigma-
Aldrich Chemical Company (St Louis, MO, USA). Distilled water was used to prepare all
solutions.
5.2.2. Extraction of pectin from banana passion fruit and characterization of the pectin
samples (LMP, MMP, and HMP)
5.2.2.1. Extraction of pectin from banana passion fruit (MMP)
Five grams of homogenized banana passion fruit epicarp (Passiflora tripartita var. mollissima)
were mixed with 30 mL of 0.1 M HCl (pH 1.0) and stirred for 60 min at 90 °C. The mixture was
neutralized to pH 7.0 with 1 M NaOH solution and then 100 mL of 95% (v/v) ethanol were added
to induce pectin precipitation. The pectin obtained after 12 h of precipitation was filtered, washed
with 100 mL of 70% (v/v) ethanol, and then dried at 45 °C for 12 h. The extraction yield was
64% (w/w). Pectin samples (LMP, MMP, and HMP) were characterized as follows:
5.2.2.2. Molecular weight and gyration radius
The average molecular weight, the molecular weight distribution, and the gyration radius (rg)
were determined using high performance size exclusion chromatography (HPSEC), using a 1260
Infinity liquid chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA). One-hundred
microliters of 0.5% (w/w) pectin samples were injected into a packed column (OHpak SB-806M
HQ, 8.0 mm × 300 mm, Shoko America Inc., Torrance, CA, USA) and the elution was performed
using 200 mM NaCl at a flow rate of 1 mL min-1
for 25 min at 20 °C. An Optilab T-rex
differential refractive index detector (Wyatt Technology Co., Santa Barbara, CA, USA) at 40 °C;
and a Dawn Heleos-II multi-angle laser light scattering detector (MALLS, Wyatt Technology
Chapter 5
124
Co., Santa Barbara, CA, USA) at 30 °C were used to monitor the eluents. Both multi-angle laser
light scattering signal at 90° and dextran analytical standards (25, 150, and 410 kDa; 0.5% w/w)
were used to estimate the average molecular weight, molecular weight distribution, and gyration
radius of the pectin samples. Although the multi-angle light scattering detector has 18 optimized
scattering angles, the scattered light signal at 90° was selected for the quantification of the
average molecular weight because it allows to obtain a reliable and accurate measure of the
scattered light (Yoo & Jane, 2002).
5.2.2.3. Fourier transform-infrared (FT-IR) spectra
Pectin samples were dried in a desiccator containing blue silica gel prior to FT-IR analysis. KBr-
pectin disc mixtures (90:10 w/w) were prepared and then the FT-IR spectra were collected at the
transmittance mode in a Nicolet iS10 FT-IR Spectrometer (Thermo Fisher Scientific, Waltham,
MA, USA) at a 4 cm-1
resolution. Eighty interferograms were measured to obtain a high signal to
noise ratio.
5.2.2.4. Methoxylation and acetylation degrees
The methoxylation and acetylation degrees of pectin samples were determined by ion exchange
chromatography (Voragen, Schols, & Pilnik, 1986). Pectin samples (30 mg) were suspended in 1
mL of an isopropanol-water mixture (1:1 v/v) containing 0.4 M NaOH and stored at room
temperature for 2 h. The suspension was centrifuged (20 min, 18000 g, 4 °C) and then 20 L of
the clear supernatant was injected into the column. A model LC-20AT liquid chromatograph
(Shimadzu Corporation, Kyoto, Japan) equipped with an Aminex HPX-87H column (300 × 7.8
mm × 9 m, Bio-Rad Laboratories, Hercules, CA, USA) was used. The column was operated at
room temperature and a flow rate of 0.6 mL min-1
with 4 mM H2SO4 as the eluent. Components
eluting from the column were detected using a RID-10A refractive index detector (Shimadzu
Corporation, Kyoto, Japan) thermostated at 40 °C. The amounts of methanol and acetic acid
released after saponification were determined using an external standard method. Calibration
lines were obtained at concentrations ranging from 5 to 40 mM, and from 0.1 to 0.8 mM for
Chapter 5
125
methanol and acetic acid, respectively. The methoxylation and acetylation degrees were
expressed as moles of methyl and acetyl esters, respectively, per 100 mol of uronic acid, and
were corrected for free methanol and acetic acid. The uronic acid content was determined
spectrophotometrically (van den Hoogen, van Weeren, Lopes-Cardozo, van Golde, Barneveld, &
van de Lest, 1998). Briefly, an aliquot of 400 L of each pectin sample solution (100 g mL-1
)
was mixed with 2 mL of 98% (w/w) H2SO4 containing 120 mM sodium tetraborate
(Na2B4O710H2O) and incubated for 60 min at 80 °C. After cooling down to room temperature,
the background absorbance of the samples was measured at 540 nm. Then, 400 L of m-
hydroxydiphenyl reagent (prepared by mixing 100 L of 100 mg mL-1
m-hydroxydiphenyl in
dimethyl sulfoxide with 4.9 mL of 80% (w/w) H2SO4) was added and mixed with the samples.
After 15 min, the absorbance of the pink-colored samples was measured at 540 nm. A calibration
line was obtained using GalA at final concentrations ranging from 0.1 to 1.0 g mL-1
.
5.2.3. Solutions and emulsions preparation
5.2.3.1. Pectin stock solutions
Pectin stock solutions (2.0% w/w) were prepared by dispersing 1 g of powdered pectins (LMP,
MMP, and HMP) into 49 g of 5 mM phosphate buffer (pH 7.0). The solutions were stirred at 800
rpm overnight at room temperature to ensure complete dispersion and dissolution. Stock
solutions were finally adjusted to pH 7.0 using 1 M NaOH solution.
5.2.3.2. Stock emulsion
A stock emulsion was prepared by mixing 20% (w/w) corn oil and 80% (w/w) buffered emulsifier
solution (5 mM phosphate buffer pH 7.0, containing 2.5% (w/w) Tween 80) together for 5 min
using a bio-homogenizer (Speed 2, Model MW140/2009-5, Biospec Products Inc., ESGC,
Switzerland). The coarse emulsion obtained was then passed 5 times through a high-pressure
homogenizer (Microfluidizer M-110L processor, Microfluidics Inc., Newton, MA, USA)
operating at 11,000 psi (75.8 MPa).
Chapter 5
126
5.2.3.3. Pectin-emulsion mixtures
Pectin-emulsion mixtures were prepared by mixing the stock emulsion containing 20% (w/w)
corn oil with buffered stock solutions of 2.0% (w/w) pectin (mass ratio 1:9), to obtain emulsions
containing 2.0% (w/w) corn oil and 1.8% (w/w) pectin. The pectin-emulsion mixtures were then
stirred with a high-speed stirrer (Fisher Steadfast Stirrer, Model SL 1200, Fisher Scientific Inc.,
Pittsburgh, PA, USA) at 800 rpm and stored overnight (approximately 12 h) at room temperature.
The pectin-emulsion mixtures were characterized to obtain the initial phase, prior to subjection to
the static in vitro digestion model.
5.2.4. Static in vitro digestion model
Each emulsion sample (initial phase) was passed through a simulated static in vitro digestion
model that consisted of oral, gastric, and intestinal phases. Measurements of emulsion
microstructure and stability, particle size distribution, particle charge, and viscosity were
performed after each phase. The standardized static in vitro digestion model used in this study
was a slight modification of that described previously (Espinal-Ruiz, Parada-Alfonso, Restrepo-
Sanchez, Narvaez-Cuenca, & McClements, 2014; Minekus, Alminger, Alvito, Ballance, Bohn,
Bourlieu, et al., 2014).
5.2.4.1. Oral phase
Simulated saliva fluid (SSF, pH 6.8) containing 3.0% (w/w) mucin was prepared according to the
composition shown in Table 5.1. Each emulsion (20 mL of initial phase) was mixed with 20 mL
of SSF and the resulting mixture containing 1.0% (w/w) corn oil and 0.9% (w/w) pectin was used
for characterization after the incubation period. The oral phase consisted of a flask containing
emulsion-SSF mixture incubated at 37 °C with continuous shaking at 100 rpm for 10 min in a
temperature controlled air incubator (Excella E24 Incubator Shaker, New Brunswick Scientific
Co., New Brunswick, NJ, USA) to mimic the conditions in the mouth. Although 10 min of
incubation time is somewhat longer than in vivo (approximately 1 min), however, accuracy and
Chapter 5
127
Table 5.1. Chemical composition of simulated saliva fluid (SSF) used to simulate oral conditions.
Compound Chemical formula Concentration (g L-1
)1
Sodium chloride NaCl 1.594
Ammonium nitrate NH4NO3 0.328
Potassium dihydrogen phosphate KH2PO4 0.636
Potassium chloride KCl 0.202
Potassium citrate K3C6H5O7•H2O 0.308
Uric acid sodium salt C5H3N4O3Na 0.021
Urea H2NCONH2 0.198
Lactic acid sodium salt C3H5O3Na 0.146
Porcine gastric mucin (Type II) ---- 30
1The SSF was prepared in double distilled water and then pH 6.8 was adjusted using 0.1 M NaOH.
reproducibility in a laboratory situation may be compromised if using any shorter digestion time.
In addition, it has been recommended an oral digestion time of 10 min in order to compensate the
lack of a proper mechanical action for static models, which in most cases is difficult to simulate
(Minekus, et al., 2014). The resulting oral phase (bolus) was used in the gastric phase.
5.2.4.2. Gastric phase
Simulated gastric fluid (SGF) was prepared by adding 2.0 g NaCl, 7.0 mL concentrated HCl
(37% w/w), and 3.2 g pepsin A (from porcine gastric mucose, 250 units mg-1
) to a flask and then
diluting with double distilled water to a volume of 1.0 L, and finally adjusting to pH 1.2 using 1
M HCl. Samples taken from the oral phase (20 mL bolus) were mixed with 20 mL of SGF so that
the final mixture contained 0.50% (w/w) corn oil and 0.45% (w/w) pectin. These mixtures were
then adjusted to pH 2.5 using 1 M NaOH and incubated at 37 °C with continuous shaking at 100
rpm for 2 h. Samples were taken for characterization at the end of the incubation period (gastric
phase). The resulting gastric phases (chyme) were used in the intestinal phase.
Chapter 5
128
5.2.4.3. Intestinal phase
Samples obtained from the gastric phase (30 mL chyme containing 0.50% (w/w) corn oil and
0.45% (w/w) pectin) were incubated for 2 h at 37 °C in a simulated small intestine fluid (SIF)
containing 2.5 mL pancreatic lipase (24 mg mL-1
), 3.5 mL bile extract solution (54 mg mL-1
), and
1.5 mL salt solution containing 0.25 M CaCl2 and 3.0 M NaCl, to obtain a final composition of
the intestinal fluid in the reaction vessel of 0.40% (w/w) corn oil and 0.36% (w/w) pectin. The
lipolytic reaction was conducted at constant (pH 7.0) using an automatic titration unit (pH stat
titration unit, 835 Titrando, Metrohm USA, Inc., Riverview, FL, USA) and then the FFAs
released were monitored by determining the amount of 0.1 M NaOH needed to maintain the
constant pH within the reaction vessel. All additives were dissolved in 5 mM phosphate buffer
solution (pH 7.0) before use. Lipase addition and initialization of the titration program were
carried out only after the addition of all pre-dissolved ingredients and balancing the pH to 7.0.
Samples were taken for physicochemical and structural characterization at the end of the
digestion period (intestinal phase). The volume of 0.1 M NaOH added to the emulsion was
recorded over time and then was used to calculate the concentration of FFAs generated by
lipolysis. The amount of FFAs released was calculated using the following equation:
(
) (5.1)
Here, VNaOH is the volume of NaOH (in L) titrated into the reaction vessel to neutralize the FFAs
released, assuming that all TAGs are hydrolyzed in two molecules of FFAs and one molecule of
MAG, CNaOH is the concentration of the sodium hydroxide (0.1 M), MWLipid is the average
molecular weight of corn oil (872 g mol-1
), and wLipid is the initial weight of corn oil in the
intestinal phase (0.15 g). Titration blanks were performed by inactivating pancreatic lipase
solution in boiling water for 15 min prior to initialization of the titration program.
Chapter 5
129
5.2.5. Emulsion characterization
5.2.5.1. Gravitational separation
Ten milliliters of each sample were transferred into a glass test tube, sealed with a plastic cap,
and then stored at room temperature for 24 h. Digital photographs (Lumix DMC-ZS8 Digital
Camera, Panasonic Corporation, Newark, NJ, USA) of the samples were taken after storage to
record their stability to gravitational separation.
5.2.5.2. Microstructure
The microstructure of the samples was characterized by confocal fluorescence microscopy. An
optical microscopy (C1 Digital Eclipse, Nikon Co., Tokyo, Japan) with a 60× objective lens was
used to capture images of the emulsions. Emulsions were gently stirred to form a homogeneous
mixture without introducing air bubbles and then the emulsions were stained with fat soluble
fluorescent dye Nile Red (0.1% (w/w) dissolved in 90% (v/v) ethanol) to visualize the location of
the oil phase. A small aliquot of the stained emulsions (5 L) was then transferred to a glass
microscope slide and covered with a glass cover slip. The cover slip was fixed to the slide using
nail polish to avoid evaporation. A small amount of immersion oil (Type A, Nikon Co., Melville,
NY, USA) was placed on the top of cover slip. All fluorescence confocal images were taken
using an excitation argon laser (543 nm) and emitted light was collected between 555 to 620 nm,
and then characterized using the instrument software (EZ CS1 version 3.8, Niko Co., Melville,
NY, USA).
5.2.5.3. Apparent viscosity
The apparent viscosity of samples was measured using a dynamic shear rheometer (Kinexus
Rotational Rheometer, Malvern Instruments Ltd., Worcestershire, United Kingdom). A cup and
bob geometry consisting of a rotating inner cylinder (diameter 25.0 mm) and a static outer
cylinder (diameter 27.5 mm) was used. The samples were loaded into the rheometer measurement
Chapter 5
130
cell and allowed to equilibrate at 37 °C for 5 min before the beginning of all experiments.
Samples underwent a constant shear treatment (10 s-1
for 10 min) prior to analysis to standardize
the shear rate of each sample. The apparent viscosity () was then obtained from measurements
with a shear rate of 10 s-1
selected to mimic oral conditions (Pal, 2011).
5.2.5.4. Particle size distribution
The samples were diluted to a droplet concentration of approximately 0.005% (w/w) using buffer
solution at the appropriate pH prior to analysis to avoid multiple scatterings effects. The particle
size distribution of emulsions was then measured using a static light scattering instrument
(Mastersizer 2000, Malvern Instruments Ltd., Worcestershire, United Kingdom). Refractive
indices of 1.47 (corn oil) and 1.33 (water) were used for the calculations of the particle size
distribution. Background corrections and system alignment were performed prior to each
measurement when the measurement cell was filled with the appropriate buffer solution. Particle
sizes were reported as particle size distribution profiles (volume fraction (%) vs. particle diameter
(μm)) and surface-weighted mean diameter (d32, nm).
5.2.5.5. Surface electrical charge
The surface electrical charge (-potential, mV) of emulsions was determined using a particle
micro-electrophoresis instrument (Zetasizer NanoSeries, Malvern Instruments Ltd.,
Worcestershire, United Kingdom). The emulsions were diluted to a droplet concentration of
approximately 0.005% (w/w) using buffer solution at the appropriate pH prior to analysis. Diluted
emulsions were injected into the measurement chamber, equilibrated for 120 s and then the -
potential was determined by measuring the direction and velocity that the droplets moved in the
applied electric field. Each -potential measurement was calculated from the average of 20
continuous readings made per sample. To determine the effect of pH on the surface electrical
charge (-potential) of pectin solutions (0.5% w/w), a titration between pH 2.0 to 8.0 with 0.25 M
NaOH was performed with an automatic titration unit (Multi Purpose Titrator MPT-2, Malvern
Chapter 5
131
Instruments Ltd., Worcestershire, United Kingdom). The -potential was recorded at each pH
after 60 s equilibrium.
5.2.6. Data analysis
All digestions and measurements were performed at least three times using freshly prepared
samples. Averages and standard deviations were calculated from these triplet measurements.
5.3. Results and discussion
5.3.1. Characterization of the pectin samples
5.3.1.1. FT-IR analysis of functional groups
The FT-IR spectra of the different pectin samples are shown in Figure 5.1. It was observed the
presence of the monosaccharide units making up pectin such as GalA, xylose, arabinose, and
rhamnose, which exhibit intense signals between 1200 and 950 cm-1
wavenumber values and
constituting the fingerprint region specific for each polysaccharide. However, the most
representative signals of the FT-IR spectra of pectin samples are those related to the carboxyl (–
COOH) and carbomethoxyl (–COOCH3) groups (Manrique & Lajolo, 2002). Common features
of all the spectra were: a peak around 3410 cm-1
due to an O-H stretching vibration; a peak
around 2900 cm-1
due to C-H stretching of –CH2 groups; and two peaks at 1610 and 1410 cm-1
due to symmetrical stretching vibrations of the O=C‒O structure. The signal that appears at 1730
cm-1
can be assigned to the C=O stretching vibration of carbomethoxyl group (and also, if
present, of protonated carboxylic group) and shows clear evidence that HMP and MMP (higher
signal strength) were more methoxylated than LMP. FT-IR spectra of aliphatic carboxylic acids
(anionic form) exhibit a characteristic pair of strong intensity signals at 1610 and 1410 cm-1
corresponding, respectively, to asymmetrical and symmetrical stretching vibrations of the
Chapter 5
132
Figure 5.1. Fourier Transform Infrared (FT-IR) spectra of low methoxylated pectin (LMP), medium
methoxylated pectin (MMP) isolated from banana passion fruit (Passiflora tripartita var. mollisima), and
high methoxylated pectin (HMP). The scale was shifted upwards by 100, and 200% for MMP, and HMP
respectively.
carboxylate group (–COO⊝). Considering that a total ionization of –COOH groups could be
attained in the partially methoxylated pectin (e.g. MMP), the 1730 cm-1
signal would be
generated exclusively by the carbomethoxyl group ( ac ur ov , Cape , asin ov , ellner,
Ebringerová, 2000). The similarity of the fingerprint region of MMP with commercial HMP and
LMP pectins, and the relative intensity of the signal at 1730 cm-1
(intermediate intensity between
LMP and HMP) demonstrated that the polysaccharide obtained by acidic extraction from
Passiflora tripartita var. mollisima corresponds to medium methoxylated pectin (MMP).
5.3.1.2. Electrical characteristics of pectin samples
In this series of experiments, we used chemical analysis and micro-electrophoresis to establish
differences in the electrical characteristics of the three pectins. The degree of methoxylation of
the three pectins was 71, 52, and 30% (mol/mol) for the HMP, MMP and LMP samples,
respectively (Table 5.2).
0
50
100
150
200
250
300
5001500250035004500
Tra
nsm
ita
nce
(%
)
Wavenumber (cm-1)
HMP
MMP
LMP
3410
2900 1730 16101410
Chapter 5
133
Table 5.2. Physicochemical properties of high methoxylated pectin (HMP), medium methoxylated pectin
(MMP) isolated from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated
pectin (LMP).
Parameter HMP MMP LMP
Average molecular weight (kDa)1 181 148 130
Methoxylation degree (% mol/mol) 71 52 30
Acetylation degree (% mol/mol) 0.4 6.6 0.1
Surface charge at pH 7 (, mV) -28.2 -34.8 -47.5
1Obtained from the multi-angle laser light scattering detector (reported
according to the signal at 90°).
Measurements of the -potential versus pH profiles of the three different pectin samples are
shown in Figure 5.2. In general, all of the samples had their highest negative charges close to pH
8.0, and became less negatively charged (more positive charge) as the pH was decreased with the
steepest change in charge occurring below pH 4.5. This effect can be attributed to protonation of
the carboxyl groups (–COO⊝ + H3O⊕ –COOH + H2O) on the pectin molecules when the pH
is reduced below their pKa values (typically around pH 3.5). As expected, the magnitude of the
negative charge increased with decreasing methoxylation (HMP, = -28.2 mV; MMP, = -34.8
mV; and LMP, = -47.5 mV), since then there were more non-esterified carboxyl groups present
that could be ionized (Table 5.2). It is also important to consider that only MMP had a
considerably degree of acetylation (6.6 % mol/mol) compared to HMP and LMP. Because of the
neutral nature of the acetyl group (–COCH3) this parameter is not expected to contribute to the
overall charge of pectin. Acetyl groups, however, play an important role in the structural
conformation of pectin since this residue is related to controlling the formation of branched
structures (Mohnen, 2008).
5.3.1.3. Molecular weight of pectin samples
The molecular weight distribution of the three pectin samples was determined by HPSEC
(Figure 5.3) and the average molecular weight was calculated. The average molecular weight
values reported in the Table 5.2 were obtained from the multi-angle laser light scattering detector
Chapter 5
134
Figure 5.2. Influence of the pH on the electrical charge (-potential) of high methoxylated pectin (HMP),
medium methoxylated pectin (MMP) isolated from banana passion fruit (Passiflora tripartita var.
mollisima), and low methoxylated pectin (LMP).
signal at 90° (MALLS). The values obtained from the dextran analytical standards were fairly
similar than those obtained from MALLS. All three pectins had mono-modal distributions, but
the width of the distribution was broader for MMP than for LMP and HMP. The average
molecular weights of the three pectins were fairly similar, with all being in the range of 130 to
181 kDa. In general, HMP had the higher average molecular weight (181 kDa), followed by
MMP (148 kDa) and LMP (130 kDa).
5.3.2. Influence of pectin type on gastrointestinal fate of emulsified lipids
In this section, the influence of pectin type on the potential gastrointestinal fate of corn oil-in-
water emulsions was determined using an in vitro digestion model that simulated oral, gastric,
and small intestinal phases. Changes in particle size, electrical charge, microstructure,
appearance, and rheology of the emulsions were measured after their exposure to each stage of
the gastrointestinal tract (Figures 5.4 to 5.8).
-50
-40
-30
-20
-10
0
2 3 4 5 6 7 8
-P
ote
nti
al
(mV
)
pH
HMP
MMP
LMP
Chapter 5
135
Figure 5.3. High performance size exclusion chromatography (HPSEC) profiles of high methoxylated
pectin (HMP), medium methoxylated pectin (MMP) isolated from banana passion fruit (Passiflora
tripartita var. mollisima), and low methoxylated pectin (LMP). The signal corresponds to the differential
refractive index (DRI) detector. Molecular weight scale on top x-axis is based on dextran standards (25,
150, and 410 kDa).
5.3.2.1. Particle size, microstructure, and appearance of emulsions
Initially, all of the emulsions had similar particle size distributions and mean particle diameters
(Figures 5.4 and 5.8a), which suggested that the droplets were stable to coalescence or Ostwald
ripening. However, the confocal microscopy images indicated that all the emulsions containing
pectin were highly flocculated (Figure 5.5), and photographs of the emulsions showed that they
were highly susceptible to creaming (Figure 5.6). The initial oil droplets were coated with a non-
ionic surfactant (Tween 80), and therefore it seems likely that the origin of aggregation was
depletion flocculation rather than bridging flocculation (Blijdenstein, Winden, Vliet, Linden, &
van Aken, 2004). Indeed, calculations of the strength of the osmotic attraction between the
droplets in the presence of pectin support this hypothesis (Section 5.3.3). When the emulsions
were diluted for particle size analysis by laser light scattering the flocs would have been disrupted
0.0
0.5
1.0
1.5
2.0
2.5
0 5 10 15 20 25
DR
I (x
10
5)
Time (min)
HMP
MMP
LMP
25150410
Chapter 5
136
Figure 5.4. Influence of high methoxylated pectin (HMP), medium methoxylated pectin (MMP) isolated
from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated pectin (LMP) on
the particle size distribution of emulsions under simulated gastrointestinal conditions consisting of initial
(a), oral (b), gastric (c), and intestinal phases (d). Control corresponds to the emulsions without addition of
pectin. The scale was shifted upwards by 25, 50, and 75% for HMP, MMP, and LMP respectively.
because the amount of pectin present would have fallen below the critical flocculation
concentration (McClements, 2000). After exposure to oral conditions, all of the emulsions
(including the ones containing no pectin) were highly flocculated (Figure 5.5) and exhibited
some creaming (Figure 5.6), but the individual droplets remained relatively small after dilution
0
20
40
60
80
100
10 100 1000 10000
Vo
lum
e F
ract
ion
(%
)
Particle Diameter (nm)
LMP
MMP
HMP
Control
0
20
40
60
80
100
10 100 1000 10000
Vo
lum
e F
ract
ion
(%
)
Particle Diameter (nm)
LMP
MMP
HMP
Control
a. b.
0
20
40
60
80
100
10 100 1000 10000
Vo
lum
e F
ract
ion
(%
)
Particle Diameter (nm)
LMP
MMP
HMP
Control
0
20
40
60
80
100
10 100 1000 10000
Vo
lum
e F
ract
ion
(%
)
Particle Diameter (nm)
LMP
MMP
HMP
Control
c. d.
Chapter 5
137
Figure 5.5. Influence of high methoxylated pectin (HMP), medium methoxylated pectin (MMP) isolated
from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated pectin (LMP) on
the microstructure of emulsions observed by confocal fluorescence microscopy under simulated
gastrointestinal conditions consisting of initial (a), oral (b), gastric (c), and intestinal (d) phases. Control
corresponds to the emulsions without addition of pectin.
(Figures 5.4 and 5.8a). This result suggests that the mucin molecules present within the SSF
promoted droplet flocculation through depletion and/or bridging flocculation (Vingerhoeds,
Blijdenstein, Zoet, & van Aken, 2005). Again, the most likely mechanism is depletion
flocculation due to the fact that the fat droplets had a low negative charge, and the flocs easily
dissociated upon dilution for particle size measurements (Jenkins & Snowden, 1996; Klinkesorn,
Sophanodora, Chinachoti, & McClements, 2004; McClements, 2000). After exposure to gastric
conditions, the particle size distribution became broader with many larger particles being present
(Figures 5.4 and 5.8a), and there was evidence of flocculation (Figure 5.5) and creaming
separation (Figure 5.6). The structure and shape of the flocs observed in the gastric phase
LMP MMP HMP
a. Initial
b. Oral
c. Gastric
d. Intestinal
Control
Chapter 5
138
Figure 5.6. Influence of high methoxylated pectin (HMP), medium methoxylated pectin (MMP) isolated
from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated pectin (LMP) on
the creaming stability of emulsions under simulated gastrointestinal conditions consisting of initial (a),
oral (b), gastric (c), and intestinal (d) phases. Control (C) corresponds to the emulsions without addition of
pectin.
was quite different from those present in the oral phase (Figure 5.5). In the gastric phase, there
appeared to be a greater number of smaller flocs than in the oral phase. This effect may have
occurred because of the dilution of the emulsions or because of changes in environmental
conditions that changed the nature of the colloidal interactions, such as pH and ionic strength
(Hur, Lim, Decker, & McClements, 2011; Singh, Ye, & Horne, 2009). After exposure to small
intestine conditions, there was evidence of a broad range of different sized particles in both the
particle size distributions (Figure 5.4d) and confocal microscopy images (Figure 5.5). Lipid
digestion may result in numerous different kinds of colloidal particles being present in the
intestinal fluids, including undigested fat droplets, mixed micelles assembled from FFAs, DAGs,
a. Initial b. Oral
c. Gastric d. Intestinal
C HMP MMP LMP C HMP MMP LMP
C HMP MMP LMP C HMP MMP LMP
Chapter 5
139
bile salts, phospholipids, and insoluble calcium soaps formed from long chain FFAs and calcium
(Golding & Wooster, 2010; McClements & Li, 2010). However, it is not possible to discern the
exact nature of these different particles from the light scattering or confocal images.
5.3.2.2. Rheological properties of emulsions
The presence of dietary fibers in the emulsions is likely to alter the rheological properties of the
gastrointestinal fluids, which may impact the rate and extent of lipid digestion by altering mixing
and mass transport processes (Langhout, Schutte, Van Leeuwen, Wiebenga, & Tamminga, 1999).
The apparent viscosity of the gastrointestinal fluids was therefore measured after the emulsions
were exposed to each stage of the simulated GIT (Figure 5.7). Initially, all of the emulsions
containing pectin had a higher viscosity than the control emulsions due to the ability of pectin
molecules to increase the effective volume fraction of the dispersed phase (Mohnen, 2008;
Ridley, O'Neill, & Mohnen, 2001).
Figure 5.7. Influence of high methoxylated pectin (HMP), medium methoxylated pectin (MMP) isolated
from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated pectin (LMP) on
the apparent viscosity () of emulsions under simulated gastrointestinal conditions consisting of initial,
oral, gastric, and intestinal phases. Control corresponds to the emulsions without addition of pectin.
0.0
0.2
0.4
0.6
0.8
1.0
Initial Oral Gastric Intestinal
(P
a s
)
Phase
Control
HMP
MMP
LMP
Chapter 5
140
Table 5.3. Molecular characteristics of the high methoxylated pectin (HMP), medium methoxylated pectin
(MMP) isolated from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated
pectin (LMP) molecules used in the theoretical calculations of the depletion interactions.
Parameter HMP MMP LMP
Average molecular weight (kDa)1 181 148 130
n2 1006 822 722
rg (nm)3 6.8 4.3 3.1
RV4 12 10 10
1Obtained from the multi-angle laser light scattering detector. (Reported according to the signal at 90°). 2Average number of monomers per molecule (n = MW/MW0). MW is the average molecular weight of the pectin
molecules, and MW0 is the molecular weight of a galacturonic acid monomer unit ( 180 g mol-1). 3Effective radius of the pectin molecules in solution (gyration radius) obtained from the multi-angle laser light scattering
detector (reported according to the signal at 90°). 4Volume ratio (dimensionless). It was assumed that pectin molecules were random coil in conformation.
The extent of the increase in viscosity decreased in the following order HMP > MMP > LMP.
This effect may have been due to differences in the average molecular weight of the different
pectins: HMP > MMP > LMP (Table 5.2), which led to corresponding differences in the radius
of gyration (Table 5.3). Extended polymers with higher molecular weights tend to have higher
effective volume fractions in aqueous solutions, and therefore cause larger increases in viscosity
(McClements, 2000). As the emulsions passed through the successive stages of the simulated
gastrointestinal system there was a progressive decrease in the apparent viscosities of the
emulsions, which can be attributed to the dilution of the systems leading to a lower effective
disperse phase volume fraction. However, in each phase the viscosities still decreased in the same
order for the different pectins: HMP > MMP > LMP. The relatively high viscosities of the
emulsions containing pectin may also have been due to some flocculation of the emulsion
droplets promoted by the biopolymer (e.g. depletion or bridging flocculation) or due to formation
of hydrogel particles (e.g., calcium pectinate) (Jenkins & Snowden, 1996; McClements, 2000).
Interestingly, the viscosities of all the samples were relatively low once they reached the small
intestine phase, which can be attributed to the fact that the emulsions had undergone appreciable
dilution. Consequently, one might not expect a large influence of viscosity on the lipid digestion
process in the small intestine (Golding & Wooster, 2010).
Chapter 5
141
5.3.2.3. Electrical characteristics of emulsions
Initially, the electrical charge on the control emulsions (containing no pectin) was around -7 mV
(Figure 5.8b), which can be attributed to the presence of some anionic impurities in the corn oil
phase (such as DAGs, MAGs, FFAs or phospholipids) as well as surfactant ingredients, to the
ionization of the hydroxyl groups of Tween 80 (Tween 80 is a nonionic surfactant with the
presence of some weakly ionizable hydroxyl groups), specific ion adsorption from bulk solution
onto lipid droplets surfaces, or due to the preferential adsorption of hydroxyl ions (OH⊝), rather
than hydronium ions (H3O⊕) from water by the lipid droplet surfaces (Nikiforidis &
Kiosseoglou, 2011). The addition of pectin to the emulsions led to an increase in the measured
negative charge, with the magnitude of the effect increasing with decreasing degree of
methoxylation. This effect can be attributed to the contribution of the anionic pectin molecules to
the measured signal used to calculate the -potential.
Figure 5.8. Influence of high methoxylated pectin (HMP), medium methoxylated pectin (MMP) isolated
from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated pectin (LMP) on
the volume-surface mean diameter (d32, a) and the electrical charge (-potential, b) of emulsions under
simulated gastrointestinal conditions consisting of initial, oral, gastric, and intestinal phases. Control
corresponds to the emulsions without addition of pectin.
-40
-30
-20
-10
0
Initial Oral Gastric Intestinal
-P
ote
nti
al
(mV
)
Phase
Control
HMP
MMP
LMP0.0
0.3
0.6
0.9
1.2
Initial Oral Gastric Intestinal
d3
2(
m)
Phase
Control
HMP
MMP
LMP
a. b.
Chapter 5
142
The electrical charge became slightly more negative in all of the emulsions after exposure to the
oral conditions, which can be attributed to the presence of anionic mucin molecules in the SSF
(Espinal-Ruiz, Parada-Alfonso, Restrepo-Sanchez, Narvaez-Cuenca, & McClements, 2014;
Vingerhoeds, Blijdenstein, Zoet, & van Aken, 2005).
The magnitude of the negative charge on the particles decreased appreciably when exposed to
simulated stomach phase, which may be due to the relatively low pH and high ionic strength of
the gastric fluids (Singh, Ye, & Horne, 2009). The acidic pH of the gastric fluids reduced the
negative charge on the pectin molecules, as well as on the non-ionic surfactant coated oil
droplets. Finally, the negative charged increased appreciably after exposure to the simulated
small intestine conditions, which can be attributed to the anionic nature of the molecules that
assemble the colloidal particles in this phase, i.e., FFAs, bile salts, and phospholipids (Hur, Lim,
Decker, & McClements, 2011; McClements & Li, 2010). The electrical properties of the
emulsions were affected by pectin samples according to their methoxylation degree (related to
electrical charge). For all gastrointestinal phases, the measured negative charge of the emulsions
increased with decreasing the degree of pectin methoxylation, which can be attributed to the
higher negative charge density of the pectin molecules (Figure 5.8b). In addition, the pectin
molecules are not digested within the upper gastrointestinal tract, and therefore, they should
remain in the gastrointestinal fluids of each phase, thereby contributing to the measured electrical
properties (Ridley, O'Neill, & Mohnen, 2001).
5.3.2.4. Digestion of emulsified lipids
In this section, the influence of the different kinds of pectin on the rate and extent of lipid
digestion was determined. In general, there was a rapid increase in the amount of FFAs released
within the first few minutes, followed by a more gradual increase at later times (Figure 5.9a). For
the control sample, the amount of FFAs formed eventually reached around 100% (w/w)
indicating that all of the TAGs were hydrolyzed by the lipase. For the emulsions containing
pectin samples, the lipid digestion profile depended on the nature of the pectin molecules in the
system. The final extent of lipid digestion decreased in the following order:
Chapter 5
143
Figure 5.9. Influence of high methoxylated pectin (HMP), medium methoxylated pectin (MMP) isolated
from banana passion fruit (Passiflora tripartita var. mollisima), and low methoxylated pectin (LMP) on
free fatty acids (FFA) released after the digestion process. Kinetic profile of intestinal release of FFA (a),
and FFA released after 2 hours of intestinal digestion (b). Control corresponds to the emulsions without
addition of pectin.
100, 92, 70 and 47% (w/w) for the control, LMP, MMP, and HMP samples, respectively (Figure
5.9b). These results suggested that the extent of lipid digestion decreased as the degree of
methoxylation and the molecular weight of the pectin molecules increased. It should be stressed
that the results obtained using simple simulated GIT models should be treated with caution, since
they cannot represent the compositional, structural, and dynamic complexity of the processes
occurring within the human GIT. Nevertheless, they may provide some useful insights into the
potential physicochemical mechanisms occurring within the GIT.
In principle, there are numerous ways that pectin methoxylation can alter the lipid digestion
process. An increase in methoxylation leads to an increase in the number of non-polar
(hydrophobic) groups on the molecules, and a decrease in the number of negative groups
(Dongowski, Lorenz, & Proll, 2002). An increase in the number of non-polar groups may lead to
increased binding of bile salts through hydrophobic attraction (Dongowski, 1995; Espinal-Ruiz,
Parada-Alfonso, Restrepo-Sánchez, Narváez-Cuenca, & McClements, 2014; Wilde & Chu,
0
20
40
60
80
100
0 20 40 60 80 100 120
FF
A R
elea
sed
(%
w/w
)
Digestion Time (min)
Control
LMP
MMP
HMP
0
20
40
60
80
100
Control LMP MMP HMP
Fin
al
Dig
esti
on
(F
FA
% w
/w)
Sample
a. b.
Chapter 5
144
2011). The binding of bile salts upon addition of pectin may inhibit lipid digestion because they
can no longer interact with the fat droplet surfaces (thereby inhibiting lipase adsorption) or
because they can no longer solubilize FFAs generated at the fat droplet surfaces and thereby,
inhibiting lipase activity (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, Narváez-Cuenca, &
McClements, 2014). An increase in methoxylation would therefore be expected to decrease the
extent of lipid digestion through this effect (Reis, Holmberg, Watzke, Leser, & Miller, 2009). A
decrease in the number of anionic carboxyl groups on the pectin chains (e.g., increased
methoxylation) may lead to decreased binding with cations, such as calcium ions (Willats, Knox,
& Mikkelsen, 2006). Calcium ions play an number of important roles in the lipid digestion
process: (i) a minimum level is required for proper lipase functioning; (ii) they precipitate long
chain FFAs, thereby removing them from the fat droplet surfaces and avoiding the adsorption of
lipase; and (iii) they form insoluble soaps with long chain FFAs thereby decreasing their
absorption (Wilde & Chu, 2011). An increase in methoxylation degree (decreased negative
charge) might therefore be expected to alter the extent of lipid digestion. Differences in the
ability of pectin molecules to promote lipid droplet flocculation may have altered the digestion
extent (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sanchez, Narvaez-Cuenca, & McClements,
2014). Flocculated fat droplets may be digested more slowly than non-flocculated ones, because
the surface area of lipids exposed to the lipase in the aqueous phase is reduced (Reis, Holmberg,
Watzke, Leser, & Miller, 2009). In this study, all of the pectins used promoted flocculation in the
mouth, stomach, and small intestine and therefore had potential to inhibit digestion through this
mechanism. Nevertheless, there may have been differences in the nature of the flocs formed, e.g.,
the packing of the fat droplets within the flocs (Figure 5.5). There are a number of
physicochemical phenomena that might account for the observed decrease in lipid digestion with
increasing methoxylation of the pectin molecules, such as binding of bile salts to the non-polar
groups. However, further studies would be required to characterize the importance of this
mechanism.
Besides the contribution of the methoxylation degree, the molecular weight (as stated above in
Section 5.3.2.2) is also an important parameter that contributes to the overall inhibition of lipid
digestion. The viscosity of the gastrointestinal phases increased with increasing molecular weight
Chapter 5
145
of pectin samples (HMP > MMP > LMP, Figure 5.7). It is well known that the higher the
molecular weight of pectin, the greater its capacity to form complex structures (e.g., gels and
hydrated networks) which are able to trap water and other components such as lipids in their
inner structures (Willats, Knox, & Mikkelsen, 2006). An increase in the viscosity of the gel
causes a restriction on the diffusive processes of lipids and lipases, inhibiting their capacity to
interact to each other and consequently, reducing the lipolytic reaction extent. Furthermore, as the
lipids are trapped inside pectin gels, lipases will not be able to access the lipid surfaces and thus,
the lipolytic reaction extent will be also reduced (Espinal-Ruiz, Parada-Alfonso, Restrepo-
Sanchez, Narvaez-Cuenca, & McClements, 2014). Although both the methoxylation degree and
molecular weight are important parameters determining the physicochemical properties of pectin
molecules, other structural parameters such as monosaccharide composition and degree of
branching may also influence their functionality, and therefore their influence on the
gastrointestinal fate of lipids. Further studies are therefore needed to clarify the importance of
specific molecular characteristics on pectin functionality.
5.3.3. Calculation of the depletion attraction between the lipid droplets
In this section, we provide a theoretical rationalization for the influence of pectin type (HMP,
MMP, and LMP) on the depletion flocculation of the emulsions in terms of the characteristics of
the different pectin molecules (Klinkesorn, Sophanodora, Chinachoti, & McClements, 2004;
McClements, 2000). The presence of non-adsorbed pectin molecules in the aqueous phase (bulk
solution) of an emulsion is known to increase the osmotic attraction between the lipid droplets
through a depletion mechanism (McClements, 2000). The magnitude of this attractive interaction
can be calculated using the following equations (Klinkesorn, Sophanodora, Chinachoti, &
McClements, 2004):
[ (
)
(
)
(
)
(
)] (5.2)
(
) (5.3)
Chapter 5
146
Here, wdepletion(h) is the inter-droplet pair potential due to depletion interactions at a surface-to-
surface droplet separation of h, r is the lipid droplet radius, POsm is the osmotic pressure arising
from the exclusion of non-adsorbed pectin molecules from a narrow region ( rg) surrounding the
lipid droplets, and rg is the effective radius of the pectin molecules in solution (gyration radius).
In addition, c, MW, and m are the concentration, molecular weight, and molecular density of the
pectin molecules in the aqueous solution, respectively. NA is the Avogadro´s number, k is the
Boltzmann´s constant, and T is the absolute temperature. The parameter, RV, is referred to as the
volume ratio, which is equal to the effective volume of a pectin molecule in solution divided by
the actual volume of the constituent atoms making up the molecule (McClements, 2000). If a
pectin molecule adopts a compact spherical conformation, like a globular protein, then RV 1.
However, pectin molecules entrain large quantities of water as they rotate in solution, then RV ⪢
1. The effective volume of pectin in solution should be considerably greater than the volume
occupied by the atoms that make up the pectin chain because it sweeps out a large volume of
solvent as it rapidly rotates due to Brownian motion (Jenkins & Snowden, 1996; McClements,
2000). In this model, we have assumed that pectin molecules behave like random coil in solution,
so that:
⁄
(5.4)
Here, n is the number of monomer units per molecule (n = MW/MW0), MW is the molecular
weight of the whole pectin molecules, MW0 is the molecular weight of a GalA monomer unit (
180 g mol-1
), l is the length of the monomer unit ( 0.47 nm), and m is the density of the pectin
chain ( 2000 kg m-3
). The molecular characteristics of the pectin molecules used in our
calculations are shown in Table 5.3. It should be noted that w(h)/kT = 0 for h 2rg and that the
strongest interaction between the fat droplets occurs when they come into contact (h = 0). So that,
Equation 5.2 is applicable for the boundary condition h < 2rg and when the separation between
the lipid droplets is small compared to their size (h ⪡ r).
Chapter 5
147
Equations 5.2, 5.3, and 5.4 were used to calculate the influence of pectin type (HMP, MMP, and
LMP) on the depletion attraction between lipid droplets (r = 100 nm), assuming that pectin
molecules behave as random coil in aqueous solution. The variation of the droplet attraction
potential (w(h)/kT) with the droplet separation (h) for different pectin types, but the same overall
aqueous concentrations (1.8% (w/w) equivalent to the initial phase) is shown in Figure 5.10a.
Both the magnitude (depletion attraction w(h)/kT) and the range (lipid droplet separation h) of the
attractive depletion attraction between lipid droplets increased with increasing molecular weight
and methoxylation degree: HMP > MMP > LMP > Control. An estimate of the overall strength of
the depletion attraction in a particular system can be obtained by calculating the magnitude of
wdepletion (h = 0) when the droplets are in contact:
*
+ (5.5)
The dependence of wdepletion(h = 0)/kT on pectin type was calculated (Figure 5.10b). The strength
of the depletion attraction increases progressively with the simultaneous increase of the
molecular weight and methoxylation degree (HMP > MMP > LMP > Control). In the absence of
pectin (control), the lipid droplets are prevented from flocculating because the repulsive droplet-
droplet interactions (e.g., steric, electrostatic, and hydration repulsion) dominate the attractive
interactions (e.g., van der Waals and electrostatic) (McClements, 2000). Addition of the pectin
molecules to the emulsion increases the depletion attraction between the lipid droplets, until
eventually the overall attractive interactions overcome the repulsive interactions and the droplets
flocculate. As the molecular weight and the methoxylation degree of the pectin molecules
increased simultaneously, a smaller amount of pectin needs to be added to the emulsion in order
to generate the additional attraction (depletion force) required to promote droplet flocculation
(Jenkins & Snowden, 1996). These calculations suggest that each of the pectins tested can
promote depletion flocculation in the emulsions used in this work. In particular, Equation 5.2
suggests that the strength of the depletion interaction is highly dependent on the molecular weight
of the pectin molecules as well as the surface electrical charge (methoxylation degree), which can
be represented by the gyration radius (rg).
Chapter 5
148
Figure 5.10. Inter-droplet pair potential attraction due to depletion interactions of lipid droplets containing
high methoxylated pectin (HMP), medium methoxylated pectin (MMP) isolated from banana passion fruit
(Passiflora tripartita var. mollisima), and low methoxylated pectin (LMP), related to the thermal energy
(kT) of the system. Inter-droplet pair potential (w(h)/kT) due to depletion interactions at a surface-to-
surface droplet separation of h (a), and inter-droplet pair potential (w(h=0)/kT) when the droplets are in
contact (b). The model corresponds to the depletion interactions of lipid droplets in the initial phase (1.8%
w/w pectins) at 37 °C, prior to subjection to the static in vitro digestion model. Control corresponds to the
emulsions without addition of pectin.
In addition, it should be stressed that the electrical properties of pectin molecules may also
indirectly influence the depletion interaction by altering the effective size (gyration radius) of the
colloidal particles and the depletion zone. For example, increasing the number of negative
charges on a pectin molecule by either decreasing the methoxylation degree or increasing the pH,
can increase its effective size. In the one hand, increasing the number of negative groups usually
causes the pectin molecules to become more extended because of electrostatic repulsion between
negatively charged groups (–COO⊝). On the other hand, decreasing the number of negative
charges on a pectin molecule by either increasing the methoxylation degree or reducing the pH,
can decrease its effective size. Thus, the strength of the depletion interaction may depend on the
electrical properties of the pectin molecules as well as the environmental conditions such as pH,
temperature, and ionic strength (Furusawa, Ueda, & Nashima, 1999). Additional calculations
were conducted to evaluate the relative contribution of the molecular weight and the
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.01 0.1 1 10
w(h
)/k
T
h (nm)
Control
LMP
MMP
HMP-2.5
-2.0
-1.5
-1.0
-0.5
0.0
Control LMP MMP HMP
w(h
=0
)/k
TSample
a. b.h
Chapter 5
149
Figure 5.11. Schematic representation of the inhibition of lipid digestion by pectin. High methoxylated
pectin (HMP) and medium methoxylated pectin (MMP) isolated from banana passion fruit (Passiflora
tripartita var. mollisima) form a closed structure around the lipid droplets (depletion flocculation) which
restricts the access of lipase to their surfaces and therefore, preventing the lipid digestion (a), whereas low
methoxylated pectin (LMP) forms an open structure due to its electrostatic repulsion with negatively
charged lipid droplets, allowing the access of lipase to their surfaces and promoting lipid digestion (b).
methoxylation degree to the overall magnitude of the depletion attraction. These calculations
were performed by fixing the rg value (rg = 4.7 nm) and changing the molecular weight
(according to Table 5.3), and by fixing the molecular weight (MW = 153 kDa) and changing the
rg value (according to Table 5.3 as well). These calculations allowed us to establish that the
molecular weight of pectin molecules had a significant impact on the magnitude of the depletion
attraction (w(h=0)/kT of -0.3, -1.1, and -1.7 for LMP, MMP, and HMP, respectively) while the
degree of methoxylation (represented by rg) had a smaller contribution to the overall magnitude
of the depletion attraction (w(h=0)/kT of -0.04, -0.07, and -0.09 for LMP, MMP, and HMP,
respectively). Finally, we can suggest that both HMP and MMP form a closed structure around
the lipid droplets which restricts the access of lipase to their surfaces and therefore, preventing
Lipase Lipid dropletHMP
MMPLMP
a. b.
Chapter 5
150
lipid digestion (Figure 5.11a), whereas LMP forms an open structure due to its repulsion with
negatively charged lipid droplets, allowing the access of lipase to their surfaces and promoting
lipid digestion (Figure 5.11b).
5.4. Conclusions
The objective of this work was to study the impact of different types of pectin on the
physicochemical characteristics and microstructure of emulsified lipids during passage through a
simulated gastrointestinal model. Three pectins with different molecular characteristics were
studied: LMP and HMP from citrus fruit and MMP from banana passion fruit. These pectins
differ in their molecular weights and degrees of methoxylation, which led to differences in their
molecular dimensions (radius of gyration) and electrical characteristics (-potential). All three
pectins promoted flocculation of the fat droplets in the emulsions, which was attributed to a
depletion flocculation mechanism, associated with exclusion of the biopolymers from the fat
droplet surfaces. The pectin molecules decreased the extent of lipid digestion with increasing
degree of methoxylation and molecular weight: HMP > MMP > LMP. These effects may have
been due to the impact of the pectin molecules on the rheological properties of the
gastrointestinal fluids, binding of key digestive components (such as calcium, free fatty acids,
and bile), alteration in the droplet aggregation state, or entrapment of the lipid droplets by pectin
microgels. Further studies are clearly required to establish the relative contribution of the
methoxylation degree and the molecular weight to the overall inhibitory effect, as well as to
identify the precise molecular origin of this inhibition. This information may be useful for the
design of emulsion-based functional foods that give healthier lipid profiles and thereby promote
health and wellness.
Acknowledgments
We are grateful to Departamento Administrativo de Ciencias, Tecnología e Innovación
(COLCIENCIAS) and Vicerrectoría Académica of Universidad Nacional de Colombia for
Chapter 5
151
providing a fellowship to Mauricio Espinal-Ruiz supporting this work. We also thank the United
States Department of Agriculture (USDA), NRI Grants (2011-03539, 2013-03795, 2011-67021,
and 2014-67021); and Red Nacional para la Bioprospección de Frutas Tropicales
COLCIENCIAS-RITFRUBIO (Contrato 0459-2013) for supporting this research. We are grateful
to student Mayra Alejandra Quintero from Departamento de Química, Universidad Nacional de
Colombia, for supporting both the extraction and characterization of Passiflora tripartita var.
mollissima pectin (MMP) sample.
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Chapter 6
Interaction of a dietary fiber (pectin) with gastrointestinal components (bile
salts, calcium, and lipase): A calorimetry, electrophoresis, and turbidity study
Published as:
Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E.,
& McClements, D. J. Journal of Agricultural and Food Chemistry. 62 (2014): 12620 – 12630.
Chapter 6
155
Abstract
An in vitro gastrointestinal model consisting of oral, gastric, and intestinal phases was used to
elucidate the impact of pectin on the digestion of emulsified lipids. The interaction of pectin with
gastrointestinal components may be related to the formation of microgel structures which are able
to promote the flocculation of lipid droplets by depletion interactions and thereby, reducing their
digestibility. It was found that pectin reduced the extent of lipid digestion, which was attributed
to its interactions with gastrointestinal components. The interaction of pectin with bile salts,
lipase, CaCl2, and NaCl was therefore investigated by turbidity, microstructure, electrophoresis,
and isothermal titration calorimetry (ITC) at pH 7.0 and 37 °C. ITC showed that the interaction of
pectin was endothermic with bile salts, but exothermic with CaCl2, NaCl, and lipase.
Electrophoresis, microstructure, and turbidity measurements showed that anionic pectin formed
electrostatic complexes with calcium ions, which may have decreased lipid digestion due to
increased lipid flocculation or microgel formation. This research provides valuable insights into
the physicochemical and molecular mechanisms of the interaction of pectin with gastrointestinal
components that may impact the rate and extent of lipid digestion.
Keywords: Pectin, gastrointestinal tract, lipid digestion, isothermal titration calorimetry,
turbidity, flocculation.
Chapter 6
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6.1. Introduction
There has been an appreciable increase in the total amount of calories consumed by humans
during the past few decades, which is believed to be an important contributing factor to increases
in obesity, diabetes, and cardiovascular diseases (Bray & Popkin, 1998). Fat has the highest
calorie density of the major food components (fat, protein, and carbohydrates) and so there has
been a major focus on the identification of effective strategies to reduce the fat content of foods,
while maintaining their desirable quality attributes. Several studies have suggested that certain
types of dietary fiber can inhibit lipid digestion and absorption in the small intestine (Beysseriat,
Decker, & McClements, 2006; Gunness & Gidley, 2010; Howarth, Saltzman, & Roberts, 2001;
Hur, Lim, Decker, & McClements, 2011; Li & McClements, 2014; McClements & Li, 2010a,
2010b; Mun, Decker, Park, Weiss, & McClements, 2006). Increased consumption of dietary fiber
may therefore be one approach for reducing some of the adverse affects associated with eating
high-fat food products.
Dietary fibers can be classified as either water-soluble or water-insoluble (Anderson, Baird,
Davis Jr, Ferreri, Knudtson, Koraym, et al., 2009). Water-insoluble fibers, such as lignins,
celluloses, and some hemicelluloses, play an important role in regulating intestinal peristalsis
(Kritchevsky, 1988). Water-soluble fibers, such as pectin, carageenan, xanthan gum, and alginate,
influence the gastrointestinal fate of ingested foods due to their ability to bind water, thicken or
gel intestinal fluids, and interact with specific food and gastrointestinal components (Theuwissen
& Mensink, 2008). Pectin is a water-soluble dietary fiber that is used widely in the
pharmaceutical, biotechnology, and food industries as a functional ingredient (Thakur, Singh,
Handa, & Rao, 1997). Pectin is a polysaccharide that has linear anionic regions formed by D-
galacturonic acid (GalA) monomers linked by -(1,4) glycosidic bonds, and branched regions
primarily formed by various types of neutral monosaccharides (mainly rhamnose, xylose,
mannose, and arabinose) linked together. The GalA units have carboxyl groups, which may be
present as free carboxyl groups or methyl esterified groups depending on the orign, isolation, and
processing of pectin (Caffall & Mohnen, 2009; Mohnen, 2008; Ridley, O'Neill, & Mohnen, 2001;
Chapter 6
157
Thakur, Singh, Handa, & Rao, 1997; Yapo, 2011). Pectin has been successfully used for many
years in the food and beverage industries as a thickening and gelling agent, as well as a colloidal
stabilizer (Maxwell, Belshaw, Waldron, & Morris, 2012; Thakur, Singh, Handa, & Rao, 1997).
The gelling characteristics of pectin have been utilized to form hydrogel delivery systems for a
range of pharmaceutical and food bioactive compounds (Maxwell, Belshaw, Waldron, & Morris,
2012; Munarin, Tanzi, & Petrini, 2012).
Previous studies have shown that pectin reduces the rate and extent of lipid digestion by
presumably interacting with various food and intestinal components (Beysseriat, Decker, &
McClements, 2006; Gunness & Gidley, 2010; Li & McClements, 2014). A number of different
physicochemical and physiological mechanisms have been proposed to account for this effect.
Pectin can interact with bile salts and phospholipids in the small intestine, which may alter lipid
digestion by reducing the amount of surface-active components available to stabilize the lipid
droplets (triacylglycerols), or to solubilize and transport lipid digestion products (free fatty acids
and monoacylglycerols) from the droplet surfaces to the epithelium cells (Hur, Lim, Decker, &
McClements, 2011; McClements & Li, 2010a). The binding of bile salts to pectin in the small
intestine has also been proposed as one of the major mechanisms responsible for the ability of
pectin to reduce cholesterol levels (Pfeffer, Doner, Hoagland, & McDonald, 1981). Pectin
molecules may interfere with the re-absorption of bile salts in the small intestine, thereby
reducing the amount of cholesterol absorbed and transported to the blood (Pfeffer, Doner,
Hoagland, & McDonald, 1981). Furthermore, the conformation and aggregation state of pectin in
aqueous solutions may be altered due to its interactions with certain gastrointestinal components,
which leads to changes in solution rheology that might impact lipid digestion, e.g., by altering
gastric emptying times or the mass transport of digestive enzymes (Beysseriat, Decker, &
McClements, 2006).
Pectin may also directly interact with other components in the gastrointestinal tract such as
CaCl2, NaCl, and digestive enzymes (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, &
Narváez-Cuenca, 2014; McClements, 2000b; McClements & Li, 2010a; Simo, Mao, Tokle,
Decker, & McClements, 2012). Finally, pectin is known to promote depletion flocculation of
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lipid droplets, which may reduce lipid digestion by protecting them from attack by lipolytic
enzymes (Jenkins & Snowden, 1996b; McClements, 2000a).
Pectin ingredients obtained from different natural sources using different isolation and processing
methods may vary widely in their molecular and physicochemical characteristics, such as
backbone length, electrical charge, hydrophobicity, surface-activity, conformation, self-
association, viscosity, and binding capacity (Yapo, 2011). In addition, the molecular and
functional characteristics of pectins may be altered appreciably in response to changes in solution
and environmental conditions such as pH, composition, ionic strength, and temperature
(Maxwell, Belshaw, Waldron, & Morris, 2012; Mohnen, 2008; Munarin, Tanzi, & Petrini, 2012;
Ridley, O'Neill, & Mohnen, 2001). For this reason, the study of the nature of the interactions that
pectin may have with the components of the gastrointestinal tract is important in understanding
the effect of pectin on digestive processes. An improved understanding of the origin and nature of
these interactions would lead to the design of functional foods with improved nutritional,
physicochemical, and sensory properties.
In this study, isothermal titration calorimetry (ITC) was used to study the interactions between
pectin and gastrointestinal components (bile salts, pancreatic lipase, CaCl2, and NaCl). ITC
measures the heat absorbed or evolved when one solution is titrated into another solution (Doyle,
1997; Freire, Mayorga, & Straume, 1990; Leavitt & Freire, 2001). Previous studies have shown
that ITC is an extremely valuable tool for studying chemical interactions of polysaccharides with
other molecules because it provides valuable data concerning binding enthalpies, critical
aggregation concentrations, and binding stoichiometries (Chang, McLandsborough, &
McClements, 2011; Wangsakan, Chinachoti, & McClements, 2004). In addition, electrophoresis,
turbidity, and microstructural observations were used to provide additional information about the
nature of the interactions between pectin and gastrointestinal components. The results of this
study may aid the rational design of functional foods designed to improve human health and
wellness by controlling lipid digestion within the gastrointestinal tract.
Chapter 6
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6.2. Materials and methods
6.2.1. Chemicals
Corn oil was purchased from a commercial food supplier (Mazola, ACH Food Companies Inc.,
Memphis, TN) and stored at 4 °C until use. The manufacturer reported that this oil contained
approximately 14, 29, and 57% (w/w) of saturated, monounsaturated, and polyunsaturated fatty
acids, respectively. Commercial powdered high methoxylated pectin (Genu Pectin (Citrus),
USP/100) was kindly donated by CP Kelco (Lille Skensved, Denmark) and was used without
further purification. The manufacturer reported that the powdered pectin ingredient contained
6.9% moisture, 89.0% galacturonic acid, and 8.6% methoxyl groups, with a degree of
methoxylation of approximately 62%. The average molecular weight was reported to be 200 kDa.
Lipase from porcine pancreas (Type II, L3126, triacylglycerol hydrolase E.C. 3.1.1.3), bile
extract (porcine, B8631), mucin from porcine stomach (Type II, M2378, bound sialic acid ≤
1.2%), and pepsin A from porcine gastric mucose (P7000, endopeptidase E.C. 3.4.23.1, activity ≥
250 units/mg solid) were purchased from Sigma-Aldrich Chemical Company (St Louis, MO,
USA). The supplier reported that the lipase activity was 100-400 units mg-1
protein (using olive
oil) and 30-90 units mg-1
protein (using triacetin) for 30 min incubation (one unit of lipase
activity was defined as the amount of enzyme required for the release of 1 eq of fatty acid from
either triacetin (pH 7.4) or olive oil (pH 7.7) in 1 h at 37 °C). The composition of the bile extract
was reported as 49% (w/w) total bile salt (BS), containing 10-15% glycodeoxycholic acid, 3-9%
taurodeoxycholic acid, 0.5-7% deoxycholic acid, 1-5% hydrodeoxycholic acid, and 0.5-2% cholic
acid; 5% (w/w) phosphatidyl choline (PC); Ca2+ ≤ 0.06% (w/w); critical micelle concentration of
bile extract 0.07 0.04 mM; and mole ratio of BS to PC being around 15:1. All other chemicals
were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). Double distilled
water was used to prepare all solutions.
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6.2.2. Simulated gastrointestinal studies
6.2.2.1. Solutions and emulsions preparation
Pectin stock solution (4.0% w/w) was prepared by dispersing 4 g of powdered pectin into 96 g of
5 mM phosphate buffer solution (pH 7.0). The resulting solution was stirred at 800 rpm for 12 h
(overnight) at room temperature to ensure complete dispersion and dissolution. Pectin stock
solution was then adjusted to pH 7.0 and equilibrated for 10 min before analysis.
A stock emulsion was prepared by mixing together 20% (w/w) corn oil and 80% (w/w) buffered
emulsifier solution (5 mM phosphate buffer, pH 7.0; containing 2.5% (w/w) Tween 80) for 5 min
using a bio-homogenizer (Speed 2, Model MW140/2009-5, Biospec Products Inc., ESGC,
Switzerland). The resulting coarse emulsion was then passed 5 times through a high-pressure
homogenizer (Microfluidizer M-110L processor, Microfluidics Inc., Newton, MA, USA)
operating at 11,000 psi (75.8 MPa) to reduce the particle size further.
Pectin-emulsion mixtures were then prepared by mixing stock emulsion (containing 20% (w/w)
corn oil) with pectin stock solution (containing 4.0% (w/w) pectin), to obtain systems of varying
composition: 2.0% (w/w) corn oil and 0.2-3.6% (w/w) pectin. The pectin-emulsion mixtures were
then stirred with a high-shear mixer (Fisher Steadfast Stirrer, Model SL-1200, Fisher Scientific,
Pittsburgh, PA) at 1000 rpm and stored overnight at room temperature. Pectin-emulsion mixtures
were then characterized to obtain the initial phase, prior to subjection to the in vitro
gastrointestinal model.
6.2.2.2. Simulated gastrointestinal tract model
Each emulsion sample (initial phase) was passed through a simulated in vitro gastrointestinal
tract that consisted of oral, gastric, and intestinal phases. This method is based on a static model
utilized in previous studies (Hur, Decker, & McClements, 2009; Salvia-Trujillo, Qian, Martin-
Belloso, & McClements, 2013).
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6.2.2.2.1. Oral phase
Simulated saliva fluid (SSF, pH 6.8) containing 3% (w/w) mucin was prepared according to the
composition shown in Table 6.1. Each emulsion (initial phase) was mixed with SSF (ratio 1:1
w/w) to obtain a mixture containing 1% (w/w) corn oil and 0.1-1.8% (w/w) pectin. The simulated
oral phase consisted of a conical flask containing emulsion-SSF mixture incubated at 37 °C with
continuous shaking at 100 rpm for 10 min in a temperature controlled air incubator (Excella E24
Incubator Shaker, New Brunswick Scientific, NJ, USA). The mixture resulting from processing
of the initial emulsions in the oral phase (bolus) was used in the gastric phase.
6.2.2.2.2. Gastric phase
Simulated gastric fluid (SGF) was prepared by adding 2.0 g NaCl, 7.0 mL concentrated HCl
(37% w/w), and 3.2 g pepsin A (from porcine gastric mucose, 250 units mg-1
) to a flask, and then
diluting with double distilled water to a volume of 1.0 L, and finally adjusting to pH 1.2 using 1.0
M HCl. Samples from the oral phase (bolus) were mixed with SGF (ratio 1:1 w/w) so that the
final mixture contained 0.5% (w/w) corn oil, and 0.05-0.90% (w/w) pectin.
Table 6.1. Chemical composition of simulated saliva fluid (SSF) used to simulate oral conditions.
Compound Chemical formula Concentration (g L-1
)1
Sodium chloride NaCl 1.594
Ammonium nitrate NH4NO3 0.328
Potassium dihydrogen phosphate KH2PO4 0.636
Potassium chloride KCl 0.202
Potassium citrate K3C6H5O7•H2O 0.308
Uric acid sodium salt C5H3N4O3Na 0.021
Urea H2NCONH2 0.198
Lactic acid sodium salt C3H5O3Na 0.146
Porcine gastric mucin (Type II) ---- 30
1The SSF was prepared in double distilled water and then pH 6.8 was adjusted using 0.1 M NaOH.
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162
This mixture was then adjusted to pH 2.5 using 1.0 M NaOH and incubated at 37 °C with
continuous shaking at 100 rpm for 2 h. The mixture resulting from processing of the emulsions in
the gastric phase (chyme) was used in the intestinal phase.
6.2.2.2.3. Intestinal phase
Samples obtained from the gastric phase (20.0 mL) were incubated for 2 h at 37 °C in a simulated
small intestine fluid (SIF) consisting of 2.5 mL pancreatic lipase (24 mg mL-1
), 3.5 mL bile
extract solution (54 mg mL-1
), and 1.5 mL salt solution containing 0.25 M CaCl2 and 3.0 M
NaCl, to obtain a final composition in the reaction vessel of 0.36% (w/w) corn oil, and 0.05-
0.65% (w/w) pectin. The free fatty acids (FFAs) released were monitored by determining the
amount of 0.10 M NaOH needed to maintain a constant pH 7.0 within the reaction vessel using
an automatic titration unit (pH stat titrator, 835 Titrando, Metrohm USA, Inc., Riverview, FL,
USA). All components were dissolved in 5 mM phosphate buffer solution (pH 7.0) before use.
Lipase addition and initialization of the titration program were carried out only after the addition
of all pre-dissolved ingredients and balancing the pH to 7.0. The volume of 0.10 M NaOH added
to the emulsion for balancing the pH to 7.0 was recorded over time and used to calculate the
FFAs generated by lipolysis. The amount of FFAs released over time were calculated using the
following equation:
(
) (6.1)
Where, CNaOH is the concentration of the sodium hydroxide (0.10 M), MWLipid is the average
molecular weight of corn oil (872 g mol-1
), WLipid is the initial weight of corn oil in the intestinal
phase (0.10 g), and VNaOH is the volume of NaOH (L) titrated into the reaction vessel to
neutralize the FFA released, assuming that all triacylglycerols are hydrolyzed in two molecules of
FFA and one molecule of monoacylglycerol. Titration blanks were performed by inactivating
lipase in boiling water for 15 min prior to initialization of the titration program.
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6.2.2.3. Creaming stability measurements of initial emulsions
Ten milliliters of emulsions (initial phase) were transferred into a test tube (internal diameter 15
mm, height 125 mm), tightly sealed with a plastic cap, and then stored at room temperature for 24
h, after which appreciable phase separation into an opaque layer at the top, a turbid layer in the
middle, and a transparent layer at the bottom was observed in some of the systems. We defined
the serum layer to be the sum of the turbid and transparent layers. The total height of the
emulsion (HE) and the height of the serum layer (HS) were measured using a laser vertical
profiling system (Turbiscan Classic MA2000, Formulaction, Wynnewood, PA). The extent of
creaming was then characterized by the creaming index (CI), defined as CI = 100 × (HS / HE).
The creaming index provided indirect information about droplet aggregation, since an increase in
particle size (e.g., due to flocculation) leads to faster creaming (provided the droplet
concentration is not too high).
6.2.3. Interaction of pectin with gastrointestinal components
6.2.3.1. Solutions preparation
Stock solutions of 0.9% (w/w) pectin, 2.7% (w/w) bile salts, and 1.1% (w/w) lipase were
individually prepared by dispersing them in 5 mM phosphate buffer solution (pH 7.0) at room
temperature for 12 h (overnight), to ensure their complete dispersion and dissolution. Salt
solutions consisting of 55 mM CaCl2 or 545 mM NaCl were prepared freshly before each
experiment. A mixture containing the above components (at the same initial concentrations of the
individual solutions), was also prepared. The initial concentration of this mixture was referred as
100% relative concentration of each component.
To establish the interaction of pectin with specific gastrointestinal components, individual
solutions of CaCl2, NaCl, lipase, bile salts, and the mixture of all components, were titrated into
either buffer solution (blank) or pectin at 37 °C. Twenty-nine 100 L aliquots of individual
solutions containing 2.7% (w/w) bile salts, 1.1% (w/w) lipase, 55 mM CaCl2, 545 mM NaCl, or
Chapter 6
164
the mixture of all components (100% relative concentration), were added sequentially into a test
tube containing 14.5 mL of either buffer (5 mM phosphate buffer, pH 7.0) or 0.9% (w/w) pectin
solutions. Turbidity, -potential, and microstructure (observed by optical microscopy) of these
solutions were then characterized throughout the titration process.
6.2.3.2. Solutions characterization
6.2.3.2.1. Optical turbidity
Optical turbidity of the solutions was determined by measuring their absorbance at 600 nm
(=A600 nm) using a UV-Visible spectrophotometer (Ultrospec 3000 pro Pharmacia Biotech,
Biochrom Ltd., Cambridge, UK) at 37 °C. The samples were contained within 1 cm path length
optical cells, and buffer solution was used as a control. The change in turbidity was defined as
= Pectin Buffer. Triplicate measurements of turbidity were carried out on each sample.
6.2.3.2.2. Electrical surface charge
Electrical surface charge (-potential) of the solutions was determined using a particle
microelectrophoresis instrument (Zetasizer NanoSeries, Malvern Instruments Ltd.,
Worcestershire, UK). Solutions were injected into the measurement chamber, equilibrated for
120 s and then the -potential was determined by measuring the direction and velocity that the
particles moved in the applied electric field. Each individual -potential measurement was
calculated from the average of 20 continuous readings made per sample. The -potential was
recorded at each pH after 60 s equilibrium.
6.2.3.2.3. Microstructure
Microstructure of the solutions was characterized by optical microscopy. An optical microscope
(C1 Digital Eclipse, Niko, Tokyo, Japan) with a 60× objective lens was used to capture images of
Chapter 6
165
the solutions. Solutions were gently stirred to form a homogeneous mixture without introducing
any air bubbles. A small aliquot of the solutions (5 L) was then transferred to a glass
microscope slide and covered with a glass cover slip. The cover slip was fixed to the slide using
nail polish to avoid evaporation. A small amount of immersion oil (Type A, Nikon, Melville, NY,
USA) was placed on the top of the cover slip. All optical images were taken using a digital
camera, and then characterized using the instrument software (EZ CS1 version 3.8, Niko,
Melville, NY, USA).
6.2.3.3. Isothermal titration calorimetry measurements
An isothermal titration calorimeter (ITC) instrument (Microcalorimeter VP-ITC, MicroCal Inc.,
Northampton, MA, USA) was used to measure the enthalpy (H) resulting from titration of
individual solutions of CaCl2, NaCl, lipase, bile salts, and a mixture of all components into either
buffer solution (blank) or pectin. Twenty-nine 10 L aliquots of individual solutions of 2.7%
(w/w) bile salts, 1.1% (w/w) lipase, 55 mM calcium chloride, 545 mM sodium chloride, or a
mixture of all components (100% relative concentration) were injected sequentially into a 1450
L titration cell initially containing either buffer (5 mM phosphate buffer, pH 7.0) or 0.9% (w/w)
pectin solutions (pectin and gastrointestinal component concentrations mimic approximately the
concentrations in the intestinal phase of the simulated gastrointestinal model). Each injection
lasted 24 s and there was an interval of 240 s between successive injections. The temperature of
the solution in the titration cell was 37 °C and the solution was stirred at a speed of 315 rpm
throughout the experiments. Triplicate measurements of enthalpy were carried out on each
sample and they were reproducible to better than 5%.
6.2.4. Data analysis
All measurements were performed at least three times using freshly prepared samples. Averages
and standard deviations were calculated from these triplet measurements.
Chapter 6
166
6.3. Results and discussion
6.3.1. Properties of initial emulsion – pectin mixtures
Initially, the properties of the initial emulsions used to prepare the emulsion-pectin mixtures were
characterized (5 mM phosphate buffer, pH 7.0). The initial emulsions had a relatively small mean
particle diameter immediately after preparation (d32 = 200 10 nm), which did not change during
the course of the experiments (Espinal-Ruiz, Parada, Restrepo-Sanchez, Narvaez-Cuenca, &
McClements, 2014). This can be attributed to the relatively strong steric repulsion between the
non-ionic surfactant-coated lipid droplets resulting from the polyoxyethylene headgroups on the
Tween 80 molecules (Almgren, 2000). The lipid droplets initially had a small negative charge (
= -5.8 0.2 mV), even though they were stabilized by a non-ionic surfactant, which can be
attributed to anionic impurities in the oil or surfactant ingredients (such as free fatty acids and
phospholipids) or to preferential adsorption of hydroxyl ions from water (OH⊝) (Singh, Ye, &
Horne, 2009; Thiam, Farese Jr, & Walther, 2013). The droplets in the initial emulsions
containing no pectin were stable to creaming throughout the experimental time frame (Figure
6.1), which is due to the fact that the droplets were small and did not associate with each other
(Weiss, Takhistov, & McClements, 2006).
Emulsions containing relatively low levels of added pectin (≤ 0.4% w/w) were still observed to be
stable to gravitational separation, i.e., they maintained a uniform white appearance (Figure 6.1).
The stability of the emulsions in presence of low levels of pectin can be evidenced according to
the slight increase of the particle size (d32 = 210 nm) of the lipid droplets in the initial phase
(containing 0.2% (w/w) pectin), compared to emulsions without pectin (d32 = 200 nm). However,
emulsions containing higher levels of pectin clearly separated into a creamed layer and a serum
layer, indicating that the droplets had moved upwards due to gravity. The origin of this effect is
depletion flocculation induced by the presence of non-adsorbed pectin molecules within the
aqueous phase (Dickinson, Semenova, Antipova, & Pelan, 1998; Jenkins & Snowden, 1996b).
Chapter 6
167
Figure 6.1. Influence of the concentration of pectin on creaming stability of 2% (w/w) corn oil-in-water
emulsions stabilized with 0.2% (w/w) Tween 80 before the digestion process (initial phase). Pectin
concentrations are referred to the initial phase.
The depletion flocculation of the lipid droplets in presence of high levels of pectin can be
evidenced according to the increase of the particle size (d32 = 350 nm) in the initial phase
(containing 2.4% (w/w) pectin), as compared to emulsions without pectin (d32 = 200 nm). Pectin
molecules should not be attracted to the surfaces of the non-ionic surfactant-coated lipid droplets
because of electrostatic and steric repulsion effects (the carboxyl groups of pectin are completely
dissociated at pH 7.0 since its pKa is 3.5). There is therefore a narrow region around each lipid
droplet (approximately equal to the radius of hydration of the pectin molecules) from which the
pectin molecules are excluded (Jenkins & Snowden, 1996a). Consequently, there is a pectin and
water concentration gradient between this exclusion zone and the surrounding bulk aqueous
phase, which leads to an osmotic pressure. This osmotic pressure generates an attraction between
the lipid droplets (depletion force) since when they come into close contact, the volume of the
thermodynamically unfavorable exclusion zone is reduced (McClements, 2000a). At low pectin
concentrations, the osmotic attraction is not large enough to overcome the various repulsive
forces in the system (e.g., steric and electrostatic repulsion), and the emulsion remains stable to
droplet aggregation. However, once a critical pectin concentration is exceeded, the attractive
0
20
40
60
80
100
0 1 2 3 4
Cre
am
ing
In
dex
(%
)
Pectin (% w/w)
Pectin Concentration (% w/w)
0.0 0.4 1.2 2.4 3.6
Chapter 6
168
forces exceed the repulsive forces and droplet flocculation occurs (Jenkins & Snowden, 1996b).
It should be noted that the main thermodynamic driving force for depletion flocculation is
entropy (Méndez-Alcaraz & Klein, 2000; Reiffers-Magnani, Cuq, & Watzke, 2000). The reason
that the thickness of the creamed layer increases (CI decreases) as the pectin concentration
increases above the critical flocculation concentration (Figure 6.1) is that the depletion attraction
is higher and so the droplets are held more strongly into a particle gel network (Dickinson, 1995).
In summary, these measurements showed that the degree of droplet flocculation in the initial
emulsion-pectin mixtures depended on the amount of pectin present in the system.
6.3.2. Influence of pectin on lipid digestion
In a recent study, we examined the influence of pectin addition on the gastrointestinal fate of
emulisified lipids (Espinal-Ruiz, Parada, Restrepo-Sanchez, Narvaez-Cuenca, & McClements,
2014). This study showed that pectin influenced the aggregation state of the lipid droplets in
different regions of the simulated gastrointestinal tract (mouth, stomach, and small intestine). It
also showed that pectin addition reduced the rate and extent of lipase-catalyzed lipid digestion in
the small intestine phase. In the current study, the same pH-stat method was used to measure the
influence of pectin on lipid digestion. We could directly compare these results with the studies of
pectin interactions with various gastrointestinal components using the same constituents.
In general, the amount of FFAs produced increased rapidly during the first few minutes of
digestion, and then increased more slowly at longer times, until a relatively constant value was
reached after 25 min of digestion (Figure 6.2a). The final amount of FFAs produced at the end of
the two hour digestion period decreased with increasing pectin concentration (Figure 6.2b),
which is in good agreement with our earlier study (Espinal-Ruiz, Parada, Restrepo-Sanchez,
Narvaez-Cuenca, & McClements, 2014). The ability of pectin to decrease the rate and extent of
lipid digestion (principally the extent rather than rate) in corn oil-in-water emulsions could be due
to several physicochemical and physiological mechanisms.
Chapter 6
169
Figure 6.2. Influence of the concentration of pectin on free fatty acids (FFA) released after the digestion
process. Kinetic profile of intestinal release of FFA (a), and FFA released after 2 hours of intestinal
digestion (b). Pectin concentrations are referred to the intestinal phase.
Pectin may have interacted with calcium ions and formed a highly viscous solution or gel
network that impeded the diffusion of GIT components (such as lipase or bile salts), thereby
retarding the lipid digestion process (Braccini & Pérez, 2001; Leroux, Langendorff, Schick,
Vaishnav, & Mazoyer, 2003; Willats, Knox, & Mikkelsen, 2006). Pectin-calcium interactions
may also have inhibited lipid digestion due to the important role that calcium ions play in
removing FFAs from lipid droplet surfaces (Devraj, Williams, Warren, Mullertz, Porter, &
Pouton, 2013). Calcium ions normally form insoluble soaps with long chain FFAs that help to
remove them from the lipid droplet surfaces and thereby allow the lipase to keep working (Ye,
Cui, Zhu, & Singh, 2013). If the calcium ions are tightly bound to pectin molecules, then the
FFAs may accumulate at the lipid droplet surfaces, thereby inhibiting further digestion. Pectin
may also have bound to bile salts or phospholipids, and so prevented them from from adsorbing
to lipid droplet surfaces or from solubilizing lipid digestion products (Anderson, et al., 2009;
Leroux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003; Maxwell, Belshaw, Waldron, &
Morris, 2012). The solubilization of long chain FFAs in mixed micelles is another important
means of removing them from the lipid droplet surfaces and thereby allowing lipase to keep
60
70
80
90
100
0.00 0.07 0.15 0.55 0.65
Fin
al
FF
A R
elea
sed
(%
w/w
)Pectin (% w/w)
0
20
40
60
80
100
0 50 100 150
FF
A R
elea
sed
(%
w/w
)
Time (min)
0.00%
0.07%
0.15%
0.55%
0.65%
a. b.
Chapter 6
170
functioning (Almgren, 2000). The adsorption of bile salts and phospholipids onto lipid droplet
surfaces often facilitates the subsequent adsorption of lipase molecules. Pectin may also be able
to alter lipid digestion by interacting directly with lipase molecules (de Roos & Walstra, 1996;
Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, & Narváez-Cuenca, 2014). Finally, the
presence of pectin in the system may alter the aggregation state of the lipid droplets through
either depletion or bridging flocculation (Biggs, Habgood, Jameson, & Yan, 2000).
The digestion of highly flocculated lipid droplets is often less than that of non-flocculated ones
because the lipase molecules have to diffuse through the flocs before they can reach the lipid
droplets in their interiors (Li, Hu, & McClements, 2011; Simo, Mao, Tokle, Decker, &
McClements, 2012). Clearly, the potential influence of pectin on the lipid digestion process
within the GIT is complex since pectin is able to interact with many constitiuents within the GIT
fluids. The purpose of this study was therefore to provide further insights into the potential origin
of these effects by studying the interactions between pectin and gastrointestinal components.
6.3.3. Interactions of pectin with mixed gastrointestinal components
In this series of experiments, we used ITC, electrophoresis, turbidity, and microstructure
measurements to examine the interactions of pectin with a solution containing a mixture of all the
major GIT components: sodium, calcium, bile salts, and lipase. The microstructure of the
emulsion-pectin mixtures changed appreciably after exposure to the mixed gastrointestinal
components (Figure 6.3). In the absence of pectin (control), the individual lipid droplets were too
small to be observed by optical microscopy, but confocal microscopy images have shown that
they are evenly distributed throughout the system (Espinal-Ruiz, Parada, Restrepo-Sanchez,
Narvaez-Cuenca, & McClements, 2014). After digestion, there were clearly some large
aggregates in the control samples, which may have been lipid digestion products or remnants
from the various GIT components (such as calcium, sodium, bile salts, and lipase). In the
presence of pectin, the lipid droplets were trapped within large clusters prior to digestion (lipid
droplets size of d32 = 350 nm for an emulsion containing 2.4% (w/w) pectin, compared to lipid
droplets size of d32 = 200 nm for an emulsion without pectin), which can be attributed to
Chapter 6
171
Figure 6.3. Influence of 2.4% (w/w) pectin on the microstructure (observed by optical microscopy) of 2%
(w/w) corn oil-in-water emulsions stabilized with 0.2% (w/w) Tween 80 before (initial phase) and after
(intestinal phase) of the digestion process. The scale bar corresponds to 20 m.
depletion flocculation induced by the non-adsorbed pectin molecules (Figure 6.3) (McClements,
2000a). After digestion, there were a relatively high number of large aggregates (undigested lipid
droplets) remaining in the samples containing pectin, which could have been due to interactions
of the pectin molecules with various components in the simulated GIT fluids. Turbidity (), -
potential, and enthalpy change (H) were measured when increasing amounts of mixed GIT
fluids (0 to 100% of mixture) were titrated into a pectin (0.9% w/w) solution at pH 7.0 (Figure
6.4). The 100% GIT fluids contained the levels of the NaCl, CaCl2, bile salts, and lipase in the
final simulated small intestinal fluids. There was a progressive increase in the turbidity when the
amount of mixed GIT fluids titrated into the pectin solution increased (Figure 6.4a). The -
potential of the mixed system moved from highly negative (-19.0 mV) to less negative (-2.0 mV)
as increasing amounts of GIT fluids were added (Figure 6.4a), which may have occurred due to
electrostatic screening effects induced by the presence of salts or due to binding of cations in the
GIT fluids to anionic pectin molecules (Israelachvili, 2011).
Pectin
Before
Digestion
After
Digestion
Control
Chapter 6
172
Figure 6.4. Influence of the relative concentration (0 to 100%) of simulated gastrointestinal fluids on
optical turbidity (=A600 nm) and -potential (a), and interaction enthalpy (H) (b) of solutions containing
either buffer solution or an initial concentration of 0.9% (w/w) pectin. The initial simulated
gastrointestinal fluids (100%) contained 0.45 % (w/w) bile salts, 0.18 % (w/w) lipase, 9 mM CaCl2, and 91
mM NaCl. Optical turbidity () was defined as = Pectin - Buffer.
There was also an appreciable difference in the enthalpy profiles of samples with either buffer or
pectin solutions (Figure 6.4b). In the absence of pectin, the enthalpy change was relatively low,
and may be attributed to heat of dilution effects associated with the various molecules and/or
particles moving further apart when the GIT fluids were injected into the reaction cell (Chang,
McLandsborough, & McClements, 2011; Doyle, 1997; McClements, 2000b).
In the presence of pectin, there was a large exothermic enthalpy change when the first few
aliquots of the mixed GIT fluids were injected into the reaction cell containing the pectin
solution. The enthalpy change became progressively less exothermic when 0 to 20% of the GIT
fluids were added, until it eventually reached a relatively constant endothermic value at higher
GIT levels. These results suggest that the pectin molecules interacted with some of the
components in the GIT fluids, possibly through electrostatic interactions, and formed aggregates
that were large enough to scatter light strongly.
-20
-15
-10
-5
0
0.0
0.5
1.0
1.5
2.0
0 20 40 60 80 100
-P
oten
tial (m
V)
(A6
00
nm
)
Relative concentration (%)
-200
-100
0
100
200
0 20 40 60 80 100
H
(k
cal/
inje
ctio
n)
Relative concentration (%)
Pectin
Buffer
a. b.
Chapter 6
173
6.3.4. Interactions of pectin with specific gastrointestinal components
In this series of experiments, ITC, electrophoresis, turbidity, and microstructure measurements
were used to characterize the interactions of pectin with specific GIT components (NaCl, CaCl2,
bile salts, or lipase). Measurements were made as increasing amounts of GIT components were
individually added to a pectin solution (0.9% w/w).
Figure 6.5. Influence of the concentration of NaCl (a), CaCl2 (b), bile salts (c), and lipase (d) on optical
turbidity (=A600 nm) and -potential of solutions containing an initial concentration of 0.9% (w/w) pectin.
Optical turbidity () was defined as = Pectin - Buffer.
-20
-15
-10
-5
0
0.0
0.5
1.0
1.5
2.0
0 2 4 6 8 10
-P
oten
tial (m
V)
(A6
00
nm
)
CaCl2 (mM)
-20
-15
-10
-5
0
0.0
0.5
1.0
1.5
2.0
0 20 40 60 80 100
-P
oten
tial (m
V)
(A6
00
nm
)
NaCl (mM)
-20
-15
-10
-5
0
0.0
0.5
1.0
1.5
2.0
0.0 0.1 0.2 0.3 0.4 0.5
-P
oten
tial (m
V)
(A6
00
nm
)
Bile salts (% w/w)
a.
-20
-15
-10
-5
0
0.0
0.5
1.0
1.5
2.0
0.00 0.05 0.10 0.15 0.20
-P
oten
tial (m
V)
(A6
00
nm
)
Lipase (% w/w)
b.
c. d.
Chapter 6
174
In addition, the turbidities and -potentials (Figure 6.5) reached at the end of these titrations
(after the injection of twenty-nine aliquouts) are reported, so that the different samples can easily
be compared (Figure 6.6).
6.3.4.1. NaCl
Addition of increasing amounts of NaCl to the pectin solutions caused an appreciable decrease in
the magnitude of the -potential of the pectin solutions (Figure 6.5a), which can be attributed to
electrostatic screening effects, i.e., accumulation of sodium ions around the negative groups on
the pectin molecules (Israelachvili, 2011). There was no evidence of an increase in aggregate
formation within the microstructure images (Figure 6.8) and there was little change in the
general appearance (Figure 6.7) and optical turbidity (Figure 6.5a) of these samples upon NaCl
addition. We did observe a slight increase in the turbidity at the highest NaCl levels used, which
suggested that the NaCl may have caused a slight amount of pectin aggregation, presumably
through screening of the electrostatic interactions (Furusawa, Ueda, & Nashima, 1999). The ITC
measurements indicated that there was not a strong interaction (H = -102 kcal/injection)
between the pectin and the NaCl (Figure 6.9a), since the enthalpy versus salt concentration
profiles were fairly similar. Overall, these results suggest that pectin did not have a major
interaction with the NaCl in the GIT fluids.
6.3.4.2. CaCl2
Addition of CaCl2 to the pectin solutions had a more pronounced influence on their
physicochemical properties than NaCl addition. Addition of increasing amounts of CaCl2 caused
the -potential of the pectin solutions to go from around -19.0 mV to -6.0 mV (Figure 6.5b),
which can be attributed to electrostatic screening and ion binding effects (Israelachvili, 2011).
Multivalent counter-ions are more effective at screening electrostatic interactions than
monovalent ones, as they have a greater tendency to bind strongly to oppositely charged groups
(Winzor, Carrington, Deszczynski, & Harding, 2004).
Chapter 6
175
Figure 6.6. Influence of 91 mM NaCl, 9 mM CaCl2, 0.45% (w/w) bile salts (BS), 0.18% (w/w) lipase (L),
and a mixture of all components (M, 100% relative concentrations) on optical turbidity (=A600 nm) (a)
and -potential (b) of solutions containing an initial concentration of 0.9% (w/w) pectin (P).
The electrical change in the -potential with calcium addition was fairly similar to that observed
with addition of the mixed GIT solution (Figure 6.6b), which suggests that calcium ions play an
important role in determing the overall electrical interactions. The ITC measurements also
indicated that there was a strong interaction (H = -241 kcal/injection) between the calcium ions
and pectin (Figure 6.9b). When calcium ions were injected into buffer solution there was an
exothermic enthalpy change that decreased in magnitude with increasing CaCl2 concentration,
which can be attributed to heat of dilution effects (McClements, 2000b). However, when calcium
ions were injected into pectin solution there was initially a strong exothermic reaction (from 0 to
4 mM), then a relatively strong endothermic reaction (from 4 to 6 mM), followed by an enthalpy
change close to zero after 6 mM CaCl2 (Figure 6.9b). The shape of the ITC curve suggests that
there may have been several events occurring sequentially, such as calcium binding, pectin
conformational changes, and/or pectin aggregation. Nevertheless, it is not possible to determine
the molecular origin of these events from ITC measurements alone, since they just provide the
overall enthalpy change.
0.0
0.5
1.0
1.5
2.0
P P+NaCl P+L P+BS P+CaCl2 P+M
(A6
00
nm
)
-20
-15
-10
-5
0P P+BS P+L P+NaCl P+CaCl2 P+M
-P
ote
nti
al
(mV
)
a. b.
Chapter 6
176
Figure 6.7. Influence of the concentration of NaCl, CaCl2, bile salts, lipase, and a mixture of all
components (% relative concentrations) on turbidity of solutions containing an initial concentration of
0.9% (w/w) pectin.
The addition of calcium ions to the pectin solutions caused a large increase in their optical
turbidity (Figures 6.5b and 6.6a), which can be attributed to the appearance of large aggregates,
as observed within the optical microscopy images (Figure 6.8). We postulate that these
aggregates were microgels formed by pectin in the presence of calcium (Braccini & Pérez, 2001;
Willats, Knox, & Mikkelsen, 2006). The calcium ions may have cross-linked the anionic pectin
molecules through electrostatic bridging (Thakur, Singh, Handa, & Rao, 1997). As mentioned
earlier, the binding of calcium ions to pectin may be important for a number of reasons.
NaCl (mM)
Lipase (% w/w)
Mixture (%)
Bile salts (% w/w)
CaCl2 (mM)
0 27 49 70 91 0 2.7 4.9 7.0 9.0
0 0.15 0.25 0.35 0.45
0 23 45 65 100
0 0.04 0.09 0.14 0.18
Chapter 6
177
Figure 6.8. Influence of 91 mM NaCl, 9 mM CaCl2, 0.45% (w/w) bile salts, 0.18% (w/w) lipase, and a
mixture of the previous components (100% relative concentrations) on the microstructure (observed by
optical microscopy) of solutions prepared on either buffer solution or 0.9% (w/w) pectin. The scale bar
corresponds to 20 m.
Buffer
Bile salts
NaCl
Lipase
Mixture
CaCl2
Pectin
Chapter 6
178
Figure 6.9. Influence of the concentration of NaCl (a), CaCl2 (b), bile salts (c), and lipase (d) on
interaction enthalpy (H) of solutions containing either buffer solution or an initial concentration of 0.9%
(w/w) pectin.
Firstly, pectin-calcium microgels may trap lipid droplets inside, thereby restricting the access of
lipase to the lipid substrates (Simo, Mao, Tokle, Decker, & McClements, 2012). Second, the
formation of these electrostatic complexes may prevent the calcium ions from forming insoluble
soaps with long-chain FFAs at the lipid droplet surfaces, thereby preventing the lipase from
functioning properly (Ye, Cui, Zhu, & Singh, 2013). Although the interaction of pectin with
NaCl had not a significant impact, the presence of NaCl may influence the interaction of pectin
-200
-100
0
100
200
0.0 0.1 0.2 0.3 0.4 0.5
Hea
t (k
cal/
inje
ctio
n)
Bile salts (% w/w)
Pectin
Buffer
-200
-100
0
100
200
0.00 0.05 0.10 0.15 0.20
Hea
t (k
cal/
inje
ctio
n)
Lipase (% w/w)
Pectin
Buffer
-200
-100
0
100
200
0 2 4 6 8 10
Hea
t (k
cal/
inje
ctio
n)
CaCl2 (mM)
Pectin
Buffer
-200
-100
0
100
200
0 20 40 60 80 100
Hea
t (k
cal/
inje
ctio
n)
NaCl (mM)
Pectin
Buffer
a. b.
c. d.
Chapter 6
179
with other gastrointestinal components such as CaCl2. In general, salts are known to screen
electrostatic interactions in aqueous solutions and would therefore be expected to influence the
formation and properties of electrostatic complexes. For instance, it can be postulated that
electrostatic interaction between pectin and CaCl2 can be weakened in the presence of NaCl,
which means that more CaCl2 had to be added to reach charge neutralization. The addition of
NaCl also would reduce the high turbidity reached with CaCl2, since the formation of calcium-
pectin complexes would be partially suppressed by electrostatic screening interactions with NaCl.
There may be considerable variations in the type and concentration of ions surrounding the lipids
droplets, which may impact the electrostatic interactions in the system through electrostatic
screening or binding effects. For example, long chain FFAs and bile salts may precipitate in the
presence of calcium ions, thereby removing them from the lipid droplet surface facilitating their
further digestion, but which may also reduce their subsequent absorption due to calcium soap
formation. Sufficiently high concentrations of monovalent (NaCl) and multivalent ions (CaCl2)
can promote extensive flocculation of emulsions containing electrically charged droplets, which
may restrict the access of lipase to the oil-water interface and slow down digestion. Certain types
of ions are capable of promoting the gelation of biopolymers, which would affect the ability of
digestive enzymes to reach any entrapped lipid droplets. For example, pectin form strong gels if
there are sufficiently high levels of calcium ions present in solution (McClements & Li, 2010a).
6.3.4.3. Bile salts
The addition of bile salts to the pectin solutions had a limited influence on their physicochemical
properties. Addition of increasing amounts of bile salts only caused a relatively small decrease in
the negative charge on the pectin molecules (Figure 6.5c), which can probably be attributed to
electrostatic screening effects (Hur, Lim, Decker, & McClements, 2011; McClements & Li,
2010a). There was an appreciable increase in the optical turbidity of the pectin solutions when
bile salts were added (Figures 6.5c and 6.6a) and the solutions appeared visibly cloudier (Figure
6.7). These effects can be attributed to the formation of large aggregates within the bile salts-
pectin solutions as observed by optical microscopy (Figure 6.8). The ITC measurements also
Chapter 6
180
indicated that the bile salts interacted with the pectin molecules. In the absence of pectin, there
was a relatively large endothermic enthalpy change (H = +204 kcal/injection), which
progressively decreased with increasing bile salt concentration (Figure 6.9c). This effect may
have been due to the breakdown of bile salt micelles within the reaction chamber, resulting in the
exposure of a greater number of non-polar groups to water (Thongngam & McClements, 2005).
In the presence of pectin, there was a large endothermic peak (from 0.05 to 0.15 % w/w) followed
by a value that was close to zero at higher bile salt concentrations (after 0.2% (w/w), Figure
6.9c). The large difference between the two curves suggests that there was an interaction between
pectin and bile salts, e.g., bile salt micelles may have bound to pectin molecules and/or promoted
their aggregation (Wangsakan, Chinachoti, & McClements, 2006).
One might not expect a strong interaction between bile salts and pectin molecules because they
are both usually negatively charged at neutral pH. However, bile salts may be able to interact
with pectin molecules through hydrophobic interactions (Dongowski, 1997). In general, the
hydrophobic interactions of pectin with bile salts are known to increase with the degree of
methoxylation of pectin molecules (by increasing its hydrophobicity) and the molecular weight
(related to viscosity) of the pectin molecules (Pfeffer, Doner, Hoagland, & McDonald, 1981).
These experiments clearly show that bile salts can interact with pectin molecules, which may
have important implications for the effects of pectin on lipid digestion observed in Figure 6.2. If
the bile salts form microgel particles with pectin, then they may not be available to absorb onto
the surfaces of the lipid droplets or to solubilize lipid digestion products, as discussed earlier. In
addition, if any lipid droplets are trapped inside the microgels then the rate of digestion may be
decreased because the lipase molecules cannot easily reach the lipid droplet surfaces
(McClements & Li, 2010a).
6.3.4.4. Lipase
Finally, we examined the influence of lipase addition to the pectin solutions on their overall
physicochemical properties. The addition of increasing amounts of lipase caused a slight change
in the -potential of the pectin (Figure 6.5d), which may have been due to some binding of lipase
Chapter 6
181
molecules to the pectin molecules. In addition, there was an appreciable difference between the
ITC curves of the control and system containing pectin (Figure 6.9d), which also suggests that
some form of interaction occurred (H = -129 kcal/injection) (Espinal-Ruiz, Parada-Alfonso,
Restrepo-Sánchez, & Narváez-Cuenca, 2014). In the absence of pectin, there was a relatively
large exothermic change observed when the lipase solution was titrated into the buffer solution.
This effect can probably be attributed to the heat of dilution associated with salts in the lipase
solution. In the presence of pectin, there was a large exothermic change observed at low lipase
levels (0.0 to 0.1% w/w), followed by a much smaller exothermic change at higher lipase levels.
The large difference between the curves in the presence and absence of pectin suggests that there
was a strong interaction between the lipase and the pectin, although again the molecular origin of
this effect cannot be established from the ITC measurements. There was a slight increase in the
optical turbidity of the lipase-pectin solutions when high levels of lipase were added (Figure
6.5d), but there was little change in their overall visual appearance (Figure 6.7) or their
microstructure (Figure 6.8). These results suggest that either any interactions between the lipase
and pectin did not lead to extensive aggregation or that the lipase-pectin complexes formed were
soluble in buffer solution.
Finally, the molecular mechanisms governing the interaction between pectin and the
gastrointestinal compounds evaluated in this study (NaCl, CaCl2, bile salts, and pancreatic lipase)
are represented schematically in the Figure 6.10. The interaction between pectin and NaCl is
mainly due to electrostatic screening effects, whereas the interaction between pectin and CaCl2 is
mainly due to electrostatic crosslinking and the further formation of egg-box structures. The
interaction between pectin and bile salts can be attributed to hydrophobic interactions. In
addition, we suggested that the interaction between pectin and pancreatic lipase is mainly due to
both electrostatic and hydrophobic interactions. Furthermore, the molecular complexes formed
after the molecular interaction between pectin and the gastrointestinal compounds was observed
to be soluble for NaCl and pancreatic lipase, and insoluble for CaCl2 and bile salts, leading to the
formation of the microgel-like structures observed in the Figure 6.8.
Chapter 6
182
Figure 6.10. Molecular mechanisms governing the interaction between pectin and gastrointestinal tract
(GIT) compounds (NaCl, CaCl2, bile salts, and pancreatic lipase). The solubility of the complexes formed
after the interaction of GIT compounds with pectin is also showed (highly schematic).
ITC is an advantageous technique in terms of thermodynamic studies of biomolecular systems.
The magnitude (H) and the sign (endothermic or exothermic) of the interaction allows to
improve the understanding of the molecular effects exerted by pectin on the digestion of lipids.
The determination of the relative contribution of the individual components to the overall
interaction may lead to the design of highly structured pectin-based food systems that selectively
contribute to the control of the lipid digestion. In addition, ITC is a convenient technique for
these studies since it allows to obtain reliable information from non-covalent interactions such as
hydrophobic, hydrogen bonding, electrostatic, and van der Waals interactions, which are
responsible of the interactions between pectin and gastrointestinal components. Other analytical
techniques to evaluate interactions are currently available.
Bile salts
NaCl
Lipase
CaCl2
⊕
⊝
⊝⊝
⊕
⊕
⊝
⊝
⊝
2⊕
⊝
⊝
⊝
2⊕
2⊕
Electrostatic screeningSoluble
Electrostatic crosslinking
―Egg box structures‖Insoluble
Hydrophobic interactionsInsoluble
⊝
⊝
⊝
⊝
⊝
⊝
Both electrostatic and
hydrophobic interactionsSoluble
Chapter 6
183
High Performance Size Exclusion Chromatography (HPSEC) allows to evaluate the formation of
molecular species arising from the interaction between pectin and the components of the
gastrointestinal system. However, this technique only allows to obtain qualitative information of
the interactions, disregarding the affinities which leads to stablish the relative contribution of
each component to the overall interaction.
6.4. Conclusions
The purpose of this study was to investigate the interaction of pectin molecules with some of the
major constituents within gastrointestinal fluids, i.e., NaCl, CaCl2, bile salts, and lipase. Our
measurements have shown that a number of these components interact strongly with pectin,
which may have important implications for understanding the influence of pectin on lipid
digestion. In particular, calcium ions and bile salts appear to promote the formation of pectin
microgels that can lead to flocculation of lipid droplets in the gastrointestinal tract, decreasing the
ability of lipids to be reached by gastrointestinal lipases and thereby, inhibiting their digestibility.
If appreciable amounts of calcium and bile salts are trapped within these microgels, then they will
not be able to remove long chain FFAs from lipid droplet surfaces, which would inhibit lipid
digestion. In addition, if lipid droplets are trapped within pectin microgels, then it may be more
difficult for lipase molecules to access the surfaces of the lipid substrate, again inhibiting lipid
digestion. These results have important implications for the rational design of dietary fiber-based
functional foods that may modulate lipid digestion within the gastrointestinal tract.
Acknowledgments
The authors are grateful to COLCIENCIAS and Universidad Nacional de Colombia for providing
a fellowship to Mauricio Espinal-Ruiz supporting this work. This material is partly based upon
work supported by United States Department of Agriculture, NRI Grants (2011-03539, 2013-
03795, 2011-67021, and 2014-67021).
Chapter 6
184
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Chapter 7
Effect of pectins on the mass transfer kinetics of monosaccharides,
amino acids, and a corn oil-in-water emulsion in a Franz diffusion cell
Published as:
Espinal-Ruiz, M., Restrepo-Sánchez, L. P., & Narváez-Cuenca, C. E.
Food Chemistry. 209 (2016): 144 – 153.
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188
Abstract
The effect of high (HMP) and low (LMP) methoxylated pectins (2% w/w) on the rate and extent
of the mass transfer of monosaccharides, amino acids, and a corn oil-in-water emulsion across a
cellulose membrane was evaluated. A sigmoidal response kinetic analysis was used to calculate
both the diffusion coefficients (rate) and the amount of nutrients transferred through the
membrane (extent). In all cases, except for lysine, HMP was more effective than LMP in
inhibiting both the rate and extent of the mass transfer of nutrients through the membrane. LMP
and HMP, e.g., reduced 1.3 and 3.0 times, respectively, the mass transfer rate of glucose, as
compared to control (containing no pectin), and 1.3 and 1.5 times, respectively, the amount of
glucose transferred through the membrane. Viscosity, molecular interactions, and flocculation
were the most important parameters controlling the mass transfer of electrically neutral nutrients,
electrically charged nutrients, and emulsified lipids, respectively.
Keywords: Pectin, methoxylation degree, diffusion, viscosity, Franz diffusion cell.
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7.1. Introduction
There is a growing interest among consumers about the nutritional, therapeutic, and functional
properties of foods consumed in the diet (Mohamed, 2014). The current trend of consumers is
based on the consumption of foods with functional components beneficial for human health such
as phytosterols, polyphenolics, anthocyanins, carotenoids, and dietary fibers (Bigliardi & Galati,
2013). In recent years, dietary fiber has received an important attention from the food industry
and consumers due to the health benefits associated with the consumption of dietary fiber-rich
foods (Brownlee, 2014). Dietary fiber components possess distinctive physicochemical
characteristics determining their functionality, and they can be categorized as either insoluble or
soluble dietary fibers (Phillips, 2013). The consumption of insoluble dietary fiber has been
associated with increasing the bulkiness of the digesta content and improving the gastrointestinal
tract (GIT) transit (Edwards, Johnson, & Read, 1988), whereas the consumption of soluble
dietary fiber has been associated with a wider variety of physiological functions such as
controlling postprandial glycemic and lipid response (Pasquier, Armand, Guillon, Castelain,
Borel, Barry, et al., 1996), decreasing blood lipid and glucose levels (Ye, Arumugam,
Haugabrooks, Williamson, & Hendrich, 2015), inhibiting the GIT enzyme activities (Espinal-
Ruiz, Parada-Alfonso, Restrepo-Sánchez, & Narváez-Cuenca, 2014), controlling the rate and
extent of lipid digestion (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sanchez, Narvaez-Cuenca, &
McClements, 2014; Espinal-Ruiz, Restrepo-Sánchez, Narváez-Cuenca, & McClements, 2016),
increasing the viscosity of the GIT content (Edwards, Johnson, & Read, 1988; Elleuch, Bedigian,
Roiseux, Besbes, Blecker, & Attia, 2011), and retarding the mass transfer process of nutritional
compounds to be absorbed by enterocytes (Fabek, Messerschmidt, Brulport, & Goff, 2014;
Srichamroen & Chavasit, 2011). Among the aforementioned properties of soluble dietary fiber,
the ability to modulate the viscosity of the GIT content stands out because this feature is related
to the control of the mobility of nutrients and their further digestion and absorption (Fabek,
Messerschmidt, Brulport, & Goff, 2014).
The functional properties of soluble dietary fibers (e.g., gums, mucilages, and pectins) rely on
their ability to thicken into swollen hydrated networks and their subsequent increasing of
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190
viscosity, determining their potential to exert physiological effects along the GIT, specifically
through the stomach and small intestine (Brownlee, 2014). The proposed mechanism by which
viscosity may induce physiological responses includes increasing of luminal bulk (Ye,
Arumugam, Haugabrooks, Williamson, & Hendrich, 2015) and inhibiting nutrient diffusion
across the unstirred water layer of the mucosal membrane (Fabek, Messerschmidt, Brulport, &
Goff, 2014; Gunness, Flanagan, Shelat, Gilbert, & Gidley, 2012). Viscous soluble fibers may
hinder the mass transfer process of nutritional compounds from the lumen to the unstirred water
layer coating the small intestine by reducing the mixing among them and reducing the time
available for intestinal absorption by enterocytes (Fabek, Messerschmidt, Brulport, & Goff, 2014;
Gunness, Flanagan, Shelat, Gilbert, & Gidley, 2012). Therefore, an increase in digesta viscosity
arising from the soluble dietary fiber consumption might influence the processes occurring during
the nutrient digestion and adsorption (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sanchez, Narvaez-
Cuenca, & McClements, 2014).
The effect of different sources of dietary fiber [e.g. barley (Hordeum vulgare) -glucans (Gs)
and wheat (Triticum aestivum) arabinoxylans (AXs)] was evaluated in in vitro models (Fabek,
Messerschmidt, Brulport, & Goff, 2014; Gunness, Flanagan, Shelat, Gilbert, & Gidley, 2012;
Srichamroen & Chavasit, 2011). Both Gs and AXs proved to be effective in retarding both the
rate and extent of the mass transfer process of bile salts through a dialysis membrane. The
inhibition of the mass transfer process of bile salts through a dialysis membrane by increasing the
viscosity of the solution upon addition of Gs and AXs was suggested to be a possible
mechanism controlling the mass transfer process (Gunness, Flanagan, Shelat, Gilbert, & Gidley,
2012). In other study it was shown that the alkaline-extracted malva nut (Sterculia lychnophora)
gum reduced significantly the amount of glucose transferred through a dialysis membrane in an
in vitro experiment, as compared to control (containing no dietary fiber) (Srichamroen &
Chavasit, 2011).
Among the different sources of soluble dietary fiber available, pectin is a polysaccharide obtained
from fruits and vegetables and it is widely used in the pharmaceutical and food industries for its
thickening, gelling, and texturing properties (Maxwell, Belshaw, Waldron, & Morris, 2012).
Chapter 7
191
Pectin is a biopolymer mainly formed by galacturonic acid (GalA) units joined in chains by -D-
(1,4) glycosidic linkages. Three pectic structures [homogalacturan (HG), rhamnogalacturonan-I
(RG-I), and rhamnogalacturonan-II (RG-II)] have been isolated and structurally characterized
(Mohnen, 2008). HG corresponds to the linear chain of -(1,4) GalA units in which the carboxyl
group (–COO⊝) of GalA moieties can be partially esterified with methanol (methoxylated),
forming the carbomethoxyl group (–COOCH3). An important characteristic of HG, present in
pectins, is the methoxylation degree, defined as the percentage of carboxyl groups which have
been methoxylated. If more than 50% of the carboxyl groups are methoxylated, the pectin is
called high methoxylated pectin (HMP), and less than that methoxylation degree are called low
methoxylated pectin (LMP). RG-I is a pectic structure containing a linear backbone of the
disaccharide [4)--D-GalA-(12)--L-Rha-(1], where Rha corresponds to rhamnose, and
RG-II consists of a HG backbone substituted with side branches consisting of twelve different
types of monosaccharides in up to twenty different linkages (Maxwell, Belshaw, Waldron, &
Morris, 2012). The most abundant pectic structure is HG that comprises 65% (mol/mol) of
pectin, whereas RG-I and RG-II comprise 25 and 10% (mol/mol), respectively (Yapo, 2011).
Many functional properties of pectin (e.g., viscosity, solubility, and gelation capacity) are
dependent on its structural parameters such as molecular weight, methoxylation degree and the
distribution pattern of methoxylation within the GalA chains (Mohnen, 2008; Ryden,
MacDougall, Tibbits, & Ring, 2000; Yapo, 2011). In previous studies, we have demonstrated that
pectin inhibits the activity of some digestive enzymes (Espinal-Ruiz, Parada-Alfonso, Restrepo-
Sánchez, & Narváez-Cuenca, 2014) and interferes with the digestion of emulsified lipids
(Espinal-Ruiz, Parada-Alfonso, Restrepo-Sanchez, Narvaez-Cuenca, & McClements, 2014;
Espinal-Ruiz, Restrepo-Sánchez, Narváez-Cuenca, & McClements, 2016) by interacting with the
GIT components (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, Narváez-Cuenca, &
McClements, 2014).
Although studies regarding the mass transfer control of some nutritional compounds have been
carried out, no information is currently available concerning the influence of pectins on the mass
transfer process of the primary nutritional compounds consumed in the diet or produced after
digestion such as monosaccharides, amino acids, and lipids. The objective of this study was,
Chapter 7
192
therefore, to determine the effect of HMP and LMP on the rate and extent of the mass transfer
process of the main nutritional compounds consumed in the diet or obtained after digestion, such
as monosaccharides, amino acids, and lipids (represented by a corn oil-in-water emulsion). The
mass transfer profiles of monosaccharides, amino acids, and a corn oil-in-water emulsion in the
absence (control) or presence of pectins (LMP or HMP) was reported and analyzed to suggest a
possible interaction mechanism between pectin and the evaluated nutritional compounds. These
results might lead to the design, formulation, and fabrication of functional foods developed to
control the postprandial blood concentration of the monosaccharides, amino acids, and lipids
consumed in the diet.
7.2. Materials and methods
7.2.1. Chemicals
D-(+)-glucose, D-(+)-galactose, D-(–)-fructose, and D-(–)-ribose were purchased from Panreac
Química SLU (Barcelona, Spain). L-lysine, L-glycine, L-aspartic acid, L-tyrosine, ninhydrin
monohydrate, Tween 80, and a dialysis tubing cellulose membrane (cut-off 14 kDa) were
purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). Corn oil was
purchased from a commercial food supplier (Grasco LTDA, Bogotá DC, Colombia) and stored in
darkness at room temperature until use. The manufacturer reported that the corn oil contained
approximately 14, 35, and 51% (w/w) of saturated, monounsaturated, and polyunsaturated fatty
acids, respectively. Commercial powdered LMP was donated by TIC Gums Inc. (Belcamp, MA,
USA) and was used without further purification, with methoxylation degree and average
molecular weight previously reported as 30% (mol/mol) and 130 kDa, respectively (Espinal-Ruiz,
Restrepo-Sánchez, Narváez-Cuenca, & McClements, 2016). Commercial powdered HMP (Genu
Citrus Pectin USP/100) was donated by CP Kelco Co. (Lille Skensved, Denmark) and was also
used without further purification, with methoxylation degree and average molecular weight
previously reported as 71% (mol/mol) and 181 kDa, respectively (Espinal-Ruiz, Restrepo-
Sánchez, Narváez-Cuenca, & McClements, 2016). All other chemicals were purchased from
Merck KGaS (Darmstadt, Germany). Deionized water was used to prepare all solutions. Franz
Chapter 7
193
diffusion cells (Figure 7.1) were fabricated by Siliser LTDA (Bogotá DC, Colombia). The
volumes of the donor and receptor compartments were 3 and 17 mL, respectively. The area of
mass transfer (A) was 1.33 cm2.
7.2.2. Sample preparation
LMP and HMP stock solutions (4% w/w) were prepared separately by dispersing 2 g of powdered
pectins into 48 g of deionized water. These solutions were stirred at 1,000 rpm overnight at room
temperature to ensure complete dispersion and dissolution. LMP and HMP solutions were then
adjusted to pH 7.0 by using 0.1 M NaOH.
A monosaccharide stock solution (280 mM of each compound) was prepared by dissolving
together glucose, galactose, fructose, and ribose in deionized water. The monosaccharide stock
solution (2.5 mL) was then mixed with either 2.5 mL deionized water (control)
Figure 7.1. Structural design of a Franz diffusion cell. Donor compartment with a nominal volume of 3
mL (a), receptor compartment with a nominal volume of 17 mL (b), position for a semipermeable
cellulose membrane (c) with an effective area of mass transfer (A) of 1.33 cm2 (d = 1.30 cm), sampling
port (d), magnetic stir bar (e), thermostated chamber at 37 °C (f), water inlet (g), and water outlet (h).
a
b
e
f
g
h
c d
Chapter 7
194
or 2.5 mL of 4% (w/w) pectin stock solutions (LMP or HMP), to obtain monosaccharide working
solutions containing 140 mM of each monosaccharide and 2% (w/w) pectin. Monosaccharide
working solutions were then adjusted to pH 7.0 by using 0.1 M NaOH.
Amino acid stock solutions (4 mM of each compound) were prepared by dissolving separately
lysine, glycine, aspartic acid, or tyrosine in deionized water. Each amino acid stock solution (2.5
mL) was then mixed separately with either 2.5 mL deionized water (control) or 2.5 mL of 4%
(w/w) pectin stock solutions (LMP or HMP), to obtain amino acid working solutions containing 2
mM of each amino acid and 2% (w/w) pectin. Each amino acid working solution was then
adjusted to pH 7.0 by using 0.1 M NaOH.
A stock emulsion (20% w/w) was prepared by mixing together 20 g corn oil and 80 g buffered
emulsifier aqueous solution (5 mM phosphate buffer pH 7.0, containing 2.5% (w/w) Tween 80)
for 5 min using an Ultra-Turrax homogenizer (Speed 35,000 min-1
; Model Miccra D-9; ART
Prozess & Labortechnik GmbH & Co. KG; Müllheim; Germany). A dilution was prepared by
mixing 10 g stock emulsion with 90 g buffer solution pH 7.0, to obtain a diluted emulsion
containing 2% (w/w) corn oil. Finally, the diluted emulsion (2.5 mL) was mixed with either 2.5
mL deionized water (control) or 2.5 mL of 4% (w/w) pectin stock solutions (LMP or HMP), to
obtain working emulsions containing 1% (w/w) corn oil and 2% (w/w) pectin samples. Each
working emulsion was then adjusted to pH 7.0 by using 0.1 M NaOH.
7.2.3. Effect of pectins on the mass transfer kinetics of monosaccharides, amino acids and a
corn oil-in-water emulsion in a Franz diffusion cell
Seventeen milliliters of deionized water (adjusted to pH 7.0) were transferred to the receptor
compartment of the Franz diffusion cell (Figure 7.1), and incubated at 37 °C for 15 min. Then, 3
mL of the thermostated working solutions (37 °C) containing monosaccharides, amino acids, or
the corn oil-in-water emulsion, with the absence (control) or presence of pectin samples (LMP or
HMP), were transferred separately to the donor compartment. The Franz diffusion cell was
thermostated at 37 ° C throughout the experiment. At regular times intervals (0, 2, 4, 6, 8, 10, 24,
Chapter 7
195
26, 28, 30, 32, 34, and 48 h) an aliquot of 500 μL was collected from the receptor compartment
and then, 500 μL of deionized water (adjusted to pH 7.0 and thermostated to 37 °C) were added
immediately to reconstitute the total volume of the cell. Samples collected from the Franz
diffusion cell over time were analyzed for their monosaccharide, amino acid or emulsion
contents, as described below. Contents of monosaccharides, amino acids, and the corn oil-in-
water emulsion were corrected by the dilution factor introduced at each sampling time.
7.2.4. Analysis of monosaccharides, amino acids, and the corn oil-in-water emulsion
7.2.4.1. Analysis of monosaccharides
Monosaccharide samples collected from the receptor compartment were analyzed by high
performance liquid chromatography coupled to a refractive index detector. An UHPLC+ Focused
Dionex Ultimate 3000 liquid chromatograph (Thermo Scientific Inc., Waltham, MA, USA)
equipped with an ion exchanger resin column [MetaCarb Ca Plus column (300 × 7.8 mm × 9 μm,
Agilent Technologies, Santa Clara, CA, USA)] was used. The column was operated at 70 °C and
at a flow rate of 0.5 mL min-1
with deionized water (adjusted to pH 7.0) as eluent.
Monosaccharide samples were centrifuged (18,000 g; 20 min; 4 °C) and then 10 μL of the clear
supernatant was injected into the column. Components eluting from the column were detected
using a Shodex RI-101 refractive index detector (Showa Denko Corporation, Tokyo, Japan)
thermostated at 35 °C. The amount of monosaccharides transported through the cell was
determined using an external standard method. Calibration lines were obtained at concentrations
ranging from 2 to 20 mM for each monosaccharide (n=10; r2=0.995, 0.998, 0.999, and 0.990 for
glucose, galactose, fructose, and ribose, respectively).
7.2.4.2. Analysis of amino acids
Amino acid samples collected from the receptor compartment (500 μL) were mixed with 500 μL
of ninhydrine solution [2% (w/v) prepared in 100 mM citrate buffer pH 4.0] and then incubated at
92 °C (boiling water) for 10 min to generate the ninhydrine chromophore (Ruhemann´s purple)
Chapter 7
196
(Friedman, 2004). The mixtures were cool down to room temperature and then the absorbance
was recorded at 570 nm with a Genesys 10uv Spectrophotometer (Thermo Scientific Inc.,
Waltham, MA, USA). The amount of amino acids transported through the cell was determined
using an external standard method. Calibration lines were obtained at concentrations ranging
from 35 to 350 μM for each amino acid (n=10; r2=0.997, 0.989, 0.992, and 0.995 for glycine,
aspartic acid, lysine, and tyrosine, respectively).
7.2.4.3. Analysis of the optical turbidity of the corn oil-in-water emulsion
The optical turbidity (at 600 nm) of the emulsion samples collected from the receptor
compartment was measured using a Genesys 10uv Spectrophotometer (Thermo Scientific Inc.,
Waltham, MA, USA) at room temperature. The amount of the emulsion transported through the
cell was determined using an external standard method. A calibration line was obtained at
concentrations of the corn oil-in-water emulsion ranging from 0.02 to 0.20% (w/w) (n=10;
r2=0.998).
7.2.5. Kinetic model
A sigmoidal response kinetic model (Equation 7.1) was used to fit the experimental data of the
mass transfer profiles of monosaccharides, amino acids, and a corn oil-in-water emulsion through
the Franz diffusion cell. Both the effective diffusion coefficients (the rate of the mass transfer
process) and the maximum amount of the compound transferred through the Franz diffusion cell
(the extent of the mass transfer process), in the absence (control) or presence of 2% (w/w) HMP
or LMP were calculated. This model represented by Equation 7.1 assumes that the rate of the
mass transfer process is directly proportional to the concentration of the nutritional compound to
be transferred across the membrane (Edwards, Johnson, & Read, 1988), as follows:
(
) (7.1)
Chapter 7
197
Where C is the concentration of the nutritional compound transferred at time t, k is the steepness
of the curve, and CMax is the maximum observable value for C. The relative transfer percentage
[RT (%)] of the nutritional compound can be conveniently expressed as the ratio of C and CMax.
The integration of this first-order differential equation leads to Equation 7.2, as follows:
( ( ( )))
(7.2)
Where RT (%) corresponds to the relative transfer percentage of the nutritional compound, and
t1/2 represents the time in which the 50% of the total concentration of the nutritional compound
has been transferred across the membrane. In addition, the effective diffusion coefficient (DEff)
can be defined as follows:
A (7.3)
Where A corresponds to the effective area of the mass transfer process.
7.2.6. Apparent viscosity of pectin solutions
Pectin solutions (2% w/w) for apparent viscosity measurements were prepared by dissolving 400
mg pectin samples (LMP or HMP) in 19.6 g of 25 mM NaCl aqueous solution with gentle stirring
for 18 h at room temperature. Then, pectin solutions were centrifuged (3,000 g; 15 min; 4 °C) to
devoid the entrapped air bubbles. Afterward, pectin solutions were stored at 4 °C overnight
before measurements. The apparent viscosity measurements of pectin solutions were determined
using an Ares Discovery HR-1 Rheometer (TA Instruments, New Castle, DE, USA) with a
parallel plate geometry with a diameter of 40 mm and a gap of 1 mm. Samples were placed in a
temperature-controlled Peltier plate and allowed to equilibrate at 25 °C for 5 min prior to
conducting the measurements. All apparent viscosity measurements were performed using a
series of fixed shear rates that consecutively increased from 0.002 to 10 s-1
and recorded at 25 °C.
Chapter 7
198
The Power Law Model (Equation 7.4) was used to analyze the rheological profiles of pectin
samples (Marcotte, Taherian Hoshahili, & Ramaswamy, 2001) by calculating both the K and n
values, as follows:
(7.4)
Where is the apparent viscosity, is the shear rate, K is the consistency index (which
corresponds to the apparent viscosity of a fluid behaving as Newtonian), and n is the flow index
(which indicates the degree of deviation from Newtonian behavior).
7.2.7. Emulsion characterization
7.2.7.1. Microstructure
The microstructure of the emulsions was characterized by optical microscopy. An optical
microscopy (C1 Digital Eclipse, Nikon Co., Tokyo, Japan) with a 60x objective lens was used to
capture images of the emulsions. A small aliquot of the emulsions (5 μL) was transferred to a
glass microscope slide and covered with a glass cover slip. Then, the cover slip was fixed to the
slide using sealing resin (Ted Pella Inc., Redding, CA, USA) to avoid evaporation. Next, a small
amount of immersion oil (Type A, Nikon Co., Melville, NY, USA) was placed on the top of
cover slip. All optical microscopy images were recorded by using the instrument software (EZ
CS1 version 3.8, Nikon Co., Melville, NY, USA).
7.2.7.2. Particle size distribution
The particle size distribution of emulsions was measured using a dynamic light scattering
instrument (Zetasizer Nano ZSP, Malvern Instruments Ltd., Worcestershire, United Kingdom).
Refractive indices of 1.467 (corn oil) and 1.333 (water) were used for the calculations of the
particle size distribution. Particle sizes were reported as particle size distribution profiles [volume
fraction (% v/v) vs. particle diameter (nm)].
Chapter 7
199
7.2.8. Data analysis
All experiments were performed at least three times using freshly prepared solutions. Apparent
viscosity measurements of pectin solutions were performed by quadruplicate. Averages and
standard deviations were calculated from these replicated measurements. Statistical analysis was
performed by using STATGRAPHICS Centurion XVI version 16.1.11 for Windows. A Fisher´s
Least Significance Difference (LSD) test was conducted to detect any significant differences
among the pectin samples. A p-value<0.05 was considered as statistical significance.
7.3. Results
7.3.1. Mass transfer profiles of monosaccharides
The functional properties of soluble dietary fibers (e.g., gums, mucilages, and pectins) rely on
their ability to thicken into swollen hydrated networks and their subsequent increasing of
viscosity, determining their potential to exert physiological effects along the GIT, specifically
through the stomach and small intestine. Pectins may hinder the mass transfer process of
nutritional compounds from the lumen to the unstirred water layer coating the small intestine by
reducing the mixing among them and reducing the time available for intestinal absorption by
enterocytes. Therefore, an increase in digesta viscosity arising from the pectin consumption
might influence the processes occurring during the nutrient digestion and adsorption. The results
obtained in these experiments showed that pectins, especially HMP, were able to inhibit both the
rate and extent of the mass transfer process of monosaccharides through the Franz diffusion cell
(especially the extent rather than rate). Figure 7.2 shows the mass transfer profiles of
monosaccharides [glucose (a), galactose (b), fructose (c), and ribose (d)]. Both the rate and
extent of the mass transfer process of monosaccharides through a Franz diffusion cell in the
absence (control) or presence of pectin (LMP or HMP) were characterized by calculating DEff and
maximum RT (%) values, respectively (Table 7.1). The maximum RT (%) value is related to the
amount of nutrients which can be effectively transferred through the cellulose membrane
Chapter 7
200
Figure 7.2. Effect of low (LMP) and high (HMP) methoxylated pectins on the mass transfer kinetics of
glucose (a), galactose (b), fructose (c), and ribose (d) through a Franz diffusion cell. Control corresponds
to the sample without addition of pectin. Points correspond to experimental data and lines correspond to
fitted data according to Equation 7.2.
(the extent of the mass transfer process), whereas the DEff value is related to the rate at which the
mass transfer process occurs. The RT (%) values increased with time and tended towards a
plateau after 36 h for each of the tested monosaccharides (Figure 7.2). When a pectin sample
(LMP or HMP) was added, all of the tested monosaccharides diffused slower as compared to
control (Table 7.1, DEff), reaching a lower maximum RT (%) value [Table 7.1, maximum RT
(%)]. The inhibitory effect of the mass transfer process was higher with HMP than LMP.
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
a. b.
c. d.
Chapter 7
201
Table 7.1. Effect of low (LMP) and high (HMP) methoxylated pectins on the effective diffusion coefficient (DEff) and the maximum relative
transfer percentage [maximum RT (%)] of monosaccharides, amino acids, and a corn oil-in-water emulsion through a Franz diffusion cell. Control
corresponds to the sample without addition of pectin. Different letters indicate significant differences. Lowercase letters indicate significant
differences of the same compound among treatments (Control, LMP, and HMP; comparison between columns), and uppercase letters indicate
significant differences of the same treatment among the type of compound (comparison within columns) at the p<0.05 level.
Compound DEff x10
5 (cm
2 s
-1)1 Maximum RT (%)
Control LMP HMP Control LMP HMP
Monosaccharides
Glucose 1.44 0.04a, B
1.14 0.04b, A
0.48 0.04c, A
94.7 0.9a, A
71.5 2.4b, B
63.3 1.6c, B
Galactose 1.44 0.04a, B
1.18 0.04b, A
0.48 0.04c, A
94.5 0.7a, A
68.5 1.6b, B
62.4 3.7c, B
Fructose 1.40 0.04a, B
1.14 0.04b, A
0.48 0.04c, A
96.0 1.6a, A
67.4 1.5b, B
60.6 2.1c, B
Ribose 3.91 0.41a, A
1.11 0.11b, A
0.52 0.04c, A
80.4 0.8b, B
86.1 3.8a, A
86.0 1.7a, A
Amino acids
Glycine 3.83 0.04c, B
4.65 0.15b, B
5.24 0.04a, B
87.8 3.3a, B
17.2 1.3b, C
10.6 0.7c, B
Aspartic acid 3.87 0.18a, B
3.91 0.26a, C
4.20 0.04a, C
96.0 1.8a, A
81.6 2.6b, A
39.3 1.9c, A
Lysine 4.06 0.04a, A
0.59 0.07c, D
3.54 0.15b, D
90.8 1.2a, B
7.0 0.7c, D
40.2 2.7b, A
Tyrosine 3.87 0.04b, B
6.75 0.29a, A
6.45 0.04a, A
95.2 1.7a, A
69.4 3.4b, B
36.7 1.8c, A
Lipids
Emulsion 3.47 0.26a 3.43 0.15
a 3.24 0.15
a 82.9 1.9
a 43.1 0.3
b 6.8 0.1
c
1Example: for Glucose (Control): DEff x 105 (cm2 s-1) = 1.44 should be read as DEff = 1.44 x 10-5 cm2 s-1.
Chapter 7
202
For example, for the diffusion of glucose (Figure 7.2a and Table 7.1), DEff values were 1.44 x
10-5
, 1.14 x 10-5
, and 0.48 x 10-5
cm2 s
-1 for control, LMP, and HMP, respectively; and the
maximum RT (%) values were 94.7, 71.5, and 63.3% for control, LMP, and HMP, respectively.
Similar trends were observed for galactose (Figure 7.2b), fructose (Figure 7.2c), and ribose
(Figure 7.2d).
7.3.2. Mass transfer profiles of amino acids
As observed with the mass transfer of monosaccharides, the rate and the extent of amino acids
transported towards the Franz diffusion cell were reduced by the presence of pectins, with a
higher effect of HMP towards glycine, aspartic acid, and tyrosine, and a higher effect of LMP
towards lysine. Figure 7.3 shows the mass transfer profiles of amino acids [glycine (a), aspartic
acid (b), lysine (c), and tyrosine (d)]. Although the mass transfer profiles obtained for the amino
acids were fairly similar to those obtained for monosaccharides, a very important difference in
the mass transfer profiles of amino acids was observed: a sigmoidal kinetic behavior in the
control and pectin containing experiments was obtained. A lag time of approximately 10 h was
observed for the mass transfer process of each of the tested amino acids in both absence (control)
and presence of pectins (LMP and HMP). Before the lag time (t<10 h), no significant differences
in the RT (%) values were observed among pectin samples for each of the tested amino acids, as
compared to control (the amount of amino acids transferred through the membrane was less than
3% after 10 h in both absence and presence of pectins). The RT (%) values increased with time
and tended towards a plateau after 36 h for each of the tested amino acids (Figure 7.3). When a
pectin sample (LMP or HMP) was added, different results for glycine, aspartic acid, and tyrosine
were observed, in comparison to lysine.
Upon addition of pectins, glycine, aspartic acid, and tyrosine diffused faster (Table 7.1, DEff), but
they reached a lower maximum RT (%) value, as compared to control [Table 7.1, maximum RT
(%)]. For example, for the diffusion of glycine (Figure 7.3a and Table 7.1), DEff values were
3.83 x 10-5
, 4.65 x 10-5
, and 5.24 x 10-5
cm2 s
-1 for control, LMP, and HMP, respectively; and the
maximum RT (%) values were 87.8, 17.2, and 10.6% for control, LMP, and HMP, respectively.
Chapter 7
203
Figure 7.3. Effect of low (LMP) and high (HMP) methoxylated pectins on the mass transfer kinetics of
glycine (a), aspartic acid (b), lysine (c), and tyrosine (d) through a Franz diffusion cell. Control
corresponds to the sample without addition of pectin. Points correspond to experimental data and lines
correspond to fitted data according to Equation 7.2.
Similar trends were observed for aspartic acid (Figure 7.3b and Table 7.1) and tyrosine (Figure
7.3d and Table 1). These results indicate that pectins, especially HMP, were able to inhibit the
amounts of glycine, aspartic acid, and tyrosine that can be effectively transferred through the
Franz diffusion cell (the extent of the mass transfer process), but they were not able to reduce the
rate at which the mass transfer process occurs.
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
a.
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
b.
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
c.
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
Tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
d.
Chapter 7
204
Figure 7.4. Effect of low (LMP) and high (HMP) methoxylated pectins on the mass transfer kinetics of a
corn oil-in-water emulsion through a Franz diffusion cell (a). Control corresponds to the sample without
addition of pectin. Points correspond to experimental data and lines correspond to fitted data according to
Equation 7.2. Particle size distribution of a corn oil-in-water emulsion in presence of LMP and HMP (b).
Microstructure of a corn oil-in-water emulsion observed by optical microscopy in presence of LMP and
HMP (c). The scale bars corresponds to 20 μm. Control corresponds to the emulsion without addition of
pectin.
In addition, it was observed that the inhibitory effect of the extent of the mass transfer process of
glycine, aspartic acid, and tyrosine through the Franz diffusion cell was higher with HMP than
LMP. Opposite to the behavior observed for glycine, aspartic acid, and tyrosine, the effect of
pectins towards the diffusion of lysine (Figure 7.3c and Table 7.1) was quite different. For this
amino acid, DEff were 4.06 x 10-5
, 0.59 x 10-5
, and 3.54 x 10-5
cm2 s
-1 for control, LMP, and HMP,
0
5
10
15
1 10 100 1000 10000V
olu
me
fra
ctio
n (
%)
Particle diameter (nm)
Control
LMP
HMP
0
20
40
60
80
100
0 12 24 36 48
Rel
ati
ve
tra
nsf
er (
%)
Time (h)
Control
LMP
HMP
a. b.
Control LMP HMP
c.
Chapter 7
205
respectively; and the maximum RT (%) values were 90.8, 7.0, and 40.2% for control, LMP, and
HMP, respectively. These results indicate that pectins, especially LMP, were able to inhibit both
the amount of lysine that can be effectively transferred through the Franz diffusion cell and the
rate at which the mass transfer process of lysine occurs. In addition, it was observed that the
inhibitory effect of the rate and extent of the mass transfer process of lysine through the Franz
diffusion cell was higher with LMP than HMP.
7.3.3. Mass transfer profile of the corn oil-in-water emulsion
Pectins, especially HMP, were able to inhibit both the rate and extent of the mass transfer process
of the corn oil-in-water emulsion through the Franz diffusion cell. Figure 7.4 shows the mass
transfer profile of the corn oil-in-water emulsion through the Franz diffusion cell. The mass
transfer profile obtained for the emulsion was fairly similar to that obtained for the
monosaccharides: the sigmoidal behavior was not observed and the effect of pectins on the rate
and extent of the mass transfer process followed the same order: Control>LMP>HMP. It was
observed that both the rate and extent of the mass transfer process of the emulsion was inhibited
upon addition of pectins (DEff were 3.47 x 10-5
, 3.43 x 10-5
, and 3.24 x 10-5
cm2 s
-1 for control,
LMP, and HMP, respectively; and the maximum RT (%) values were 82.9, 43.1, and 6.8 for
control, LMP, and HMP, respectively), being the inhibitory effect of the rate and extent of the
mass transfer process of the emulsion through the Franz diffusion cell higher with HMP than
LMP.
7.4. Discussion
7.4.1. Mass transfer profiles of electrically neutral nutrients
The inhibition of both the rate and extent of the mass transfer process of electrically neutral
nutrients (monosaccharides, glycine, and tyrosine) observed upon addition of pectins might be
due to the high viscosity of both LMP and HMP as compared to that of control. The apparent
viscosity profiles of 2% (w/w) LMP and HMP solutions are shown in Figure 7.5.
Chapter 7
206
Figure 7.5. Rheological profiles (apparent viscosity versus shear rate) measured at 25 °C of 2% (w/w) low
(LMP) and high (HMP) methoxylated pectin solutions prepared in 25 mM NaCl aqueous solution. Pectin
solutions were thermally stabilized at 25 °C for 5 min prior to analysis. Points correspond to experimental
data and lines correspond to fitted data according to Equation 7.4.
Both LMP and HMP solutions behaved as pseudoplastic non-Newtonian fluids because the
viscosity of the solutions decreased as the shear rate increased (Sato, Oliveira, & Cunha, 2008).
The apparent viscosity of HMP was significantly higher than the one of LMP, at low shear rates
(below to 0.01 s-1
). In addition, the flow behavior indexes for both LMP and HMP solutions were
less than 1 (n=0.292 for HMP and n=0.183 for LMP), demonstrating that pectin solutions
behaved as non-Newtonian fluids [it has been defined for Newtonian fluids, n=1 (Cross, 1965)].
Due to the high molecular weight of HMP (181 kDa) as compared to that of LMP (130 kDa), the
consistency index of HMP (281 mPa s) solution was significantly higher than that of LMP (62
mPa s). The non-Newtonian behavior of pectin solutions aggress with the presence of
macroscopic network structures which are characteristic of the hydration process of pectin
molecules (Löfgren, Walkenström, & Hermansson, 2002). Viscous fibers with hydrophilic nature
such as pectins have been reported to create complex networks by the entanglements of fully
hydrated chains of the polymer producing viscous solutions (Ryden, MacDougall, Tibbits, &
0
5000
10000
15000
0.001 0.01 0.1 1 10
Ap
pa
ren
t V
isco
sity
(m
Pa
s)
Shear Rate (s-1)
HMP
LMP
Chapter 7
207
Ring, 2000). The formation of these complex hydrated networks is commonly related to the
capacity of pectin molecules to embed nutritional compounds, thereby limiting their further
mobility (Zsivanovits, MacDougall, Smith, & Ring, 2004). Therefore, the retardation of the rate
and extent of the mass transfer process of electrically neutral nutrients (monosaccharides,
glycine, and tyrosine) through the Franz diffusion cell was higher with HMP than LMP probably
due to the high viscosity of HMP as compared to that of LMP.
It is important to consider that amino acids can be electrically charged depending on the pH, and
this electrical charge will determine their potential to interact with other charged species (such as
pectin). The net electrical charge for glycine and tyrosine was calculated by using the relative
distribution of their charged species at pH 7.0. For glycine, the neutral specie (charge 0,
⊕NH3CH(H)COO⊝) has a relative abundance of 99.8%; and for tyrosine, the neutral specie
(charge 0, ⊕NH3CH(CH2OH)COO⊝) has a relative abundance of 99.4%. These calculations
confirm that both glycine and tyrosine are neutral amino acids at pH 7.0. Because of the neutral
structure of glycine and tyrosine at pH 7.0, the electrostatic interactions between them and pectin
were not significant. Therefore, the viscosity of the pectin solutions might be the parameter
governing the retardation of the mass transfer process of all the studied electrically neutral
nutrients. For electrically neutral nutrients, however, other structural parameters such as
hydrophobicity and molecular size might probably be involved in the interactions with pectins.
Nevertheless, the effect of these parameters on the mass transfer process of electrically neutral
nutrients was unclear.
7.4.2. Mass transfer profiles of electrically charged amino acids
We suggest that the mass transfer process of electrically charged amino acids (aspartic acid and
lysine) can be influenced by both the viscosity of the pectin solutions and the electrostatic
interactions between each charged amino acid and the surface electrical charge of LMP and
HMP. It is well known that pectin molecules are anionic in nature (Mohnen, 2008) because of the
spontaneous ionization of the carboxyl group in aqueous solution (–COOH + H2O –COO⊝ +
H3O⊕). The surface electrical charge of the pectins tested in this study were previously
Chapter 7
208
characterized (Espinal-Ruiz, Restrepo-Sánchez, Narváez-Cuenca, & McClements, 2016). LMP
possess a higher surface negative charge ( = -47.5 mV at pH 7.0) as compared to that of HMP
( = -28.2 mV at pH 7.0) because a higher fraction of the carboxyl groups (–COO⊝) of LMP
remain free than in HMP (Espinal-Ruiz, Restrepo-Sánchez, Narváez-Cuenca, & McClements,
2016). Furthermore, the electrical net charge of aspartic acid and lysine at pH 7.0 is -1 and +1,
respectively (Moore, 1985).
We suggest, therefore, that the repulsive electrostatic interactions between the carboxyl group of
aspartic acid (Figure 7.3b) and the carboxyl groups of LMP prevented aspartic acid molecules to
be trapped in the structural network of LMP, and therefore, the extent of the mass transfer
process of aspartic acid through the Franz diffusion cell was not significantly hindered upon
addition of LMP. In contrast, because of the lower surface negative charges of HMP as compared
to that of LMP, the electrostatic interactions between aspartic acid and HMP were not significant,
and therefore, the inhibition of the extent of the mass transfer process observed for aspartic acid
upon addition of HMP can be attributed to viscosity effects. Conversely, the attractive
electrostatic interactions between the -amino group of lysine (–NH3⊕) and the carboxyl group of
LMP (–COO⊝) promoted significantly the entrapment of lysine molecules in the structural
network of LMP, and therefore, both the rate and extent of lysine through the Franz diffusion cell
were significantly hindered upon addition of LMP. Moreover, our results suggest that the
electrostatic attractive interactions overcomed the viscosity effects and consequently, the capacity
of LMP to inhibit the mass transfer process of lysine through the Franz diffusion cell was higher
as compared to that of HMP (Table 7.1).
We hypothesize that positive charges of lysine molecules play an important role controlling the
interaction of them with pectins because lysine was the only amino acid evaluated where the
effect of LMP was higher than HMP (Figure 7.3c). In addition, we suggest that the lag phase and
the sigmoidal behavior observed in the diffusion profiles of all of the tested amino acids (Figure
7.3) were attributed to the strong electrostatic interactions between the ionizable groups of the
amino acids (–NH3⊕, –COO⊝, and side chains) and the cellulose molecules composing the
membrane (Mahrenholz, Tapia, Stigler, & Volkmer, 2010).
Chapter 7
209
7.4.3. Diffusion profile of the corn oil-in-water emulsion
The mechanism by which pectin is able to retard the rate and extent of the mass transfer process
of the emulsion might be related to the high capacity of HMP to flocculate the lipid droplets of
the emulsion as compared to LMP (Espinal-Ruiz, Restrepo-Sánchez, Narváez-Cuenca, &
McClements, 2016).
Figure 7.6. Molecular mechanisms governing the interaction between pectin and nutritional compounds
(monosaccharides, amino acids, and emulsified lipids) and its effect on the mass transfer process of them
through the Franz diffusion cell (highly schematic). The mass transfer process of monosaccharides and
neutral amino acids (glycine and tyrosine) is controlled by the viscosity of the pectin solutions, whereas
the mass transfer process of charged amino acids is controlled by both viscosity and repulsive (aspartic
acid) and attractive (lysine) electrostatic interactions between them and pectin. Finally, pectin induces
flocculation of lipid droplets by depletion attraction and therefore, their mobility is reduced.
⊝
⊝⊝
⊝
⊝⊝
⊝⊝⊝
⊕
⊕
⊕
Monosaccharides,
glycine, and tyrosine
Viscosity
Lipids
Flocculation
Lysine
Electrostatic attraction
Aspartic acid
Electrostatic repulsion
Viscosity
Flow
Pectin
Membrane
Compound
Direction and magnitude
of the mass transfer
Chapter 7
210
The depletion interactions might be the driving force which promotes the flocculation of the
emulsified lipids, and it has been established that this force induced by polysaccharides increases
as the molecular weight of the polysaccharide increases (McClements, 2000). Consequently,
because the molecular weight of HMP (181 kDa) is higher than that of LMP (130 kDa), the
flocculation capacity of HMP was greater than that of LMP. Therefore, the inhibition of the rate
and extent of the mass transfer process of the emulsion through the Franz diffusion cell was
greater with HMP (6.8% emulsion was transferred) as compared to LMP (43.1% emulsion was
transferred), because of the higher capacity of HMP to flocculate the lipid droplets composing the
emulsion. The particle size distribution analysis (Figure 7.4b) indicated that the control emulsion
(without addition of pectin) contained small lipid droplets (the lipid droplet diameter was around
300 nm) with a monomodal distribution (only one peak was observed), suggesting that the lipid
droplets in the control emulsion were stable to aggregation (Figure 7.4c, Control). However, the
multimodal distribution of the emulsions containing LMP and HMP (Figure 7.4b) indicated that
pectins were able to induce aggregation of the lipid droplets (Figures 7.4c, LMP and HMP)
through a mechanism of flocculation [lipid droplet (flocs) diameters were 1100 and 1500 nm for
LMP and HMP, respectively]. Therefore, it can be suggested that the inhibitory effect of the rate
and extent of the mass transfer process of the corn oil-in-water emulsion through the Franz
diffusion cell was higher with HMP than LMP probably due to the high capacity of HMP to
flocculate the lipid droplets of the emulsion as compared to LMP (Figures 7.4b and 7.4c).
All in all, it can be suggested that the chemical nature of the nutritional compound
(monosaccharides, amino acids, and a corn oil-in-water emulsion) to be transferred through the
Franz diffusion cell will determine the mechanism by which the nutritional compound is able to
interact with pectin molecules (Figure 7.6). For example, it was observed that viscosity was the
most important parameter governing both the rate and extent of the mass transfer process of
monosaccharides and neutral amino acids (glycine and tyrosine), whereas electrostatic
interactions played an important role controlling the extent of the mass transfer process of
charged amino acids (repulsive for the interaction between pectin and aspartic acid, and attractive
for the interaction between pectin and lysine). Furthermore, the complex aggregated structures
formed by the flocculation of emulsified lipids leads to complex interactions between them and
Chapter 7
211
pectin molecules, being flocculation the most important parameter governing both the rate and
extent of the mass transfer process of the corn oil-in-water emulsion.
7.5. Conclusions
Viscosity of pectin solutions and electrostatic interactions between pectins and nutrients are
proposed to be the main factors influencing both the rate and extent of the mass transfer process
of nutrients across a cellulose membrane in a Franz diffusion cell. Our results suggest that the
higher the viscosity of the pectin solution, the higher the inhibition of the rate and extent of the
mass transfer of nutrients through the Franz diffusion cell. In addition, it was established that
HMP was more effective than LMP to inhibit the extent of the mass transfer process of all
evaluated nutritional compounds, except for lysine, where its strong attractive electrostatic
interaction with LMP was the dominant factor governing its mass transfer process. Our results
suggest that the inclusion of pectin in food formulations might be an effective strategy for
controlling the calorie intake by limiting the digestion and absorption of nutritional compounds.
However, the formulation of foodstuffs with high pectin contents must be done carefully in
countries whose inhabitants are deficient in minerals such as iron, calcium, and zinc, since the
consumption of pectin has been associated with a decreased bioavailability of these minerals.
Acknowledgments
We are grateful to Departamento Administrativo de Ciencia, Tecnología e Innovación de
Colombia (COLCIENCIAS) and Vicerrectoría Académica of Universidad Nacional de Colombia
for providing a fellowship to Mauricio Espinal-Ruiz supporting this work. We are also grateful to
Vicerrectoría de Investigaciones of Universidad Nacional de Colombia for fundig the project
Efectos moleculares de la pectina sobre el metabolismo de lípidos y carbohidratos (Código
Hermes 20610).
Chapter 7
212
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Chapter 8
General discussion
Chapter 8
215
Although it has been previously demonstrated that pectin has several physiological functions
beneficial to human health (Cerda, 1988), studies explaining the mechanisms by which pectin is
capable to exert its physiological functions have not been conducted. The research described in
this thesis was focused on the evaluation of the mechanisms by which pectin is capable to exert
its physiological functions.
The mechanisms evaluated in this thesis included the effect of pectin on i) the activities of the
major digestive enzyme (pancreatic lipase, -amylase, alkaline phosphatase, and protease), ii) the
rate and extent of the digestion process of emulsified lipids, and iii) the rate and extent of the
mass transfer process of some of the most important nutritional compounds (monosaccharides,
amino acids, and lipids). Because pancreatic lipase was strongly inhibited by pectin, a special
emphasis was made on the effect of pectin on the gastrointestinal fate of emulsified lipids. The
structural characteristics of pectin (e.g., molecular weight and methoxylation degree) in relation
to their ability to inhibit the digestion process of emulsified lipids were studied. The effect of the
molecular interactions of pectin with the different gastrointestinal components involved in the
lipid digestion process (e.g., pancreatic lipase, bile salts, CaCl2, and NaCl) was evaluated as well.
In this chapter, we discuss the relevance of the mechanisms involved on the physiological
functions of pectin including the effect of pectin on i) the digestive enzyme activities, ii) the
gastrointestinal fate of emulsified lipids, and iii) the mass transfer kinetics of nutritional
compounds. We also discuss some important aspects of the experimental models used to evaluate
the mechanisms by which pectin is capable to exert its physiological functions including i) the
use of artificial chromogenic substrates as a model to evaluate the in vitro activity of the major
digestive enzymes, ii) the advantages and disadvantages of using emulsions as an experimental
model, iii) the advantages and disadvantages of the simulated in vitro digestion model used to
evaluate the effect of pectin on the gastrointestinal fate of emulsified lipids, and iv) the use of a
Franz diffusion cell as an experimental model to evaluate the effect of pectin on the mass transfer
kinetics of some of the most important nutritional compounds.
Chapter 8
216
8.1. Mechanisms involved on the physiological functions of pectin
8.1.1. Effect of pectin on the digestive enzyme activities
In chapter 3, we evaluated the effect of the methoxylation degree (MD) of pectin on the activity
of the digestive enzymes. Alkaline hydrolysis of high methoxylated pectin was performed to
obtain pectins with different MD. The MD of the pectins obtained after alkaline hydrolysis
ranged from 7.1% to 87.4% (mol/mol) (Table 3.1). This allowed us to evaluate the effect of both
low methoxylated (LMP) and high methoxylated (HMP) pectins on the activities of the digestive
enzymes. We found that increasing the concentration of pectin decreased the activity of all of the
tested digestive enzymes by means of a non-competitive inhibition mechanism (Figures 3.2 and
3.3). We also found that HMP was more efficient in inhibiting the activity of all of the tested
digestive enzymes than LMP. Interestingly, each enzyme was inhibited with different efficiencies
(Figure 3.5). Pancreatic lipase was the most likely to be inhibited by pectin, followed by -
amylase, alkaline phosphatase, and protease. Therefore, because pectins, especially HMP,
exhibited a high capacity to inhibit the activity of pancreatic lipase, it was expected that the
digestion process of lipids was also affected by the addition of pectin. Therefore, the following
research was focused in evaluating the effect of pectin on the gastrointestinal fate of lipids. A
simulated in vitro digestion model was used to evaluate the effect of pectin on the gastrointestinal
fate of lipids by using a corn oil-in-water emulsion as the experimental model.
8.1.2. Effect of pectin on the gastrointestinal fate of emulsified lipids
The effect of pectin on the gastrointestinal fate of emulsified lipids was evaluated by using a
simulated in vitro digestion model designed to mimic the oral, gastric, and small intestine phases
of the human gastrointestinal tract (GIT). In chapter 4, the influence on the dietary fiber type
(chitosan, methyl cellulose, and pectin at different concentrations) on the gastrointestinal fate of
emulsified lipids was examined. The aim of that chapter was to compare the effect of pectin on
the rate and extent of the digestion process of emulsified lipids with other sources of dietary fiber
Chapter 8
217
(methyl cellulose and chitosan) with different structural characteristics, especially, the electrical
charge. We found that both the rate and extent (principally the extent of the lipid digestion rather
than rate) of the lipid digestion process were inhibited with increasing chitosan, methyl cellulose,
and pectin concentrations (Figures 4.7 and 4.8). However, the magnitude of the inhibition was
different with each dietary fiber: methyl cellulose had the highest capacity to inhibit the lipid
digestion process, followed by pectin and chitosan. We suggested that the physicochemical
mechanisms that may account for the observed influence of dietary fiber on the rate and extent of
the lipid digestion process are i) the modification of the viscosity of the GIT fluids, ii) the
flocculation of the emulsified lipids and iii) the electrostatic interactions between dietary fiber
and emulsified lipids.
As observed, several mechanisms by which dietary fiber, especially pectin, is able to inhibit the
lipid digestion process were proposed. However, a relationship between chemical structure and
functional properties of dietary fiber could not be established in that chapter because the majority
of the structural characteristics of the different sources of dietary fiber used in chapter 4 were
unknown. Therefore, we focused on studying the effect of the structural characteristics of pectin
(e.g., molecular weight and methoxylation degree) on the rate and extent of digestion process of
emulsified lipids. In chapter 5, the effect of pectin properties (e.g., methoxylation degree and
molecular weight) on the gastrointestinal fate of emulsified lipids was evaluated. Medium
methoxylated pectin (MMP) was isolated from banana passion fruit (Passiflora tripartita var.
mollisima) and then, the impact of MMP on the rate and extent of the digestion process of
emulsified lipids was compared to that of commercial LMP and HMP. We found that all three
pectins promoted flocculation of the lipid droplets, which can be attributed to a depletion
flocculation mechanism. Calculations of the inter-droplet pair potential due to depletion attraction
of lipid droplets (Figure 5.10) in emulsions containing LMP, MMP, and HMP revealed that the
strength of the depletion attraction increases with the simultaneous increase of both molecular
weight and methoxylation degree in the following order: HMP>MMP>LMP. Therefore, it can be
suggested that HMP exhibited the highest ability to inhibit the lipid digestion process probably
due to its higher capacity to flocculate the lipid droplets, followed MMP and LMP (Figure 5.9).
Chapter 8
218
Although flocculation was identified as a mechanism controlling the rate and extent of the lipid
digestion process, the results obtained so far did not give any information concerning the
physicochemical nature of the interactions occurring between pectin and the various GIT
components associated with the digestion process of emulsified lipids. Therefore, in the next
section we provide a discussion on the effect of the molecular interactions occurring between
pectin and the various GIT components and their relationship with the rate and extent of the
digestion process of emulsified lipids.
8.1.3. Molecular interactions between pectin and the GIT components
We showed that pectin is able to reduce the rate and extent of the digestion process by interacting
with emulsified lipids. However, the interaction of pectin with the different components of the
GIT involved in the digestion process of emulsified lipids (e.g., pancreatic lipase, bile salts, and
electrolytes) may also contribute to the overall inhibitory effect. In chapter 6, isothermal titration
calorimetry (ITC) was used to study the interactions of pectin with the major GIT components
involved in the digestion process of emulsified lipids (e.g., pancreatic lipase, bile salts, CaCl2,
and NaCl). In addition, microelectrophoresis, turbidity, and microstructural observations were
used to provide additional information about the nature of the interactions between pectin and the
aforementioned GIT components.
We found that the addition of NaCl (Figure 6.5a), bile salts (Figure 6.5c), and pancreatic lipase
(Figure 6.5d) to pectin solutions caused a small decrease in the magnitude of the zeta-potential,
as compared to the large decrease caused upon addition of CaCl2 (Figure 6.5b). Consequently,
the addition of CaCl2 to pectin solutions caused the highest increase of the optical turbidity
because of the formation of large aggregates (Figure 6.8), followed by bile salts, pancreatic
lipase, and NaCl. This observation allowed us to suggest that both CaCl2 and bile salts play a
determinant role in the overall interaction of pectin with the GIT components. In particular,
CaCl2 and bile salts promoted the formation of pectin gels (Figure 6.8) that can lead to the
flocculation of lipid droplets and decrease the ability of emulsified lipids to be reached by
pancreatic lipase, thereby inhibiting the digestion process.
Chapter 8
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The formation of gel-like structures resulting from the interaction between pectin and the GIT
components can lead to the flocculation of lipid droplets and thereby inhibiting the digestion
process of emulsified lipids. However, the increase of the viscosity upon addition of pectin may
also affect the mobility of both the GIT components and the nutritional compounds to be
digested. Therefore, the effect of pectin on the rate and extent of the mass transfer process of the
most important nutritional compounds (monosaccharides, amino acids, and lipids) was evaluated
in chapter 7 as another possible mechanism controlling the GIT processes.
8.1.4. Effect of pectin on the mass transfer of nutrients
We found that the mass transfer process of electrically neutral nutrients (monosaccharides,
glycine, and tyrosine, all neutral at pH 7.0) was governed by the viscosity of the pectin solutions,
whereas the mass transfer process of electrically charged nutrients (aspartic-negatively charged at
pH 7.0 acid and lysine-positively charged at pH 7.0) was governed by both viscosity and the
electrical interactions of these nutrients with pectin. In addition, flocculation was proposed to be
the most important mechanism controlling the mass transfer process of emulsified lipids. Pectins,
especially HMP, were able to inhibit both the rate and extent of the mass transfer process of
electrically neutral nutrients (monosaccharides, glycine, and tyrosine). The apparent viscosity
profiles of LMP and HMP (Figure 7.5) revealed that the viscosity of HMP was significantly
higher than the one of LMP. Therefore, the retardation of the rate and extent of the mass transfer
process of electrically neutral nutrients was higher with HMP than LMP probably due to the
higher viscosity of HMP as compared to that of LMP (Figures 7.2, 7.3a, and 7.3d). Interestingly,
the mass transfer process of electrically charged nutrients (aspartic acid and lysine) was governed
by both the viscosity of the pectin solutions and the electrical interactions of these nutrients with
pectins. The repulsive interactions between aspartic acid and LMP prevented aspartic acid
molecules to be trapped in the structural network of LMP, and therefore, the extent of the mass
transfer process of aspartic acid was not significantly hindered by LMP. Conversely, the
attractive interactions between lysine and LMP promoted significantly its entrapment in the
structural network of LMP, and therefore, both the rate and extent of the mass transfer process of
lysine was significantly hindered upon addition of LMP.
Chapter 8
220
Finally, we suggested that the mechanism by which pectin is able to retard the rate and extent of
the mass transfer process of the emulsified lipids might be related to the high capacity of HMP to
flocculate the lipid droplets of the emulsion as compared to LMP (chapter 5). Figure 8.1 shows
schematically that several mechanisms were proposed in this thesis for controlling the
physiological properties of pectin, including the inhibition of the digestive enzymatic activities
and the modulation of both the mass transfer of nutrients and the digestion of emulsified lipids.
Structural characteristics of pectin (methoxylation degree and molecular weight) significantly
influenced the ability of pectin to interact with nutrients as well as the GIT components.
However, further studies are required to discriminate the relative contribution of each mechanism
to the overall effect, as well as the relative contribution of the individual structural characteristics
of pectin, including the methoxylation degree and molecular weight.
Figure 8.1. Schematic representation of the mechanisms proposed in this thesis for controlling the
physiological properties of pectin. i) Non-competitive inhibition of digestive enzymes, ii) flocculation of
lipid droplets, and iii) hindered diffusion of enzymatic products to epithelial cells (enterocytes). Red,
substrate; blue, product; green, enzyme; and grey-red stick, pectin.
i) Non-competitive inhibition
ii) Flocculation
iii) Hindered diffusion
Ephitelial cells (enterocytes)
Chapter 8
221
8.2. Using artificial chromogenic substrates for evaluating the
activity of digestive enzymes
Pancreatic lipase, -amylase, and protease are the main enzymes responsible for the digestion of
lipids, carbohydrates, and proteins, respectively (Campbell, 2015). These enzymes were studied
because of their biological relevance on the digestives processes. Alkaline phosphatase was also
studied because it participates in the dephosphorylation of nutrients, which is a necessary process
for their further digestion (Vaishnava & Hooper, 2007). In chapter 2, the optimization of the
reaction conditions affecting the activity of these digestive enzymes by using artificial
chromogenic substrates was carried out. The reaction conditions obtained in that chapter allowed
obtaining rapid, reproducible, and reliable measurements of the activities of each of the tested
digestive enzymes. The best reactions conditions obtained in chapter 2 for the measurement of
the aforementioned digestive enzyme activities (conditions summarized in Table 2.1) were then
used in chapter 3 to evaluate the inhibitory effect of pectin upon the activity of these enzymes by
using artificial chromogenic substrates.
Enzymes are often highly specific to its native substrate (Qian, 2008). However, some enzymes
are able to exhibit activity toward several substrates, showing a characteristic known as enzyme-
substrate promiscuity (Hult & Berglund, 2007). In particular, pancreatic lipase and proteases have
been reported to exhibit enzyme-substrate promiscuity by accepting a wide variety of substrates
(decrease in specificity) with which they have higher affinities (increase in selectivity) as
compared to native substrates (Khersonsky, Roodveldt, & Tawfik, 2006). These substrates are
e.g. chromogenic artificial substrates and the high selectivity of digestive enzymes by these
substrates allows obtaining high enzymatic activities in short period of time. In the one hand, the
high selectivity of pancreatic lipase towards p-nitrophenyl ester substrates (e.g., p-nitrophenyl
palmitate) has been reported to be related with the lack of chirality of the alcohol moiety of the
ester substrate (Kapoor & Gupta, 2012). In the other hand, the high selectivity of protease
towards p-nitrophenyl acetate has been reported to be related to the lack of the peptide bond of
the substrate (which makes p-nitrophenyl acetate a suitable substrate for esterases in general) and
Chapter 8
222
because the structural similarity of the substrate with aromatic amino acids (Gossrau, Lojda,
Smith, & Sinha, 1984). The high selectivity of alkaline phosphatase and -amylase towards p-
nitrophenyl phosphate and 2-chloro-p-nitrophenyl--D-maltotrioside, respectively, has been
related to the structural similarity of those substrates with their analogous native substrates
(Copley, 2003; Hult & Berglund, 2007; Khersonsky, Roodveldt, & Tawfik, 2006). Therefore, the
advantages of using artificial chromogenic substrates are the velocity, reproducibility, and
reliability of the results that can be obtained as compared to those obtained when native
substrates are used. Nevertheless, it is important to stress that the use of native substrates is
preferred because of the biological relevance of the results that can be obtained, but native
substrates are often difficult to obtain and the heterogeneity of their chemical structures (e.g.,
chemical composition, molecular weight, and three-dimensional conformation) often prevent
obtaining reproducible results (Jedrzejas, 2000; MacGregor, Janeček, & Svensson, 2001).
One of the disadvantages of using artificial chromogenic substrates is that it is necessary to adjust
the measurement parameters because the experimental conditions reported in literature for native
substrates often differ to those required for artificial ones. For example, the experimental
conditions adjusted for pancreatic lipase in chapter 2 were used in chapter 3 to evaluate the
effect of pectin on the activity of this enzyme in model solutions. However, these experimental
conditions were not suitable to be used to evaluate the effect of pectin on the gastrointestinal fate
of emulsified lipids in chapters 4, 5, and 6 because of the differences on both the chemical nature
of the substrates used in each system and the environmental conditions of the enzymatic
reactions. It has been established that pancreatic lipase has low specificity towards soluble
substrates (e.g., p-nitrophenyl palmitate) but it exhibits a high specificity when the substrate (e.g.,
triacylglyceride) is able to form an interface between the aqueous and the oil phases of an
emulsion (Reis, Holmberg, Miller, Leser, Raab, & Watzke, 2009; Reis, Holmberg, Watzke,
Leser, & Miller, 2009). Therefore, the differences among the chemical nature of the substrate
(e.g., solubility, polarity, hydrophobicity, and three-dimensional conformation) as well as the
environmental factors (e.g., pH, temperature, ionic strength, and cofactors) affecting the rate of
the enzymatic reaction make necessary to adjust the experimental conditions for each reaction
depending on the nature of the system that will be used.
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223
8.3. Emulsified lipids as an experimental model: advantages and
disadvantages
The selection of emulsions as an experimental model representing the wide variety of lipids that
can be consumed in the diet was a critical factor to be considered for obtaining reproducible and
reliable results of the gastrointestinal fate of emulsified lipids by using a simulated in vitro
digestion model. In the oral phase of the in vitro digestion model, dietary lipids (fats and oils) are
mixed with saliva in the mouth. The shear effect of the mastication process (which was simulated
by using mechanical agitation) and the presence of mucin in saliva induced the formation of lipid
droplets which are dispersed in the aqueous phase, leading to the formation of an oil-in-water
emulsion (Vingerhoeds, Blijdenstein, Zoet, & van Aken, 2005). Depending on the chemical
nature and the concentration of both the oil phase (e.g., corn oil) and the surfactant (e.g., Tween
80) as well as the residence time that the emulsion spends in the oral phase, emulsified lipids are
susceptible of suffering flocculation and coalescence induced by mucin (Vingerhoeds,
Blijdenstein, Zoet, & van Aken, 2005). Therefore, the oral phase modifies the structural
characteristics of emulsified lipids and defines their subsequent behavior in the digestion process.
Regardless of the origin, chemical nature, and physical state of the lipids consumed in the diet,
lipids will be structured as an oil-in-water emulsion in the oral phase (Singh, Ye, & Horne, 2009).
This behavior allowed us to use the emulsified lipids as a suitable model for studying the
gastrointestinal fate of lipids when they are subjected to the digestion process. Therefore,
emulsified lipids provide a fundamental model which allows understanding the relationship
between the initial physicochemical properties of the emulsion (e.g., composition, concentration,
particle size, electrical charge, and interfacial characteristics) and its behavior toward the
digestion process (e.g., stability, appearance, rheology, and spatial distribution). Among the
advantages of using emulsified lipids as an experimental model are that they represent the
structure of the wide variety of lipids that can be consumed in the diet, they are easy to prepare
being possible to replicate their initial physicochemical characteristics, they usually have a high
long-term stability to gravitational separation and aggregation, and they are susceptible to present
Chapter 8
224
structural changes depending on the environmental conditions of the digestive process (e.g., pH,
ionic strength, mechanical forces, and the presence of dietary fiber such as pectin). Nevertheless,
some disadvantages of using emulsified lipids as an experimental model are that there are a
limited number of food-grade emulsifiers that can be used to prepare them (e.g., milk proteins,
phospholipids, monoacylglycerides, and diacylglycerides) and that expensive equipment for their
homogenization and particle size reduction is required (McClements, 2010).
8.4. Simulated in vitro digestion of emulsified lipids: Advantages and
disadvantages
The effect of pectin on the gastrointestinal fate of emulsified lipids (chapters 4 and 5) was
evaluated by using a simulated in vitro digestion model designed to mimic the oral, gastric, and
small intestine phases of the human gastrointestinal tract (GIT). These phases involved mixing of
the sample (a corn oil-in-water emulsion mixed with different pectin samples and other sources
of dietary fibers at different concentrations) with simulated digestive fluids of variable
composition depending on the simulated phase. The temperature of the in vitro digestion model
was maintained at 37 °C to mimic human corporal temperature. An accurate simulation of the
human GIT should involve mimicking the exact composition and dynamics of the GIT fluids.
However, this is usually complicated to be implemented because the model would be difficult to
design and operate. Nevertheless, the utilization of the key components of the GIT fluids which
are able to impact the gastrointestinal fate of emulsified lipids as well as the simulation of the
GIT dynamics (e.g., mastication and both gastric and intestinal peristalsis were simulated by
orbital shaking) were taken into account for understanding the mechanisms involved in the lipid
digestion process (Hur, Lim, Decker, & McClements, 2011). Because emulsified lipids are fully
digested in the small intestinal phase (Li, Hu, & McClements, 2011; Singh, Ye, & Horne, 2009),
the simulation of the colonic phase was not performed. Furthermore, the colonic phase is usually
difficult to mimic because simulation of the anaerobic conditions requires special instrumentation
and the wide variety of colonic bacteria representing the colonic microbiota are often difficult to
preserve (Hur, Lim, Decker, & McClements, 2011).
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225
The advantage of using a simulated in vitro digestion model is that it provides a useful alternative
to in vivo models (e.g., cell culture, animal, and human models) by rapidly screening the effect of
different sources of dietary fibers in the gastrointestinal fate of emulsified lipids. Although in
vivo models often provide accurate results, they are time consuming, poorly reproducible,
expensive, and several ethical considerations should be taken into account when using animal
models (Li, Kim, Park, & McClements, 2012). Results obtained with in vitro models are often
different to those obtained with in vivo models because of the high complexity of the dynamics,
structure, and composition of the in vivo models (Koven, Henderson, & Sargent, 1997).
Consequently, a simulated in vitro digestion model must be designed to find an appropriate
balance between the accuracy and biological relevance of the results with the cost and the
affordable operability of the model (Hur, Lim, Decker, & McClements, 2011; McClements & Li,
2010).
The disadvantage of using a simulated in vitro digestion model is that the results are highly
dependent of the experimental conditions (McClements & Li, 2010). For example, the selection
of the type and concentration of enzymes (e.g., selecting between pepsin, trypsin, or
chymotrypsin), ion metal cofactors (e.g., selecting between CaCl2, MgCl2, ZnCl2, or NaCl), and
surface-active compounds (e.g., selecting between phospholipids or bile salts) is a key factor
defining the efficiency of the digestion process of emulsified lipids as well as the reliability and
the biological relevance of the results (Li, Hu, & McClements, 2011). Minor changes in these
parameters can significantly affect the final results and also limit their comparability with those
obtained with other experimental models (Bonnaire, Sandra, Helgason, Decker, Weiss, &
McClements, 2008).
For this reason, the simulated in vitro digestion model used in this thesis was selected because
this model has been widely studied (Hur, Lim, Decker, & McClements, 2011; McClements & Li,
2010; Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, et al., 2014), validated (Bonnaire,
Sandra, Helgason, Decker, Weiss, & McClements, 2008; Li, Hu, & McClements, 2011), and the
results obtained with this model have been shown to correlate with in vivo models (Li, Kim, Park,
& McClements, 2012).
Chapter 8
226
8.5. Franz diffusion cell as an in vitro model for evaluating the mass
transfer of nutrients
In chapter 7, the effect of both low (LMP) and high (HMP) methoxylated pectins on the rate and
extent of the mass transfer process of the primary nutritional compounds consumed in the diet or
produced after digestion such as monosaccharides (e.g., glucose, galactose, fructose, and ribose),
amino acids (e.g., glycine, aspartic acid, lysine, and tyrosine), and lipids (represented by a corn
oil-in-water emulsion) was evaluated. Both viscosity of the pectin solutions and the electrical
interactions between pectin and the nutritional compounds were proposed to be the main factors
determining the rate and extent of the mass transfer process. Franz diffusion cells have been used
successfully for several years in the pharmaceutical industry to evaluate transdermal drug
administration (Ng, Rouse, Sanderson, Meidan, & Eccleston, 2010). No studies had been
reported concerning the use of the Franz diffusion cells for studying the mass transfer kinetics of
nutritional compounds. Results presented in chapter 7 are among those few studies related to the
effect of pectin on the mass transfer kinetics of nutritional compounds (monosaccharides, amino
acids, and emulsified lipids) by using the Franz diffusion cell model. There are several
experimental parameters to be considered when using a Franz diffusion cell including i) the cell
design, ii) the agitation of the receptor compartment, and iii) the physicochemical nature of the
barrier membrane. These parameters are related to the reproducibility, reliability, and biological
relevance of the results.
i) The Franz diffusion cell is an experimental setup widely used for studying drug permeation
through several tissues. This has led to a great demand of these devices as well as the emergence
of a wide variety of designs depending on the requirements of each experiment. However, the
design of the Franz diffusion cell was standardized by the U.S. Food and Drug Administration
(FDA) to reduce the variability between the measurements, to reduce the cost of the experimental
setup, and to make the results comparable to other studies (Skelly, Shah, Maibach, Guy, Wester,
Flynn, et al., 1987). It has been established that the minimum volume of the donor and receptor
compartments of the Franz diffusion cell should be 1 and 10 mL, respectively, to prevent
Chapter 8
227
significant evaporation in long-period experiments (Bonferoni, Rossi, Ferrari, & Caramella,
1999). In our experimental setup, the volume of the donor and receptor compartments were 3 and
17 mL, respectively, allowing us to ensure that our results (e.g., effective diffusion coefficients)
are reliable and they may be comparable to other experiments. For example, we found that the
effective diffusion coefficients of glucose through the Franz diffusion cell were 1.44x10-5
and
0.48x10-5
cm2 s
-1 for control (containing no pectin) and 2% (w/w) HMP, respectively, at a
temperature of 37 °C, whereas the effective diffusion coefficients of glucose through a dialysis
membrane were reported to be 1.21x10-5
and 0.27x10-5
cm2 s
-1 for control (containing no malva
nut gum) and 1% (w/w) malva nut gum, respectively, at the same temperature (Srichamroen &
Chavasit, 2011). Therefore, our results concerning the effect of pectin on the mass transfer
process of nutritional compounds through a Franz diffusion cell can be compared to those
obtained with other experimental models.
ii) Agitation of the receptor compartment is a critical parameter for the maintenance of both
uniform nutrient distribution through the cell as well as temperature equilibrium (Ng, Rouse,
Sanderson, Meidan, & Eccleston, 2010). Nevertheless, agitation introduces a convective
parameter that also has an effect on the overall mass transfer process. It is important to consider
that the mass transfer process in the unstirred water layer of the intestinal lumen (where the
absorption process of nutrients takes place) is strictly controlled by diffusion, while convection
can be considered as negligible (Smithson, Millar, Jacobs, & Gray, 1981; Thomson & Dietschy,
1984). Therefore, agitation may have caused an overestimation on the effective diffusion
coefficients (related to the rate at which the mass transfer process occurs) because convective
effects often accelerates the mass transfer process of nutrients (Cussler, 2009). However,
agitation does not necessarily affect the extent of the mass transfer process of nutrients, it only
affects the time at which the mass transfer process can be completed (Cussler, 2009).
iii) Another key parameter affecting the mass transfer process of nutrients relates to the barrier
membrane used to separate the donor and the receptor compartment of the Franz diffusion cell.
Usually, the membranes used to simulate the absorption of nutrients must be biological
membranes capable to mimic both passive and active diffusion mechanisms occurring in in vivo
Chapter 8
228
systems (Schulthess, Lipka, Compassi, Boffelli, Weber, Paltauf, et al., 1994). The most common
biological membranes used to simulate the intestinal absorption process of nutrients are rat
intestinal microvillus membrane and pig small intestine brush border (Deglaire & Moughan,
2012; Gill, Smith, Wissler, & Kunz, 1989). However, biological membranes are often difficult to
obtain and preserve, in addition to the reproducibility of the results is sometimes unacceptable
(Schulthess, et al., 1994; Smithson, Millar, Jacobs, & Gray, 1981). Synthetic membranes (e.g.,
cellulose membrane) allow the permeation process to occur by simple diffusion (in the absence of
membrane proteins) rather than passive and active diffusion, which involves the presence of
membrane proteins (Lack & Weiner, 1961). Usually, simple diffusion is faster than both passive
and active diffusion because there are not proteins restricting the mass transfer process of
nutritional compounds through the membrane (Ng, Rouse, Sanderson, Meidan, & Eccleston,
2010). Therefore, the presence of a cellulose membrane in our experimental setup may have also
contributed to the overestimation on the effective diffusion coefficients of the nutritional
compounds because of the lack of selectivity of the cellulose membrane.
8.6. Future prospects
The effect of pectin on the activity of digestive enzymes was conducted by using artificial
chromogenic substrates (chapter 3). These substrates are structurally similar to their analogous
native substrates and they allowed obtaining fast, reliable, and reproducible results. However,
artificial substrates should only be used for comparison purposes because the enzymatic activities
obtained with these substrates are not necessarily comparable to those obtained by using native
substrates. Therefore, the evaluation of the inhibitory effect of pectin on the activity of the
digestive enzymes by using native substrates might lead to obtain results with greater biological
relevance as compared to those obtained with artificial substrates.
Comparisons of the functional properties of different pectins must be done carefully because of
the diversity of pectins that can be obtained from different plant sources. In further studies,
detailed information on the chemical structure of pectins must be obtained. For example, it has
been pointed out that structural features of pectins such as the acetylation degree, branching
Chapter 8
229
degree, contents of rhamnose, arabinose and galactose, and the water holding capacity are closely
related to their functional properties, besides methoxylation degree and molecular weight (James,
1986). In addition, the evaluation of the functional properties of several pectin samples with
different structural features is required to identify the relative contribution of the individual
structural characteristics to the overall effect. Furthermore, those physicochemical properties
must be independent to each other to discriminate the relative effect produced by each parameter
individually.
Simulated in vitro gastrointestinal digestion of emulsified lipids is widely employed in many
fields of the food science and nutrition research. Several in vitro digestion models have been
proposed depending on the purpose of the research and the availability of resources. Therefore,
the possibility to compare results with other investigations is often difficult because of the
variability of experimental conditions used in each model. For example, a large variety of
digestive enzymes from different sources have been employed. In addition, differences in pH,
ionic strength, and digestion times may also affect the results of the digestion process. Therefore,
we propose to use the recently published standardized in vitro digestion model (Infogest
protocol) coming from an international consensus that it is suitable for application in several
experimental situations and allowing to obtain more comparable results in future research
(Minekus, et al., 2014). In addition, the sophistication level of the simulated in vitro digestion
model can significantly affect the reliability and biological relevance of the results (Hur, Lim,
Decker, & McClements, 2011; McClements & Li, 2010). Analytical instruments designed to
simulate the full digestion process are currently available, e.g., the TIM (Intestinal Tract Model)
lipid absorption system (TNO Innovation for Life, Zeist, The Netherlands) (Dickinson, Abu
Rmaileh, Ashworth, Barker, Burke, Patterson, et al., 2012). The TIM model has been developed
to simulate the full dynamic and physicochemical complexity of the human GIT. The TIM model
allows performing studies concerning the digestibility and bioaccessibility of several food
components including lipids, proteins, minerals, and vitamins (Dickinson, et al., 2012).
Therefore, analytical instruments for simulating the dynamics of the GIT would lead to obtain
reliable results with significant biological relevance.
Chapter 8
230
The rate and extent of the in vitro digestion process of emulsified lipids, as well as the
bioaccessibility and bioavailability of emulsified lipids must be correlated with in vivo
experiments by using cell or animal models. For example, the human intestinal Caco-2 cell line
has been extensively used as a model of the intestinal barrier to simulate the absorptive properties
of the intestinal mucosa and to evaluate both bioaccessibility and bioavailability of food
components (Sambuy, De Angelis, Ranaldi, Scarino, Stammati, & Zucco, 2005). In addition,
animal models have been used for determining several food components digestibility,
bioaccesibility, and bioavailability (Deglaire & Moughan, 2012). For example, Wistar rat (Rattus
norvegicus) is usually considered as a suitable animal model for digestion experiments because
its upper GIT is anatomically and physiologically similar to that of the humans (Gill, Smith,
Wissler, & Kunz, 1989). In addition, Wistar rats are usually easy to rise and relatively
inexpensive to maintain, and the results that can be obtained are biologically relevant. However,
it is important to consider the large variability of the results that can be obtained between
experimental units depending on gender, age, feeding, and environmental conditions, besides the
ethical considerations that must be taken into account when using animals as experimental
models (Deglaire & Moughan, 2012; Gill, Smith, Wissler, & Kunz, 1989). As aforementioned,
the correlation of the results obtained in this study by using in vitro models must be correlated
with results obtained by in vivo models (e.g., cell lines or Wistar rats) to validate the biological
relevance of the results obtained here and to enhance the possibility to extrapolate these results to
more complex models (e.g., clinical trials with humans).
Finally, the results obtained in this thesis would lead to the rational design of pectin-based
functional and nutraceutical foods targeted for populations with different susceptibilities of
suffering several cardiovascular diseases (e.g., adult and elderly populations). It is well known
that pectins are able to significantly impact the sensory and physicochemical properties of pectin-
enriched foodstuffs; therefore, the rational design of pectin-based functional foods would lead to
obtain a functional food which retains its desirable sensory and textural properties.
Chapter 8
231
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Concluding remarks
233
Concluding remarks
The results obtained in this thesis showed that pectin is able to exert its physiological functions
by several mechanisms including the inhibition of the activity of the major digestive enzymes
(pancreatic lipase, -amylase, alkaline phosphatase, and protease) and the modulation of the rate
and extent of both the digestion of emulsified lipids (predominantly the extent of the digestion
process of emulsified lipids rather than the digestion rate) and the mass transfer of the most
important nutritional compounds (monosaccharides, amino acids, and emulsified lipids). These
effects may have been due to i) the impact of pectin on the activity of the digestive enzymes
(pectins behave as non-competitive inhibitors of the digestive enzymes), ii) the rheological
properties of the gastrointestinal fluids (pectins inhibited both the rate and extent of the mass
transfer process of nutrients by increasing the viscosity), iii) the interaction of pectin with key
gastrointestinal components [e.g., pancreatic lipase, electrolytes (Na⊕ and Ca2⊕), and bile salts],
and iv) the alteration in the lipid droplet aggregation state (pectin promoted depletion flocculation
of emulsified lipids). In addition, the structural characteristics of pectin (e.g., methoxylation
degree and molecular weight) have a significant effect on the magnitude by which such effects
may occur. However, further studies are required to discriminate the relative contribution of each
mechanism to the overall inhibitory effect as well as the relative contribution of the individual
structural characteristics of pectin, including the methoxylation degree and molecular weight. The
results obtained in this thesis have important implications for the rational design of pectin-based
functional and nutraceutical foods that may modulate lipid digestion within the gastrointestinal
tract. The design of pectin-based functional and nutraceutical foods is focused to give healthier
lipid profiles and thereby promoting health and wellness of the consumers. The inclusion of
pectin in food formulations might be also an effective strategy for controlling the calorie uptake
by limiting the digestion and absorption of nutritional compounds. However, the formulation of
foodstuffs with high pectin contents must be done carefully in countries whose inhabitants are
deficient in vitamins (e.g., B-complex vitamins and vitamin E) and minerals (e.g., iron, calcium,
and zinc) since the consumption of pectin has been associated with a decreased bioaccesibility of
these nutritional compounds.
Academic production
234
Academic production
List of publications
1. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., & Narváez-Cuenca, C. E.
(2014). Inhibition of digestive enzyme activities by pectic polysaccharides in model
solutions. Bioactive Carbohydrates and Dietary Fibre. 4: 27-38.
2. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E., &
McClements, D. J. (2014). Impact of dietary fibers [methyl cellulose, chitosan, and pectin] on
digestion of lipids under simulated gastrointestinal conditions.
Food & Function. 5: 3083-3095.
3. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E., &
McClements, D. J. (2014). Interaction of a dietary fiber (pectin) with gastrointestinal
components (bile salts, calcium, and lipase): A calorimetry, electrophoresis, and turbidity
study. Journal of Agricultural and Food Chemistry. 62: 12620-12630.
4. Espinal-Ruiz, M., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E., & McClements, D. J.
(2016). Impact of pectin properties on lipid digestion under simulated gastrointestinal
conditions: Comparison of citrus and banana passion fruit (Passiflora tripartita var.
mollisima) pectins. Food Hydrocolloids. 52: 329-342.
5. Espinal-Ruiz, M., Restrepo-Sánchez, L. P., & Narváez-Cuenca, C. E. (2016). Effect of
pectins on the mass transfer kinetics of monosaccharides, amino acids, and a corn oil-in-
water emulsion in a Franz diffusion cell. Food Chemistry. 209: 144-153.
Academic production
235
Conferences and meetings
1. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., & Narváez-Cuenca, C. E.
(2014). Inhibition of digestive enzyme activities by pectic polysaccharides. Presented as Oral
communication. Food Structure and Functionality Forum Symposium: From Molecules to
Functionality. March 30 to April 02. Amsterdam, The Netherlands.
2. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., & Narváez-Cuenca, C. E.
(2014). Inhibition of digestive enzyme activities by pectic polysaccharides in model
solutions. Presented as Poster. International Food Physics Symposium. May 08. Amherst
(MA), United States.
3. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E., &
McClements, D. J. (2014). Impact of polysaccharides on digestion of lipids under simulated
gastrointestinal conditions. Presented as Oral communication. 2014 Annual Conference &
Exhibition: Functional Foods, Nutraceuticals, Natural Health Products, and Dietary
Supplements. October 14 to 17. Istanbul, Turkey.
4. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E., &
McClements, D. J. (2015). Interaction of pectin with gastrointestinal components: A
calorimetry, microelectrophoresis, and turbidity study. Presented as Oral communication.
Delivery of Functionality in Complex Food Systems: Physically-Inspired Approaches from
the Nanoscale to the Microscale. July 14 to 17. Paris, France.
5. Espinal-Ruiz, M., Restrepo-Sánchez, L. P., Narváez-Cuenca, C. E., & McClements, D. J.
(2016). Impact of pectin properties on lipid digestion under simulated gastrointestinal
conditions: Comparison of citrus and banana passion fruit (Passiflora tripartita var.
mollisima) pectins. Presented as Oral communication. 2nd
Food Structure and Functionality
Forum Symposium: From Molecules to Functionality. February 28 to March 02. Singex,
Singapore.
Acknowledgements
236
Acknowledgements
I would like to express my gratitude to those who have contributed to this thesis, either directly or
indirectly. First of all, I would like to thank my director, Carlos-Eduardo Narváez-Cuenca, for his
continuous guidance during the PhD program and for his contribution to the development of the
work presented here. Thank you for all your feedback and support, not only within the thesis
project, but also for my personal development.
I also want to thank to professors Luz-Patricia Restrepo-Sánchez and Fabián Parada-Alfonso for
giving me the chance to carry out my doctoral thesis. I am proud having both of you as “co-
directors”. I would like to thank to professors Cecilia Anzola and Laura Ortiz for their
contributions to the thesis. For all involved in the research group: Thanks for the collaboration we
had, it was a great experience working in such a special team: Eliana, Katherine, Elizabeth,
Mayra, Jonathan, Nelson, Mónica, Diego, Paola, Catalina, and Patricia.
A special thanks to Dr. David Julian McClements (Department of Food Science, University of
Massachusetts Amherst) and his team for having received me in the Food Biopolymers and
Colloids Research Laboratory and for helping me to achieve the objectives proposed in this
thesis: Jean, Iris, Amir, Ben, Cheryl, Zachary, Vanessa, Gabriel, Penny, Bengü, Becca, Dr. Hu,
Laura, Tommy, Patrick, Izlia, Eric, Cynthia, Jennifer, Saehun, Jenny, Xuan, and Bicheng.
I would like to thank to my granters for funding me along the thesis: COLCIENCIAS-
COLFUTURO (Convocatoria No. 528, Beca Francisco José de Caldas para estudios de doctorado
en Colombia) and Vicerrectoría Académica from Universidad Nacional de Colombia (Beca
Estudiante Sobresaliente de Posgrado).
Finally, I would like to say a very special thank you for my family (my parents and brothers), and
for my wife. Jeimmy, Meeting you is the best, unexpected thing that happened to me. Thanks for
supporting me and understanding me all these years. I would like to dedicate this thesis to you.
Of course, Gretta could not miss. Thanks for brightening our lives.
