The Influence of Guar Gum on Lipid Emulsion Digestion and Beta-Carotene
Bioaccessibility
By
Jamal Amyoony
A thesis
presented to
The University of Guelph
In partial fulfillment of requirements for the degree of Master of Science
In
Human Health and Nutritional Sciences
Guelph, Ontario, Canada
© Jamal Amyoony, December 2013
ABSTRACT
The Influence of Guar Gum on Lipid Emulsion Digestion and Beta-Carotene
Bioaccessibility
Jamal Amyoony
University of Guelph, 2013
Advisor:
Dr. Amanda J. Wright
A better understanding of how dietary fibres impact the bioavailability of fatsoluble nutrients and nutraceuticals is required. The purpose of this research was to
determine the influence of guar gum (GG) on the transfer processes impacting betacarotene (BC) bioaccessibility (transfer to the aqueous phase) from an oil-in-water
emulsion using an in vitro model simulating gastric and duodenal digestion. Canola oil
emulsions (1.5 % soy protein isolate, 10 % canola oil and 0.1 % all trans BC, D4,3~160
nm) were prepared by microfluidization (40 MPa, 4 passes) and exposed, in the presence
of 0.0, 1.0, 1.5, 2.0, or 4.0 % GG, to conditions representative of the stomach and
duodenum in the fed state. Lipolysis, BC bioaccessibility, digestate apparent viscosities,
droplet size, and bile acid (BA) binding were studied. With increasing concentration of
GG, digestate viscosity was increased and lipolysis and bioaccessibility were decreased
(P<0.05). Peak lipolysis was 56.2% vs. 21.6% for emulsions containing 0.0 % vs. 4.0 %
GG, respectively. BC bioaccessibility was also lower in the presence of GG (i.e. 29.7 vs.
6.98 % for 0.0 vs. 4.0 % GG respectively). Thus, the presence of GG impacted digestive
processes central to BC absorption. The impact of GG may be related to increased
digestate viscosity entrapping mixed micelles or BAs and decreasing diffusion leading to
decreased lipolysis and BC bioaccessibility.
.
Acknowledgements
I would like to first and foremost thank my advisor, Dr. Amanda Wright. Without
your guidance, constant support, feedback, and positive energy, this project would not
have been possible. It has been a long journey, but I’ve gained so many skills, a lot of
experience and an invaluable amount of knowledge throughout this process. Thank you
for the constant support, your patience, and taking a chance on me. I am very grateful for
this opportunity.
I would like to thank my committee members, Dr. Douglas Goff and Dr. Lindsay
Robinson. Both have contributed a great deal to the success of my project and have given
great feedback, advice, and directions to pursue.
I would also like to thank Dr. Amir Malaki Nik, who because of time constraints,
had to quickly train and teach me the basic methods that I carried on using throughout my
research. Thank you to Dr. Marangoni and Dr. Corredig for allowing me to use the
instruments in their lab.
Thanks to Sally Huynh and Lois Lin, my lab mates, for your constant
encouragement and technical support. A year into my masters you both joined the lab and
made it an enjoyable working environment. Thank you to Prabjot Parmar, the summer
student, who also brought a positive energy into the lab and for his technical support.
Last but certainly not least, thank you to my family, to my mother, father, and to
my two sisters. Nothing throughout this process would have been possible without their
constant support and love. This thesis is for you dad.
iii
Table of Contents
Abstract
Acknowledgements.......................................................................................................... iii
Table of Contents.............................................................................................................iv
List of Tables.................................................................................................................... vi
List of Figures................................................................................................................. vii
List of Abbreviations....................................................................................................... ix
1.0 Introduction:........................................................................................................................1
2.0 Literature Review: .............................................................................................................3
2.1 Functional Food Industry:........................................................................................................ 3
2.2 Carotenoids: .................................................................................................................................. 4
2.2.1 β-carotene:.............................................................................................................................................5
2.3 Carotenoid Digestion: ................................................................................................................ 6
2.4 Lipolytic Bioavailability and Bioaccessibility:.................................................................. 8
2.5 Dietary Fibre:................................................................................................................................ 9
2.6 Relationship Between Dietary Fibre and Carotenoid Bioavailability:...................11
2.8 Human Studies on Carotenoid Bioavailability:...............................................................13
2.9 In vitro Testing of the Effects of Fibre on Nutrient Bioaccessibility........................18
2.10 The Effects of Guar Gum: ......................................................................................................20
2.11 Cholesterol Lowering Effects of Dietary Fibre Consumption: ................................23
2.12 Effects of Fibre on Lipase Activity:....................................................................................25
2.13 Viscosity on Diffusion:...........................................................................................................26
2.14 Viscosity on Gastric Emptying:...........................................................................................28
2.15 Research Rationale: ...............................................................................................................29
3.0 Materials and Methods: ................................................................................................ 29
3.1 Materials:......................................................................................................................................29
3.1 Preparation of BC-Oil Stock:..................................................................................................30
3.2 Preparation of Emulsions.......................................................................................................30
3.3 Preparation of “Meal Samples”: ...........................................................................................31
3.4 Preparation of Simulated Gastric Fluid (SGF), Simulated Bile Fluid (SBF), and
Simulated Duodenal Fluid (SDF) for digestion:.....................................................................31
3.5 In vitro Gastro-Duodenal Digestion:...................................................................................32
3.6 Triacylglycerol Lipolysis During In Vitro Digestion: ....................................................33
3.7 β-carotene Bioaccessibility: ..................................................................................................33
3.8 Determination of Digestate Viscosity: ...............................................................................34
3.9 Particle Size Analysis:..............................................................................................................34
3.10 Bile Acid Binding Determination:.....................................................................................35
3.11 ζ-potential: ................................................................................................................................36
3.12 Replication and Statistics: ...................................................................................................36
4.0 Results and Discussion: ................................................................................................ 36
iv
4.1 Lipid Hydrolysis Results:........................................................................................................38
4.2 BC Bioaccessibility Results: ...................................................................................................42
4.3 Digestate Viscosity:...................................................................................................................45
4.4 Changes in Particle Size During Digestion: ......................................................................47
4.5 Bile Acid Binding: ......................................................................................................................55
4.6 Investigations with Cholestyramine and Cellulose:......................................................57
4.6.1 Lipid Hydrolysis: .............................................................................................................................. 58
4.6.2 BC Bioaccessibility:.......................................................................................................................... 60
4.6.3 Viscosity Effects:............................................................................................................................... 61
4.6.4 Particle Size: ....................................................................................................................................... 63
4.6.5 ζ-potential Results:.......................................................................................................................... 66
4.6.6 Bile Acid Binding:............................................................................................................................. 68
5.0 Conclusions and Future Directions: ......................................................................... 69
6.0 References:........................................................................................................................ 74
v
List of Tables
Table 1: Summary of human studies reporting the effects of dietary fibre intake on
carotenoid absorption.___________________________________________________ 15
Table 2: Summary of studies investigating the effects of GG on lipemic and glycemic
marker bioavailability and the underlying mechanisms. ________________________ 21
Table 3: Apparent viscosities (Pa.s) at 47.6 and 94.9 s-1 at the beginning of duodenal
digestion (0 min) for the different concentrations of GG1._______________________ 46
Table 4: Average D4,3 and D3,2 (μm) (volume/mass moment mean and surface area
moment mean, respectively) of the emulsion “meal samples” with 0.0, 1.0, 1.5, 2.0, and
4.0 % GG prior to digestion1. _____________________________________________ 50
Table 5: Summary of particle size distribution peak diameters for emulsion “meal
samples” prior to and at the beginning and end of gastric and duodenal digestion stagesa,b
in the absence (Control, 0.0%) and presence of 1.0, 1.5, 2.0, and 4.0% GG1. ________ 55
Table 6: Apparent viscosities (Pa.s) of digestate samples at 47.6 and 94.9 s-1 at the
beginning of simulated duodenal digestion (0 min) for Control sample containing no fibre
and samples containing 4.0% GG, CH, or CE1. _______________________________ 63
Table 7: Summary of particle size distribution peak diameters for emulsion “meal
samples” prior to and at the beginning and end of gastric and duodenal digestion stagesa
in the absence (Control, 0.0%) and presence of 4.0% GG, 4.0% CE or 4.0% CH1. ___ 64
vi
List of Figures
Figure 1: Molecular structure of all trans beta-carotene (Sourced From: Health Canada,
2013). ________________________________________________________________ 6
Figure 2: A: Lipid digested (%) during 2 h duodenal digestion determined based on FFA
release at 2, 5, 10, 15, 30, 60, 90, and 120 min. B: Lipid digested (%) during the first 30
min of duodenal digestion determined based on FFA release at 2, 5, 10, 15, and 30 min.
Data shown is the mean ± SEM, n=6._______________________________________ 39
Figure 3: FFA release (%) at the end (120 min) of duodenal digestion in the presence of
different concentrations of GG. Data shown is the mean ± SEM, n=6. Different letters
indicate significant differences (P<0.05) between means. _______________________ 41
Figure 4: BC bioaccessibility (%) at the end of duodenal digestion (120 min) in the
presence of 0-4 % GG. Data shown is the mean of six independent experiments ± SEM.
Different letters indicate a statistical difference (P<0.05) between treatment means. __ 44
Figure 5: BC bioaccessibility (%) at the end of duodenal digestion (120 min) as a
function of the lipid hydrolysis (%) at the end of duodenal digestion (120 min) of the
Control “meal sample.” Data shown is the mean of 3-6 independent experiments.____ 44
Figure 6: Viscosity (Pa.s) of duodenal digestate samples at the start of simulated duodenal
digestion determined by shear rate sweeps (30 to 200 s-1) using an acrylic cone (6 cm, 2 o,
50 μm truncation gap). Data shown is the mean of three independent trials._________ 45
Figure 7: Particle size (μm) distribution of (A) the initial Control emulsion and (B) of the
Control emulsion "meal sample" at the beginning and end of gastric digestion (C) and of
the Control emulsion "meal sample" at the beginning and end of duodenal digestion. Data
shown is representative of the particle size distributions observed during an in vitro
digestion of the Control emulsion. _________________________________________ 52
Figure 8: Particle size (μm) distributions of the Control emulsion, 1.0, 1.5, 2.0, and 4.0 %
GG in 5 mL of water. Data shown is representitive of three replicates samples.______ 53
Figure 9: Particle size (μm) distributions of the initial “meal samples” and digestate
samples with 1.0 (A), 1.5 (B), 2.0 (C) and 4.0 % GG (D) at the beginning and end of
gastric and duodenal digestion.____________________________________________ 54
Figure 10: Bile acid concentration (μmol/mL) of the aqueous phase after duodenal
digestion of the emulsion “meal samples’ containing 0-4 % GG. Data shown is the mean
± SEM, n=6. No significant (P>0.05) differences were found between treatment means.
_____________________________________________________________________ 57
Figure 11: (A) Lipid digested (%) throughout 2 h duodenal digestion determined based
on FFA released at 2, 5, 10, 15, 30, 60, 90, and 120 min for the Control and in the
vii
presence of 4.0 % GG, 4.0 % CE, or 4.0 % CH. (B) Lipid digested during the first 30
min of duodenal digestion. Data shown is the result of three independent experiments ±
the SEM, n=6. _________________________________________________________ 59
Figure 12: Percent FFA released at the end of duodenal digestion (120 min) for the
Control and in the presence of 4.0 % GG, 4.0 % CE or 4.0 % CH. Data shown is the
mean of six independent experiments ± the SEM. Different letters indicate a statistical
difference (P<0.05) between treatment means.________________________________ 60
Figure 13: BC bioaccessibility (%) at the end of duodenal digestion (120 min) for the
Control and in the presence of 4.0 % GG, 4.0 % CE or 4.0 % CH. Data shown is the
mean of six independent experiments ± the SEM. Different letters indicate a statistical
difference (P<0.05) between treatment means.________________________________ 61
Figure 14: Viscosity (Pa.s) of duodenal digestate samples at the start of simulated
duodenal digestion, as determined by shear rate sweeps (30 to 200 s-1) with an acrylic
cone (6 cm, 2o, 50 μm truncation gap). Data shown is the mean of three independent
trials. ________________________________________________________________ 62
Figure 15: Particle size (μm) distributions of (A) the emulsion “meal sample” containing
4.0 % GG, (B) 4.0 % CE, or (C) 4.0 % CH, at the beginning and end of gastric and
duodenal digestion. Data shown is representitive of three replicates for each in vitro
digestion._____________________________________________________________ 65
Figure 16: Particle size (μm) distributions of the Control emulsion, 4.0 % CE, and 4.0 %
CH in 5 mL of water. Data shown is representitive of three replicates samples. ______ 66
Figure 17: Bile acid concentration (μmol/mL) of the aqueous phase after duodenal
digestion for Control emulsion “meal samples’ and containing 4.0 % GG, 4.0 % CE or
4.0% CH. Data shown is the mean of six independent trials ± the SEM. Different letters
indicate statistical differences (P<0.05) between treatment means. ________________ 69
viii
List of Abbreviations
BA- Bile acid(s)
BC- Beta-carotene
BS- Bile salt(s)
CE- Cellulose
CH- Cholestyramine
D4,3- Volume weighted/mass moment mean
D3,2- Surface weighted mean
FA- Fatty acid(s)
FFA- Free fatty acid(s)
FF- Functional food(s)
GG- Guar gum
GI- Gastrointestinal
GIT- Gastrointestinal tract
LD- Lipid digestion
MAG-Monoacylglyceride(s)
PL- Phospholipid(s)
ix
PTL- Pancreatic triacylglyceride lipase
SBF- Simulated bile fluid
SDF- Simulated duodenal fluid
SEM- Standard error of the mean
SGF- Simulated gastric fluid
TAG- Triacylglyceride(s)
β-carotene- Beta-carotene
ζ-potential- Zeta-potential
x
1.0 Introduction:
The functional food (FF) industry exists because consumers want foods that go
beyond meeting basic nutritional requirements (Statistics Canada, 2007). However,
whether the intended health benefits of functional food products are realized needs to be
further examined and verified (Roberfroid, 2002). Specifically, added nutrients and
nutraceutical compounds are not always available for absorption, thus impacting their
efficacy. In particular, the bioavailability of lipophilic molecules tends to be low and
variable and can be impacted by a variety of factors (McClements et al., 2009). Factors
such as the food matrix, lipase inhibitors, bile salt (BS) concentrations, processing
effects, and intrinsic factors related to health status can all affect bioavailability.
Bioaccessibility refers to the fraction of a consumed compound that gets released from
the food matrix, incorporated into the gastrointestinal (GI) lipid emulsion, and then
transferred to aqueous phase structures such as mixed BS micelles, during digestion.
Once lipophilic molecules are incorporated within the aqueous phase structures, they are
able to cross the unstirred water layer to approach the enterocyte for absorption.
The carotenoid beta-carotene (BC) is a nutraceutical and vitamin A precursor. It
has antioxidant properties and is important for vision, cell differentiation, and immune
system regulation. However, its bioavailability from foods and supplements tends to be
low and highly variable. A major food trend that could impact the bioavailability of BC
and other lipophilic molecules is fortification with dietary fibre. Dietary fibre includes a
diverse group of soluble and insoluble edible and non-digestible carbohydrates (Anderson
et al., 2009; Lattimer and Haub, 2010). Fibres have important functional properties in
foods such as thickening, stabilizing, and emulsifying. However, they also have widely
1
recognized important health benefits. Specifically, they have been shown to reduce
cholesterol levels, to help prevent colon cancer, to help modulate body weight (Anderson
et al., 2009; Fernandez, 1995), and may decrease blood glucose levels and postprandial
rises in insulin (Torsdottir et al., 1989). Despite these potential benefits, some dietary
fibre may also negatively impact pre-absorptive events that are central to digestive
processing of lipophilic molecules such as BC, thereby decreasing their bioaccessibility
(Borel et al., 1996; Riedl et al., 1999). In order for lipid digestion to be efficient, the
formation of stable fine emulsion droplets is required providing plenty of surface area for
the adsorption of digestive enzymes (Bauer et al., 2010) and providing a lipid-water
interface required for the insoluble lipids to interact with water-soluble lipases (Carey et
al., 1983; Armand et al., 1996). Lipid bioaccessibility could be impacted by the presence
of soluble dietary fibre through a variety of mechanisms, including direct inhibition of
pancreatic triglyceride lipase activity, by binding BSs and thereby minimizing micelle
formation and emulsifying activity, by forming a physical barrier around mixed micelles,
or by increasing viscosity and affecting diffusion rates of enzymes (McClements et al.,
2009). These processes can be studied using in vitro digestion models.
Guar gum (GG) is a viscosity-inducing galactomannan soluble fibre (Kawamura,
2008) commonly used by the food industry. It is an approved emulsifier in places such as
Japan, Australia, Europe and the USA and is an approved food additive in Canada
(Kawamura, 2008). There is some evidence that GG may reduce cholesterol absorption
and attenuate postprandial rises in blood glucose and insulin (Diez et al., 1998; Jenkins et
al., 1978). However, there are very few reports of the impacts of GG, or other soluble
fibres, on dietary lipid digestion. A better understanding of how dietary fibres such as GG
2
affect lipid digestive processes that impact bioavailability will support food-based
strategies to promote the delivery of lipophilic micronutrients and bioactives, thereby
optimizing their health benefits. This is particularly important in light of the trend
towards fibre fortified foods. The project will also support a better understanding of how
dietary fibres can impact lipid digestibility, with relevance for strategies to modulate
postprandial lipemia.
Thus, the objective of this work is to investigate whether and how the presence of
GG impacts TAG lipolysis and BC bioaccessibility in an in vitro model of upper GIT
digestion. It is hypothesized that GG will decrease lipolysis and BC bioaccessibility.
2.0 Literature Review:
2.1 Functional Food Industry:
Malnutrition remains a significant concern, particularly in developing nations. For
example, it is estimated that, annually, between 250 and 300 million children go blind
due to vitamin A deficiency (Food and Nutrition Board, Institute of Medicine, 2001).
However, even in industrialized societies where foods are in ample supply, maintaining a
nutritionally balanced diet remains a challenge and diet-related diseases, particularly
those associated with aging and overconsumption, are contributing to growing healthcare
costs (Roberfroid, 2002).
Thus, today’s nutritional markets are focusing on developing concepts such as
optimized nutrition (Milner, 2000). The development of nutritionally “optimized foods”
is becoming popular, as seen in the growth of the FF category. FFs are foods, not drugs,
and are aimed at reducing the risk of disease rather than treating disease (Roberfroid,
3
2002). According to Health Canada’s (2013) definition, a FF must be “similar in
appearance to, or may be, a conventional food that is consumed as part of a usual diet,
and is demonstrated to have physiological benefits and/or reduce the risk of chronic
disease beyond basic nutritional functions.” The FF industry has become over a billiondollar industry (Statistics Canada, 2007). Consumers want foods that go beyond meeting
their basic nutritional requirements. However, there is a need for more evidence on the
intended health benefits of these products (Roberfroid, 2002).
2.2 Carotenoids:
Carotenoids are commonly occurring plant pigments found in yellow, orange, and
red flowers and fruits (Britton, 1995). However, this group of isoprenoid compounds
occurs in a much wider distribution, including animals and microorganisms (Britton,
1995). In fact, over 600 different carotenoids have been identified, isolated, and
characterized from natural sources (Kull and Plandar, 1995). Carotenoids play critical
roles in photosynthesis and are important nutritionally (Britton, 1995). The two main
roles of carotenoids in human health are based on their antioxidant activity and their role
as vitamin A precursors (Liu et al., 2004). Of note, only 10% of the 600 known
carotenoid species have pro-vitamin A activity (Bendich and Olson, 1989). Carotenoid
consumption has been linked to enhanced immune function, prevention of the
development of atherosclerosis, and inhibition of low-density lipoprotein oxidation (Diaz
et al., 1997; Steinberg et al., 1989; Berliner and Heinecke, 1996). Carotenoids are
hydrophobic molecules, which limits their water solubility and restricts them to
hydrophobic areas in a cell, such as the inner core of membranes (Tydeman et al., 2010b;
Britton, 1995), although the association with some proteins may allow carotenoids to
4
access an aqueous environment. Importantly, their hydrophobic nature means that
carotenoids can have low bioavailability, which minimizes their potential health benefits.
For further explanation, refer to Section 2.4.
2.2.1 β-carotene:
One common carotenoid is β-carotene (BC), the structure of which is shown in
Figure 1. BC has been of particular interest to biomedical researchers because its
established role as a precursor for vitamin A means it can be important for meeting basic
nutritional requirements (Brubacher and Weiser, 1985). BC also has potential as a
nutraceutical supplement or for incorporation into a food to make it a FF (Bendich,
1989). Of note, there has been some controversy surrounding BC supplementation with
respect to its cancer prevention potential, with some research also suggesting it may be
associated with lung cancer in individuals whom smoke (Druesne-Pecollo1 et al., 2009).
BC absorption is not very efficient and its bioavailability from foods or
supplements may be low and highly variable (Faulks and Southon, 2001; Wang et al.
2012). Due to their hydrophobic nature, carotenoids, including BC, can be dissolved
within an oil emulsion after being released from a food matrix (Borel et al., 1996) and
must then get incorporated into the aqueous phase structures. This is an essential step in
terms of potential bioavailability.
5
Figure 1: Molecular structure of all trans beta-carotene (Sourced From: Health Canada,
2013).
2.3 Carotenoid Digestion:
The digestion of carotenoid-containing foods begins in the mouth as the food
mixes with saliva changing its pH, ionic strength and temperature and involves
interactions with digestive enzymes (Malone et al., 2003a; Malone et al., 2003b). The
food is degraded through mastication (Charman et al. 1997), an important step in forming
an emulsion, which increases the food’s surface area. Humans consume fat in multiple
forms. Whether bulk or emulsified fat is consumed (i.e. oil-in-water emulsions like milk,
or water-in-oil emulsions like butter), the first step of lipid digestion involves the
formation of emulsion droplets. This creates a larger surface area for lipase adsorption
and activity (Golding and Wooster, 2010). When a carotenoid is contained within a lipidbased food, preduodenal lipases (lingual lipase) may initiate the digestion process (Bauer
et al., 2005; Mu and Hoy, 2004). Once food, in the form of a bolus, reaches the stomach,
it mixes with acidic digestive juices containing gastric enzymes, minerals and surfaceactive compounds and is subjected to mechanical action due to stomach contractions
(Weisbrodt, 2001a; Weisbrodt, 2001b). Lipids present in a food begin to form an
emulsion in the aqueous phase of the stomach. These lipid emulsions get broken down
into smaller lipid droplets with diameters between 10 and 20 μm, primarily due to the
6
grinding and contracting actions of the stomach (Bauer et al., 2005; Fave et al., 2004).
During gastric digestion, researchers have observed that the majority of large lipid
droplets (which generally measured 70-100 μm) were transformed into more fine droplets
ranging in size from 1-10 μm (Golding and Wooster, 2010; Bauer et al., 2005).
In adults, the majority of lipid digestion occurs in the small intestine, although 1030% may occur in the stomach as well (Armand, 2007; Fave et al., 2004; Bauer et al.,
2005) partly due to the low stomach pH and also due to the action of gastric and
continued lingual lipase at the lipid-water-interface (Bauer et al., 2005). The action of
these enzymes on the lipids releases fatty acids (FAs), which are generally hydrolyzed
from the dietary triacylglycerols (TAGs) at the sn-1 and sn-3 positions. The products of
lipid digestion (i.e. FFA, MAG, and DAG) promote pancreatic lipolysis by changing the
interfacial composition of emulsified lipid drops and by aiding in lipid emulsification in
the stomach (Golding and Wooster, 2010). Once the food chyme has passed from the
stomach to the small intestine and mixes with BSs and phospholipids, fat digestion
continues via the action of pancreatic TAG lipase (PTL) (Fave et al., 2004; Lowe, 2002).
PTL initiates hydrolysis of TAG and DAG (Bauer et al., 2005), which is further
promoted by the presence of BSs, which are released by contraction of the gall bladder in
response to the presence of lipids in the duodenum. BSs are natural biosurfactants,
primarily based on cholic acid and synthesized from cholesterol in the hepatocytes in the
liver (Borgström, 1974). BSs help to solubilize the products of lipase action in the bulkwater phase of the intestinal contents, thereby removing them from the site of enzymatic
action and solubilizing them within the aqueous phase (Bauer et al., 2005). The digestion
of dietary TAG eventually leads to the release of lipophilic molecules, such as
7
carotenoids, from the hydrophobic core of the lipid emulsion droplet. These are
solubilized within the aqueous phase, i.e. the so-called mixed micellar phase. In reality,
the aqueous phase structures include a series of colloidal structures, including
multilamellar, unilamellar vesicles, mixed micelles, and micelles (Porter et al., 2004;
Porter and Charman, 2001). Lipophilic molecules, including carotenoids, get
incorporated into mixed BS micelle structures, which also contain phospholipids,
cholesterol, and products of lipid digestion such as FFAs and vesicles necessary for the
lipid digestion products transport to the intestinal membranes (Bauer et al., 2005). The
micelle structures can then pass through the unstirred water layer to approach the
enterocyte where the lipid contents are absorbed (Charman and Porter, 1997). Therefore,
the processes by which lipophilic dietary molecules become solubilized during digestion
are critical in determining their potential for absorption and bioavailability.
2.4 Lipolytic Bioavailability and Bioaccessibility:
Carotenoids and their metabolites have to be bioavailable (i.e. must be absorbed
and delivered to the target tissue for utilization or storage) to exert an intended systemic
benefit (Failla et al., 2008). In order for a carotenoid to be bioavailable, it must first be
rendered bioaccessible by the processes described in the previous section.
Bioaccessibility refers to the fraction of a nutrient that is released from the food matrix,
transferred to lipid droplets, and incorporated into the various aqueous phase structures
during digestion (McKevith et al., 2003; Tydeman et al., 2010). In contrast,
bioavailability refers to the fraction of a lipophilic compound which is absorbed and
metabolized by enterocytes and incorporated into chylomicrons for secretion into lymph
(Mckevith et al., 2003). Thus, bioaccessibility is a precursor for bioavailability.
8
Carotenoid bioavailability can be affected by a number of different factors,
including the following: 1) intrinsic factors (e.g. biliary insufficiencies; gut health;
nutritional status); 2) physiochemical properties (e.g. free vs. protein bound complexes);
3) food sources and matrix (e.g. leaf vs. flower vs. seed; food vs. supplements); 4)
processing (e.g. raw vs. processed food); 5) interactions with other dietary compounds
such as lipids and fibre during digestion or absorption; (Failla et al., 2008; Failla et al.,
2005; Yonekura and Nagao 2007). The focus of this review will be the impact of dietary
fibres on BC bioaccessibility. Dietary fibres have been implicated as negatively
impacting lipid bioavailability, as discussed in Sections 2.7 and 2.8. However, research in
this area is limited and fragmented based on fibre type, concentration, and experimental
models. A solid understanding of the mechanisms by which dietary fibre could exert an
effect does not yet exist. However, the development and advertising of foods based on
their natural, enriched, or fortified dietary fibre content is a major food trend (Health
Canada, 2011). Therefore, it is imperative that a better understanding of the impact of
fibre on lipid bioaccessibility be achieved. This would support the ability to tailor foods
and natural health products for either enhanced or decreased lipophilic nutrient
absorption. The possible mechanisms by which dietary fibres could affect
bioaccessibility, and thus bioavailability, will be discussed.
2.5 Dietary Fibre:
By the 1930s, dietary fibres had been described as non-digestible carbohydrates
(Rideout et al., 2008; McCance et al., 1936). In 2001, a more detailed and comprehensive
definition of dietary fibre was proposed by the American Association of Cereal Chemists
(AACC). Accordingly, dietary fibre was defined as being “the edible parts of plants or
9
analogous carbohydrates that are resistant to digestion and absorption in the human small
intestine with complete or partial fermentation in the large intestine.” AACC further
included polysaccharides, oligosacchaides, lignin, and associated plant substances as
dietary fibres and that dietary fibres enhance beneficial physiological effects during
laxation, blood cholesterol and glucose attenuation (AACC, 2001). Currently, the World
Health Organization and the Food and Agriculture Organization have a similar definition
of dietary fibre as the AACC, although this specifically indicates that dietary fibres must
have ten or more monomeric units, which cannot be hydrolyzed by endogenous enzymes
in the small intestine (FAO/WHO Alimentarius Commission, 2009).
Fibres can be subdivided into two types, i.e. soluble and insoluble (Lattimer and
Haub, 2010). Soluble fibres are those that dissolve in water and generally form viscous
gel like solutions, unlike insoluble fibres (Lattimer and Haub, 2010). Soluble fibres are
also fermented in the large intestine by the microflora and include gums, such as guar
gum, pectin, and inulin (Lattimer and Haub, 2010). Fermentation of insoluble fibres is
limited, although they provide many health benefits such as adding bulk to stool and
allowing food to pass more quickly through the digestive system (Lembo et al., 2010;
Slavin, 2008). Examples of insoluble fibres are cellulose, lignin, and some hemicelluloses
(Lattimer and Haub, 2010). The fiber component of foods is generally made up of onethird soluble and two-thirds insoluble fibers (Wong and Jenkins, 2007), although specific
fibre types can be characteristic of individual types of foods.
10
2.6 Relationship Between Dietary Fibre and Carotenoid Bioavailability:
The relationship between dietary fibre consumption, carbohydrate digestion,
glycemic response and diabetes continue to be thoroughly investigated. Meyer et al.
(2000) reported an inverse relationship between the intake of dietary fibre and diabetes,
showing that women who consumed 26 g of fibre per day had a lower risk of developing
diabetes compared to women who consumed only 13 g. These findings were in
agreement with the results of Schulze et al. (2004) and Hu et al. (2001), who also
observed inverse relationships between intake of dietary fibre and diabetes. Generally,
plasma cholesterol levels have also been shown to be reduced by the consumption of
dietary fibre (Brown et al., 1999; Knopp et al., 1999; Butt et al., 2007). Many researchers
have tried to clarify the mechanisms by which dietary fibres exert their effects on blood
glucose and cholesterol levels. Currently, there are hypothesized mechanisms and
evidence supporting that increased GI content viscosity can delay gastric emptying and
decrease absorption of macronutrients leading to lower postprandial blood glucose and
insulin (Jenkins et al., 1978; Hlebowicz et al., 2007). However, further research is
required to establish the relevance of dietary fibre structure, content, GI processes, and
glycemic response. The same is true with respect to dietary lipids where relatively little
research has been done
2.7 Using In vitro Colloidal Systems to Test Fibre Effects on Simulated Lipid
Digestion:
Colloid delivery systems, particularly oil-in-water emulsions, are becoming
increasingly used in the food industry in order to protect and deliver lipophilic molecules
11
such as oil-soluble vitamins, carotenoids, and phytosterols (McClements et al., 2007;
Chen et al., 2006; Kesisoglou et al., 2007). There is interest in controlling digestion of
these systems to optimize release and absorption of encapsulated lipids. One strategy has
been to modify the interfacial composition and structure through the inclusion of dietary
fibres within the emulsified structures. Tokle et al. (2012) tested the effects of dietary
fibres on emulsions using a simulated GIT experimental design. Emulsions were
stabilized with lactoferrin (LF) and coated by the dietary fibres: alginate, low methoxyl
pectin (LMP), and high methoxyl pectin (HMP). It was found that the stability of the
emulsified lipid within the simulated GIT greatly depended on the nature of the
surrounding coating. LF-alginate emulsions aggregated at the stomach and small intestine
stages, whereas the other emulsions (LF, LF-LMP, and LF-HMP) only aggregated under
the small intestinal conditions. The authors suggested that the dietary fibres may have
become detached in the small intestine or that they were permeable to digestive enzymes.
These results are significant as they suggest important fates of nutrients in terms of their
release and absorption as well as potential responses of the human body to nutrient
intake. Whether emulsions aggregate within the GIT or not will determine the ability of
digestive enzymes to access and adsorb to the surfaces of these lipid drops and to act on
them (Tokle et al. 2012). Digestive enzymes and molecules within the GIT interact with
emulsified lipids and adsorbed proteins, depending on the type, structural organizations
and interactions of the molecules present at the oil-in-water interface.
Aside from understanding how systems can be structured with dietary fibres, it is
very important to consider how the presence of dietary fibres impacts lipolysis and
bioaccessibilty in a mixed meal scenario. Soy protein stabilized emulsions were
12
previously investigated by our group in relation to their digestive release of encapsulated
BC (Malaki Nik et al., 2011). In this thesis, the impact of GG on processes impacting
TAG lipid digestion and BC bioaccessibility from the same emulsion system will be
investigated. The work aims to address gaps in our understanding of how soluble dietary
fibres impact the potential bioavailability of lipophilic nutrients and nutraceuticals.
2.8 Human Studies on Carotenoid Bioavailability:
Human studies are ideal to study carotenoid bioavailability as in vitro models and
studies with other animal models are not able to exactly mimic human conditions.
However, even in human studies of carotenoid absorption, results can vary between
individuals. Table 1 summarizes the human studies investigating the influence of dietary
fibre on carotenoid absorption. To date, not many controlled studies have reported on the
effects of dietary fibre on carotenoid absorption in humans. From Table 1, it seems that
consuming fibre-containing vegetables with high carotenoid levels, such as carrots or
broccoli, results in lower plasma carotenoid levels compared with ingesting BC capsules
(Granado et al., 2006; Brown et al., 1989; Micozzi et al., 1992). Variability in the results
summarized in Table 1 could result from a variety of factors. The condition of the food,
i.e. whether it is processed or not, can affect nutrient bioavailability. For example, Liu et
al. (2004) showed that the uptake of carotenoids by Caco-2 human cells from cooked
corn was higher than from raw corn. This was in agreement with Borel et al. (1996) who
suggested that emulsified forms of BC were more efficient in getting delivered to Caco-2
cells. These findings are also in agreement with the human study results of Rock et al.
(1998). That study indicated that β-carotene concentrations increased in human plasma
after consumption of heat-processed carrots and spinach. Tydeman et al. (2010b) found
13
that, after performing in vitro simulated digestions on carrots, more carotene partitioned
into the oil phase (which is a necessary step for lipophilic bioavailability) from the raw
carrot preparations than from the cooked tissues. Tydeman et al. suggested that, while
cells in raw vegetables tend to rupture and release nutrients like carotenes during
digestion, heating of the tissues tends to delay this release. Contradicting this work, in an
in vivo study, Tydeman et al. (2010b) found that more carotene was absorbed from
cooked carrots. This agrees with the publications of Reboul et al. (2006) and Bohm and
Bitsch (1999), who found that carotenoid bioavailability was higher from cooked pureed
tissue and from juiced tissue than from raw tissue. The severity of processing was also
found to have an effect on the amount of carotene portioning into the fat phase during
simulated digestion (Tydeman et al. 2010). Tyssandier et al. (2003) observed, in vivo, that
45 % of the carotene from pureed carrots was transferred to the lipid phase in the
stomach, whereas Tydeman et al. (2010) observed only 8.0-10.8 % carotene partitioning.
The different values obtained are likely due to different instrument sensitivities and
lengths of time applied between the different research groups.
14
Table 1: Summary of human studies reporting the effects of dietary fibre intake on carotenoid absorption.
Reference
Fibre or Meal
Details
1) 200 g
broccoli/day
(source of
insoluble fibre)
1) 30 mg capsules
of BC
2) 270 g carrots
(29 mg BC and
3.2% dietary fibre,
primarily pectin)
Study Design
Crossover, 2
weeks,
controlled diet
Micozzi et al.
(1992)
J. Clin. Nutr.
1) 30 mg capsules
of BC
2) 272 g carrots
(29 mg BC)
van het Hof et al.
(1999)
Br. J. Nutr.
Castenmiller et al.
(1999)
J. Nutr.
Granado et al.
(2006)
Exprim. Bio Med.
Brown et al.
(1989)
J. Clin. Nutr.
Results
Other Notes
Significant increase only in serum
lutein levels
Broccoli was
microwaved at
800W for 5 min
30 men, healthy,
ages 20-45 y
Carrot consumption was
associated with a plasma response
equivalent to 1/7th of the response
to BC capsules
Parallel arm, 6
weeks,
controlled diet
30 men, healthy,
ages 20-45 y
Carrots were
initially frozen,
thawed, and
cooked
according to
manufacturer’s
instructions
Carrots were
cooked
1) High vegetable
diet (490 g/d)=
2) Supplements of
BC (6 g/d) and
lutein (9 mg/d)
Parallel arm, 4
weeks,
controlled diet
55 subjects,
healthy, ages 1845 y
Maximum change in plasma BC
levels for subjects consuming
carrots was only 18% of the
response by subjects taking BC
capsules
Plasma BC and lutein responses to Suspected
the high vegetable diet were 14% conversion of
& 67%, respectively, compared to BC to retinol
pure carotenoid supplemented diet
1) 20 g of liquified
spinach + sugar
beet (10 g/kg wwper 100 g, 73 g
total fibre, 1/3
soluble including
22 g pectin)
Parallel arm, 3
weeks,
controlled diet
42 women, 30
men, healthy,
ages 18-58 y
Parallel arm, 1
week, controlled
diet
Study
Participants
7 men, 7
women, healthy,
ages 20-35 y
15
Sugar beet fibre addition had no
effect on serum carotenoid
response
The sugar beet
was added in
order to
compensate for
the loss of fibre
upon liquefying
Riedl et al. (1999)
J. Nutr.
1) 0.4 mg/kg BC +
0.15 g/kg of
dietary fibre
(pectin, guar,
alginate, cellulose,
or wheat bran)
Parallel arm, 18
days, controlled
diet
6 women,
healthy, ages 2629 y
Mean BC levels reduced
significantly (33-43%) by the
water soluble fibres pectin, guar,
and alginate
Rock &
Swendseid (1992)
Am. J. Clin. Nutr.
1) Meal with
negligible fibre +
25 mg BC capsule
2) Meal with
added 12 g of
citrus pectin +
25 mg BC capsule
Crossover, 8
days, controlled
diet
7 women,
healthy, ages 2331 y
Plasma BC levels in the
individuals that first consumed the
meal without added fibre
increased at 30 and 192 h (0.939
and 0.300 μmol/L, respectively),
whereas there were significant
decreases in B levels (0.393 and
0.061 μmol/L at 30 and 192 h,
respectively) in individuals whom
consumed the meal with pectin
Hoffmann et al.
(1999)
Eur. J. Nutr.
1) 0.4 mg/kg of
bw BC
supplement. Then
each subject
received a meal
enriched with
pectin, guar, and
cellulose (0.15
mg/kg of bw)
Crossover, 18
6 women,
days (including a healthy, ages 262 week wash-out 29 y
period), standard
diet
bw= body weight; ww= wet weight
16
BC levels decreased to 63, 9, and
14% upon enriching the meals
with pectin, guar, and cellulose,
respectively
High carotenoid
contents and
unrealistic fiber
doses were used
Granado et al. (2006) evaluated the bioavailability of carotenoids present in
broccoli in 14 healthy volunteers who were instructed to consume 200 g of broccoli along
with 10 mL of olive oil once a day for 7 days and whom were asked not to consume any
other green vegetables or fortified foods throughout the study. The broccoli was
microwaved at 800 W for 5 min prior to consumption. Biochemical and hematological
serum levels of vitamins A and E and carotenoids were determined. There were no
changes in β-carotene, α-tocopherol, or α-carotene serum levels with consuming
microwaved broccoli. This is not in agreement with the results of van het Hof and
colleagues (1999) who reported a 28 % increase in serum β-carotene levels after 4 days
of broccoli (boiled for 6 min) consumption. This may be related to difference in the
amount of broccoli consumed (containing different amounts of β-carotene, i.e. 1.4-1.8 vs.
1.7-24.6 mg/day). The participants in the Granado et al. study were also asked not to
consume fortified food or other green vegetables, and thus may have only consumed
enough broccoli that compensated for their typical daily intake of BC levels (resulting in
no change in their daily BC levels), since it was observed that serum levels of the other
carotenoids not found in the broccoli (ß-cryptoxanthin and lycopene) actually decreased.
One way in which dietary fibre may impact lipid absorption is through impacting
cholesterol metabolism (Hillman et al., 1986). The study by Hillman et al. (1986)
highlights that dietary fibres can differentially impact biliary lipids in the duodenum.
They measured biliary lipid composition after testing the effects of consuming 3 different
fibres (pectin, cellulose, and lignin) in 30 healthy subjects. Three groups of ten subjects
each consumed 15 g of one of the fibre supplements for a test period of 4 weeks.
Duodenal bile was aspirated and bilary lipid (cholesterol, phospholipids, and total bile
17
acid) contents measured by gas-liquid chromatography. Pectin, cellulose, and lignin
supplementation did not significantly alter the percentages of biliary cholesterol,
phospholipids or total BAs. However, the group that consumed the pectin supplement
showed
significant
decreases
in
total
primary
BAs
(cholic
acid
and
chenodeoxycholicacid) and a significant increase in secondary BAs, deoxycholic acid
(DCA). Cellulose had the opposite effect, i.e. levels of DCA and total secondary BAs
were significantly decreased. Subjects in the lignin group had no significant differences
in individual BAs with supplementation. Suggested mechanisms involve the decreased
production of 7α-dehydroxylase activity, associated with the decreased production of
secondary BAs. Another mechanism is the formation of gelling networks (viscosity
effects), leading to the entrapment of BAs (Hillman et al., 1986). In fact, participants in
the pectin group experienced difficulty consuming the 15 g of the fibre because of its
high thickening effects in their food. Therefore, modulation of bilary lipids is one
mechanism by which dietary fibre might impact carotenoid uptake. The impact on BA
binding, specifically, is discussed in Section 4.5.
2.9 In vitro Testing of the Effects of Fibre on Nutrient Bioaccessibility
Discrepant results, ethical and logistical considerations and costs associated with
human studies have rationalized the use of in vitro testing to investigate the effects of
dietary fibres on nutrient bioaccessibility. Furthermore, in vitro digestion models have
been used relatively successfully to determine the bioaccessibility of minerals, vitamins,
and lipophilic compounds such as cholesterol and carotenoids from different foods and
have helped in testing the effects of dietary components, by controlling factors such as
fibres, on bioaccessibility (Fernandez-Garcia et al., 2008). There are many factors to
18
consider when using in vitro digestion methods to study the digestion of lipophilic
molecules. This includes the type of oil, concentrations of the simulated gastric and
intestinal contents, including pepsin, phospholipids, pancreatin, and bile, and type of
emulsifier, all of which have been shown to have different effects on the optimal
micellization of carotenoids (Fernandez-Garcia et al., 2008; Söderlind et al., 2010). For
example, the length of the lipophilic tail and degree of esterification of an emulsifier can
impact bioaccessibility (Fernandez-Garcia et al., 2008; Söderlind et al., 2010). However,
despite all these factors, Reboul and colleagues (2006) concluded that there is a high
correlation between carotenoid bioaccessibility results obtained using in vitro and in vivo
models. Similarities exist with respect to measurements of carotenoid bioaccessibility
determined using in vitro digestion models and analysis of samples from the lumen of the
small intestine in humans consuming carotenoid-rich foods (Reboul et al., 2006; Failla et
al., 2008).
Simulated gastric and duodenal digestion experiments have been applied to study
the effects of fibres on lipid digestion and bioaccessibility from oil-in-water emulsions.
Beysseriat et al. (2006) studied fibre-droplet interactions and lipid digestion using an in
vitro simulated human digestion model. Emulsions were prepared using corn oil, Tween
80 as an emulsifier and either pectin or chitosan as the fiber by homogenizing and
sonicating. Beysseriat and colleagues found that the control, which contained no fibre,
had <3 % creaming (not significant). Emulsion microstructure appeared uniform (stable
size distribution), suggesting that no droplet flocculation, coalescence or creaming
occurred throughout exposure to the digestion model. In the presence of 0.5 % pectin,
creaming and aggregation were observed. The presence of pectin was found to reduce the
19
lipid hydrolysis (Beysseriat et al. 2006), which would potentially prevent lipophilic
molecules, such as carotenoids, from being released and solubilized. As for chitosan, the
low molecular weight form of the fibre did not promote appreciable creaming or
aggregation, whereas the high molecular weight chitosan promoted droplet aggregation.
Beysseriat et al. suggested that this was due to the highly positive charged chitosan
adsorbing to the negatively charged lipid droplet surface and retarding lipid digestion.
These results are in agreement with Hur et al. (2009) who performed an in vitro digestion
on beef patties and found that chitosan and pectin covered the lipid droplets as well as the
muscle fibres, thus reducing the extent of lipid digestion.
2.10 The Effects of Guar Gum:
GG is widely used by the food industry in products such as ice cream, puddings,
and baby food (Wielinga and Maehall, 2000). This is primarily because of its increasing
viscosity effects that help to control texture, prevent creaming and influence shelf-life
(Wielinga, 2010). GG is a galactomannan obtained from the cluster bean, Cyanopsis
tetragonoloba (Jenkins et al. 1978). It is composed of linear chains of (14)-linked β-Dmannopyranosyl units with (16)-linked α-D-galactopyranosyl residues as side chains
and has a molecular weight range between 50,000 and 8,000,000 g/mol (Kawamura,
2008). GG is known to offer buffering capacity and is very stable in a pH range of 4.010.5 (Kawamura, 2008). The cholesterol lowering and glycemia limiting effects of GG
have been tested in vitro, animal, human studies. Table 2 summarizes studies where the
effects of GG on endpoints related to lipid and carbohydrate digestion and underlying
mechanisms were investigated.
20
Table 2: Summary of studies investigating the effects of GG on lipemic and glycemic marker bioavailability and the
underlying mechanisms.
Reference
Type of
Study
9 men; 0, 2.5, 7.5 or 12.5 g HumanGG per meal, 12 h fast and Glycemia
samples collected at
intervals up to 170 min
Results
Proposed Mechanism
Rise in blood glucose was
higher for males that ingested
no GG. Increased doses of GG
resulted in decreased
postprandial rises in insulin.
Viscosity effects played an
important role. The authors tested
the effects of GG on glucose and
insulin levels after destroying the
viscosity effects by processing into
a canned product.
Russo et al.
2003
11 men & women; drink
containing 50 g glucose,
30 mL lemon juice and
270 mL water, with or
without 9 g GG, 2 days
After ingestion of the solution
with guar, blood glucose and
serum insulin levels were
lower.
Presence of guar resulted in slower
gastric emptying, resulting in the
small intestine being exposed to
glucose for a longer period of time.
Hoffmann et al.
1999
6 women; 0.4 mg/kg of bw HumanBC supplement. Then 0.15 Lipemia
mg/kg of bw of a GG
enriched meal, 18 days
The oxidative resistance of
LDL was reduced by 22 %.
Antioxidant enrichment of LDL was
reduced by the action of dietary
fibre at the absorption site.
Seal and
Mathers 2001
25 male Wistar strain rats;
0, 50 g (5 %), 100 g (10
%) GG to a semi-purified
cholesterol-free diet, 21
days
AnimalLipemia
In the caecal contents: mmol/g
of SCFA increased with GG
presence vs. control. Total
output of faecal bile acids
increased from 24 to 59
mmol/7 d in rats fed 50 g
GG/kg vs. control.
There was a two to threefold
increase in faecal bile acid output
compared to control rats, suggesting
that GG sequestered BA.
Moriceau et al.
2000
Animal8 male Wistar rats;
Control (fibre free), 5 %
Lipemia
GG, 0.25 % cholesterol, or
5 % GG/0.25 %
cholesterol, 21 days
GG significantly lowered
cholesterol levels (-13 %) and
was more potent in rats fed the
GG/cholesterol diet (-20 %).
Increasing luminal viscosity and
disturbance of micelle formation
(slowing down cholesterol transfer
across the unstirred layer and
inhibiting BA reabsorption).
Torsdottir et al.
1989
Study Design
HumanGlycemia
21
Moundras et al.
1997
10 male Wistar rats;
Control (C), 7.5 % GG,
0.3 % cholesterol (Ch), or
0.3 % cholesterol/ 7.5 %
GG,
AnimalLipemia
Plasma cholesterol levels after
C, GG, Ch and Ch/GG diets
were 1.62, 1.40, 2.44, and
1.65 mmol/L respectively
(significantly lower after
ingestion of GG).
Hypothesize that GG increased
excretion of cholesterol and BA and
also due to its viscosity and binding
properties.
Roy et al. 2000
54 Guinea Pigs, male and
female; control diet: 10
g/100 g of cellulose & 2.5
g/100 g of GG. Soluble
fibre (SF) diet: 5 g/100 g
of psyllium, 5 g/100 g of
pectin and 2.5 g/100 g GG
AnimalLipemia
SF: 44 % lower plasma LDL
cholesterol & 22 % lower
plasma TAG concentrations
compared to guinea pigs fed
the control diet.
Not discussed.
Philips 1986
10 mL mixed micelle
solution (0.3 mM monoolein, 0.6 mM oleic acid,
1.0 mM phsphatidyl
choline & 0.1 mM
cholesterol in cholorform)
+ 50 μL GG (0-0.5 %)
solutions +10 mM sucrose
In vitroLipid
Related
A concentration as low as 0.25 Due to viscosity effects.
% GG significantly reduced
the rate of cholesterol mixed
micelles.
Vahouny et al.
1980
5 mL mixed micelle
solution (5 mM
taurocholate, 625 μM
lecithin, 250 μM
monoolein, 500 μM oleic
acid, and 250 μM
cholesterol) + 40 mg GG
In vitroLipid
Related
GG bound 31.7, 27.3, and
18.3 % of the BSs,
phospholipids, and
cholesterol, respectively.
SCFA= short chain fatty acids, BA= bile acid(s)
22
Altered bulk phase viscosity, and
thus diffusion rates of micelles,
ultimately reducing the availability
for lipid digestion products to be
solubilized.
2.11 Cholesterol Lowering Effects of Dietary Fibre Consumption:
Many studies have focused on the effects of dietary fibre and their cholesterol
lowering abilities. Fernandez (1995) studied the effects of pectin, GG, and psyllium on
plasma low-density lipoproteins (LDL) in guinea pigs that were fed either high
cholesterol (HC) or low cholesterol (LC) diets. Pectin, GG, and psyllium consumption
decreased plasma cholesterol levels by 18, 21, and 32 %, respectively in the LC groups
and by 50, 38, and 55 %, respectively in the HC groups. The author suggested that the
fibres might up-regulate 7α-hydroxylase (i.e. the rate-limiting enzyme of BA synthesis)
depending on the diets fed. Other researchers including Gunness and Gidley (2010)
suggested other possible mechanisms such as the possibility that soluble fibres prevent
BS re-absorption from the small intestine leading to an excess fecal BS excretion. As a
result of BA binding by dietary fibres, lipid digestion and specifically cholesterol
metabolism can be altered (Gallaher and Schneeman, 1986) through increased BA
secretion related to cholesterol homeostasis. In order to preserve BA concentrations,
more cholesterol may be catabolized to BAs by the liver (Gallaher and Schneeman, 1986;
Li and Chiang, 2009).
Another mechanism by which BA binding can impact lipid digestion is by
decreasing the availability of BAs for mixed micelle formation. This would result in
decreased potential for solubilized lipid, including cholesterol, in the intestine, which
could ultimately lead to reduced lipid absorption (Gallaher and Schneeman, 1986;
Ishibashi et al. 1996. Gallaher and Schneeman tested the BA binding capacity of oat bran,
GG, lignin, and cholestyramine (CH) in Wistar rats. Of the fibres and sequesterants
tested, GG, lignin and cholestyramine were shown to be effective BA-binding agents
23
within the small intestine. Gallaher and Schneeman’s analysis determined that the
quantity of lipid solubilized would be altered in the presence of the molecules studied.
These findings are in agreement with Ebihara and Schneeman (1998) who found that
BAs were bound or trapped by GG, konjac mannan, and chitosan in the GIT of rats.
Another possible mechanism for the cholesterol lowering effects of dietary fibre is
potential physiological effects of fibre fermentation products, such as propionate (Bridges
et al., 1992). However, given that this relates to digestion in the large intestine, it will not
be the focus of this review.
Most studies suggest that dietary fibres could interact with BAs by either directly
binding to the BA or BS molecules (Dongowski, 2007; Sayar et al., 2006) or by
entrapping BS micelles in a viscous or gelatinous network formed by the polymer (Lia et
al., 1995; Ellegard and Andersson, 2007; Cho and Clark, 2001). Haikal et al. (2008)
suggested that increased viscosity effects of fibres could interfere with efficient emulsion
formation, specifically in forming very small mixed micelles associated with efficient
absorption from the digestive track because of increased surface area (Bauer et al., 2005).
O’Connell et al. (2008) studied the effects of 5% (w/w) oat bran, wheat bran, or pectin on
carotenoid micellization from homogenized vegetables in vitro. O’Connell and
colleagues found significant inhibition of β-carotene, β-cryptoxanthin, and lutein
micellization by oat bran and only a slight reduction in micellization by wheat bran and
pectin. Their results suggest that binding of BAs necessary for micelle formation, by the
fibre, is responsible for this pattern. Binding of BAs causes reduced availability for the
formation of micelles to solubilize other lipophilic compounds.
24
The extent to which dietary fibres bind BAs is dependent on many factors.
According to Eastwood and Mowbray (1976), effective fibre and BA binding depends on
pH and osmolality of the intestinal contents, the nature of the BA, the involvement of
micelles, and the chemistry of the fibre. Cui (2006) proposed that not only do properties
such as water solubility, viscosity enhancement, and surface activity affect the BA
binding capacity of fibres, but also the physical chemistry of the fibers. This includes
molecular weight (high vs. low) and additional methyl or carboxyl groups attached to the
fibres (Cui 2006; Eastwood and Hamilton, 1968; Kay et al., 1979).
Yamaguchi et al.
(1995) tested the effects of low (M= 66,000 g/mol) and high (M=750,000 g/mol)
molecular weight pectins on serum cholesterol levels in male Sprague-Dawley rats. The
high molecular weight pectin was more effective at lowering serum cholesterol. Kay et
al. (1979) tested the effects of different forms of lignin (differing in methoxyl content) on
BA sequestering capacity in vitro. They found no relationship between BA absorption
and methoxyl content and BA absorption, although lignin methylation was previously
reported to increase BA absorption (Eastwood and Hamilton, 1968).
2.12 Effects of Fibre on Lipase Activity:
Direct interactions of dietary fibres with digestive enzymes and reductions in
enzymatic activity are one way fibres might impact lipid digestion (Hur et al., 1999;
O’Connor et al., 2003). Pasquier et al. (1996) measured initial lipase activity in emulsion
samples containing no fiber and in samples containing 2 % gum arabic. The lipase
activity decreased from 9.2 to 8.5 μmol/min in the presence of the fibre. TAG lipolysis
(% of non-esterified FA release) was also measured and decreased significantly from 5.4
25
to 2.9 % at 15 min and from 8.8 to 4.3 % at 30 min for the control and with gum arabic,
respectively.
Another mechanism by which dietary fibre may impact lipid digestion is through
the formation of a physical protective membrane around lipid droplets, thereby
preventing digestive enzymes from coming into contact with the lipid substrate (Mun et
al., 2006; Ogawa et al., 2003). Since lipolysis is required for the release of molecules
within the droplets, such factors can decrease the potential bioaccessibility of lipophilic
molecules. Using an in vitro digestion model, Malaki Nik et al. (2011a) observed a
positive correlation between lipid digestion and bioactive release from the same soystabilized emulsion to be studied in this thesis. More extensive lipid digestion resulted in
increased molecular transfer.
This review will now focus on the effects of dietary fibre with respect to viscosity
enhancement. Many dietary fibres can increase the viscosity of the aqueous phase
surrounding the lipid droplets of an emulsion. This may alter the efficiency of droplet
disruption and lead to coalescence in the GIT. An increase in viscosity may also lead to a
slower transport of molecules to or from the lipid droplet surfaces.
2.13 Viscosity on Diffusion:
Dietary fibres are widely recognized for and used to induce viscosity in order to
retard carbohydrate digestion to moderate rises in plasma glucose levels, thus, reducing a
food’s glycemic index (Jenkins, et al., 1976; ADA, 2008). Increased viscosity reduces the
diffusion rates of molecules and other species such as lipase or mixed micelles within the
GIT. This results in decreased transport rates of the enzymes necessary for digestion to
26
the lipid surfaces, as well as decreased transport rates of lipid digestion products within
micelles to the GIT, which reduces the overall digestion and absorption rates of lipids
(Hur et al., 2009). Hur et al. (2009) suggested that increased viscosity due to the addition
of certain fibers like pectin inhibited lipid digestion during in vitro digestion of beef
patties, whereas cellulose, a fiber which did not significantly increase the viscosity of the
solution, had no effect on the lipid digestion.
Phillips (1986) examined the increased viscosity effects of increased GG
concentration on the diffusion rate of cholesterol mixed micelles. A rapid increase in
viscosity for concentrations of GG greater than 0.2 % was observed. This coincided with
the findings that the critical concentration at which molecules of GG’s galactomannan
chain structure become entangled was 0.23% (Morris et al., 1981). Phillips found that
with this increase in viscosity due to increasing GG concentration, there was a rapid
decrease in the rate of diffusion of cholesterol mixed micelles, as determined by
observing dye diffusion. He suggested that future research should concentrate on the
effects of GG and other fibres on the malabsorption of essential nutrients that are
absorbed through micellar suspensions. However, very few studies have done so.
Yonekura and Nagao (2009) investigated the effects high viscosity alginates, caboxymethylcellylose and methylcellulose, and different types of pectins on carotenoid
micellization and cellular uptake (using Caco-2 cells) in vitro. Medium and high viscosity
alginates (12.5 g/L) inhibited β-carotene uptake and micellization. Citrus and apple
pectins (12.5 g/L) also reduced β-carotene micellization. The authors suggested that this
may have been due to the formation of gel aggregates or high viscosity acid gels at the
acidic pH during the gastric phase of the in vitro digestion process.
27
2.14 Viscosity on Gastric Emptying:
The presence of dietary fibres, primarily due to viscosity enhancement, can affect
the rate of gastric emptying, defined as the rate of food passage from the stomach to the
duodenum (Charman et al., 1997). Slower rates of gastric emptying cause the stomach
contents to be in contact with gastric acid and enzymes for a longer period of time. In
turn, food digestion may be more complete, potentially leading to the higher release of
nutrients, such as carotenoids.
Bortolotti et al. (2007) tested the effects of a 3.5 g dietary fibre mixture (made up
of glucomannan, inulin, lemon fibre, psyllium, apple pectin, etc.) on gastric emptying in
human volunteers. Gastric emptying time increased from approximately 200 to 300 min
after ingestion of the fibre mixture. Rainbird and Low (1985) studied the effects of
consuming 40 g/kg of 4 different fibres (including caboxymethylcellulose (CMC) and
GG) on the rate of gastric emptying in pigs. The rate was reduced by CMC, mainly due to
reduced emptying of water. The dietary fibres which increased the viscosity of the gastric
digesta did not affect the emptying of dry matter (including total nitrogen and glucose).
As a result, it was concluded that increasing viscosity reduced the gastric emptying of the
liquid phase only. These results are in contrast to those of Morgan et al. (1993), i.e. the
rate of gastric emptying was increased in individuals who consumed 10 g of GG on 3
separate days. Decreases in digestion and TAG absorption were attributed to sequestering
of BAs by GG resulting in reduced micelle formation, thus limiting the amount of lipids
that can be solubilized and eventually absorbed. Holt et al. (1979) and van
Nieuwenhoven et al. (2001) found that GG supplementation at increasing concentrations
(from 0 - 4.5 g) did not slow gastric emptying, whereas administration of pectin did (Holt
28
et al., 1979). The different results suggest that the rate of gastric emptying depends on the
concentration and type of the dietary fibre. Variations may also arise from different
measuring techniques of in vivo systems, due to solid-liquid partitioning.
2.15 Research Rationale:
Although dietary fibre is known to impact lipid digestion and carotenoid
bioaccessibility, as discussed, some results are discrepant and the underlying mechanisms
are not clearly understood. The application of in vitro digestion methods may expand our
understanding in this regard. Therefore, this thesis will apply in vitro digestion methods
to investigate the effects of GG on processes impacting emulsion lipid digestion and BC
bioaccessibility. With the rise of fibre-fortified food, the understanding gained by such
studies could be critical to ensure functional food strategies that optimize nutrient
bioavailability.
3.0 Materials and Methods:
3.1 Materials:
Beta-carotene (all trans, Type 1 synthetic, >95% purity, powder), porcine bile
extract (containing approximately 49% wt. % BS consisting of hyodeoxycholic acid (15%), deoxycholic acid (0.5-7%), cholic acid (0.5-2%), glycodeoxycholic acid (10-15%),
and taurodeoxycholic acid (3-9%)), pyrogallol (99% A.C.S. reagent), porcine pepsin
(P7000-25G, from stomach mucosa with activity of 1020 units/mg protein) and porcine
pancreatin (P1750-100G, 4 x USP, contains amylase, tyrpsin, lipase, ribonuclease, and
protease) were purchased from Sigma Aldrich (St. Louis, MO, USA). Also purchased
from
Sigma
Aldrich
were
the
analytical
29
grade
solvents
hexane
(>95%,
spectrophotometric grade), acetone (99.5% A.C.S. reagent), and anhydrous ethanol
(A.C.S. reagent). Soybean lecithin (containing 90% phosphatidylcholine and 10%
phosphatidylethanolamine) was obtained from Lipoid (Lipoid, GmbH, Ludwigshafen,
Germany). 100% pure canola oil (No Name, TO, Canada) and GG (Sigma-Aldrich,
G4129, MW 50-8000 kg/mol) were also purchased. The non-esterified fatty acid (NEFAHR2) kit was purchased from Wako Pure Chemical Industries (Wako Diagnostics, VA,
USA) and the Bile Acid Kit was purchased from Trinity Biotech (St. Louis, MO). The
soy protein isolate (SPI) used was from the Solae Company (Solae, St. Louis, MO, USA).
Cellulose (powder) and cholestyramine (Dowex® 1X2 Cl-Form) were also purchased
from Sigma Aldrich (St. Louis, MO, USA).
3.1 Preparation of BC-Oil Stock:
A stock solution of BC-Oil was prepared by mixing 0.1 mg of BC into 100 g of
canola oil at 37 oC under reduced lighting. This mixture was stirred for 2-3 h and then
vacuum filtered using a 0.22 μm nylon filter to remove any BC crystals (MAGNA, Fisher
Scientific Inc.). This filtered solution was then transferred to an amber jar, flushed with
nitrogen and stored at -20 oC until use.
3.2 Preparation of Emulsions
A standard 10 % canola oil-in-water emulsion was prepared by dissolving 1.5 g
SPI in 88.5 mL distilled water overnight. The following day, 10 g of the BC-Oil stock
was added to the mixture which was then homogenized using a handheld mixer (UltraTurrax, IKa T18 Basic, Germany) for 1 min at 10,000 rpm. The crude emulsion was
subsequently emulsified with four passes at 40 MPa through a microfluidizer at room
30
temperature (110S Microfluidizer Processor, Microfluidics, MA, USA). The emulsion
was covered with Parafilm and stored at 4 oC until use within no more than 2 days.
3.3 Preparation of “Meal Samples”:
“Meal samples” represent the material that was digested in the experiments
discussed below. This material included 5 mL of the oil-in-water emulsion with the
appropriate amount of fibre or cholestyramine. For example, the 4.0 % GG “meal
sample” contains 0.2 g of GG in addition to 5 mL of the oil-in-water emulsion.
3.4 Preparation of Simulated Gastric Fluid (SGF), Simulated Bile Fluid (SBF), and
Simulated Duodenal Fluid (SDF) for digestion:
The synthetic juices used for the in vitro digestion model were prepared in 500
mL volumetric flasks. The inorganic solution for the gastric juice contained 175.3 g/L
NaCl, 88.8 g/L NaH2PO4, 89.6 g/L KCl, 22.2 g/L ,CaCl2.2H2O, 30.6 g/L NH4Cl, and 37%
g/g HCl. The synthetic inorganic solution for the duodenal juice contained 175.3 g/L
NaCl, 84.7 g/L NaHCO3, 8 g/L KH2PO4, 89.6 g/L KCl, 5 g/L MgCl2, and 37% g/g HCl.
The inorganic solution for the bile juice contained 175.3 g/L NaCl, 84.7 g/L NaHCO3,
89.6 g/L KCl, and 37% g/g HCl. Each solution was augmented to 500 mL with distilled
water and stirred. The pH of the gastric, duodenal, and bile fluids were adjusted to 1.30 ±
0.02, 8.1 ± 0.2, and 8.2 ± 0.2, respectively, using 1 M HCl and 1 M NaOH.
The simulated gastric, bile, and duodenal fluids were prepared freshly prior to
each digestion by mixing the above juices with the appropriate enzymes. For 3 replicate
31
jars, 30 mL of SGF was prepared by mixing 30 mL of the simulated gastric salt solution
with 160 mg of pepsin (final concentration of 3.2 mg/mL during the gastric phase which
includes 5 mL of “meal sample” + 7.5 mL of SGF) and 630 mg of pyrogallol (final
concentration of 12.6 mg/mL during the gastric phase) as an antioxidant. 30 mL of SDF
was prepared by mixing 30 mL of simulated duodenal salt solution with 470 mg of
pancreatin (final concentration of 5 mg/mL during the duodenal phase which includes 5
mL of “meal sample” + 7.5 mL SGF+ 3.5 mL SBF+ 7.5 mL SDF). SBF solutions were
prepared by mixing 14 mL of simulated BS solutions with 940 mg of BSs (final
concentration of 10 mg/mL during the duodenal phase) and 357.2 mg of phospholipids
(final concentration of 3.8 mg/mL during the duodenal phase). If needed, simulated
duodenal fluid and simulated bile fluid were adjusted to pH 8.1 ± 0.2 and 8.2 ± 0.2,
respectively.
3.5 In vitro Gastro-Duodenal Digestion:
5 mL of SPI-stabilized emulsion along with 0, 0.5, 1, 1.5, 2, or 4 wt % GG were
incubated in a water bath at 37 oC for 15 min. The gastric phase of digestion was initiated
by adding 7.5 mL of SGF solution to the amber jar which was then immersed in a 37 oC
shaking water bath at 250 rpm for 1 h. After 1 h, 3.5 mL of SBF was added to the jar and
immediately followed by the addition of 7.5 mL of SDF solution and shaking at 37 oC
(250 rpm) for 2 h. pH of the solutions at each stage of the digestion was determined and
adjusted by dropwise addition of 1M HCl or 1M NaOH if needed.
32
3.6 Triacylglycerol Lipolysis During In Vitro Digestion:
Lipolysis was quantified throughout the duodenal stage of digestion. 100 μL
aliquots of duodenal digestate were withdrawn at 2, 5, 10, 15, 30, 60, 90, and 120 min
and deposited into Eppendorf tubes containing 3 mL of hexane and 100 μL of
hydrochloric acid. The mixture was then centrifuged at 12,000 rpm (5418 laboratory
centrifuge, Eppendorf Hamburg, Germany) for 30 min. FFAs were quantified using a
non-esterified enzymatic kit purchased from Wako Pure Chemical Industries (Wako
Diagnostics, VA, USA) according to the manufacturers instructions. Briefly, wells (in a
96 well plate) were filled with 225 μL of Reagent A, followed by 5 μL of test sample.
This was incubated at 37 oC for 10 min and then 75 μL of Reagent B was added to each
well and the mixture was incubated for 15 min at 37 oC. The plates were read at an
absorbance of λmax of 550 nm in a UV-VIS micro-plate spectrophotometer (Spectramax
plus, Molecular Devices Corporation, CA, USA). FFA concentration was determined
from a standard curve based on oleic acid (0.1 to 1 mM). Lipid hydrolysis for each time
point was calculated based on the moles of FA hydrolyzed with respect to the total moles
of FA originally present.
3.7 β-carotene Bioaccessibility:
At the end of duodenal digestion (120 min), 6.5 mL of the digestate was
centrifuged at 49,000 g (1-4 h) to obtain the aqueous phase. 1 mL of the aqueous phase
(the phase between the undigested oil layer and the sediment) was portioned into a glass
vile. BC was extracted though the addition of 1 mL, 3 mL, 1 mL, and 2 mL of ethanol,
acetone, water, and hexane respectively, with vortexing for 10 s after the addition of each
33
solvent. The upper organic layer was collected using a fine tip transfer pipet and was
deposited into a glass tube. 2 additional aliquots of 2 mL of hexane were added to ensure
complete extraction of BC. The upper organic layers were pooled and evaporated just to
the point of dryness in a glass tube placed over a warm (35 °C) water bath. BC was
reconstituted in 2 mL of hexane, vortexed for 10 s, and quantified using a
spectrophotometer (Hewlett Packard 8451A Diode Array Spectrophotometer) at an
absorbance of 450 nm and using Beer’s Law. Bioaccessibility is defined as the % of BC
in the original “meal sample” transferred to the aqueous phase.
3.8 Determination of Digestate Viscosity:
Viscosities (Pa.s) of the digestion samples at the beginning of the duodenal phase
(0 min) were determined using shear rate sweeps between 30 and 200 s-1 with an acrylic
cone (6 cm, 2o, 50 μm truncation gap) on a controlled stress rheometer (AR 2000, TA
Instruments, New Castle, DE, USA) maintained at 37 oC. Apparent viscosities (Pa.s) of
the samples at 47.6 and 94.9 s-1, representing typical shear rates throughout digestion
(Borwankar, 1992), were determined and are reported.
3.9 Particle Size Analysis:
Particle size distributions were determined by laser diffraction using a Mastersizer
(2000S, Malvern Instruments Inc., Southborough, MA). Refractive indices of 1.46 and
1.333 were used for the oil and water (dispersant) respectively. The initial emulsions
were analyzed, as well as the emulsion “meal samples” (containing various
concentrations of GG, CH, or CE) at the beginning and end of the gastric phase, and at 0,
5, 10, 15, 30, 60, 90, and 120 min of duodenal digestion. Particle size measurements of
34
control mixtures of 1.0, 1.5, 2.0, and 4.0 % GG, 4.0 % CH, and 4.0 % CE in 5 mL of
water were also determined.
3.10 Bile Acid Binding Determination:
BA binding by GG (0.0, 1.0, 1.5, 2.0, and 4.0 %), CE (4.0 %) and CH (4.0 %) was
investigated based on a method adapted from Camire et al. (1993). At the end of
duodenal digestion, 6.5 mL of digestate was centrifuged at 49,000 g at 20 oC for 2.5 h.
This was done in triplicate. The aqueous phase was frozen at -20 oC overnight. 0.2 mL of
room temperature thawed aqueous phase was diluted in 6 mL of dH2O. BAs were
quantified using a BA-binding enzymatic kit (Trinity Biotech, St. Louis, MO), based on
the methodology of Mashige et al. (1981). As per the manufacturer’s instructions,
Reagents A and B were reconstituted in 10 and 5 mL of dH2O, respectively, and inverted
multiple times to mix. The Test Reagent was prepared by mixing 4 mL of Reagent A and
1 mL of Reagent B. The blank reagent was prepared by mixing 4 mL of Reagent A and 1
mL of distilled water. Both solutions were incubated at 37 oC until use. As per the
manufacturer’s protocol, tubes were filled at timed intervals with 0.2 mL of either the
Calibrator or Sample, followed by addition of 0.5 mL of either the Test or Blank solution
into the corresponding tubes. The tubes were mixed immediately and incubated at 37 oC
for 5 min, at which point 0.1 mL of Stop Reagent (consisting of 0.091 L phosphoric
acid/L H2O) was added at the same timed intervals. The absorbance was then read at 530
nm within 1 h using a spectrophotometer (Hewlett Packard 8451A Diode Array
Spectrophotometer). The BA concentration of each sample was calculated by subtracting
the Blank absorbance from the corresponding Test absorbance and using the equation of
35
the standard curve prepared based on Calibrator concentrations of 5, 25, 50, 100, and 200
μmol/mL.
3.11 ζ-potential:
ζ-potential of the initial “meal samples” was measured using a Nano Series
Zetasizer (Nano ZS-90, Malvern Instruments, Worcestershire, UK). The samples were
prepared as described in Section 3.3 and diluted 500-fold (5 μL of sample in 500 mL of 5
mM phosphate buffer at pH 6.5).
3.12 Replication and Statistics:
Emulsions were always prepared in at least triplicate. Each experiment was
preformed three to six times for each concentration of GG, CE, or CH used. Results were
analyzed by 1-way ANOVA and Bonferroni post testing (Gallaher and Schneeman,
1986), with P<0.05 using GraphPad Prism Software (Version 5.0a, CA, USA).
4.0 Results and Discussion:
In this study, an in vitro digestion model was used to test the effects of GG on
lipid digestion by measuring FFA released throughout simulated duodenal digestion and
on BC bioaccessibility by measuring the content of BC transferred to the aqueous phase
at the end of duodenal digestion. After these initial results were obtained, the mechanisms
by which GG exerts an effect, such as by increasing viscosity, effects on particle size
distribution of the emulsion “meal samples”, and BA binding were studied.
Since BC is generally consumed as part of a meal, studying in vitro digestion
effects on BC bioaccessibility with conditions resembling the fed state is most relevant
36
(Wright et al., 2008). Thus, in fed conditions, the pH during the gastric phase reaches as
low as pH 2 (McClements et al., 2009). The simulated gastric fluid pH was adjusted to
pH 1.30 ± 0.02 (Hur et al., 2009) so that when it was mixed with the “meal sample,” the
pH would reach an average of pH 2. When food reaches the stomach, contractions occur,
making low amplitude and continuous peristaltic contractions (McClements et al., 2009).
This was crudely mimicked using a shaking water bath at 250 rpm. During the gastric
phase, hydrophobic molecules such as carotenoids are transferred into the oily phase and
lipids are further dispersed and emulsified (Rich et al., 1998; Golding and Wooster,
2010). The contents that pass to the stomach mix with gastric enzymes, minerals, and
other surface-active compounds, and are subjected to mechanical agitation from stomach
contractions, resulting in a further emulsified state (Weisbrodt, 2001a; Weisbrodt, 2001b;
McClements et al., 2009). However, since an oil-in-water emulsion was prepared with
BC, the intestinal phase of digestion was the main focus of this work. Previous work has
shown that eliminating the gastric phase, mainly the action of gastric lipase on lipids,
does not significantly affect the transfer of BC to the aqueous phase (Garrett et al., 1999;
Wright et al., 2008). Furthermore, many foods and supplements consist of emulsified
lipids. Once the food chyme has entered the small intestine, specifically the duodenum,
the contents are mixed with sodium bicarbonate, BSs, phospholipids, and pancreatic
enzymes (Tso, 2000). The release of sodium bicarbonate causes the pH of the contents to
rise from pH 2 (from the acidic environment of the stomach) to pH 6.5 (Bauer et al.,
2005). After the appropriate volumes of SDF and SBF were added to the meal sample at
the end of the gastric phase, the digestate pH was adjusted to 6.5, i.e. the optimal pH for
the pancreatic enzymes to function (Bauer et al., 2005). Equally important when
37
simulating the gastric phase of human digestion, is the addition of multivalent cations
such as Ca2+ and Mg2+ (Read, 2004; Vaskonen, 2003; Karupaiah and Sundram, 2007).
Calcium helps to activate pancreatic lipase and both types of ions promote precipitation
of BSs and long chain saturated FAs (Read, 2004; Vaskonen, 2003; Karupaiah and
Sundram, 2007). Salts consisting of these ions, including CaCl2 and MgCl2, were part of
the SGF, SDF, and SBF solutions. BS concentrations, provided by the SBF solution and a
critical part of lipid digestion (McClements et al., 2009; Bauer et al., 2005) reach between
5 and 15 mM in the duodenum (Kalantzi et al., 2006). In this study, a BS concentration of
10 mM was used. 10 mM was previously found to be the optimal BS concentration to
allow maximum BC transfer to the aqueous phase in an in vitro digestion study (Wright
et al. 2008). It is important that the BC concentration in an in vitro study exceeds the
critical micelle concentration (CMC) needed to form micelles in order to solubilize the
lipophilic compounds under investigation, i.e. BC in this case (Bauer et al., 2005). The
CMC needed in the small intestine is between 1 and 2 mM (Hoffmann, 1963; Bauer et
al., 2005), and thus, the BS concentration used exceeds the CMC needed. Equally
important during the duodenal phase is the release and action of phospholipids, which
increase from a concentration of 0.2 to between 3 and 6 mM, primarily due to pancreatic
lipase release in the fed state (Porter et al., 2007). The phospholipid concentration used in
this study was 5 mM, falling within the concentrations found in vivo during the fed state.
4.1 Lipid Hydrolysis Results:
The majority of lipid digestion occurs in the small intestine, and thus the
concentration of FFAs, i.e. products of lipid digestion, was measured throughout the
38
duodenal phase. Figure 2 shows the extent of lipid digestion (%) occurring over the 120
min simulated duodenal digestion for all concentrations of GG studied.
Figure 2: A: Lipid digested (%) during 2 h duodenal digestion determined based on FFA
release at 2, 5, 10, 15, 30, 60, 90, and 120 min. B: Lipid digested (%) during the first 30
min of duodenal digestion determined based on FFA release at 2, 5, 10, 15, and 30 min.
Data shown is the mean ± SEM, n=6.
During the duodenal phase, food chyme or, in this experiment, the food “meal sample” is
mixed with sodium bicarbonate, BSs, phospholipids, and enzymes such as pancreatic
lipase which is essential for lipid digestion (Tso, 2000). These components initiate the
majority of lipid digestion, resulting in the hydrolysis of 2 out of 3 acyl chains on a TAG
molecule due to the specificity of pancreatic lipase to the sn-1 and sn-3 positions on a
TAG molecule (Golding and Wooster, 2010). Figure 2 shows that, within 2 min, 28.7 %
of the lipid was digested in the Control “meal sample” (i.e. with no GG present) and that,
within 5 min, lipolysis had reached 46.7 %. By the end of 120 min digestion, lipolysis
reached 56.2 %, showing that the majority of the lipid was digested within the first 5 min
of duodenal digestion. This is similar to the findings of Hamosh et al. (1975), who
observed, in human esophageal and gastric aspirates, that the maximal rate of lipolysis
39
occurred within 4 min of digestion. Incomplete digestion may indicate that lipolytic
products as well as the presence of BSs inhibit pancreatic lipase from having access to
the oil-water emulsion droplet (Malaki Nik et al., 2010). As described by Golding and
Wooster (2010), only 2/3rds of a TAG molecule gets hydrolyzed (approximately 67 %)
and in this experiment, lipolysis reached about 56.2 %, which is close to the expected 67
%.
Concentrations of 1.0, 1.5, 2.0, and 4.0 % GG in the meal samples resulted in
39.4, 34.6, 26.9, and 21.6%, respectively, of the lipid being digested after 120 min
(Figure 3). Thus, increasing GG concentrations resulted in decreased lipolysis. This could
be due to altered emulsion droplet interfaces (Golding and Wooster, 2010; Malaki Nik et
al., 2010), now covered by GG, preventing binding of pancreatic lipase. Potentially, the
presence of GG could affect lipolysis by physically forming a barrier around lipid
emulsions droplets, preventing lipase from binding and promoting further digestion, or by
slowing down diffusion rates of enzymes. These results agree with the findings of Kondo
et al. (2004) who found that the partially hydrolyzed GG (PHGG) decreased the amount
of FAs in the blood in a dose dependent matter in volunteers that consumed a yogurt
drink. After in vitro digestion of a yogurt drink with 0, 3, or 6 % PHGG, the amount of
bioaccessible FAs were 79.4, 70.8 and 60.1 %, respectively. In another study by Pasquier
et al. (1996), TAG lipolysis was also found to be decreased with increasing concentration
of viscous fibres present in the emulsions. Specifically, increasing the concentration of
fibre resulted in increased droplet size and decreased surface area of oil-in-water
emulsions, which in turn decreases TAG hydrolysis.
40
Figure 3 shows the amount of FFAs released at the end of duodenal digestion
(120 min). It shows that with increasing concentrations of GG, the amount of FFAs
released at 120 min decreases (P<0.05). Significantly reduced lipid digestion was
observed for the meal samples containing GG concentrations of 2.0 and 4.0 % vs. the
Control without GG. Figure 3 also shows that the error associated with the 4.0 % sample
was significantly higher than the lower GG concentrations studied, possibly related to the
viscosity effects making representative sampling more difficult.
Figure 3: FFA release (%) at the end (120 min) of duodenal digestion in the presence of
different concentrations of GG. Data shown is the mean ± SEM, n=6. Different letters
indicate significant differences (P<0.05) between means.
With 4.0 % GG present, FFA release was significantly lower. Mun et al. (2006)
observed reduced FFA release in an in vitro digestion system in the presence of chitosan.
They attributed this to the lipid droplets being surrounded by chitosan layers or trapped
within chitosan aggregates, thereby preventing lipases from reaching the surfaces. The
authors also suggested the reduced lipid digestion could be due to droplet flocculation,
caused by the chitosan layer around the lipid droplets, reducing surface area and limiting
41
the ability of lipase to adsorb to the surfaces, although its charge in acidic and basic
environments and attraction to charged lipid droplets could influence this interaction
(Mun et al., 2006). Similar mechanisms could be involved with GG.
4.2 BC Bioaccessibility Results:
Figure 4 shows the results for BC bioaccessibility. It shows the BC that was
transferred into the mixed micelles, showing that, with increasing concentrations of GG,
BC bioaccessibility was decreased. A similar trend was observed as for FFA release.
Bioaccessibility at 120 min was 29.7 ± 4.2 % for the Control. Emulsion meal samples
containing 1.0, 1.5 %, 2.0 %, and 4.0 % GG had BC bioaccessibility values of 28.9 ± 4.1
%, 22.9 ± 5.3 %, 18.0 ± 2.8, and 6.98 ± 2.0 %, respectively. The addition of 2.0 and 4.0
% GG significantly decreased BC transfer to the aqueous phase (P<0.05).
The role of BSs is critical for lipid solubilization. BSs promote lipase activity by
solubilizing lipid digestion products, including sn-2 MAGs and FFAs, thereby removing
them from the site of enzyme action at the oil-water interface (McClements et al., 2009;
Holt, 1971). BSs do this by forming micelles that act as carriers to solubilize and
transport lipolytic and lipophilic compounds (bioaccessibility) from the lipid droplets to
intestinal membranes where they are absorbed (bioavailablility) (McClements et al 2009;
Dietschy, 1978). Upon digestion of the lipid emulsion droplets that encapsulate BC, the
lipophilic compound is released and incorporated into mixed micelles and potentially
other structures in the aqueous phase. The potential mechanism of GG binding BAs and
thereby impacting BC solubilization capacity is discussed later in Section 4.5.
42
Malaki Nik et al. (2010) observed a positive correlation between lipid hydrolysis
and BC transfer to the aqueous phase with the digestion of SPI-stabilized emulsions. This
trend was similarly observed in this experiment. For example, with 0.0 vs. 4.0 % GG, the
extent of lipid digestion was 56.2 vs. 21.6 %, respectively, correlating with 29.7 vs. 6.98
% BC transfer (Figure 5). With more lipid hydrolysis, the amount of BC in the aqueous
phase (bioaccessbile) increased. The trend was linear up until a certain extent of lipolysis
(~ 30%), at which point BC bioaccessibility seemed to plateau. This could be due to a
variety of factors such as incomplete digestion of the lipid phase, less efficient BC
solubilization as it becomes more concentrated at the end of digestion, and/or fewer BS
micelles available to solubilize BC. Of relevance, BC bioaccessibility and FFA release
were measured at 150 min in separate experiments (data not shown). BC bioaccessibility
and FFA release values (24.3 % and 55.3 %, respectively) by the end of 150 min
duodenal digestion were not significantly different from the results at 120 min (29.7 %
and 56.2 % respectively), indicating a plateau (maximum transfer) of BC after 120 min of
lipolysis.
43
Figure 4: BC bioaccessibility (%) at the end of duodenal digestion (120 min) in the
presence of 0-4 % GG. Data shown is the mean of six independent experiments ± SEM.
Different letters indicate a statistical difference (P<0.05) between treatment means.
Figure 5: BC bioaccessibility (%) at the end of duodenal digestion (120 min) as a
function of the lipid hydrolysis (%) at the end of duodenal digestion (120 min) of the
Control “meal sample.” Data shown is the mean of 3-6 independent experiments.
44
4.3 Digestate Viscosity:
In order to explain the above results, and knowing that GG is viscosity-inducing,
digestate viscosity at the beginning of the duodenal stage was determined for each
sample. Figure 6 shows that, at the beginning of duodenal digestion (0 min), the Control
sample without any GG had a constant viscosity in the range of 1.0 x 10-3-1.8 x 10-3
mPa.s. However, samples containing GG displayed non-Newtonian pseudoplastic
behaviour as evidenced by the decrease in apparent viscosity with increasing shear rate
(Tipvarakarnkoon and Senge, 2008). Increasing concentrations of GG was associated
with higher viscosities of the duodenal digestate samples. Values of apparent viscosity at
47.6 and 94.9 s-1 are compared for each sample in Table 3.
Figure 6: Viscosity (Pa.s) of duodenal digestate samples at the start of simulated duodenal
digestion determined by shear rate sweeps (30 to 200 s-1) using an acrylic cone (6 cm, 2 o,
50 μm truncation gap). Data shown is the mean of three independent trials.
45
With increasing GG present in the duodenal digestate samples, apparent viscosity
values were increased. Emulsion “meal samples” with 2.0 and 4.0 % GG were
significantly more viscous than the other samples. For example, the value at 47.6 s-1 for
the digestate containing 4.0 % GG was significantly higher than with 1.0 % GG, i.e. 167
x 10-3 vs. 3.0 x 10-3 Pa.s, respectively (Table 3). Therefore, higher digestate apparent
viscosities at higher GG concentrations in the “meal samples” were associated with
significantly lower FFA release and significantly lower BC transfer to the aqueous phase.
This is in accordance with the results of Hur et al. (2009) who observed that increased in
vitro digestion viscosity due to the addition of pectin resulted in decreased lipid digestion
of beef patties. Higher digestate viscosity is known to impact glycemic index, by slowing
down the digestion and absorption of carbohydrates (Jenkins et al., 1976; ADA, 2008).
Table 3: Apparent viscosities (Pa.s) at 47.6 and 94.9 s-1 at the beginning of duodenal
digestion (0 min) for the different concentrations of GG1.
GG
Apparent Viscosity at 47.6 s-1
Apparent Viscosity at 94.9 s-1
(%)
(Pa.s)
(Pa.s)
-3a
0.0
0.759 ± 0.136 x 10
0.715 ± 0.081 x 10-3 a
1.0
3.00 ± 1.79 x 10-3 a
2.33 ± 0.87 x 10-3 a
1.5
15.0 ± 1.80 x 10-3 a
12.0 ± 1.50 x 10-3 a
2.0
44.5 ± 7.10 x 10-3 b
34.5 ± 5.70 x 10-3 b
4.0
167 ± 11.0 x 10-3 c
130 ± 6.00 x 10-3 c
1
Data shown is the mean ± SEM of three independent trials.
Different superscript letters indicate significant differences (P<0.05) between values
within each column.
a-c
A similar mechanism could be responsible with lipids. Higher digestate viscosities may
have minimized diffusion rates, slowing down enzymes and micelles needing to reach the
oil-water interface and decreasing the transport rates of digestion products incorporated
46
in mixed micelles (Hur et al., 2009). Our results also agree with Philips (1986), who
observed that, with increased viscosity due to GG, the rate of diffusion of cholesterol
mixed micelles decreased, which could eventually lead to the malabsorption of fat
soluble vitamins and carotenoids.
The initial emulsion had a monomodal size distribution with a volume/mass
moment mean of D4,3= 0.16 ± 0.01 μm. Throughout digestion, particle size increased,
eventually leading to bimodal and multimodal distributions. Yonekura and Nagao (2009)
tested the effects of viscosity-inducing soluble fibres (alginates and pectins) on
carotenoid micellization and their uptake by Caco-2 cells in vitro. They found that BC
uptake was strongly and inversely related to the viscosity of the test media. In 1971,
Heaton et al. discussed Stokes-Einstein’s equation for spherical particles. This shows that
the diffusion coefficient of spherical particles, thus, molecules like emulsion particles, is
inversely related to the medium viscosity (D=kT/6πrη; where k=Boltzmann’s constant,
T=absolute temperature (K), r=particle radius, and η=medium viscosity). As GG is added
to the “meal samples”, the viscosity of the duodenal digestate increases (Figures 6, Table
3). Therefore, according to Stokes-Einstein’s equation, higher viscosities result in slower
diffusion rates of carotenoid micelles, bile acids, enzymes, or other necessary molecules
needed for efficient digestion and bioaccessibility (Gallaher and Schneeman, 1986). To
summarize, in these experiments, increasing concentration of GG increased the digestate
apparent viscosity and decreased the FFA release and BC bioaccessibility.
4.4 Changes in Particle Size During Digestion:
The action of digestive enzymes and mechanical forces results in the formation
and structural breakdown of emulsion lipid droplets. To investigate the influence of GG
47
on these processes, changes in emulsion particle size throughout the simulated gastric and
duodenal digestion were monitored. The size distributions of the Control and emulsions
with 0.0 – 4.0 % GG during digestion are shown in Figures 7 and 9.
The original emulsion droplet sizes averaged at 0.16 μm when no GG was present
in the “meal sample” (control). There was a significant change in droplet size distribution
when the Control emulsion was incubated with SGF containing pepsin (Table 5). At the
start of the gastric phase, a bimodal distribution with peaks around 0.16 and 30.2 μm was
observed in the Control (Figure 7). By the end of the gastric phase, the majority of the
particles were in the range of 11.5 μm in diameter. This increase in size distribution
suggests that proteolysis of the stabilizing soy protein by pepsin and acidification resulted
in flocculation and/or coalescence of the droplets and is consistent with previous work
(Malaki Nik et al., 2010; Sarkar et al., 2008). Lipids are emulsified during gastric
processing, primarily due to peristalsis leading to oil droplet dispersal (Malagelada and
Azpiroz, 1989). In this experiment, a shaking water bath helped to maintain
emulsification of the droplets which decreased in mean diameter from 30.2 to 11.5 μm at
the end of the gastric phase. Armand et al. (1999) studied the effects of the gastric
environment on food emulsions containing proteins from milk and whey, observing that
emulsions of various sizes (0.7 and 10.1 μm) increased in size during the gastric phase by
at least 10 fold. Sarkar et al. (2008) studied protein-stabilized emulsions under simulated
gastric conditions in vitro. Due to the acidic environment (pH 2 during fed state) and
passage through the isoelectric point, the protein-stabilized emulsions initially underwent
extensive flocculation and these flocculated droplets were prone to coalescence due to
proteolysis of the surfactant (Sarkar et al., 2008).
48
After the gastric phase, the emulsion “meal sample” was incubated with SBF and
SDF fluids, containing BSs, phospholipids, pancreatin and other salts. During the first 4
min of the duodenal stage, the majority of the lipid droplets are digested as described
before in Section 4.6.1. As shown in Figure 7, a bimodal particle size distribution was
observed. The 2 h incubation with SBF and SDF fluids resulted in particle sizes of mean
diameters of 0.14 and 26.3 μm. Smaller droplets (0.14 μm) by the end of the 2 h duodenal
digestion could be the result of undigested lipid (as in Figure 3, maximum lipid
hydrolysis reached 56.2%). The observed particle sizes, ~ 25.3 μm, is in agreement with
the findings of Armand et al. (1996), Bauer et al. (2010) and Malaki Nik et al. (2010) that
lipid droplets in the duodenum range in size from 1-50 μm (Armand et al., 1996). During
duodenal digestion, the presence of trypsin in pancreatin causes further proteolysis of SPI
and the absorption of BS and lipolysis products results in even further changes in mean
droplet diameter (Malaki Nik et al., 2010). No significant changes in droplet diameter
were found previously to occur in a similar system in the absence of BS (Malaki Nik et
al., 2010; Sarkar et al., 2010). Similarly, Malaki Nik and colleagues as well as Sarkar et
al. concluded that, in the absence of pancreatin, BS absorption occurs without changes in
the mean lipid droplet diameter. However, as observed, incubation with BS, PL, and
pancreatin resulted in mean droplet sizes increasing.
Malaki Nik et al. (2011) prepared 1.5 % SPI oil-in-water emulsions and measured
D4,3 and D3,2 values throughout the different stages of digestion. They measured the
emulsion diluted in 7.5 mL of SGF (without the pepsin enzyme), and found slight
aggregation of the droplets and measured a D3,2 value of 0.23 ± 0.02 μm. They also found
that adding more inorganic salts (from SBF and SDF solutions without enzymes), caused
49
slightly more droplet aggregation from 0.23 ± 0.02 μm to 0.82 ± 0.1 μm. In the present
experiments, the 1.5% SPI emulsion had a D3,2 value of 0.17 ± 0.02 μm, i.e. slightly
smaller than the D3,2 value measured by Malaki Nik et al. (2011). This may be due to the
fact that Malaki Nik et al. measured their emulsion diluted in SGF (without enzymes),
where the acidic pH and the inorganic salts caused slight particle aggregation.
Figure 8 shows that control mixtures containing different concentrations of GG in
water had relatively large particle diameters (as high as 316 μm). These values correlate
with the data obtained by measuring the particle size values of the “meal samples”
containing increasing concentrations of GG (Table 5). The two peaks observed in Figure
9 for each GG-containing “meal sample” represent the emulsion droplets (0.16-0.18 μm)
and the second peak represents the GG particles that have formed a thickened solution
(measuring up to 316 μm).
Table 4: Average D4,3 and D3,2 (μm) (volume/mass moment mean and surface area
moment mean, respectively) of the emulsion “meal samples” with 0.0, 1.0, 1.5, 2.0, and
4.0 % GG prior to digestion1.
Control
1.0 % GG
1.5 % GG
2.0 % GG
4.0 % GG
D4,3
(μm)
D3,2
(μm)
1
0.16 ± 0.01
26 ± 0.006
134 ± 16.0
256 ± 93.1
278 ± 22.8
0.17 ± 0.02
0.22 ± 3.1
0.36 ± 0.14
0.76 ± 0.29
0.92 ± 0.07
Data shown is the mean ± the SEM.
Therefore, GG in the “meal samples” was responsible for the bimodal size distributions
observed. With the addition of GG to the original emulsion “meal samples”, droplet size
of the mixture increased from 0.16 μm up to 316 μm, resulting in bimodal distributions.
Though GG is a water-soluble fibre, it’s high viscosity enhancing properties result in
thick gels forming, even at concentrations as low as 1.0 % (Marianski, 2011). Table 5
50
summarizes the particle size distribution trends for the “meal samples”. With increasing
GG concentrations, both the D4,3 and D3,2 increased (Table 4). With the addition of 1.0 %
GG to the 5 mL “meal sample” increases in the D4,3 value to 26 μm (vs. 0.16 μm for the
Control) and the surface-weighted mean to increase from 0.17 μm to 0.22 μm were
observed. As more GG was added, the D4,3 and D3,2 values continued to increase.
Patterns of mean diameter of particle size changes of emulsion “meal samples”
containing GG throughout digestion were similar to the patterns observed with the
control digestion. However, distribution of the mean particle sizes of initial “meal
sample” containing GG were bimodal. With a higher the GG concentration, larger
particles were observed. This is in agreement with the findings of Huanga et al. (2001),
who observed bimodal distributions when they prepared their hydrocolloidal systems
with GG concentrations ranging from 0.5 to 1.5 %. In their case, an emulsion was
stabilized with the fiber, as opposed to in this study where GG was added to a pre-formed
protein-stabilized emulsion. In the Huanga et al. (2001) study, higher viscosities may
have led to reduced homogenization efficiency and the formation of larger emulsion
droplets. Pasquier et al. (1996) also tested the effects of viscous soluble fibres (different
GGs, pectins, and gum arabic) on lipid emulsification and TAG lipolysis. Pasquier and
colleagues found that fibre addition caused particle size and overall droplet surface area
to increase, and that this correlated with increased viscosity. In this study, with increasing
concentration of GG present, digestate viscosity was increased and particle size during
digestion was increased. These changes were associated with decreased TAG lipolysis
and BC bioaccessibility.
51
Figure 7: Particle size (μm) distribution of (A) the initial Control emulsion and (B) of the
Control emulsion "meal sample" at the beginning and end of gastric digestion (C) and of
the Control emulsion "meal sample" at the beginning and end of duodenal digestion. Data
shown is representative of the particle size distributions observed during an in vitro
digestion of the Control emulsion.
52
Figure 8: Particle size (μm) distributions of the Control emulsion, 1.0, 1.5, 2.0, and 4.0 %
GG in 5 mL of water. Data shown is representitive of three replicates samples.
GG is reported to have the highest hydration rate at pH 8.0 (Hui et al., 2006). The
molecule’s hydration is also reduced in the presence of salts (Hui et al., 2006). The
addition of SGF solution results in the pH being lowered to 2.0, causing destabilization of
the GG, especially with the presence of the salts present in SGF solution. As described
before, the presence of pepsin causes proteolysis of the SPI, leading to flocculation
followed by coalescence of the emulsion droplets and resulting in larger droplet sizes. It
has been reported that, in humans, the duodenum does not have enough mechanical
energy to allow further emulsification of the lipids (Kellow et al., 1986). By the end of
duodenal digestion, particle sizes were observed to be > 200 μm (Table 5). It is
important to note that simulated duodenal shear in vitro does not mimic human duodenal
shear.
53
Figure 9: Particle size (μm) distributions of the initial “meal samples” and digestate samples with 1.0 (A), 1.5 (B), 2.0 (C) and
4.0 % GG (D) at the beginning and end of gastric and duodenal digestion.
54
Table 5: Summary of particle size distribution peak diameters for emulsion “meal
samples” prior to and at the beginning and end of gastric and duodenal digestion stagesa,b
in the absence (Control, 0.0%) and presence of 1.0, 1.5, 2.0, and 4.0% GG1.
Control
1.0 % GG
1.5 % GG
2.0 % GG
4.0 % GG
“Meal
Sample”
(0.16, 7.7)
(0.18, 5.1)
(182, 1.8)
(0.16, 4.3)
(275, 3.7)
(0.18, 1.6)
(316, 5.2)
(0.18, 1.2)
(275, 7.6)
Start of
Gastric
(0.16, 6.1)
(30, 1.0)
(0.16, 4.5)
(2.5, 2.0)
(209, 1.3)
(5.8, 7.2)
(275, 1.2)
(2.9, 1.1)
(45.7, 2.1)
(316, 5.9)
(0.11, 0.38)
(240, 0.38)
End of
Gastric
(12, 6.9)
(0.26, 2.3)
(1.7, 2.3)
(275, 2.6)
(7.6, 7.8)
(275, 0.85)
(3.3, 2.8)
(40, 1.8)
(275, 4.3)
(6.6, 3.1)
(209, 4.5)
Start of
Duodenal
(0.36, 7.2)
(0.28, 2.9)
(209, 2.7)
(0.18, 3.9)
(240, 3.6)
(0.21, 2.7)
(240, 4.1)
(0.23, 1.8)
(240, 5.6)
End of
Duodenal
(0.14, 4.4)
(26, 4.2)
(0.26, 0.86)
(209, 4.2)
(209, 3.6)
(240, 6.9)
(0.18, 0.46)
(275, 7.2)
1
Data shown is the mean ± the SEM of three independent trials.
Data shown is (x,y), where x represents the particle size (μm) and y represents the
volume (%) of particles of that size. In cases where multiple peaks were observed, this is
indicated by multiple (x,y) coordinates in one cell.
b
Start of Gastric or Duodenal phase refers to measurements at 0 min; End of Gastric
refers to measurements made at 60 min; End of Duodenal phase refers to measurements
made at 120 min.
a
4.5 Bile Acid Binding:
One proposed mechanism by which GG may decrease BC bioaccessibility is by
binding BAs. This potential effect was investigated by measuring the amount of BA in
the aqueous phase of samples after 120 min duodenal digestion. Figure 10 shows that the
BA concentration was similar for all samples. The fact that no significant differences
were observed among the samples indicates there were no significant differences in BA
binding by the GG. However, other studies have shown that BA-binding by fibres is
55
fibre-type dependent. In 2007, Dongowski tested the binding effects of different fibres
(oat bran and the fibre from fruits and vegetables) on in vitro solutions containing
different glycoconjugated BA. Dongowski found that cereal fibres bound the di-hydroxyBAs glycochenodeoxycholic acid (GCDCA) and glycodeoxycholic acid (GDCA) more
readily than the trihydroxy-BA glycocholic acid (GCA). It was found that alcoholinsoluble substances (containing >70% fibre) from fruits and vegetables varied in their
binding effects. Alcohol-insoluble substances from apples bound all 3 BAs, whereas
alcohol-insoluble substances from red beets did not bind them as effectively. Thus, fibres
from different sources bound specifically to different BAs. It is possible that GG has no
potential for direct binding to the BAs present throughout this simulated digestive model.
Since GG is a soluble fibre, it remains in the aqueous phase after centrifugation (Gallaher
and Schneeman, 1986), and thus it was detected and measured in this phase after
simulated duodenal digestion. Since there was an observed decrease in BC
bioaccessibility with increasing concentration of GG, but no significant difference in BA
concentration in the aqueous phases of samples containing 0 or 4.0 % GG, it is possible
that GG physically entraps, rather than binds BAs (Hill, 1982), allowing them to remain
in the aqueous phase, but preventing their ability to form into micelles (Gallaher and
Schneeman, 1986), thus reducing BC transfer.
Initially, 10 mmol/mL of BAs were added to the digestive fluids, however, the
highest measured amount of BAs in the aqueous phase after digestion was measured to be
1.287 mmol/mL (1.287 x 103 μmol/mL). This significant difference may be due to the
fact that the presence of multivalent cations in the digestive fluids, such as Ca2+ and
Mg2+, have the ability to cause BS to precipitate during duodenal digestion, which
56
decreases their digestibility (Read, 2004; Vaskonen, 2003; Karupaiah and Sundram,
2007). Evidence of this precipitate is found after the centrifugation process (described in
Section 3.7) after simulated duodenal digestion is complete. More discussion about BA
binding can be found in Section 4.6.6.
Figure 10: Bile acid concentration (μmol/mL) of the aqueous phase after duodenal
digestion of the emulsion “meal samples’ containing 0-4 % GG. Data shown is the mean
± SEM, n=6. No significant (P>0.05) differences were found between treatment means.
4.6 Investigations with Cholestyramine and Cellulose:
In order to further test the mechanisms by which GG may impact BC
bioaccessibility, the results of the effects of 4.0 % GG in the emulsion “meal samples”
were compared to the effects of “meal samples” containing 4.0% CE and CH. CE is a
polysaccharide of plant cells and an insoluble fibre that does not significantly affect
viscosity (Lattimer and Haub, 2010) or sequester BAs (Ebihara and Schneeman, 1998)
CH is an anion exchange resin with a strong affinity for binding BAs and lowering
57
cholesterol levels, which therefore significantly can affect complete lipid digestion (Johns
and Bates, 1969).
4.6.1 Lipid Hydrolysis:
Emulsion “meal samples” containing 4.0 % CH or 4.0 % CE were prepared as
described in Section 3.3. Figure 11 shows the progression of lipid hydrolysis (%)
throughout the 120 min of duodenal digestion in the presence of CH and CE, compared
with the Control and with 4.0 % GG. A unique pattern with CH was observed. Within 2
min of duodenal digestion, 23.3% of the lipid was hydrolyzed. As discussed earlier, the
majority of lipid digestion often occurs quickly, within 4 min of human in vivo digestion
(Hamosh et al. (1975). However, Figure 11 shows that after 2 min of digestion, the
amount of lipid digested begins to decrease and reaches 8.2 % FFA release by 120 min
(Figure 11). CH is known to bind FFAs (Johns and Bates, 1970; Hagerman et al., 1973)
and to affect pancreatic lipase activity (Lairon et al., 1985). John and Bates (1970)
prepared solutions of FAs ranging from 0.75-5.0 mM and added 25 mg of CH. They
found that CH had a high binding affinity to FAs, particularly those with longer chain
lengths (i.e. laurate, oleate, and linoleate, in increasing order). Hagerman et al. (1973)
found that the more available unbound FAs present, the more CH would bind to them.
Thus, it is possible that some of the FFA generated by lipolysis became bound to the CH
present. As per Figure 11, this may have occurred after 23.3 % of the emulsion’s oil was
digested, until released FAs bound to the CH present in the sample. With more extensive
digestion (i.e. the more FFAs released), the more the available CH bound the FAs present
in solution. FFA release could have also been minimized if the CH rendered pancreatic
lipase inactive. Lairon et al. (1985) tested the affects of CH on lipase activity in vitro with
58
a system of BSs, phospholipids, cholesterol, and pancreatic lipase (0.6 x 10-7 M) in the
absence of oil. They concluded that CH inhibited pancreatic lipase activity by binding it.
Specifically they reported that in a 2.0 % CH solution, 47.5% of the pancreatic lipase was
bound.
Figure 11: (A) Lipid digested (%) throughout 2 h duodenal digestion determined based
on FFA released at 2, 5, 10, 15, 30, 60, 90, and 120 min for the Control and in the
presence of 4.0 % GG, 4.0 % CE, or 4.0 % CH. (B) Lipid digested during the first 30
min of duodenal digestion. Data shown is the result of three independent experiments ±
the SEM, n=6.
The patterns of duodenal lipid hydrolysis were similar between the Control and
samples containing 4.0 % CE. After 120 min, lipid hydrolysis reached 56.2 and 41.8 %
for the control and 4.0% CE, respectively (P<0.05). This was significantly higher than for
4.0 % GG and 4.0 % CH (i.e. 21.6 and 18.5 %, respectively) (P<0.05). Figure 12 shows
the amount of FFAs (%) released at the end of digestion. FFA release was 36.9 and 34.1
% for the Control and with 4.0 % CE, respectively and only 11.0 and 8.23 %, with 4.0 %
GG and 4.0 % CH, respectively (P<0.05).
Therefore, lipid digestion (%) was not
significantly impacted with 4.0 % CE present in the meal sample. It seems that CE had
little or no effect on lipolysis or pancreatic lipase activity. This is in agreement with the
59
several reports showing little or no binding affinity of CE to enzymes, fats, or salts
(Lairon et al., 1985; Vahouny et al., 1980; Hur et al. 2009). Hur et al. performed an in
vitro digestion on beef patties (90 % lean meat, 9.5 % tallow) and incorporated 0.5 % CE.
CE was reported to have no affect on lipid digestion, as there were no significant changes
in FA composition comparing to the control (no fibre added).
Figure 12: Percent FFA released at the end of duodenal digestion (120 min) for the
Control and in the presence of 4.0 % GG, 4.0 % CE or 4.0 % CH. Data shown is the
mean of six independent experiments ± the SEM. Different letters indicate a statistical
difference (P<0.05) between treatment means.
4.6.2 BC Bioaccessibility:
As observed in Figure 13, CE had little effect on BC bioaccessibility. The
proportion of BC transferred to the aqueous phase was the same with 4.0 % CE as for the
Control (i.e. 21.4 vs. 28.3 %, respectively, P>0.05). However, CH, like GG, resulted in a
significantly lower BC bioaccessibility compared with the Control (i.e. 6.98 and 14.1%
respectively). This was expected since CH is a known BA sequesterant (Johns and Bates,
60
1969). As discussed below in relation to Figure 17, this is expected to result in the
formation of fewer mixed BS micelles to solubilize and transport for lipophilic
compounds (Bauer et al., 2005) such as BC.
Figure 13: BC bioaccessibility (%) at the end of duodenal digestion (120 min) for the
Control and in the presence of 4.0 % GG, 4.0 % CE or 4.0 % CH. Data shown is the
mean of six independent experiments ± the SEM. Different letters indicate a statistical
difference (P<0.05) between treatment means.
4.6.3 Viscosity Effects:
The influence of CH and CE on digestate viscosity was investigated, as
previously with GG. Those results are shown in Figures 14 and Table 6. Generally,
duodenal digestate samples containing GG, CE, or CH displayed non-Newtonian
pseudoplastic behaviour. Samples containing 4.0 % GG were the most viscous. CH and
CE had no significant effect on viscosity of the duodenal digestate. Apparent viscosities
values for CH, CE and the Control were significantly lower than the sample containing
61
GG, at both shear rates compared. GG clearly induces viscosity, which may be the
mechanism by which it reduces lipolysis and BC bioaccessibility. Studies have tried to
describe the direct effect of dietary fibre on lipid digestive processes (Moriceau et al.,
2000; Pasquier et al., 1996). These studies suggest that viscous fibres such as GG form
viscous networks, caused by polymer packing or particle packing, which physically
entraps digestive molecules necessary for lipolysis or bioaccessibility. The formation of a
viscous network would decrease molecular diffusion rates, resulting in delayed transport
of the molecules to the interfaces of lipid particles or delayed transport of BSs to form
micelles.
Figure 14: Viscosity (Pa.s) of duodenal digestate samples at the start of simulated
duodenal digestion, as determined by shear rate sweeps (30 to 200 s-1) with an acrylic
cone (6 cm, 2o, 50 μm truncation gap). Data shown is the mean of three independent
trials.
62
Apparent viscosities at 47.6 and 94.9 s-1 of the duodenal digestate samples (0 min)
were compared and are shown in Table 6. 4.0 % GG emulsion “meal samples” at the
beginning of duodenal digestion have viscosities that are significantly higher than the
viscosities of emulsion “meal samples” containing 4.0 % CE or CH.
Table 6: Apparent viscosities (Pa.s) of digestate samples at 47.6 and 94.9 s-1 at the
beginning of simulated duodenal digestion (0 min) for Control sample containing no fibre
and samples containing 4.0% GG, CH, or CE1.
Apparent Viscosity at
Apparent Viscosity at
47.6 s-1 (Pa.s)
94.9 s-1 (Pa.s)
Control
7.59 ± 1.36 x 10-4 a
7.59 ± 1.36 x 10-4 a
4.0 % GG
1.67± 0.11 x 10-1 b
1.30 ± 0.06 x 10-1 b
4.0 % CH
1.23 ± 0.90 x 10-2 a
7.26 ± 4.47 x 10-3 a
4.0 % CE
2.37 ± 0.56 x 10-3 a
1.83 ± 0.08x 10-3 b
1
Data shown is the mean ± the SEM of three independent trials.
Different superscript letters indicate significant differences (P<0.05) between values
within each column.
a-b
4.6.4 Particle Size:
Figure 15 and Table 7 show that the addition of GG, CE, or CH to the emulsion
caused bimodal size distributions. With GG, the observed diameters were in the range of
275 μm, related to lack of complete GG solubilization. Bimodal distributions were
observed with the addition of CH or CE to the emulsion, likely also because of
insolubility of CH and CE in the emulsion. This is supported by results from control
mixtures of CE and CH in water (Figure 16). With 4.0 % CE or 4.0 % CH, the first peak
(measuring ~ 0.16 – 0.24 μm) observed for each “meal sample” is consistent with the
emulsion droplets (correlated with the values found in Table 7) and the second major
63
peak, in the range of 45.7 and 79.4 μm, represent the particle sizes for CE and CH,
respectively. With exposure of the samples to simulated gastric conditions, further
increases in particle size (i.e. up to 417 μm) were observed. After duodenal digestive
fluids were added, the emulsion drops were further digested and sizes remained as large
as 316 μm. The peak values and their respective volumes can be found in Table 7.
Table 7: Summary of particle size distribution peak diameters for emulsion “meal
samples” prior to and at the beginning and end of gastric and duodenal digestion stagesa
in the absence (Control, 0.0%) and presence of 4.0% GG, 4.0% CE or 4.0% CH1.
Control
4.0% GG
4.0% CE
4.0% CH
(0.16, 7.7)
(0.18, 1.2)
(0.21, 2.2)
(0.24, 1.7)
“Meal Sample”
(275, 7.6)
(52.5, 4.1)
(79.4, 5.2)
(0.6, 6.1)
(0.11, 0.38)
(0.14, 3.1)
(20.0, 5.2)
Start of Gastric
(240, 0.38)
(3.31, 4.7)
(417, 3.4)
(45.7, 2.2)
(11.5, 6.9)
(6.61, 3.1)
(0.16, 1.5)
(20.0, 5.4)
End of Gastric
(209, 4.5)
(3.88, 3.1)
(60.3, 4.6)
(0.36, 7.2)
(0.28, 1.8)
(0.36, 3.1)
(1.10, 2.1)
Start of
(240, 5.6)
(3.80, 0.82)
(477, 4.4)
Duodenal
(52.5, 3.2)
(0.14, 4.4)
(0.18, 0.5)
(0.14, 1.4)
(316, 8.2)
End of
Duodenal
(26.3, 4.2)
(275, 7.2)
(39.8, 6.6)
1
Data shown is the mean ± the SEM of three independent trials.
The x coordinate represents the particle size (μm) and the y coordinate represents the
volume (%) of the particles of that size.
a
Start of Gastric or Dudoenal phase is at 0 min; End of Gastric is at 60 min; End of
Duodenal phase is 120 min.
64
Figure 15: Particle size (μm) distributions of (A) the emulsion “meal sample” containing
4.0 % GG, (B) 4.0 % CE, or (C) 4.0 % CH, at the beginning and end of gastric and
duodenal digestion. Data shown is representitive of three replicates for each in vitro
digestion.
65
Figure 16: Particle size (μm) distributions of the Control emulsion, 4.0 % CE, and 4.0 %
CH in 5 mL of water. Data shown is representitive of three replicates samples.
4.6.5 ζ-potential Results:
ζ-potential is a measure of the ionic strength and is useful to characterize the
interfacial composition of emulsions. Initial colloidal stability of the emulsion with and
without GG, CH and CE was investigated and those results shown in Table 8. The
Control “meal sample” stabilized with SPI had a ζ-potential value of -38.6 ± 1.0 mV at
pH 6.5. This is similar to Malaki Nik and colleagues (2011) for the same system. For the
same SPI-stabilized emulsion, they obtained a ζ-potential measurement of -38.9 ± 0.7
mV. With the addition of 4.0 % GG, CH, or CE, ζ-potential measurements were -39.9 ±
1.7, -40.5 ± 0.7, -41.8 ± 1.7 mV, respectively. These values were not significantly
different from the Control. Thus, the addition of GG or CH did not significantly affect the
charge on the emulsion droplets. Malaki Nik et al. (2011) showed that after incubating
the emulsion with SGF at pH 2 without pepsin led to a change in ζ-potential to +10 mV.
Similar changes are expected in the case of the Control emulsion. In addition to SPI being
66
hydrolyzed by pepsin, resulting in emulsion droplets to flocculate and coalesce, droplet
flocculation, affecting overall lipolysis, could also result from the low net charge of the
oil droplets during the gastric phase (Malaki Nik et al. 2011). Thus it is likely that the
initial addition of GG, CH, or CE into the emulsion to create the “meal sample” did not
cause emulsion droplet aggregation due to changes in surface charge.
Table 8: ζ-potential (mV) of the Control emulsion “meal samples” with 1.0, 1.5, 2.0 and
4.0 % GG, or 4.0 % CH, or 4.0 % CE.1,2
Sample
ζ-potential (mV)
Control
-38.6 ± 1.0a
1.0 % GG
-42.4 ± 1.2a
1.5 % GG
-43.2 ± 2.6a
2.0 % GG
-37.9 ± 0.7a
4.0 % GG
-39.9 ± 1.7a
4.0 % CH
-40.5 ± 0.7a
4.0 % CE
-41.8 ± 1.7a
1
Data shown is the mean ± SEM, n=3.
Means with different superscript letters are significantly different from each other at
P<0.05.
2
67
4.6.6 Bile Acid Binding:
BA binding experiments were conducted with CH and CE. The results are shown
in Figure 17. The concentrations of BAs in the aqueous digestate samples with 4.0 % CE
or 4.0 % GG were not significantly different from that of the Control (i.e. the meal
sample containing no potential BA sequesterant). However, the BA concentration was
drastically lower in the case of CH (i.e. ~ 0 μmol/mL). This confirms that CH binds BAs.
Samples containing no fibre, 4.0% CE or 4.0% GG contained 812, 566, or 628 μmol/mL
BA in the aqueous phase after duodenal digestion (P<0.05). In contrast, CH is a known
bile acid sequesterant (Vahouny et al., 1980). Its presence in the “meal sample”
drastically reduced the amount of BAs left in the aqueous phase after duodenal digestion.
This supports that GG does not bind BA and that the observed impacts on lipid digestion
and bioaccessibility are not due to BA binding. Kahlon et al. (2007) prepared stock
solutions of BA (36 mmol/L) and incubated them with 26 mg of CH and observed
binding of 92% of the BAs. Their results agreed with the earlier work of Kahlon and
Chow (2000) and of Sugano and Goto (1990) who both observed that CH bound 93% of
the BA taurocholate. Vahouny and colleagues (1980) prepared solutions of mixed
micelles using 5 mM taurocholate, 625 μM lecithin, 250 μM monoolein, 500 μM oleic
acid, and 250 μM cholesterol. Vahouny et al. added 40 mg of CH to 5 mL of the micellar
solution and observed 81.6, 98.9, and 95.3 % binding of the BSs, phospholipds, and
cholesterol, respectively. As suggested previously, bound BAs are unavailable for micelle
formation (Gallaher and Schneeman, 1986), thus limiting the potential for solubilization
of BC in the aqueous phase.
68
Figure 17: Bile acid concentration (μmol/mL) of the aqueous phase after duodenal
digestion for Control emulsion “meal samples’ and containing 4.0 % GG, 4.0 % CE or
4.0% CH. Data shown is the mean of six independent trials ± the SEM. Different letters
indicate statistical differences (P<0.05) between treatment means.
5.0 Conclusions and Future Directions:
In vitro digestions of a BC-containing lipid emulsion in the presence of different
concentrations of GG were initially performed to investigate the effect of this viscosityinducing fibre on TAG lipolysis and BC bioaccessibility. At higher GG concentrations
from 0.0 - 4.0 %, the extent of lipolysis and transfer of BC to the aqueous phase were
decreased. Duodenal digestate viscosity also increased with GG concentration, and
increases in particle size were also observed. By inducing aqueous phase viscosity, GG
could entrap BSs or enzymes, impeding their ability to reach the oil-water interface and
to form micelles in which to solubilize BCs, thereby resulting in decreased lipolysis and
BC bioaccessibility. Also, by inducing viscosity, GG could slow the diffusion rates of the
BSs or enzymes, once again slowing down the formation of mixed micelles to solubilize
69
BC or slowing down lipid digestion. The addition of GG to the emulsion resulted in
viscous “meal samples.” This initially caused the emulsion “meal sample” to behave
differently than the Control “meal sample” (containing no GG). The addition of GG
resulted in larger particle sizes but no significant changes in surface charge. The Control
emulsion transformed from a uniform distribution to a multimodal distribution of sizes by
the end of simulated duodenal digestion. The addition of GG to the Control “meal
sample” resulted in larger droplet sizes, which caused further flocculation and
coalescence throughout digestion, resulting in larger surface areas, and thus less surface
area for enzymes to adsorb to the emulsion droplets to allow digestion.
Experiments were also conducted with 4.0 % CH and CE, as examples of a
known BA sequesterant (Johns and Bates, 1969) and a non-viscous forming fibre
(Lattimer and Haub, 2010), respectively. “Meal samples” containing 4.0 % CH resulted
in similar (not significantly different) results to “meal samples” containing 4.0 % GG in
terms of lipolysis and BC bioaccessibility. Emulsion “meal samples” containing 4.0 %
CH had less FFA release by the end of digestion as well as lower BC bioaccessibility. In
contrast, “meal samples” containing 4.0% CE had similar lipolysis and BC
bioacccessibilty results as the Control. CE did not affect the release of FFA by the end of
digestion, neither the amount of BC transferred to the aqueous phase after simulated in
vitro gastric and duodenal digestion.
BA binding by GG was also measured and compared to the effects of CE and CH.
The presence of GG in the aqueous phase did not affect BA concentration just as CE.
This was opposite to the affect CH had on BA concentration in the aqueous phase. CH is
a known bile acid sequesterant, and it resulted in relatively significantly less BAs in the
70
aqueous phase. The amount of BAs in the aqueous phase remaining after simulated in
vitro gastric and duodenal digestion of a “meal sample” containing 4.0 % GG was not
significantly different than the amount of BAs remaining in the aqueous phase after
simulated digestion of “meal samples” containing 0.0 % GG or 4.0 % CE. Therefore GG
does not impact lipid digestion or bioaccessibility through BA binding. Due to
significantly increased viscosity of digestate samples due to addition of GG to the initial
“meal samples,” it is most likely that GG exerts its reducing effects on lipolysis and BC
bioaccessibility by reducing diffusion rates and motility of digestive enzymes necessary
to simulate complete digestion.
An ideal model to study the effects of GG on lipid digestion and BC
bioaccessibility would be in vivo human clinical observations. However, due to ethics,
time, costs, and other potential problems, in vitro studies have proven to be a useful
alternative and have been used in the areas of food and animal science (Hur et al., 2009).
In vitro models are relatively inexpensive, fast, simple, and reproducible (Coles et al.,
2005; Failla et al., 2007). However, in vitro systems will rarely match the accuracy that
an in vivo system can offer (Hur et al., 2009; Coles et al., 2005) in terms of unique hostrelated and food related responses to foods at different stages of digestion
(Chitchumroonchokchai et al., 2004). Thus, different responses must be studied and
validated in different systems in vivo (Granado et al., 2006). However, Reboul et al.
(2006) have reported a high correlation between in vivo and in vitro results in the case of
carotenoid release from food matrices and carotenoid incorporation into emulsions and
mixed micelles (bioaccessibility). Using in vitro models allows the researcher to control
the formulations to study the conditions that yield the highest bioavailability of nutrients
71
(Fernandez-Garcia et al., 2008). In vitro models further allow one to study digestive
stability, metabolism, mechanisms, intestinal transport of enzymes, micelles and other
digestive components, and to predict food component bioavailability (Granado et al.,
2006; Chitchumroonchokchai et al., 2004; Garret et al., 1999; Sugawara et al., 2001;
Hedren et al., 2002; During et al., 2002; Tyssandier et al., 2003).
Although 10-30% of lipid digestion may occur during the gastric phase of
digestion by gastric and lingual lipases (Bauer et al., 2005; Mu and Hoy, 2004), this
portion of lipolysis tends to be more important for unhealthy individuals with
impairments in pancreatic lipase, such as patients with cystic fibrosis (Fave et al., 2004).
The gastric phase is important for promoting fat emulsification through the action of
gastric lipase and peristalsis (Armand et al., 1994; Bauer et al., 2005). In these
experiments, emulsification was achieved through sample homogenization and
microfluidization.
The recommended intake of fibre in Canada has been justified due to the growing
incidence of diseases and deficiencies (Heart and Stroke Foundation of Canada, 2003;
Lichtenstein et al., 2006). Specifically, GG has been shown to reduce risks associated
with cardiovascular diseases, such as reducing plasma cholesterol and TAG
concentrations (Roy et al., 2000; Moriceau et al. 2000). However, many studies have
focused on the effects of GG reducing lipemia and rarely have researchers focused on the
potential negative effects of this viscous fibre on the bioaccessibility of other components
within the food matrix. As presented, GG may alter lipid digestion and absorption events
of BC, and potentially of many other lipophilic molecules.
72
Further studies of the mechanisms by which GG exerts its effects on lipolysis
and BC bioaccessibility are needed. Experiments to measure the diffusion rates of bile
acids or enzymes after the addition of GG are necessary to confirm the viscosity effects
observed. Microscopic techniques could help to confirm particle size changes upon the
addition of GG to the emulsion “meal sample” and to changes in particle sizes throughout
digestion. Research should also be expanded to animal and human studies to establish the
effects of GG on nutrient and nutraceutical bioavailability since the fibre is used many
foods. In addition, the study of other dietary fibres on these same processes should be
evaluated as high fibre diets are becoming common. The ability to further understand the
release system of FFAs and BC throughout lipid digestion is an important tool for
designing future delivery systems of bioactive compounds.
73
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