Article

pubs.acs.org/JAFC

Interaction of a Dietary Fiber (Pectin) with Gastrointestinal

Components (Bile Salts, Calcium, and Lipase): A Calorimetry,
Electrophoresis, and Turbidity Study
Mauricio Espinal-Ruiz,†,‡ Fabián Parada-Alfonso,† Luz-Patricia Restrepo-Sánchez,†
Carlos-Eduardo Narváez-Cuenca,† and David Julian McClements*,‡,§
†
Departamento de Quı ́mica, Facultad de Ciencias, Universidad Nacional de Colombia, AA 14490 Bogotá, Colombia
‡
Food Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, 102 Holdsworth
Way, Amherst, Massachusetts 01003, United States
§
Department of Biochemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

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. Pectin reduced the extent of lipid digestion, which was attributed to its binding
interactions with specific 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 because this
would reduce the surface area of lipid exposed to the lipase. This research provides valuable insights into the physicochemical and
molecular mechanisms of the interaction of pectin with gastrointestinal components that may affect the rate and extent of lipid
digestion.
KEYWORDS: pectin, gastrointestinal tract, lipid digestion, isothermal titration calorimetry, turbidity, flocculation

■ 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.1 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 fibers can inhibit lipid digestion and absorption in the
small intestine.2−9 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.10 Water-insoluble fibers, such as lignins,
celluloses, and some hemicelluloses, play an important role in
regulating intestinal peristalsis.11 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.12 Pectin is a
water-soluble dietary fiber that is used widely in the
pharmaceutical, biotechnology, and food industries as a
functional ingredient.13 Pectin is a polysaccharide that has
linear anionic regions formed by D-galacturonic acid (GalA)
monomers, linked by α-(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.13−17 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.13,18 The
gelling characteristics of pectin have been utilized to form
hydrogel delivery systems for a range of pharmaceutical and
food bioactive compounds.18,19
Previous studies have shown that pectin reduces the rate and
extent of lipid digestion by interacting with various food and
intestinal components.3,6,8 A number of different physicochem-
ical 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.7,9 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.20 Pectin molecules may interfere with the
reabsorption of bile salts in the small intestine, thereby reducing
Received: October 6, 2014
Revised: November 17, 2014
Accepted: December 5, 2014
Published: December 5, 2014

© 2014 American Chemical Society 12620 dx.doi.org/10.1021/jf504829h | J. Agric. Food Chem. 2014, 62, 12620−12630
Journal of Agricultural and Food Chemistry
the amount of cholesterol absorbed and transported to the
blood.20 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 affect lipid
digestion, for example, by altering gastric emptying times or the
mass transport of digestive enzymes.3 Pectin may also directly
interact with other components in the gastrointestinal tract
such as CaCl2, NaCl, and digestive enzymes.9,21−23 Finally,
pectin is known to promote depletion flocculation of lipid
droplets, which may reduce lipid digestion by protecting them
from attack by lipolytic enzymes.24,25
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.17 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.14,15,18,19 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.26−28 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 stoichiome-
tries.29,30 In addition, electrophoresis, turbidity, and micro-
structural 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.
■
MATERIALS AND METHODS
Chemicals. Corn oil was purchased from a commercial food
supplier (Mazola, ACH Food Co. Inc., Memphis, TN, USA) 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-methoxyl 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 esterification of approximately 62%. The average
molecular weight was reported to be 200 kDa. Lipase from porcine
pancreas (type II, L3126, triacylglycerol hydrolase EC 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 EC 3.4.23.1, activity ≥ 250 units/mg
solid) were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO, USA). The supplier reported that the lipase activity was 100−400
units/mg protein (using olive oil) and 30−90 units/mg protein (using
12621
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triacetin) for 30 min of incubation (1 unit of lipase activity was defined
as the amount of enzyme required for the release of 1 μequiv 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% hydro-
deoxycholic acid, and 0.5−2% cholic acid; 5% (w/w) phosphatidylcho-
line (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 Co. Double-distilled water was used to prepare all solutions.
Simulated Gastrointestinal Studies. Solution and Emulsion
Preparation. Pectin stock solution (4% 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 and
equilibrated for 10 min before analysis.
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, containing 2.5% (w/w) Tween 80) together for 5 min using a
biohomogenizer (speed 2, model MW140/2009-5, Biospec Products
Inc., ESGC, Switzerland). The resulting coarse emulsion was then
passed five times through a high-pressure homogenizer (microfluidizer
M-110L processor, Microfluidics Inc., Newton, MA, USA) operating at
11000 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% (w/w) pectin) to obtain systems of various
compositions: 2% (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, USA) at 1000 rpm and stored overnight at room temperature. The
pectin−emulsion mixtures were characterized to obtain the initial
phase, prior to subjection to the in vitro gastrointestinal model.
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.31,32
Simulated saliva fluid (SSF, pH 6.8) containing 3% (w/w) mucin
was prepared according to the composition shown in Table 1. Each
Table 1. Chemical Composition of Simulated Saliva Fluid
(SSF) Used To Simulate Oral Conditions
concentrationa
compound chemical formula (g/L)
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
a
The SSF was prepared in double-distilled water and then pH 6.8 was
adjusted.
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 an
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, New Brunswick, NJ,
USA). The mixture resulting from processing of the initial emulsions
in the oral phase (bolus) was used in the gastric phase.
dx.doi.org/10.1021/jf504829h | J. Agric. Food Chem. 2014, 62, 12620−12630
Journal of Agricultural and Food Chemistry
Simulated gastric fluid (SGF) was prepared by adding 2 g of NaCl, 7
mL of concentrated HCl (37% w/w), and 3.2 g of pepsin A (from
porcine gastric mucose, 250 units/mg) to a flask, then diluting the
mixture with double-distilled water to a volume of 1 L, and finally
adjusting the pH to 1.2 using 1 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 0.05−0.9% (w/w) pectin. This
mixture was then adjusted to pH 2.5 using 1 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.
Samples obtained from the gastric phase (20 mL) were incubated
for 2 h at 37 °C in a simulated small intestine fluid (SIF) consisting of
2.5 mL of pancreatic lipase (24 mg/mL), 3.5 mL of bile extract
solution (54 mg/mL), and 1.5 mL of salt solution containing 0.25 M
CaCl2 and 3 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 (FFA) released were monitored by determining the
amount of 0.1 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.). All components were dissolved in
5 mM phosphate buffer solution (pH 7) before use. Lipase addition
and initialization of the titration program were carried out only after
the addition of all predissolved ingredients and balancing the pH to 7.
The volume of 0.1 M NaOH added to the emulsion was recorded over
time and used to calculate the FFAs generated by lipolysis. The
amount of FFAs released was calculated using the equation
FFA (% w/w) = 100
⎛ VNaOH (L) × C NaOH (M) × MWlipid (g/mol) ⎞
× ⎜⎜ ⎟⎟
⎝ 2 × wlipid (g) ⎠ microscopy. An optical microscope (C1 Digital Eclipse, Niko, Tokyo,
(1)
where CNaOH is the concentration of the sodium hydroxide (0.1 M),
MWlipid is the average molecular weight of corn oil (872 g/mol), wlipid
is the initial weight of corn oil in the intestinal phase (0.1 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.
Creaming Stability Measurements of Initial Emulsions. Ten
milliliters of emulsions (initial phase) 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
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, For-
mulaction, Wynnewood, PA, USA). 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, because an increase in particle size (e.g., due to
flocculation) leads to faster creaming (provided the droplet
concentration is not too high).
Interaction of Pectin with Gastrointestinal Components.
Solution 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) 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 stock solution
containing a mixture of all the above components at the same initial
concentrations as in their individual solutions was also prepared. This
stock solution was referred to as having a relative concentration of
“100%” of the digestive components. A series of mixtures containing
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lower amounts of the digestive components was prepared by diluting
the stock solution with buffer solution.
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 solution at 37 °C. Twenty-nine 100 μL aliquots of
individual solutions containing 2.7% (w/w) bile salts, 1.1% (w/w)
lipase, 55 mM CaCl2, 545 mM NaCl, or 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) or
0.9% (w/w) pectin solutions. Turbidity, ζ-potential, and micro-
structure (observed by optical microscopy) of these solutions were
then characterized throughout the titration process.
Solution Characterization. Optical turbidity of the solutions was
determined by measuring their absorbance at 600 nm (Δτ = 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 Δτ = τpectin − τbuffer. Triplicate measurements of turbidity
were carried out on each sample.
The electrical charge (ζ-potential) of the solutions was determined
using a particle microelectrophoresis instrument (Zetasizer NanoS-
eries, Malvern Instruments Ltd., Worcestershire, UK). Solutions were
injected into the measurement chamber and equilibrated for 120 s, and
then the ζ-potential was determined by measuring the direction and
velocity that the particles moved in the applied electric field. Each
individual ζ-potential measurement was calculated from the average of
20 continuous readings made per sample. The ζ-potential was
recorded at each pH after 60 s of equilibrium.
The microstructure of the solutions was characterized by optical
Japan) with a 60× objective lens was used to capture images of the
solutions. Solutions were gently stirred to form a homogeneous
mixture without introducing any air bubbles. A small aliquot of the
solutions (5 μL) was then transferred to a glass microscope slide and
covered with a glass coverslip. The coverslip 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
coverslip. All optical images were taken using a digital camera and then
characterized using the instrument software (EZ CS1 version 3.8,
Nikon).
Isothermal Titration Calorimetry Measurements. An ITC instru-
ment (Microcalorimeter VP-ITC, MicroCal Inc., Northampton, MA,
USA) was used to measure the enthalpy (ΔH) resulting from titration
of individual solutions of CaCl2, NaCl, lipase, bile salts, and a mixture
of all components (100% relative concentration) into either buffer
solution (blank) or pectin solution. Twenty-nine 10 μL 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 μL titration cell initially containing either buffer (5 mM
phosphate buffer, pH 7) or 0.9% (w/w) pectin solutions. The range of
concentrations of pectin and gastrointestinal components used was
selected to approximately mimic the concentrations in the small
intestinal phase of the gastrointestinal tract. 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 profiles were carried out on each
sample, and they were reproducible to better than 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.
■
RESULTS AND DISCUSSION
Properties of Initial Emulsion−Pectin Mixtures.
Initially, the properties of the initial emulsions used to prepare
the emulsion−pectin mixtures were characterized (5 mM
dx.doi.org/10.1021/jf504829h | J. Agric. Food Chem. 2014, 62, 12620−12630
Journal of Agricultural and Food Chemistry
phosphate buffer, pH 7). 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.33 This can be attributed to the relatively strong
steric repulsion between the nonionic surfactant-coated lipid
droplets resulting from the polyoxyethylene headgroups on the
Tween 80 molecules.34 The lipid droplets initially had a small
negative charge (ζ = −5.8 ± 0.2 mV), even though they were
stabilized by a nonionic surfactant, which can be attributed to
anionic impurities in the oil or surfactant ingredients (such as
free fatty acids) or to preferential adsorption of hydroxyl ions
from water (OH−).35,36 The droplets in the initial emulsions
containing no pectin were stable to creaming throughout the
experimental time frame (Figure 1), which is due to the fact
that the droplets were small and did not associate with each
other.37
Figure 1. Influence of pectin concentration on creaming stability of 2%
(w/w) corn oil-in-water emulsions stabilized with 0.2% (w/w) Tween
80 before the digestion process.
Emulsions containing relatively low levels of added pectin
(≤0.4% w/w) were still observed to be stable to gravitational
separation; that is, they maintained a uniform white appearance
(Figure 1). They also had good stability to droplet aggregation
with the mean particle diameter (d32) increasing only from
around 200 to 210 nm when the pectin concentration was
Figure 2. Influence of pectin concentration on free fatty acids (FFA) released during lipid digestion process (pH stat): (a) kinetics; (b) FFA released
after 2 h. Pectin concentrations refer to those present in the intestinal phase.
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increased from 0 to 0.2% (w/w). However, emulsions
containing higher levels of pectin clearly separated into a
creamed layer and a serum layer, indicating that the droplets
moved upward due to gravity. The origin of this effect is
depletion flocculation induced by the presence of nonadsorbed
pectin molecules within the aqueous phase.24,38 There was an
appreciable increase in mean particle diameter in the samples in
which depletion flocculation occurred, for example, d32 = 350
nm in the sample initially containing 2.4% (w/w) pectin. This
effect can be attributed to the fact that the droplets were
pushed together in the flocs due to the depletion attraction,
which promoted coalescence. Pectin molecules should not be
attracted to the surfaces of the nonionic surfactant-coated lipid
droplets because of electrostatic and steric repulsion effects (the
carboxyl groups of pectin are completely dissociated at pH 7
because 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.39 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) because when they come
into close contact, the volume of the thermodynamically
unfavorable exclusion zone is reduced.25 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), and so the emulsion remains stable to
droplet aggregation. However, once a critical pectin concen-
tration is exceeded (∼0.5% w/w), the attractive forces exceed
the repulsive forces and droplet flocculation occurs.24 It should
be noted that the main thermodynamic driving force for
depletion flocculation is entropy.40,41 The reason that the
thickness of the creamed layer increases (CI decreases) as the
pectin concentration increases above the critical flocculation
concentration (Figure 1) is that the depletion attraction is
higher and so the droplets are held more strongly into a particle
gel network.42 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.
Influence of Pectin on Lipid Digestion. In a recent
study, we examined the influence of pectin addition on the
dx.doi.org/10.1021/jf504829h | J. Agric. Food Chem. 2014, 62, 12620−12630
Journal of Agricultural and Food Chemistry
gastrointestinal fate of emulisified lipids.33 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 so that 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 2a). The final
amount of FFAs produced at the end of the 2 h digestion
period decreased with increasing pectin concentration (Figure
2b), which is in good agreement with our earlier study.33
The ability of pectin to decrease the rate and extent of lipid
digestion in corn oil-in-water emulsions could be due to several
physicochemical and physiological mechanisms. 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.43−45 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.46 Calcium ions normally form insoluble soaps with
long-chain FFAs that help remove the FFAs from the lipid
droplet surfaces and thereby allow the lipase to keep working.47
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 adsorbing
to lipid droplet surfaces or from solubilizing lipid digestion
products.10,18,44 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
functioning.34 The adsorption of bile salts and phospholipids to
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.21,48
Finally, the presence of pectin in the system may alter the
aggregation state of the lipid droplets through either depletion
or bridging flocculation.49 The digestion of highly flocculated
lipid droplets is often less than that of nonflocculated ones
because the lipase molecules have to diffuse through the flocs
before they can reach the lipid droplets in their interiors.23,50
Clearly, the potential influence of pectin on the lipid digestion
process within the GIT is complex because pectin is able to
interact with many constituents within the GIT fluids.
The purpose of the current study was to provide further
insights into the potential origin of these effects by studying the
interactions between pectin and specific gastrointestinal
components.
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 3). In the absence of
pectin (control), the individual lipid droplets were too small to
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Figure 3. Influence of pectin on the microstructure (observed by
optical microscopy) of emulsions before (initial phase) and after
(intestinal phase) of lipid digestion. The initial emulsions contained
2% (w/w) corn oil, 2% (w/w) Tween 80, and 0 or 2.4% (w/w) pectin.
The scale bar corresponds to 20 μm.
be observed by optical microscopy, but confocal microscopy
images have shown that they are evenly distributed throughout
the system.33 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, which can be attributed to
depletion flocculation induced by nonadsorbed pectin mole-
cules (Figure 3).25 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 (Δτ), ζ-potential, and enthalpy change (ΔH) 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 (Figure 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 4a). The ζ-
potential of the mixed system moved from highly negative
(−19 mV) to less negative (−2 mV) as increasing amounts of
GIT fluids were added (Figure 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.51 There was also an appreciable difference in
the enthalpy profiles of samples with either buffer or pectin
solutions (Figure 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.22,27,30 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 0−20% of the GIT
fluids was added, until it eventually reached a relatively constant
endothermic value at higher GIT levels (∼40%). 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.
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Journal of Agricultural and Food Chemistry Article

Figure 4. Influence of relative concentration (0−100%) of simulated gastrointestinal fluids on (a) optical turbidity (Δτ = A600 nm) and ζ-potential
and (b) interaction enthalpy (ΔH) 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
(Δτ) was defined as Δτ = τpectin − τbuffer.

Figure 5. Influence of the concentrations of NaCl, CaCl2, bile salts, and lipase on optical turbidity (Δτ = A600 nm) and ζ-potential of solutions
containing an initial concentration of 0.9% (w/w) pectin.

Interactions of Pectin with Specific Gastrointestinal
Components. In this series of experiments, ITC, electro-
phoresis, 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 5).
12625
In addition, the turbidities and ζ-potentials reached at the end
of these titrations were reported so that different samples could
be compared more easily (Figure 6).
NaCl. Addition of increasing amounts of NaCl to the pectin
solutions caused an appreciable decrease in the magnitude of
the ζ-potential of the pectin solutions (Figure 5, NaCl), which
can be attributed to electrostatic screening effects, that is,
dx.doi.org/10.1021/jf504829h | J. Agric. Food Chem. 2014, 62, 12620−12630
Journal of Agricultural and Food Chemistry
Figure 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 (Δτ = A600 nm) (a) and ζ-potential (b) of solutions containing an initial concentration of 0.9% (w/w)
pectin (P).
accumulation of sodium ions around the negative groups on the
pectin molecules.51 There was no evidence of an increase in
aggregate formation within the microstructure images (Figure
8, NaCl), and there was little change in the general appearance
(Figure 7, NaCl) and optical turbidity (Figure 5, NaCl) of these
Figure 7. Influence of the concentrations of NaCl, CaCl2, bile salts,
lipase, and a mixture of all components (% relative concentrations) on
the turbidity of solutions containing an initial concentration of 0.9%
(w/w) pectin.
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.52 The ITC measurements indicated that there was
not a strong interaction (ΔH = −102 kcal) between the pectin
12626
Article
Figure 8. 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 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
μm.
and the NaCl (Figure 9, NaCl), because 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.
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 ζ-potential of the pectin solutions to go from around −19
mV to around −6 mV (Figure 5, CaCl2), which can be
attributed to electrostatic screening and ion binding effects.51
Multivalent counterions are more effective at screening
electrostatic interactions than monovalent ones, and they
have a greater tendency to bind strongly to oppositely charged
groups.53 The change in the ζ-potential with calcium addition
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Journal of Agricultural and Food Chemistry
Figure 9. Influence of the concentrations of NaCl, CaCl2, bile salts, and lipase on interaction enthalpy (ΔH) of solutions containing either buffer
solution or an initial concentration of 0.9% (w/w) pectin.
was fairly similar to that observed with the addition of the
mixed GIT solution (Figure 6b), which suggests that calcium
ions play an important role in determining the overall electrical
interactions. The ITC measurements also indicated that there
was a strong interaction (ΔH = −241 kcal) between the
calcium ions and pectin (Figure 9, CaCl2). 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.22 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 9, CaCl2). 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. Never-
theless, it is not possible to determine the molecular origin of
these events from ITC measurements alone because they
provide only the overall enthalpy change.
The addition of calcium ions to the pectin solutions caused a
large increase in their optical turbidity (Figures 5 (CaCl2) and
6a), which can be attributed to the appearance of large
aggregates, as observed within the optical microscopy images
(Figures 7 and 8, CaCl2). We postulate that these aggregates
were microgels formed by pectin in the presence of
calcium.43,45 The calcium ions may have cross-linked the
anionic pectin molecules through electrostatic bridging.13 As
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Article
mentioned earlier, the binding of calcium ions to pectin may be
important for a number of reasons. First, pectin−calcium
microgels may trap lipid droplets inside, thereby restricting the
access of lipase to the lipid substrates.23 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.47 In practice, there will be other types of salts in the
gastrointestinal fluids that may alter the interactions of calcium
with pectin through electrostatic screening or binding effects. It
should be noted that there may have been some sodium or
calcium present in the ingredients used in this study (such as
pectin), and these ions could also contribute to the observed
electrostatic interactions.
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 caused only a
relatively small decrease in the negative charge on the pectin
molecules (Figure 5, bile salts), which can probably be
attributed to electrostatic screening effects.7,9 There was an
appreciable increase in the optical turbidity of the pectin
solutions when bile salts were added (Figures 5 (bile salts) and
6a), and the solutions appeared visibly cloudier (Figure 7, bile
salts). These effects can be attributed to the formation of large
aggregates within the bile salts−pectin solutions as observed by
optical microscopy (Figure 8, bile salts). The ITC measure-
ments also indicated that the bile salts interacted with the
pectin molecules. In the absence of pectin, there was a relatively
dx.doi.org/10.1021/jf504829h | J. Agric. Food Chem. 2014, 62, 12620−12630
Journal of Agricultural and Food Chemistry
large endothermic enthalpy change (ΔH = 204 kcal), which
progressively decreased with increasing bile salt concentration
(Figure 9, bile salts). 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 nonpolar
groups to water.54 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 9, bile salts). The large difference
between the two curves suggests that there was an interaction
between pectin and bile salts; for example, bile salt micelles may
have bound to pectin molecules and/or promoted their
aggregation.55
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 inter-
actions.56 In general, the hydrophobic interactions of pectin
with bile salts are known to increase with the degree of
esterification of pectin molecules (by increasing its hydro-
phobicity) and the molecular weight (viscosity) of the pectin
molecules.20 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 2. If the bile salts form microgel particles with pectin,
then they may not be available to absorb to 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.9
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 ζ-potential of the pectin (Figure 5,
lipase), which may have been due to some binding of lipase
molecules to the pectin molecules. In addition, there was an
appreciable difference between the ITC curves of the control
and the system containing pectin (Figure 9, lipase), which also
suggests that some form of interaction occurred (ΔH = −129
kcal).21 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 (0−0.1 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 5, lipase), but there was little
change in their overall visual appearance (Figure 7, lipase) or
their microstructure (Figure 8, lipase). These results suggest
either that 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.
For simple systems involving the binding of a ligand to
another molecule, ITC is a powerful tool for determining
thermodynamic properties, such as the number and strength of
binding sites and the nature of the enthalpy change (ΔH)
12628
Article
associated with the interaction (endothermic or exothermic).
For more complex systems, the interpretation of ITC data is
more difficult because many different molecular and
physicochemical events contribute to the overall enthalpy
changes, such as binding, conformation changes, and
aggregation. Consequently, it is not possible to establish the
precise nature of the different contributions to the measured
ITC signal. Nevertheless, ITC measurements do still provide
valuable information, because they indicate that one or more
types of interaction are taking place between two molecules,
and they show the range of concentrations over which these
intearctions occur. Typically, ITC measurements should be
combined with other techniques to provide a more detailed
understanding of the nature of the interactions involved, such
as chromatography, scattering, spectrometry, or spectroscopy
methods.
The purpose of this study was to investigate the interaction
of pectin molecules with some of the major constituents within
gastrointestinal fluids, that is, 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 emulsified lipids to be accessed
by gastrointestinal lipases and thereby inhibiting digestion. If
appreciable amounts of calcium and bile salts are trapped within
these microgels, then they will not be able to remove long-chain
fatty acids 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 delaying 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.
■
AUTHOR INFORMATION
Corresponding Author
*(D.J.M.) E-mail: mcclements@foodsci.umass.edu. Phone:
(413) 545-1019. Fax: (413) 545-1262.
Funding
We are grateful to COLCIENCIAS and Universidad Nacional
de Colombia for providing a fellowship to M.E.-R. supporting
this work. This material is partly based upon work supported by
the U.S. Department of Agriculture, NRI Grants (2011-03539,
2013-03795, 2011-67021, and 2014-67021).
Notes
The authors declare no competing financial interest.
■
ABBREVIATIONS USED
FFA, free fatty acid; GalA, D-galacturonic acid; GIT, gastro-
intestinal tract; ITC, isothermal titration calorimetry; SSF,
simulated saliva fluid; SGF, simulated gastric fluid; SIF,
simulated intestinal fluid
■
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