Download Aqueous protein based extraction of oat beta

Aqueous protein based extraction of oat beta glucan and its physiological
effects on satiety and glycaemic responses in healthy adults
by
Joseph Nicholas Katongole
A Thesis
presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Science
In
Food Science
Guelph, Ontario, Canada
© Joseph N. Katongole, December, 2011
ABSTRACT
AQUEOUS PROTEIN BASED EXTRACTION OF OAT BETA GLUCAN AND ITS
PHYSIOLOGICAL EFFECTS ON SATIETY AND GLYCAEMIC RESPONSES IN
HEALTHY ADULTS
Joseph Nicholas Katongole
University of Guelph, 2011
Advisor:
Professor H. D. Goff
β-D-Glucan has been proposed to suppress appetite related perceptions
thus contribute favourably to the regulation of energy intake and the increasing
obesity problem in North America. Due to its low concentrations in grains, the
challenge has been to produce β-glucan concentrates that can be incorporated
into foods without adversely affecting product attributes. Therefore in the first
part of the study, a protocol for the concentration of β-glucan, based on proteinpolysaccharide incompatibility, was investigated. The extract obtained was
utilized in the second part, where the effect of beverages with increased β-glucan
content on perceived satiety and blood glucose, at different fibre concentrations
was studied. Twenty nine healthy adults participated in this study. 5 beverage
pre-loads, containing between 0-2.5 g of β-glucan in 500 mL of the sample, were
ingested 120 min before the given meal. Results showed a trend towards a
decrease in appetite scores with increasing β-glucan content of the beverages, as
well as differences in the blood glucose readings, though these were not
significant, and could not solely be attributed to β-glucan content due to
differences in beverage composition.
ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. H. D. Goff, for having given me the opportunity to
undertake this graduate research in the Department of Food Science at the University of
Guelph. This will always be appreciated.
I would as well like to say thank you to Dr. A. Duncan, and Dr. S. Tosh for their
contribution towards the completion of my research.
The contribution of Dr. H. Anderson and his team at the University of Toronto was truly
appreciated, and I say thank you to this group of researchers.
Special thanks and gratitude go towards my parents, Mr. and Mrs. Katongole, and my
in-laws, Mr. and Mrs. Gosal, for always supporting me.
Words cannot truly express how much gratitude and appreciation that I have for
someone special, that has always believed in me, and who has been with me through
my highs and lows. To my wife Aneeta Katongole……I thank you.
iii
Table of Contents
Chapter I ................................................................................................................................................ 1
Introduction............................................................................................................................................ 1
1.1
An overview of dietary fibre .................................................................................................. 2
A historical account of dietary fibre research .................................................................. 4
1.1.1
Chapter II ............................................................................................................................................... 6
Literature Review .................................................................................................................................. 6
2.1
Dietary fibre ............................................................................................................................ 7
2.1.1
Canadian regulations ........................................................................................................ 8
2.1.2
Recommended intake ....................................................................................................... 9
2.2
Oats ...................................................................................................................................... 11
2.3
β-glucan................................................................................................................................ 12
2.3.1
Oat β-glucan..................................................................................................................... 14
2.3.2
Aqueous extraction of β-glucan from oat bran .............................................................. 17
2.4
Benefits of dietary fibre consumption, and suggested mechanisms of action ............... 18
Cholesterol and cardiovascular diseases ...................................................................... 19
2.4.1
2.4.1.1
Oat and lipids ............................................................................................................... 21
2.4.2
Diabetes ........................................................................................................................... 23
2.4.3
Weight Control ................................................................................................................. 24
2.4.4
Constipation and diarrhea ............................................................................................... 26
2.4.5
Diverticulosis .................................................................................................................... 27
2.4.6
Irritable Bowel Syndrome (IBS) ...................................................................................... 27
2.5
Protein polysaccharide mixtures ........................................................................................ 28
2.5.1
Polymer interaction .......................................................................................................... 29
2.5.1.1
Segregative phase separation .................................................................................... 30
2.5.1.2
Associative phase separation ..................................................................................... 30
2.5.1.3
Thermodynamics aspects ........................................................................................... 31
2.5.2
Factors affecting protein polysaccharide phase separation......................................... 31
2.5.2.1
pH .................................................................................................................................. 32
2.5.2.2
Ionic strength ................................................................................................................ 32
iv
2.5.2.3
Biopolymer ratio and biopolymer concentration ........................................................ 32
2.5.2.4
Charge density ............................................................................................................. 34
2.5.2.5
Processing factors ....................................................................................................... 35
Phase separation kinetics ............................................................................................... 35
2.5.3
Chapter III ............................................................................................................................................ 36
Concentration of β-glucan from oat bran .......................................................................................... 36
Abstract................................................................................................................................................ 37
3.1
Introduction .......................................................................................................................... 38
3.2
Materials and methods........................................................................................................ 40
3.2.1
Materials ........................................................................................................................... 40
3.2.2
Laboratory- scale aqueous protein-based β-glucan extraction ................................... 40
3.2.3
Pilot plant β-glucan extraction process .......................................................................... 42
3.2.4
β-glucan, protein and starch tests .................................................................................. 44
3.2.4.1.1
Extractable β-Glucan ................................................................................................... 44
3.2.4.1.2
Total β-Glucan.............................................................................................................. 44
3.2.4.2
Protein........................................................................................................................... 44
3.2.4.3
Starch............................................................................................................................ 45
3.2.5
Molecular Weight Determination .................................................................................... 45
3.2.6
Rheological properties .................................................................................................... 45
3.2.7
Structural investigation .................................................................................................... 46
3.3
Results and discussion ....................................................................................................... 46
3.3.1
Developed extraction process ........................................................................................ 46
3.3.2
Phase behaviour .............................................................................................................. 49
3.3.3
Rheological properties .................................................................................................... 51
3.3.4
Structural analysis ........................................................................................................... 54
3.4
Conclusion ........................................................................................................................... 56
Chapter IV ........................................................................................................................................... 57
Satiety and related blood glucose effects of Oat β-glucan supplemented dairy beverages ........ 57
Abstract................................................................................................................................................ 58
4.1
Introduction .......................................................................................................................... 59
4.2
Materials and methods........................................................................................................ 60
4.2.1
Subjects ............................................................................................................................ 60
v
4.2.2
Materials ........................................................................................................................... 62
4.2.3
Preparation of beverages................................................................................................ 62
4.2.4
The meal........................................................................................................................... 63
4.3
Statistical analysis ............................................................................................................... 64
4.4
Results and discussion ....................................................................................................... 64
4.4.1
Satiety ............................................................................................................................... 64
4.4.2
Blood glucose .................................................................................................................. 67
4.4.3
Meal intake ....................................................................................................................... 69
4.5
Conclusion ........................................................................................................................... 71
Chapter V ............................................................................................................................................ 72
General Discussion ............................................................................................................................ 72
5.1
Dietary fibre .......................................................................................................................... 73
5.2
The extraction ...................................................................................................................... 74
5.3
The clinical trial .................................................................................................................... 76
References .......................................................................................................................................... 79
vi
List of Tables
Table 2.1:
Dietary reference intake.............................................................................9
Table 2.2:
Dietary fiber content of commonly consumed fruits, vegetables, and
grains........................................................................................................10
Table 2.3:
Taxonomic information..............................................................................11
Table 2.4:
β-Glucan content of commonly available oat based foods.......................12
Table 2.5:
Summary of representative studies involving consumption of
β-Glucan from oats....................................................................................21
Table 3.1:
Process extraction mass flow....................................................................47
Table 3.2:
Typical shear rates observed in some common processes relevant to
food emulsions..........................................................................................53
Table 4.1:
Composition of the oat β-Glucan fortified beverages................................63
Table 4.2:
Macro-nutrient composition of beverages.................................................63
vii
List of Figures
Figure 2.1:
Different β-Glucan sources and their structures........................................13
Figure 2.2:
Polymer of β-(1-4)-D-glycopyranosyl units separated by single β-(1-3)D-glycopyranosyl units..............................................................................14
Figure 2.3:
How dietary fibre affects physiologic measurements................................19
Figure 2.4:
Schematic representation of factors affecting phase separation in
protein-polysaccharide mixtures...............................................................33
Figure 3.1:
Schematic of the laboratory-scale extraction process...............................41
Figure 3.2:
Schematic of the pilot plant-scale extraction process...............................43
Figure 3.3:
Freeze dried extract..................................................................................48
Figure 3.4:
(a) A phase diagram representing initial mixtures.....................................49
(b) A bi-phasic system showing an upper liquid phase and lower gel like
phase........................................................................................................49
Figure 3.5:
Phase separation of oat β-glucan / whey protein isolate aqueous
dispersions................................................................................................50
Figure 3.6:
Mechanical spectra for 0.2-1% (w/v) extract solutions and their
apparent viscosity……………………………………………………………...52
Figure 3.7:
Selected shear rates for 0.2-1% (w/v) extract solutions............................54
Figure 3.8:
An SEM image showing the extract microstructure..................................55
Figure 4.1:
Baseline adjusted changes in the VAS ratings for pre-meal and postmeal appetite.............................................................................................65
Figure 4.2:
Baseline adjusted changes in the measurement ratings for pre-meal
and post-meal blood glucose....................................................................68
Figure 4.3:
Food intake and cumulative food intake....................................................69
viii
List of Appendices
Appendix A: Information sheet and consent form……………………………………..103
Appendix B: Food acceptability form……………………………………………………112
Appendix C: Recent food intake and activity form……………………………………..114
Appendix D: Visual analogue scales – motivation to eat……………………………...115
Appendix E: Visual analogue scales – physical comfort………………………………116
Appendix F: Visual analogue scales – energy / fatigue and stress…………………..117
Appendix G: Fibre enriched beverage study session schedule………………………118
ix
Chapter I
Introduction
1
1.1
An overview of dietary fibre
Current market trends indicate that the main factors that drive research in the
area of functional foods are higher health care cost, greater life expectancy, and desire
for an improved quality of life (Vasiljevic et al., 2007). As consumers become more
aware of the link between diet and health there is an ever increasing demand for health
promoting foods. This consequently has increased interest within the food industry in
functional foods and nutraceuticals as a source of value added products (Fagan et al.,
2006).
The beneficial effects of healthy diets on quality of life and on the costeffectiveness of health care has prompted the food industry to face the challenge of
developing new food products with special health-promoting characteristics. Meeting
this challenge involves the identification of new sources of nutraceuticals, as well as
other nutritional and natural materials with the desirable functional characteristics.
Barley and oats are examples of such sources and could be good bases for functional
food products. Cereals are an important source of dietary fibre, contributing to about
50% of the fibre intake in Western countries (Nyman et al., 1989).The hemicellulosic
polysaccharides of rye and wheat are composed mainly of pentosans (arabinoxylans),
whereas those in oats and barley are composed mainly of β-glucans (Selvendran and
Verena, 1990). Both barley and oats have been reported to be effective in lowering total
serum and LDL-cholesterol in humans and animals, the effect being attributed to the
content of β-glucans (Maier et al., 2000; Bell et al., 1999; Behall et al., 1997; McIntosh
et al., 1991).
2
Plant cell wall materials, primarily cellulose, other non-starch polysaccharides
and lignin, are components of dietary fibre. The only common feature of these polymers
is that they are non-digestible, which is the principal criterion for being classified as a
component of dietary fibre (Sikorski et al., 2008). Therefore, not only do natural
components of foods contribute to dietary fibre, so also do gums that are added to
provide the functionalities that are attributed to dietary fibre. The key characteristic is
that the substance not be digested in the human small intestine, so non-digestible
oligosaccharides (e.g. raffinose and stachyose) are included as dietary fibre
substances.
Oligo- and polysaccharides may be digestible, partially digestible, or nondigestible. When digestive hydrolysis to monosaccharides occurs, the products of
digestion are absorbed and metabolised. Those carbohydrates not digested to
monosaccharides by human enzymes in the small intestine may be metabolized by
microorganisms in the large intestine, producing low molecular weight acids that are
partially absorbed and catabolised for energy (Sikorski et al., 2008). Dietary fibre
increases intestinal and fecal bulk, which lowers colonic transit time and helps prevent
constipation, and its presence in foods induces satiety at meal time.
However, in Western countries dietary fibre intake is considered low. The U.S.
Food and Nutrition Board of the Institute of Medicine (IOM) set an adequate daily intake
of total fibre in the diet at 25 g and 38 g for women and men respectively, based on the
intake level observed to protect against coronary heart disease (IOM, 2005). In Canada,
surveys of nutrient intakes from foods indicate that the mean dietary fibre intake ranged
from 14.3 to 16.6 g/d for women and from 16.5 to 19.4 g/d for men, in 2002 (Table 8.13,
3
CCHS 2.2, Health Canada and Statistics Canada, 2004). This intake is well below the
IOM recommendations for dietary fibre and reflects a limited consumption of whole grain
cereals, fruits, vegetables, and pulses considered to be the best natural sources of
dietary fibres.
1.1.1 A historical account of dietary fibre research
Sixty three years ago, the concept of dietary fibre was introduced by Hipsley
(1952) to designate non-digestible plant cell wall constituents (Malkki, 2004; Hipsley,
1953). Ten years later Groot et al., (1963), presented very promising results on the
reduction of blood cholesterol in rats and in young human volunteers after eating large
doses of oatmeal. At the beginning of the 1970s researchers such as Burkitt and
Trowell (Burkitt et al., 1974; Trowell, 1972), correlated diets among people in some
African countries and in Western industrialized countries to the prevalence of some
diseases. Conclusions that were drawn on the prevalence of some so-called civilization
diseases opened the eyes of nutritionists, other food scientists, and a part of the
medical profession, to study the underlying facts and the possibilities they could open
(Malkki 2004).
An intensive research soon followed regarding effects of foods rich in dietary
fibre and of isolated fibre components. Effects of various fibre materials on blood
glucose and insulin were found in several studies (Jenkins et al., 1978), and the
influence of dietary fibre on blood cholesterol and insulin were investigated by several
groups (Malkki, 2004). Active research was also performed by several groups on the
effects of fibre on the physiology of the gastrointestinal tract (Cummings and Englyst,
4
1995) and on its various diseases. In 1988, according to Malkki (2004), an official
recommendation to increase consumption of whole grain foods and cereals was
included in the report of the Surgeon General by the U. S. Department of Health and
Human Services.
5
Chapter II
Literature Review
6
2.1
Dietary fibre
Dietary fibre consists of both soluble and insoluble fibre. Both types are important
to health in different ways. Soluble fibre includes gums, mucilages, pectin and some
hemicelluloses. Cellulose, lignin and the rest of the hemicelluloses, are all insoluble
fibres. Water-soluble fibre in cereals is composed of non-starchy polysaccharides such
as β-glucan. Water-soluble dietary fibre can form viscous solutions. Increased viscosity
in the intestine slows intestinal transit, delays gastric emptying and slows down glucose
and sterol absorption in the intestine (Anderson and Chen, 1986).
The soluble fibre from oatmeal and oat bran is very effective in lowering blood
cholesterol and normalizing blood sugar levels (Kahlon and Chow, 1997; Wood et al.,
1990). Insoluble fibre contains lignin as well as non-starchy polysaccharides. Lignin is
not a polysaccharide but is a lipophilic, phenolic polymer, which can absorb bile acids.
Insoluble dietary fibers usually have high water holding capacity, which contributes to
increased fecal bulk. Associated with dietary fibre are antinutrients, such as phytic acid
and oxalic acid, and proteins which could affect to a certain extent mineral bioavailability
by binding and trapping minerals within dietary fibre particles or shortening the transit
time of nutrients through the intestine. There are several in vivo and in vitro studies
which indicate that dietary fibre might have important impacts on mineral balance
(Haack et al., 1998; Idouraine et al., 1996).
In 2002, the Institute of Medicine published a set of definitions for dietary fibre.
The definition suggested that the term dietary fibre would describe the non-digestible
carbohydrates and lignin that are intrinsic and intact in plants, whereas functional fibre
consists of the isolated nondigestible carbohydrates that have beneficial physiological
7
effects in human beings. Total fibre would then be the sum of dietary fibre and
functional fibre. Nondigestible would designate fibre that is not digested and absorbed in
the human small intestine (American Dietetic Association, 2008).
2.1.1 Canadian regulations
A guideline developed by the Bureau of Nutritional Sciences, Food Directorate,
Health Products and Food Branch, Health Canada (Proposed Policy: Definition and
Energy Value for Dietary Fibre 2010), sought to address potential safety issues unique
to novel sources of fibre and a desire that the product not be misrepresented to the
Canadian public. It stated that if a novel fibre source or novel fibre containing product
were not safe for human consumption, it would be in violation of Subsection 4(a) of the
Food and Drugs Act. In addition to this, if a product was to be represented as containing
dietary fibre, but did not have the beneficial physiological effects expected of dietary
fibre, then the product would be in violation of Subsection 5(1) of the Food and Drugs
Act. The guideline indicated that both the safety and the efficacy of the fibre source
must be established in order for the product to be identified as a source of dietary fibre
in Canada, and the physiological efficacy must be demonstrated through experiments
using human subjects. Health Canada identified three physiological effects (improving
laxation or regularity, normalization of blood lipid levels, and attenuation of blood
glucose responses), at least one of which must be demonstrated by a novel fibre to be
accepted as dietary fibre. Dietary fibre was therefore defined as that which consists of
naturally occurring edible carbohydrates (DP>2) of plant origin that are not digested and
8
absorbed by the small intestine and includes accepted novel dietary fibres (Health
Canada 2010).
2.1.2 Recommended intake
Dietary Reference Intakes (DRIs) for total fibre by life stage group are shown in
Table 2.1. The AIs for total fibre are based on the intake level observed to protect
against CHD based on epidemiological and clinical data. The Adequate Intake (AI) was
developed by the American Dietetic Association (2008), and this was done because
data was not available to determine Estimated Average Requirements and thus
Recommended Dietary Allowance for total fibre. The AI was based on the median
intake level observed to achieve the lowest risk of coronary heart disease (CHD).
Table 2.1: Dietary Reference Intakes (American Dietetic Association, 2008)
Dietary Reference Intakes (DRI) for total fibera by life group stage and DRI values (g/1000
kcal/d)b
Adequate Intake
Life stage group
(y)
Men
g/1000 kcal/d
Women
g/1000 kcal/d
1-3
4-8
9-13
14-18
19-30
31-50
51-70
>70
14
14
14
14
14
14
14
14
19
25
31
38
38
38
30
30
14
14
14
14
14
14
14
14
19
25
26
26
25
25
21
21
a
Total fiber is the combination of dietary fiber (the edible, nondigestible carbohydrate and lignin
components in plant foods) and functional fiber (isolated, extracted, or synthetic fiber that has proven
health benefits)
b
Values are examples of the total grams per day of total fiber calculated from g/1000 kcal multiplied by
the median energy intake (kcal/1000 kcal/day) from the Continuing Survey of Food Intakes by
Individuals 1994-1996,1998.
9
The reduction of risk of diabetes can be used as a secondary endpoint to support the
recommended intake level. The DRI development panel suggested the recommended
intakes of total fibre may also help ameliorate constipation and diverticular disease,
reduce blood glucose and lipid levels, and provide a source of nutrient-rich, low energydense foods that could contribute to satiety, although these benefits were not used as
the basis for the AI (American Dietetic Association, 2008).
Many popular American foods contain little dietary fibre. Servings of commonly
consumed grains, fruits, and vegetables contain only 1 to 3 g dietary fibre (Marlett and
Cheung, 1997). Major sources of dietary fibre in North America include grains and
vegetables (Fungwe et al., 2007). Legumes are very rich in dietary fibre, but because of
low consumption only provide about 6% of the fibre in the US diet. Fruits provide only
10% of the fibre in the overall US diet because of low fruit consumption and the low
amount of fibre in fruits, except for dried fruits.
Table 2.2: Dietary fiber content of commonly consumed fruits, vegetables, and grains
(American Dietetic Association, 2008)
Food
Fruits
Orange
Apple, large with skin
Banana
Vegetables
Beans, kidney, canned
Peas, green, canned
Grains
Bread, white wheat
Bread, whole wheat
Oat bran muffin
Serving size
Total dietary fiber (g/serving)
1 orange
1 apple
1 banana
3.1
3.7
2.8
½c
½c
4.5
3.5
1 slice
1 slice
1 muffin
0.6
1.9
2.6
10
Vegetables and cereal grains are especially rich in water insoluble fibre, with the highest
amounts in wheat and corn (Theuwissen and Mensink, 2008). The natural gel-forming
or viscous fibres (pectins, gums, mucilages, algal polysaccharides, some storage
polysaccharides, and some hemicelluloses) are water-soluble. Foods rich in watersoluble fibre include oats, barley, dried beans and some fruits and vegetables. Of total
dietary fibre intake, approximately 20% is water-soluble and 80% is water-insoluble
(Bazzano et al., 2003).
2.2
Oats
Oat (Avena sativa) is distinct among the cereals due to its multifunctional
characteristics and nutritional profile. It is a good source of dietary fibre especially βglucan, minerals and other nutrients. Oat bran in particular is a good source of B
complex vitamins, protein, fat, minerals besides heart healthy soluble fibre β-glucan.
Table 2.3 : Taxonomic information (Sadiq Butt et al., 2008)
Botanical name
Kingdom
Subkingdom
Superdivision
Division
Class
Subclass
Order
Family
Genus
Species
Avena sativa
Plantae: plants
Tracheobionta: vascular plants
Spermatophyta: seed plants
Magnoliophyta: flowering plants
Liliopsida: monocotyledons
Commelinidae
Cyperales
Poaceae: grass family
Avena: oat
A.sativa: common oat, A.byzantina, A. fatua,
A. diffusa, A. orientalis
Russia and Canada are the biggest producers of oats. Available figures on world
production estimate that 24.6 million metric tonnes of oats were harvested in 2005 with
11
3 million metric tonnes going into the human food chain (United States Department of
Agriculture, 2008). Oat products include breakfast cereals, oatmeal and breads made
with rolled oats, oat flour or oat bran, with oats mainly used in their natural wholegrain
state (Ruxton and Derbyshire, 2008). In 2004, the Joint Health Claims Initiative (JHCI)
of the United Kingdom approved a health claim which permitted oat products, containing
at least 0.75 g β-glucan per serving, to display the following wording: “The inclusion of
oats as part of a diet low in saturated fat and a healthy lifestyle can help reduce blood
cholesterol” (JHCI, 2004). Table 2.4 shows some commonly available oat based foods
and their β-glucan content.
Table 2.4: β-Glucan content of commonly available oat-based foods (Ruxton and
Derbyshire, 2008)
Food
Oat bran
Oatmeal (dry weight)
Muesli
Cereal bar
Oat biscuits
Porridge oats (dry weight)
Oat flake breakfast cereal
Oat biscuit breakfast cereal
Bread made with oat flour
Pasta (dry weight)
2.3
Serving size (g)
40
60
50
25
50
35
30
2 biscuits
50
100
Β-Glucan content (g)
3.00
3.00
1.50
0.75
0.75
1.75
0.90
1.75
0.75
2.00
β-glucan
β-Glucans are carbohydrates consisting of linked glucose molecules, which are
major structural components of the cell walls of yeast, fungi and some bacteria (Volman
et al., 2008). Also, cereals such as barley and oat contain β-glucans as part of their
12
endosperm cell walls. Depending on the source, there are clear differences in
macromolecular structure between β-glucans (Fig: 2.1).
Fig 2.1: Different β-glucan sources and their structure (Volman et al., 2008)
The cell wall β-glucans of yeast and fungi consist of 1,3 β-linked glycopyranosyl
residues with small numbers of 1,6 β-linked branches (Volman et al., 2008). In contrast,
the oat and barley cell walls contain unbranched β-glucans with 1, 3 and 1,4 β-linked
glycopyranosyl residues, whereas β-glucans from bacterial origin are unbranched 1,3 βlinked glycopyranosyl residues (Brown et al., 2003; Estrada et al., 1997).
Besides differences in type of linkage and branching, β-glucans can vary in
solubility, molecular mass, tertiary structure, degree of branching, polymer charge and
solution conformation (triple or single helix or random coil). All these characteristics may
influence their immune modulating effects. Brown et al., (2003) suggested that high
molecular weight (MW) and / or particulate β-glucans from fungi directly activate
leukocytes, while low MW β-glucans from fungi only modulate the response of cells
when they are stimulated with, for instance, cytokines. With respect to the
characteristics of the β-glucans, it should be noted that the isolation method may
13
influence these characteristics. Consequently, differences can be expected between
various β-glucans differentially isolated from the same source (Volman et al., 2008).
Fig. 2.2: Polymer of β-(1-4)-D-glycopyranosyl units separated by single β-(1-3)-D-glycopyranosyl units
2.3.1 Oat β-glucan
Purified oat β-glucan is a linear, unbranched polysaccharide composed of 1-4-Olinked (70%) and 1-3-Olinked (30%) β-D-glucopyranosyl units (Sadiq Butt et al.,
2008).The 1-3-linkages occur singly and most of the 1-4-linkages occur in groups of two
or three leading predominantly to a structure of β-(1-3)-linked cellotriosyl and
cellotetraosyl units (Dawkins and Nnanna, 1995)(Fig. 2.2). β-glucan has outstanding
functional and nutritional properties exhibiting high viscosities at relatively low
concentrations. β-glucan solutions (1%) have a low flow behaviour index and a high
consistency index in the power law model (Autio et al., 1987). Its viscosity is stable over
a wide range of pH (2–10) but it decreases with increasing temperature (Dawkins and
Nnanna, 1995). β-glucan can be used as a thickening agent in the food industry (Lyly et
14
al., 2003; Wood, 1984); it may influence the sensory quality of beverages (Lyly et al.,
2003) and is of particular importance in human nutrition (Malkki, 2004).
Molecular weight and concentration have a great influence on the viscosity and
the rheological behaviour of β-glucans in aqueous solution and in the intestinal tract.
Differences in rheological properties of β-glucan from several oat varieties was found at
the same β-glucan concentration due to difference in molecular weight (Autio et al.,
1987). β-glucans extracted from oat bran has higher viscosities than those from oat
endosperm (Wikstrom et al., 1994). Similarly β-glucans extracted from enhanced oat
lines are more viscous than those from traditional lines (Colleoni-Sirghie et al., 2004).
In order to be physiologically active and form viscous solutions in the gut, β-glucan must
be soluble, and the concentration and molecular weight must be sufficiently high. The
molecular weight of β-glucan in oat / barley products is reported to be smaller than the
molecular weight of β-glucan in the raw material (Aman et al., 2004; Kerckhoffs et al.,
2002; Beer et al., 1997; Sundberg et al., 1996). The molecular weight of β-glucan in oat
bread, for example, was reduced as compared with the molecular weight of β-glucan in
oat bran. Raw material, endogenous β-glucanase activity, processing, and storage
conditions affect the amount, solubility, molecular weight, and structure of β-glucan in
the products (Degutyte-Fomins et al., 2002; Zhang et al., 1992). There are some
indications that the molecular weight of β-glucan may partly be reduced during its
passage through the upper gastrointestinal tract (Robertson et al., 1997).
Oat products of different composition, for example, oat meal, oat bran, pretreated by different methods, for instance, extrusion, autoclavation or even untreated
when used in diets, have shown beneficial effects in nutritional studies with humans and
15
rats (Drzikova et al., 2005). Highly water-soluble β-glucan, with moderate to high
molecular weight, may reduce serum LDL cholesterol levels better than β-glucan with a
low water-solubility and low molecular weight. This difference in effect is explained by
the hypothesis that a higher intestinal viscosity lowers the reabsorption of bile acids,
leading to an increased excretion of bile acids. Increased bile acid excretion promotes
bile acid synthesis from cholesterol, which will increase LDL cholesterol uptake in the
liver.
The Food and Drug Administration (FDA) of the United States approved in
January 1997 a health claim that “Water-soluble fibre from oatmeal, as part of a low
saturated fat, low cholesterol diet, may reduce the risk of heart disease” (Kerckhoffs et
al., 2002). The FDA determined that 3 g of β-glucan must be consumed per day to
achieve a clinically relevant serum cholesterol-lowering effect. The whole oat containing
food should provide at least 0.75 g of water-soluble fibre per serving (Ruxton and
Derbyshire, 2008).
While oats contain around 4 % β-glucan, 5 % to 10 % β-glucan is present in
barley. Despite its naturally higher β-glucan content, fewer trials have investigated
barley fibre since barley is less palatable than oats and a less common dietary
component. Nevertheless, the FDA did conclude, based on the totality of available
scientific evidence that whole grain barley and dry milled barley products, such as
flakes, grits, flour and pearled barley, are appropriate sources of β-glucan water-soluble
fibre to claim that they reduce the risk of heart disease (FDA, 1997).
16
2.3.2 Aqueous extraction of β-glucan from oat bran
In the aqueous alcohol based enzymatic process (Vasanthan and Temelli, 2002)
grain flour or bran is initially slurried in aqueous ethanol and screened to remove other
components, primarily starch and protein, with the filtrate. The fibre particles that are
enriched in β-glucan are retained on the screen. This β-glucan concentrate on the
screen is re-slurried in alcohol and then treated with enzyme preparations (protease and
thermo-stable α-amylase) to hydrolyze protein and starch that are bound to fibre
particles. With this the β-glucan remains intact within the cell wall and is not solubilised.
Subsequently, the β-glucan rich cell wall fibre particulates that are free from bound
starch and protein are recovered by simple screening techniques.
The aqueous enzymatic (Inglett, 1992) and aqueous thermomechanical (Inglett,
2000) approaches have also been used to yield β-glucan concentrates in oat and barley
processing. In the aqueous enzymatic process oat flour or bran is initially mixed with
water containing amylase enzyme (thermostable α-amylase) and heated to boiling
temperatures. During this step, both β-glucan and starch solubilize and the amylase
enzyme selectively hydrolyses starch into dextrin. The solubilized β-glucan and dextrin
are recovered from the slurry by centrifugation and dried to a powder which can as a
„„fat replacer” in various food products. In the aqueous thermo-mechanical process flour
or bran is slurried in water and mechanically sheared while the slurry is heated to
boiling. In the end, the solubilised β-glucan and gelatinized starch are recovered by
centrifugation and dried to a powder.
Wood et al., (1989) developed the aqueous alkali process which essentially
involves: slurrying of flour in aqueous alkali media, centrifugation of the slurry to
17
separate the insoluble solid particles, containing starch and insoluble fibre, from the
liquid phase, containing solubilised β-glucan and proteins; precipitation of proteins at
their isoelectric point by the addition of acid and their removal from the liquid phase by
centrifugation, and, recovery of β-glucan concentrate from the liquid / aqueous phase by
alcohol precipitation and centrifugation, followed by drying.
A look at the wet separation processes above shows that they either employ the
use of ethanol, and or enzymes, to obtain an extract with increased β-glucan content.
Hydrolysis of proteins through the use of proteases can lead to either depolymerisation
of the extracted β-glucan as it may contain enzymes with β-glucanase activity
(Immerstrand et al., 2009) and or denaturation of the proteins within the developed
products consequently leading to shorter shelf life of such products. Non-utilization of
alcohol during extraction will substantially reduce production costs as the energy
intensive recycling step will be done away with. Therefore the objective in this section of
my study is to create a process that will produce a high molecular weight, and soluble,
extract, without using either enzymes or alcohol.
2.4
Benefits of dietary fibre consumption, and suggested mechanisms of
action
Fig. 2.3 shows a summarised illustration of the way dietary fibre affects
physiologic measurements and the consequent benefits of dietary fibre inclusion in
one‟s diet.
18
Fig. 2.3: How dietary fibre affects physiologic measurements (Slavin 2005)
2.4.1 Cholesterol and cardiovascular diseases
Coronary artery disease is the major cause of death in the United States and in
most Western countries, and blood cholesterol is a major risk factor (Kannel et al.,
1971). Dietary and pharmacologic reductions in total and LDL cholesterol decrease the
risk of the malady (Sacks et al., 1996; Byington et al., 1995) . Oat bran exerts a small
but potentially useful effect on plasma lipoprotein risk factors for cardiovascular disease
(Sadiq Butt et al., 2008). In subjects with mild hypercholesterolemia and normal blood
pressure fed different diets containing dietary fibre from wheat, oat, and rice bran, the
oat bran was found to be the only fibre source that significantly lowered total and lowdensity-lipoprotein (LDL) cholesterol levels. Although all three brans were found to
slightly increase high-density-lipoprotein (HDL) cholesterol levels there were no
19
significant changes in blood pressure, blood glucose, or serum insulin responses to a
test meal on any of the bran supplemented diets (Kestin et al., 1990).
The cholesterol lowering potential varies extensively among different fibre
sources (Sadiq Butt et al., 2008). The reasons for such sample variations include small
sample sizes, different dosages of fibre, different background diets, concurrent changes
in body weight, varying dietary control, and different types of subjects. Certain fibres
lower cholesterol more effectively than others and oat is an example in this regard
(Anderson, 1995; Ripsin et al., 1992; Bell et al., 1990; Kris-Etherton et al., 1988).This
recommendation is supported by the results of studies that demonstrate significant,
although variable, reductions in serum cholesterol after ingestion of various oat brancontaining products (Ripsin et al., 1992; Anderson et al., 1984; Kirby et al., 1981). A
number of oat based studies are summarized in Table 2.5.
The mechanism for cholesterol reduction with oat bran diets can include an
increased excretion of bile acid (Schrijver et al., 1992), which in turn stimulates the liver
to utilize available cholesterol to produce more bile acid (Kahlon et al., 1992; Kahlon et
al., 1990). Intake of oat bran results in increased lipid excretion and percent digestibility.
In general, when a soluble fibre that is not viscous is evaluated or the fibre is treated to
reduce viscosity sufficiently, the cholesterol-lowering ability is lost (Anderson et al.,
1993; Everson et al., 1992). As components in foods are digested and absorbed from
the small intestine, fibre becomes a major component in the gut lumen, making the
viscosity evident. This viscosity interferes with bile acid absorption from the ileum
(Marlett et al., 1994; Everson et al., 1992). In response, LDL cholesterol is removed
20
from the blood and converted into bile acids by the liver to replace the bile acids lost in
the stool (Hillman et al., 1986).
2.4.1.1
Oat and lipids
Dietary fibre reduces fasting lipoproteins and postprandial lipoproteins (Anderson
et al., 1991; Redard et al., 1990). Oat bran significantly decreased serum total
cholesterol and LDL cholesterol compared with control values. Anderson et al., (1991)
compared the effects of soluble-fibre (oat bran) and insoluble fibre (wheat bran) intakes
on serum lipids, apolipoproteins, and lipoprotein fractions for 20 hypercholesterolemic
men on a metabolic ward for 21 days. They postulated that the interactions of four
separate processes may contribute to the hypocholesterolemic effect of oat bran. First,
oat bran significantly increases fecal bile acid excretion and alters bile acid metabolism.
Secondly, oat bran may alter lipoprotein metabolism, possibly by increasing hepatic LDL
receptors. Oat bran tends to selectively lower LDL cholesterol to a greater extent than
does HDL cholesterol. Thirdly, oat bran is fermented in the colon into short chain fatty
acids such as acetate, propionate, and butyrate and after absorption into the portal vein
propionate may inhibit hepatic cholesterol synthesis (Wright et al., 1990). Lastly,
decreases in insulin secretion associated with fibre such as oat bran could lead to
reduction in cholesterol synthesis (Jenkins et al., 1989). A daily intake of 106 g oat bran
providing 15.3 g TDF (total dietary fibre) and 7.6 g soluble fibre over 21 days was
accompanied by significant reduction in total cholesterol, LDL cholesterol, and
apolipoprotein B-100 in hypercholesterolemic men. An equivalent intake of TDF from
wheat bran providing 1.3 g soluble-fibre did not significantly reduce these contents.
21
Table 2.5: Summary of representative studies involving consumption of β-glucan from oats
(Ryan et al., 2007)
Subjects
Healthy adults (n 62)
Male adults (moderate
hypercholesterolaemia) (n 52)
Oat product
Fermented oat
products
Oat milk
Healthy adults (n 62)
Oat bran concentrate
Adults
(hypercholesterolaemic) (n
112)
National cholesterol
Education Program
Step 1 diet plus snack
bar, cereal and
beverage (containing
phytosterols and Bglucan)
National Cholesterol
Education Program
Step 1 diet plus oats
and milk, or oats and
soya, or wheat and
milk, or wheat and
soya (cooked oatmeal
or oat bran cereal)
Ready to eat oat
cereal
Female adults (n 127)
Healthy adults (n 152)
Male overweight subjects
(hypercholesterolaemic) (n
235)
Healthy adults (n 30)
Oat bran (incorporated
in bread, sauces and
desserts)
Oatmeal and oatbran
ready to eat cereal
Healthy adults (n 50)
Rolled oats
Healthy adults (typically n 10)
Oat gum
Adults with elevated serum
total cholesterol (n 36)
Oat bran
Outcome
Significant reduction
in total cholesterol
Significant reduction
in total and LDL
cholesterol
No significant
difference in total or
LDL cholesterol,
glucose and insulin
between test and
placebo (wheat
product)
Significant reduction
in total and LDL
cholesterol
Reference
(Martensson et al.,
2005)
(Onning et al., 1999)
Significant reduction
in total and LDL
cholesterol for oatssoya and oats-milk
groups only
(Van Horn et al.,
2001)
Significant reduction
in total and LDL
cholesterol
Significant reduction
in total and LDL
cholesterol, apo B
No significant change
in flow mediated
vasolidation after
acute or sustained
ingestion
Oat ingestion
prevented endothelial
dysfunction induced
by acute fat ingestion
79-96% of change in
plasma glucose and
insulin attributable to
viscosity
Serum total
cholesterol declined
transiently
(Karmally et al., 2005)
(Lovegrove et al.,
2000)
(Maki et al., 2003)
(Berg et al., 2003)
(Katz et al., 2001a)
(Katz et al., 2001b)
(Wood et al., 1994)
(Uusitupa et al., 1997)
22
2.4.2 Diabetes
A fibre-rich meal is processed more slowly and nutrient absorption occurs over a
greater time period (Jenkins et al., 2002). As such a diet of foods providing adequate
fibre is usually less energy dense and larger in volume than a low-fibre diet that may
limit spontaneous intake of energy (Rolls et al., 1999).
This larger mass of food takes longer to eat and its presence in the stomach may
bring a feeling of satiety sooner, although this feeling of fullness is short term. A diet of a
wide variety of fibre-containing foods also is usually richer in micronutrients. When
viscous fibres are isolated and thereby concentrated, their effects on digestion are
frequently easier to detect. When these types of fibres are added to a diet, the rate of
glucose appearance in the blood is slowed and insulin secretion is subsequently
reduced. These beneficial effects on blood glucose and insulin concentrations were
most evident in individuals with diabetes mellitus (American Dietetic Association, 2008).
Considerable experimental evidence demonstrates that the addition of viscous dietary
fibres slow gastric emptying rates, digestion, and the absorption of glucose to benefit
immediate postprandial glucose metabolism (Anderson et al., 1999) and long-term
glucose control in individuals with diabetes mellitus (Chandalia et al., 2000; Vuksan et
al., 1999).
The mechanism by which fibre affects insulin requirements or insulin sensitivity is
still up for debate. Glucagon- like peptide 1 reduced gastric emptying rates, promoted
glucose uptake and disposal in peripheral tissues, enhanced insulin-dependent glucose
disposal, inhibited glucagon secretion, and reduced hepatic glucose output in animals
and human beings (D‟Alessio, 2000). These multiple effects of glucagon-like peptide 1
23
may reduce the amount of insulin required by individuals with impaired glucose
metabolism when consuming a high-fibre diet (American Dietetic Association, 2008).
2.4.3 Weight Control
Heaton (1973) proposed that fibre acts as a physiological obstacle to energy
intake by at least three mechanisms: fibre displaces available energy and nutrients from
the diet; fibre increases chewing, which limits intake by promoting the secretion of saliva
and gastric juice, resulting in an expansion of the stomach and increased satiety; and
fibre reduces the absorption efficiency of the small intestine (American Dietetic
Association, 2008).
Human beings may consume a constant weight of food and as such, a constant weight
of lower energy (high fibre) food per unit weight may promote a reduction in weight
(Rolls, 2000). High-fibre foods have much less energy density compared to high-fat
foods. Consequently, high-fibre foods can displace other energy sources. The bulking
and viscosity properties of dietary fibre are predominantly responsible for influencing
satiation and satiety. Fibre-rich foods usually are accompanied by increased efforts
and/or time of mastication, which leads to increased satiety through a reduction in rate
of ingestion. Intrinsic, hormonal, and colonic effects of dietary fibre decrease food intake
by promoting satiation and/or satiety (Slavin, 2005). Satiation herein defined as the
satisfaction of appetite that develops during the course of eating and which eventually
results in the cessation of eating, and satiety as the state in which further eating is
inhibited and which occurs as a consequence of having eaten. Dietary fibre also
24
decreases gastric emptying and/or slows energy and nutrient absorption leading to
lower postprandial glucose and lipid levels.
Howarth et al., (2001) reviewed the effects of dietary fibre on hunger, satiety,
energy intake, and body weight. The majority of studies with controlled energy intake
reported an increase in post meal satiety and a decrease in subsequent hunger with
increased dietary fibre. With ad libitum energy intake, the average effect of increasing
dietary fibre across all the studies indicated that an additional 14 g fibre per day resulted
in a 10% decrease in energy intake and a weight loss of more than 1.9 kg through about
3.8 months of intervention. In addition, the effects of increasing dietary fibre were
reported to be even more impressive in individuals with obesity. This group concluded
that increasing the population‟s mean dietary fibre intake from the current average of
about 15 g / day to 25 to 30 g / day would be beneficial and may help reduce the
prevalence of obesity.
In the prospective Nurses Health Study, women who consumed more fibre
weighed less than women who consumed less fibre (Liu et al., 2003) .In addition,
women in the highest quintile of dietary fibre intake had a 49% lower risk of major
weight gain. Howarth et al., (2005) examined the association of dietary composition
variables with body mass index among US adults aged 20 to 59 years in the Continuing
Survey of Food Intakes by Individuals 1994-1996. For women, a low-fibre, high-fat diet
was associated with the greatest increase in risk of overweight or obesity compared
with a high-fibre, low-fat diet.
25
2.4.4 Constipation and diarrhea
Constipation is defined as three or fewer spontaneous bowel movements per
week (Lederle et al., 1990). The longer feces remain in the large intestine, the more
water is absorbed into the intestinal cells, resulting in hard feces and increased
defecation difficulty. The rectum becomes distended, which may then cause abdominal
discomfort and other adverse symptoms such as headache, loss of appetite, and
nausea (American Dietetic Association, 2008; Widmaier and Raff, 2006). Clinical
diarrhea is defined as an elevated stool output (200 to 250 g / day); watery, difficult to
control bowel movements; and more than three bowel movements per day (McRorie et
al., 2000). Laxation refers to a slight increase in the frequency of bowel movements and
a softer consistency of feces (Livesey, 2001). Other symptoms that are associated with
laxation include increased stool weight and water content, decreased gastrointestinal
transit time, loose stools, bloating and distention, abdominal discomfort, and flatus
(Flood et al., 2004). Carbohydrates that reach the large intestine are fermented to
different degrees, depending on the degree of polymerization, solubility, and structure of
the carbohydrates (Nyman, 2002). Fermentation of the carbohydrates in the large
intestine produces gases, which may cause bloating, distention, and flatulence. If the
carbohydrates are not fermented in the large intestine, either because the bacteria do
not metabolize the carbohydrates or because intake exceeds the fermentation capacity
of the bacteria, the water remains bound to the carbohydrates that are eliminated in the
feces, which increases fecal bulk, but also may produce a watery stool or diarrhea. The
total amount of poorly digested carbohydrates in the diet will therefore affect tolerance.
26
2.4.5 Diverticulosis
The movement of material through the colon is stimulated in part by the presence
of residue in the lumen. When chronic insufficient bulk characteristic of a low-fiber diet
occurs in the colon, the colon responds with stronger contractions to propel the smaller
mass distally. This chronic increased force leads to the creation of diverticula, which are
herniations of the mucosal layer through weak regions in the colon musculature
(American Dietetic Association, 2008). Adequate intake of dietary fiber may prevent the
formation of diverticula by providing bulk in the colon so that less forceful contractions
are needed to propel it. Although few clinical studies have been conducted on dietary
fiber and diverticular disease, case-control studies and case studies report success with
high-fiber intakes (Aldoori and Ryan-Harshman, 2002). A high-fiber diet is standard
therapy for diverticular disease of the colon (Eglash et al., 2006).
Formed diverticula will not be resolved by a diet adequate in fiber, but the bulk
provided by such a diet may prevent the formation of additional diverticula, lower the
pressure in the lumen, and reduce the chances that one of the existing diverticula will
burst or become inflamed (American Dietetic Association, 2008).
2.4.6 Irritable Bowel Syndrome (IBS)
IBS may disturb gastrointestinal motility and reduce small intestinal absorption,
resulting in an increase in water that reaches the large intestine and diarrhea if the large
intestinal lumen can not absorb the excess water; other disruptions to motility may
cause constipation. In addition to diarrhea and constipation, symptoms of IBS include
27
bloating, straining, urgency, feeling of incomplete evacuation, and passage of mucus
(Bijkerk et al., 2004).
The composition and health of colonic microflora affect the fermentation of
carbohydrates. Antibiotic treatments may alter colonic bacteria, reducing fermentation
and causing diarrhea. In addition, viral or bacterial infections can cause secretory
diarrhea in which increased chloride ions and water are secreted into the small intestine
but not reabsorbed (American Dietetic Association, 2008). Although large doses of
fermentable carbohydrates may cause diarrhea, people may adapt over time, likely
because the fermentation capacity of the colonic bacteria increases. Individuals with
inflammatory bowel disease may experience exudative diarrhea when nutrient
absorption is diminished, which adds to the increased osmotic load from the presence
of mucus, blood, and protein from an inflamed gastrointestinal tract. Dietary fiber intake
may improve symptoms of patients with the aforementioned ailments.
2.5
Protein polysaccharide mixtures
In the food industry, proteins and polysaccharides are often used simultaneously
and as such interactions between these biopolymers play an important role in the
structure and stability of processed foods. Therefore the control and or manipulation of
these macromolecular interactions are key factors in the development of novel food
processes and products (Ye, 2008).
From an industrial perspective, protein-polysaccharide interactions are important
for applications such as micro- and nano-encapsulation processes (Champagne and
Fustier, 2007), the design of multi-layers structures (Noel et al., 2007), the formation
28
and stabilization of food emulsions, the formation of new food gels (Haug et al., 2004)
and the recovery of proteins from industrial by-products (Montilla et al., 2007)
(Damianou and Kiosseoglou, 2006). These mixtures generally lead either to phase
separation through thermodynamic incompatibility or complex coacervation (Turgeon et
al., 2003).
2.5.1 Polymer interaction
In many biopolymer mixtures, the entropic contribution is often greater than the
enthalpic one, so that phase separation of biopolymers is generally the rule (Doublier et
al., 2000). Two phase separation phenomena can be observed, depending on the
affinity between the different biopolymers and the solvent. Thermodynamic
incompatibility or segregative phase separation is generally observed and it appears
when the Flory-Huggins interaction parameter χ23 (accounting for the biopolymer 1 biopolymer 2 interactions) is positive, indicating a net repulsion between the
biopolymers. Clearly, solvent – biopolymer 1(biopolymer 2) interactions are favoured to
the detriment of biopolymer 1 – biopolymer 2 and solvent-solvent interactions, so that
the system finally demixes into two phases, each enriched with one of the two
biopolymers (Schmitt et al., 1998). Associative phase separation occurs when the
interactions between the two biopolymers are favoured (χ23 < 0). This occurs when both
polymers carry an opposite charge, for instance at a pH slightly lower than the
isoelectric point of the protein, while the polysaccharide still carries a negative charge.
Complexation then takes place, which can yield either the formation of soluble
complexes or an aggregative phase separation. In associative phase separation, the
29
two coexisting phases have a rich solvent phase with very small amounts of biopolymer
and a rich biopolymer phase forming the so-called coacervate (Doublier et al., 2000).
2.5.1.1
Segregative phase separation
Thermodynamic incompatibility generally arises in conditions when the protein is
in the presence of a neutral polysaccharide or of an anionic polysaccharide bearing a
charge of the same sign as the protein (Doublier et al., 2000). The result is that the
phase separation of a mixed protein-polysaccharide solution requires a total polymer
concentration usually higher than 4 % while depletion-flocculation takes place at a total
concentration of less than 1% (Doublier et al., 2000).
2.5.1.2
Associative phase separation
Associative phase separation between proteins and polysaccharides refers to a
demixing phenomena induced either by direct interactions between biopolymers, e.g.
electrostatic interactions (complex coacervation phenomenon) or hydrogen bonding
(Antipova and Semenova, 1997). Basically, associative phase separation implies the
formation of primary soluble macromolecular complexes that interact to form electrically
neutralised aggregates, then unstable liquid droplets and/or precipitates that ultimately
sediment to form the coacervated phase containing both biopolymers (Schmitt et al.,
1998; Tolstoguzov, 1995).
30
2.5.1.3
Thermodynamics aspects
The formation of macromolecule–macromolecule complexes takes place
spontaneously when the total Gibbs free energy change ΔG decreases (ΔG < 0),
regardless of the actual amount of favourable free energy change accumulated by direct
molecular contact between the associating macromolecules (Turgeon et al., 2007;
Jelesarov and Bosshard, 1999). A delicate balance between large favourable entropic
(−TΔS) and large unfavourable enthalpic (ΔH) contributions determine the value of ΔG,
and therefore the possibility or not for biopolymers to form a complex. Favourable
entropic contributions mainly include the release of counterions (Laugel et al., 2006; de
Kruif et al., 2004; Jelesarov and Bosshard, 1999).
Unfavourable entropic contributions originate from the decreased mobility of
biopolymers upon binding and, possibly, from the ordering of water at the complex
interface (Jelesarov and Bosshard, 1999). Due to the complexity of the water structure
and the subtlety of the processes involved, it is difficult to estimate the total entropic
contribution (Laugel et al., 2006).
2.5.2 Factors affecting protein polysaccharide phase separation
As the interactions occur in solution, complex formation and thermodynamic
incompatibility are primarily influenced by pH, ionic strength, conformation, charge
density and the concentration of both protein and polysaccharide (Ye, 2008; Schmitt et
al., 1998).
31
2.5.2.1
pH
pH plays a key role in the formation of protein–polysaccharide complexes
because it influences the degree of ionisation of the functional side groups carried by
the biopolymers (i.e. amino and carboxylic groups) (Schmitt et al., 1998). At pHs below
the IEP of the protein, an anionic polysaccharide and a protein carry opposite net
charges, resulting in a maximum electrostatic attraction. There is a critical pH for phase
separation, which corresponds to a net protein charge that is opposite to that of the
polysaccharide.
2.5.2.2
Ionic strength
The net charge carried by the proteins and polysaccharides is reduced by interaction
with the microions, resulting in a decrease in the electrostatic attraction between the
macromolecules at high salt concentration. At high ionic strength, screening the charges
of the proteins and polysaccharides also lead to reduce electrostatic interactions and
hence affect the formation of complexes (Weinbreck et al., 2003). At low ionic strength,
the microion concentration has only a small effect on protein–polysaccharide
complexes. The number of charges present on the proteins and polysaccharides is
sufficient to allow electrostatic interaction.
2.5.2.3
Biopolymer ratio and biopolymer concentration
The ratio of protein and polysaccharide in the mixture will influence the charge balance
of complexes, hence affecting the behaviour of complexes. For a mixture, maximum
complexation is obtained at a specific ratio of protein to polysaccharide at a given
32
Fig. 2.4: Schematic representation of factors affecting phase separation in protein–
polysaccharide mixtures (Turgeon et al., 2003)
condition (pH and ionic strength). When one of the components (protein or
polysaccharide) in the mixture is in excess, soluble complexes may be obtained
because of the presence of non-neutralised charges. At high biopolymer concentrations,
when the polysaccharide or the protein is in excess in the solution, no complexation
occurs (Ye et al., 2006; Weinbreck et al., 2003).
Weinbreck et al., (2003) explained that increasing the biopolymer concentration
favours the release of more counterions in solution, which screen the charges of the
33
biopolymers, suppressing complexation and increasing the solubility of the complexes.
Moreover, at high biopolymer concentrations, the system will show phase separation
through thermodynamic incompatibility because of the competition between the
macromolecules for the solvent (Tolstoguzov, 1997).
2.5.2.4
Charge density
The interaction between oppositely charged biopolymers is enhanced when the
net opposite charges of the biopolymers are increased and the ratio of net charges of
the biopolymer reactants approaches unity. Proteins and polysaccharides possess a
large number of ionisable and other functional side-chain groups. They differ in shape,
size, conformation, flexibility and net charge at a given pH and ionic strength
(Tolstoguzov, 1997). The strength of attractive coulombic interactions between proteins
and polysaccharides depends to a large extent on the macromolecular charge densities.
As such sulphated polysaccharides such as carrageenan will interact more strongly with
proteins than carboxylated polysaccharides such as alginates and pectin (Kato, 1996). It
has been reported that flexible proteins, e.g. caseins or gelatin, bind polysaccharides
more strongly than globular proteins, e.g. BSA or β-lactoglobulin, and that the thermal
denaturation of the latter enhances their binding affinity. The explanation proposed is
that flexible molecules are able to form a maximum number of contacts with the other
oppositely charged molecules, i.e. an increase in local concentration of interacting
groups is favoured (Ye, 2008) .
34
2.5.2.5
Processing factors
Processing factors, including temperature, shearing rate and time, and pressure,
can affect protein–polysaccharide interaction (Dickinson and Pawlowsky, 1997).
Changes in environmental conditions may induce protein or polysaccharide
conformational changes and modifications in protein–solvent or polysaccharide–solvent
interactions. For instance, it has been demonstrated that hydrophobic interactions may
overcome electrostatic interactions when hydrophobic groups have been anchored
along the polymer backbone (Borrega et al., 1999). An increase in temperature
enhances hydrophobic interactions and covalent bonding, whereas low temperature is
conducive to hydrogen bond formation. At high temperature, globular protein
denaturation and polysaccharide conformational changes cause the exposure of more
reactive sites and hence favour the complexation interactions between the functional
side groups of the biopolymers.
2.5.3 Phase separation kinetics
The general models conveniently used to describe phase separation kinetics are
nucleation and growth (NG) or spinodal decomposition (SD) (Turgeon et al., 2003). The
former is characterized by initial short-range high-amplitude concentration fluctuations,
whereas SD proceeds through long-range small-amplitude fluctuations (Maugey et al.,
2001). NG generally ends up with spherical droplets dispersed in a continuous phase,
whereas SD exhibits a 3D interconnected network (Turgeon et al., 2003).
35
Chapter III
Concentration of β-glucan from oat bran
36
Abstract
(1/3) (1/4) β-D-glucan is a component of cereal grains such as oat and barley.
Various studies have demonstrated a link between the regular consumption of foods
containing cereal β-glucan and reduced incidence of the metabolic syndrome.
Due to its low concentrations in grains, β-glucan incorporation into regular foods at
physiologically effective levels without compromising the sensory attributes of foods
would be desirable but is still a challenge. One way of addressing this challenge is to
produce β-glucan concentrates that can then be incorporated into foods without
adversely affecting sensorial attributes. Therefore in this study a new procedure for the
concentration of oat β-glucan was developed. The procedure was based on the
thermodynamic incompatibility between polysaccharides and proteins, and all
processes were carried out at room temperature. The polymer demixing process was
„arrested‟ by chain aggregation events, leading to a bi-phasic system consisting of an
upper liquid phase and a lower gel-like phase. After freeze drying, rheological and
scanning electron microscopy (SEM) investigations were carried out on the lower phase
extract solution and dry extract respectively. Pseudoplastic flow was demonstrated by
the solutions and SEM revealed a seemingly new microstructure that was created by
the concentration procedure.
Keywords: Oats; (1/3) (1/4) β-D-glucan; Thermodynamic incompatibility; Bi-phasic
system; Rheology; Microstructure
37
3.1
Introduction
The (1/3) (1/4) β-D-glucans are linear homopolysaccharides of consecutively
linked (1/4)-β-D-glucosyl residues separated by single (1/3)-linkages. The presence of
such β-1/3 linkages leads to kinks in the straight chain polymer, allowing water to get in
between the chains and making β-glucan soluble in water. In general, the storage
polysaccharides, exudate gums, and bacterial capsular polysaccharides are easier to
extract than the structural or matrix polysaccharides of the cell walls of plants and
microorganisms (BeMiller, 1996). For instance, the cell wall polysaccharides have to be
extracted from the insoluble cell wall material, while xanthan gum produced by
Xanthomonas campestris is released directly into the culture medium and can be easily
isolated from the culture broth by precipitation with ethanol (Izydorczyk, 2005). Cell wall
constituents can be extracted with many solvents, though water at various temperatures
is usually the first choice for extraction of neutral polysaccharides. Normally the
extractability of polysaccharides increases with increasing temperature of the aqueous
solvent. Acidic solutions are usually avoided for the extraction of polysaccharides
because of the risk of hydrolysis of the glycosidic linkages. As such alkali solutions are
used extensively to extract the cell wall polysaccharides. It is suggested that under the
alkali conditions, the ester and other covalent and noncovalent linkages are broken and
the initially unextractable polysaccharides are released from the complex network of the
cell walls. Once the polysaccharides are released from the cell wall, they become
soluble in water. Consequently these polysaccharides become available for use such as
in the fortification of food products.
38
The United States Food and Drug Administration (FDA) allowed a health claim
indicating that regular consumption of oat and barley products containing 3 g of βglucan per day may lower the risk of heart disease (FDA, 2005; FDA, 1997). To qualify
for this claim, food products must contain 0.75 g of β-glucan per serving. This relatively
high intake (3 g / day), required to achieve physiologically effective levels, presents
some challenges in the development of food formulations. As such, various processing
technologies have been developed to obtain β-glucan concentrates. Some of these
processes include: the aqueous alkali extraction (Wood et al., 1989), the aqueous
enzymatic method (Inglett, 1992), and the aqueous alcohol based enzymatic process
(Vasanthan and Temelli, 2002) .
The separation processes mentioned either employ the use of ethanol and / or
enzymes to obtain an extract with increased β-glucan content. As such, hydrolysis of
proteins through the use of proteases can lead to either depolymerisation of the
extracted β-glucan as it may contain enzymes with β-glucanase activity (Immerstrand et
al., 2009) and / or denaturation of the proteins within the developed products
consequently leading to shorter shelf life of such products. Non utilization of alcohol
during extraction will substantially reduce production costs as the energy intensive
recycling step will be done away with. Consequently, the objective of this study was to
create processing technology that would produce a high molecular weight, and soluble,
extract, without the use of either enzymes or alcohol. Therefore, based upon the
thermodynamic incompatibility of binary β-glucan / protein systems in the liquid state, in
particular, the phase behaviour of mixed β-glucans / protein systems (Kontogiorgos et
39
al., 2009), an aqueous protein based processing technology was developed for the
concentration of β-glucan from oat bran.
Whey protein isolate (WPI) was the protein source utilized in this study. WPI is a
milk protein product, with ever increasing usage in the food industry, and the major
proteins in this milk fraction are β-lactoglobulin (β-Lg, ~ 50%), and α-lactalbumin (α-La,
~20%). The structures of β-lactoglobulin and α-lactalbumin are typical of those of other
globular proteins, and similar to caseins, they have a net charge at the pH of milk
(Swaisgood, 2008). They have relatively low molecular weights with β-lactoglobulin
approximately 18 kDa, and α-lactalbumin approximately 14 kDa. These two proteins
make up approximately 70 % of protein in whey protein isolate (WPI) and determine its
physicochemical properties.
3.2
Materials and methods
3.2.1 Materials
Oat bran (Oatwell® 28%) was donated by CreaNutrition (a subsidiary of SOF
Swedish Oat Fiber AB) with the following specifications: 28 % β-glucan, 25 % protein,
and 6 % moisture. Whey protein isolate (WPI) was purchased from Davisco (BiPro,
Davisco Foods International, Inc., MN) with the following specifications: 97.8 % (dry
basis) protein, 4.3 % moisture, and 2.0 % ash.
3.2.2 Laboratory- scale aqueous protein-based β-glucan extraction
Oat bran was added to an aqueous solution of sodium hydroxide (0.25 N), and
mixed for 1 hr at 250C. The resultant slurry was then centrifuged (RC5C plus, Mandel
40
Oat bran
β-glucan and
proteins are
solubilised in
water under
alkali conditions
0
Mix with aqueous protein solution
(20% w/v)
Centrifuge – 10000g, 20min, 250C
1:1 ratio, at 25 C
Supernatant
Add 0.25N sodium hydroxide
(1.2%w/v slurry) at 250C. Stir
for 30min
Freeze dry residue
Centrifuge – (10000g, 20min, 250C)
Separate the insoluble
solid particles,
containing starch and
insoluble fibre
Extract (β-glucan enriched)
Supernatant
Neutralize supernatant
Fig. 3.1: Schematic of the laboratory-scale extraction process
Scientific) at 10000 g for 20 min at 250C. After centrifugation, the supernatant was
separated from the residue by decanting. The supernatant pH was then adjusted to 7.
An aqueous protein solution (20 % w/v) was made by dissolving whey protein isolate
(WPI) powder in micropore filtered water at 250C, and mixed for 1 hr. The solution was
then centrifuged to ensure removal of any insolubles. The „polysaccharide‟ supernatant
was then added to the protein solution, mixed vigorously for 30 min and left to stand for
another 30 min. The mixture was then centrifuged at 10000 g for 20 min at 250C, after
41
which the supernatant and residue were separated by decanting, and the residue
(extract) was then freeze dried.
3.2.3 Pilot plant β-glucan extraction process
Oat bran was mixed with sodium hydroxide solution (0.25 N) to form a 1.2 %
(w/v) slurry. This was done in a jacketed mixer at room temperature. The slurry was
mixed for 1 hr. When all the bran was hydrated, the slurry was then centrifuged in a „two
stage‟ process so as to get rid of insoluble material. A basket centrifuge was used for
this process, with the 1st stage centrifuge using a sieve with coarse pores (25 µm). The
filtrate from the first centrifuge was then used as the solution for the second centrifuge
process (2nd stage) using a sieve with fine pores (5µm). The filtrate from the 2 nd stage
was then neutralized using phosphoric acid. This polysaccharide solution was then
mixed with a previously prepared whey protein isolate (20 % w/v) solution in a 1:1 ratio.
The protein-polysaccharide mixture was then stirred for 2 hr, and then left to stand
overnight at 40C. A gel like layer formed as „sediment‟ in this mixture, and this was then
isolated using a suction pump, by sucking off as much of the „top‟ layer (liquid portion)
as possible. The remaining gel like layer was then freeze dried to give the extract that
was then used in the formulation of the beverages (Fig. 3.2). All materials used in this
extraction were food grade.
42
Oat bran
Mix with 0.25N sodium hydroxide to form 0.2 % (w/v) slurry. Stir for 1 hr at 250C
Residue
1st stage centrifuge – coarse basket sieve
Filtrate
2nd stage centrifuge – fine basket sieve
Residue
Filtrate
Neutralize filtrate with phosphoric acid
Mix the neutralized filtrate with the protein solution in a 1: 1 ratio. Mix for 2 hr, at 250C
Leave the protein-polysaccharide mixture to stand overnight at 40C
Use a suction pump to separate the upper liquid layer from the lower gel-like layer that forms as sediment
‘Sediment ‘
‘Liquid’
Freeze dry
Extract
Fig. 3.2: schematic of the pilot plant scale extraction process
43
3.2.4 β-glucan, protein and starch tests
3.2.4.1.1
Extractable β-Glucan
Solubilized β-glucan was measured by flow-injection analysis (FIAstar 5010
Analyzer, Foss Analytical, Denmark) equipped with a fluorescent detector essentially as
described previously (Jorgensen and Aastrup, 1988). Essentially, the solution is mixed
with the fluorescent dye calcofluor (Calcofluor White M2R New, American Cyanamid,
Brookbound, NY, Fluorescent agent #28) in TRIS buffer (Sigma-Aldrich, St. Luis, MO) at
pH 8.0, and a standard curve was used to determine the concentration of β-glucan from
the increase in fluorescence intensity of calcofluor bound to β-glucan.
3.2.4.1.2
Total β-Glucan
The total β-glucan was determined by the using the McCleary method (AOAC
Method 995.16) in which a specific (1/3) (1/4)-β-D-glucan-4-glucanohydrolase (EC
3.2.1.73; lichenase) and β-glucosidase (EC 3.2.1.21) were used to hydrolyze β-glucan
to glucose. The glucose concentration was then measured spectrophotometrically with
an automated glucose oxidase procedure.
3.2.4.2
Protein
The protein content of samples was measured using Dumas method (LECO, FP528, Mississauga, ON, Canada) using EDTA as the standard for calibration of the
instrument. The protein content was calculated using 6.38 as the conversion factor for N
determination according to approved AACC method 46-30.
44
3.2.4.3
Starch
Starch content was determined using the total starch assay procedure
(Amyloglucosidase / α-Amylase method), AOAC Method 996.11. The principle here is
that thermostable α-amylase hydrolyzes starch into soluble branched and unbranched
maltodextrins. Amyloglucosidase then quantitatively hydrolyses maltodextrins to Dglucose, which is then oxidized to D-gluconate with the release of hydrogen peroxide.
The hydrogen peroxide is then quantitatively measured in a colourimetric reaction
employing peroxidase and the production of a quinoneime dye.
3.2.5 Molecular Weight Determination
The peak molecular weight (Mp) of β-glucan was determined using highperformance size-exclusion chromatography (HPSEC) with post-column calcofluor
addition as described by (Wood et al., 1991). HPSEC was performed using two columns
(300 × 7.5 mm) in series (Shodex OHpak KB806M, Waters Ultrahydrogel; Waters,
Milford, MA). A Perkin-Elmer ISS 100 autosampler and injector were used with an
injection volume of 100 μL. The β-glucan was eluted with 100 mM tris buffer (pH 8.0) at
1 mL/min at 40°C. β-Glucan molecular weight standards were used to construct a
calibration curve (retention time vs. log Mp) (Beer et al., 1997).
3.2.6 Rheological properties
Rheological measurements were performed using the Advanced Rheometrics
Expansion System (ARES, TA Instruments, New Castle, DE), which is a controlled
strain rheometer, using the operational software accompanying the instrument (TA
45
Orchestrator, TA Instruments, Waters LLC, USA). Samples were analyzed using cone
and plate geometry (angle 0.04 rad, 50 mm diameter). Two types of rheological
measurements were performed: (a) flow behavior by measuring steady state shear
viscosity (η) over a range of shear rates ( ) of 0.01-500 s-1 at 50C (b) oscillatory
measurements of G‟ (storage modulus), G” (loss modulus), and tan δ (G”/G‟) were
performed with a strain of 0.1% and a 0.1-10 Hz frequency range.
3.2.7 Structural investigation
Samples were mounted on specimen stubs with the help of double-sided scotch
tape, and sputter coated using an Emitech K550 (Emitech Ltd, Kent, England) with goldpalladium (20 mA, 150 s). The preparations were then viewed and photographed with a
Hitachi S-570 (Hitachi High Technologies, Tokyo, Japan) scanning electron microscope.
3.3
Results and discussion
3.3.1 Developed extraction process
A high molecular weight extract with a concentration of ~30 % β-glucan was
attained in this study (Fig. 3.3), and due to the deliberate non-utilization of either alcohol
nor enzymes, further purification using these procedures was not undertaken. The mass
flow Table 3.1 shows us the concentrations of β-glucan, protein, and starch along given
stages of the process. Notably significant is the increase of the β-glucan concentration
in the final extract as compared to its concentration in the polysaccharide-protein
mixture. This increase is essentially the cornerstone of this extraction protocol, and it
46
Table 3.1: Process extraction mass flow
β-glucan
(g)
0.84
1st
residue
0.1
Protein
(g)
Starch
(g)
TDF (g)
Dry
weight
(g)
Volume
(mL)
Starch
conc.
(g/mL)
Protein
conc.
(g/mL)
β-glucan
Conc.
(g/mL)
0.75
0.14
0.61
0.53
0.27
0.02
0.25
0.25
1.52a
-
n.d
1.13
n.d
2.88
n.d
n.d
244
19
225
394*
375
18.18
0.0011
0.0010
0.0011
0.0013*
0.0003
0.0050
0.0030
0.0074
0.0027
0.1013*
0.0873
0.0545
0.0034
0.0053
0.0033
0.0016*
0.0003
0.0297
Slurry
1st
supernatant
0.74
Pos.#
solution
0.65
WPI
solution
-
Final
supernatant
0.1
Extract
39.4
32.74
0.99
0.12
0.09
n.d
40.05
0.54b
1.69
n.d
0.54
Pos.# polysaccharide
a
b
TDF Total dietary fibre ( includes soluble and insoluble fibre. includes only soluble fibre)
n.d = not determined
*Mixture of the polysaccharide / protein solutions
arises due to the thermodynamic incompatibility between polysaccharide and the
protein contained within the mixture. The increase in concentration was approximately
19 fold, and this fell within the range reported earlier by Lazaridou and Biliaderis (2009)
who investigated phase separation in oat β-glucan-sodium caseinate / pullulan mixtures.
In this particular oat β-glucan-WPI mixture, its suggested that the increase in
concentration due to phase separation could have been even higher, which was
47
desirable, but the effects arising from an „incomplete‟ phase separation led to this „lower
than expected‟ increase in β-glucan concentration.
Fig. 3.3: Freeze dried extract
A „complete‟ phase separation process would therefore theoretically lead to a higher βglucan content in the final extract, with consequent decreases in the „un-desired‟
material such as protein and starch. Also notable in this protocol is the change in total
dietary fibre (TDF) containing both soluble and insoluble fractions in the oat bran at the
start of the protocol, to TDF basically containing only soluble β-glucan in the final
extract. This attribute augurs well for incorporation of increased amounts of such an
extract into products without drastically affecting palatability and mouthfeel of such
products. According to Izydorczyk (2005) dilute solutions of sodium hydroxide have
been used to extract xyloglucans, xylans, β-glucans, and pectins. And once the
polysaccharides are released from the cell wall by extraction with alkali, they become
soluble in water. This kind of extraction usually results in solutions containing a mixture
of components (other polysaccharides, non carbohydrate material), which then have to
be further purified to isolate the polysaccharide of interest. So in essence this was what
was done in this study, with use of sodium hydroxide in the early stages of the protocol
thereafter followed by further separation based on protein-polysaccharide incompatibility
48
when the polysaccharide solution is mixed with the protein solution. In this regard both
the laboratory based extraction and the scaled up pilot plant protocols were the same,
with the main constituents as β-glucan, protein, and starch at ~30 %, ~60 %, ~5 %; and
~10 %, ~70 %, and ~6 % for the laboratory and pilot plant extracts respectively. The
differences in β-glucan and protein content were thought to be a consequence of the
lack of an appropriate centrifugation step after mixing of the polymers in the pilot plant.
3.3.2 Phase behaviour
Various concentrations of the initial mixtures were investigated during the course
of this study. All initial mixtures investigated resulted into a two phase system with the
upper phase in a liquid state, while the lower phase existing in a gel like state (Fig:
3.4b). Similar behaviour by immiscible biopolymer mixtures has been reported by
several investigators (Lazaridou and Biliaderis, 2009; Tolstoguzov, 2003; Manoj et al.,
1996; Tolstoguzov, 1995; Garnier, et al., 1995; Kasapis, et al., 1993).
a
b
Fig. 3.4: (a) A phase diagram representing initial mixtures that all resulted as indicated in (b)
(b) A bi-phasic system showing an upper liquid phase and lower gel like phase
49
It is likely that phase separation phenomena induce intermolecular associations
of β-glucan chains and thereby gel network formation due to increasing polysaccharide
concentration in the lower phase. The addition of another hydrocolloid (gel-forming or
not) to a solution of a gel-forming polymer decreases the critical concentration and
increases the rate of gelation due to polymer immiscibility (Tolstoguzov, 2003;
Tolstoguzov, 1995). Although oat β-glucan aqueous dispersions can gel at room
temperature depending on the concentration and the molecular weight of the
polysaccharide (Lazaridou et al., 2003), neither the WPI nor the oat β-glucan solution
used here formed a gel (even after 3 weeks of aging at 5 0C) when left to stand.
However, when mixed, the lower phase of the system „gelled‟.
Figure 3.5 shows what occurs in this protein-polysaccharide mixture, with the protein
only marginally increased in comparison to β-glucan (exhibiting as much as a 10 fold
increase), from intial (one phase) to final (bi-phasic) systems (final referring to the lower
gel like phase). This essentially indicates that this process does lead to, and could
therefore be used for, the effective concentration of β-glucan.
15
WPI (%)
10
5
0
0
10
20
30
40
 -glucan (%)
Fig. 3.5: Phase separation of oat β-glucan / whey protein isolate aqueous dispersions. Similar symbols
represent initial (clear) and final (filled) concentrations respectively, of a particular mixture.
50
Lazaridou and Biliaderis (2009) similarly reported a less than 10% increase in the upper
phase and a 5-110 % increase in the lower phase for a sodium caseinate / β-glucan
mixture. It is likely that the β-glucan is preferentially hydrated, even as it is concentrated
in the lower phase (or due to excluded volume effect), leading to effective concentration
of the entrapped protein above the solubility point hence precipitation of the protein
within its microdomains. As such, phase separation seems to coexist and compete with
chain aggregation-gelation phenomena, and as reported by Lazaridou and Biliaderis
(2009), phase diagrams of such systems are diagrams of state rather than typical phase
diagrams established under equilibrium conditions.
3.3.3 Rheological properties
The mechanical spectra of 0.2-1 % (w/v) solutions of the extract are depicted in
Fig. 3.6 (a-e). The 0.2 % (a) exhibited concentrated solution behaviour in that at lower
frequencies G” > G‟ with a crossover at higher frequency showing a tendency for more
solid like behaviour at higher frequencies. The trend shown at the lower frequency was
typical of the viscoelastic behaviour of a macromolecular dispersion. The 0.4-1 %
concentrations (b-e) showed gel behaviour with G‟ > G‟‟ throughout the determined
frequency range, with both moduli increasing with increasing frequency. It was also
observed that at 0.4 % concentration, G” was less dependent on the frequency as
compared to G‟. A lesser frequency dependence of the moduli would indicate that the
viscoelastic properties are dominated by an established network structure (Vaikousi and
Biliaderis, 2005), and as such it was thought that in the case of graphs (b) to (e) it was
more the case of a „developing‟ or weak network structure.
51
G'
0.1
G"
tan δ
0.01
0.001
G', G", tan δ
G"
tan δ
10
0.1
c
G'
1
G"
tan δ
0.1
1
10
10
G', G", tan δ
1
10
d
G'
1
G"
tan δ
0.1
0.1
1
10
10
G', G", tan δ
G'
1
0.1
0.1
0.1
G'
1
G"
tan δ
1
10
10
e
η(Pa.s)
G', G", tan δ
1
b
10
G', G", tan δ
a
10
f
1
0.2
0.1
0.4
0.6
0.01
0.8
1
0.001
0.1
0.1
1
f (Hz)
10
0.0
1
0.1
1
10
100
(s-1)
Fig. 3.6: Mechanical spectra of (a) 0.2 %, (b) 0.4 %, (c) 0.6 %, (d) 0.8 %, (e) 1% (w/v)
extract solutions; and their apparent viscosity (f).
52
For 0.2 % and 0.4 % curves, tan δ decreased with increasing frequency while for the 0.6
%-1 % curves, tan δ only showed a slight decrease. The tendency of a dilute solution
and a concentrated solution to exhibit more fluid or solid like behaviour with increasing
frequency can be examined by considering the frequency dependence of tan δ. High
values of tan δ at low frequencies will indicate a tendency toward more fluid like
behaviour for both dilute and concentrated solutions at low deformation rates (Steffe,
1992). More solid like behaviour is observed for these solutions at the high deformation
rates associated with high frequencies. The tan δ for a gel is practically constant
indicating consistent solid like behaviour over the entire frequency range. This therefore
correlates with what was observed for the G‟ and G” moduli, in that, for the 0.2 %,
higher initial values of tan δ with decreasing values at higher deformation rates depicts
a concentrated solution to a more gel like (solid) behaviour. For especially the 0.6-1 %
curves, the gel like behaviour would be further evidenced by the more or less constant
tan δ readings.
Fig. 3.6 (f) shows the viscosity for the 0.2-1.0 % solutions, and as indicated, all
concentrations exhibited shear thinning (pseudoplastic) behaviour which is typical of
cereal β-glucans (Lazaridou et al., 2004). Apparent viscosity readings increased from
the 0.2 % to the 1 % concentration as would be expected. Values for all the
concentrations at typically relevant shear rates are depicted in Fig. 3.7.
53
1
(s-1)
0.1
η(Pa.s)
5
10
50
0.01
100
0.001
0.2
0.4
(%) 0.6
0.8
1
Fig. 3.7: Selected shear rates ( ) for 0.2-1 % (w/v) extract solutions
Table 3.2: Typical Shear rates observed in some common processes relevant to food
emulsions
Process
Shear rate (s-1)
Pumping
100 - 103
Mixing and stirring
101 - 103
Chewing and swallowing
Pouring
101 - 102
10-2 - 102
3.3.4 Structural analysis
Images obtained from scanning electron microscopy of the freeze dried extract (Fig.
3.8) showed a seemingly new type of microstructure formed, with identical cylindrical
shaped bodies of about 1 µm in length and 0.5 µm in diameter. These may possibly
represent micro-domains with the β-glucan encapsulating the protein as a result or
manifestation of the „arrested‟ or incomplete phase separation that arose due to the
gelation of β-glucan in the lower phase of the mixture, though the exact mechanism
remains to be elucidated. Chemical and physical interactions between constituents,
54
Fig. 3.8: An SEM image showing the extract microstructure
such as water, proteins, lipids, saccharides, fibre, mineral compounds, and dissolved
and dispersed gases, can produce various macroscopic and microscopic structures in
natural and formulated foods, and food ingredients. The size and shape of such
structures and their stability, distribution, and interactions, have a large impact on many
quality attributes, especially on the texture and appearance of foods (Sikorski et al.,
2008). Turgeon et al., (2003) indicate that the presence of a bi-phasic morphology
allows control of the final product microstructure, and that a key parameter is shear
treatment of these biphasic systems. The flow history applied during processing will as
well influence the morphology of the mixture, and this in turn modifies its rheological and
55
sensorial properties. Other authors also reported effects of shear histories on the
morphology development within particular systems, with resultant structures such as
formation of elongated droplets with narrow size distribution, fibrillar morphology,
percolated structures, and emulsion like spherical inclusions (Van Puyvelde et al., 2003;
Walkenstrom and Hermansson, 2002; Turgeon and Beaulieu, 2001; Michel et al., 2001).
3.4
Conclusion
The objective in this study was essentially to concentrate β-glucan, with oat bran
as the starting material, based on an alternate approach that would utilize the
thermodynamic incompatibility between proteins and polysaccharides as a driving force.
In essence this was achieved, as the β-glucan concentration achieved was relatively
high, though an „incomplete‟ phase separation occurred in this particular biopolymer
mixture, resulting in a higher than expected protein content in the extract. Without
further alcohol and or enzyme based purification of this extract, it could be referred to as
a biopolymer „composite‟ as opposed to a complex due to the nature of interaction
between the polymers, though this remains to be further elucidated. As stated by
Doublier et al., (2000), interbiopolymer complexes (or composites as in this study) can
be regarded as a new type of food biopolymer, the functional properties of which may
differ markedly from those of the macromolecular reactants.
56
Chapter IV
Satiety and related blood glucose effects of Oat β-glucan supplemented dairy
beverages
57
Abstract
Soluble fibre has been proposed to suppress appetite related perceptions and it
could thus contribute favourably to the regulation of energy intake and the increasing
obesity problem in North America. Therefore the objective of this study was to
investigate the effect of a beverage with increased β-glucan content on perceived
satiety and blood glucose, at different fibre concentrations. 29 healthy male and female
subjects, 20-30 years old and a BMI of 20-24.9 kg / m2, participated in the study.
Measurement of subjective perceptions (desire to eat something/ fullness), and blood
glucose levels were carried at given time points throughout the 260 min pre- and postmeal period. There were 5 beverage pre-loads in total, containing between 0-2.5 g of βglucan in 500 mL of pre-load, ingested by the subjects 120 min before the meal. The
order of the samples was randomised for each subject and evaluated separately with a
1 week washout period. There was an observable trend towards a pre-meal decrease in
appetite scores with increasing β-glucan content of the pre-loads, though this was not
significant. The post meal responses showed a similar trend, but no significant
differences were seen. There were observable differences in pre-meal blood glucose
readings but due to the composition of the pre-loads, it could not be concluded that the
effect was effectively due to the increasing β-glucan content. Post meal differences in
blood glucose could not be attributed to the pre-load ingestion. In conclusion, a β-glucan
containing beverage may induce post meal satiety though this effect was not
conclusively proven in this study.
Keywords: β-glucan, satiety, blood glucose, pre-meal, post-meal
58
4.1
Introduction
Obesity is becoming an increasingly common health problem worldwide (Lyly et
al., 2009; WHO, 2003). In addition, obesity is one of the risk factors in metabolic
syndrome and type II diabetes, requiring a lot of resources from the public health care
system (WHO, 2003) and as such effective tools for stopping this progress are actively
being sought (Lyly et al., 2009).
Maintenance of body weight is related to the short term choices in meal size and
frequency and therefore, important in this regard are the processes of satiation that
bring a meal to an end and the state of satiety, which determines the interval of time
following a meal and the amount consumed during the next meal (van Aken, 2010).
The intake of dietary fibre has been linked to the regulation of energy intake and satiety
and therefore could contribute favourably to the obesity problem. A meta-analysis of 22
studies concluded that a 14 g increase in fibre intake was linked to a 10 % reduction in
energy intake and 1.9 kg reduction in weight during 3.8 months (Howarth et al., 2001).
Increased consumption of whole grains (Koh Banerjee et al., 2004) or dietary fibre (Liu
et al., 2003) has been shown to protect from weight gain. An inverse relationship
between dietary fibre intake and body mass index (BMI, kg/m2) has been shown in
women (Howarth et al., 2001). The satiating effects of fibre have been tested in many
short-term studies, using a variety of fibres, doses and food matrices. Results have
varied depending on the type of fibre used. Among soluble, viscous fibres such as guar
gum (Chow et al., 2007; Pasman et al., 1997) psyllium (Delargy et al., 1997; Delargy et
al., 1995), pectin (Tiwary et al., 1997) and β-glucan (Kim et al., 2006) , some of the
studies have shown reduced hunger and / or appetite perceptions after consumption of
59
the test food compared to low / no fibre controls (Chow et al., 2007; Pasman et al.,
1997; Tiwary et al., 1997). The soluble, non-viscous fibres have shown only very limited
effects or no effects on perceived satiety even at very large doses (Lyly et al., 2009;
Slavin and Green, 2007). There are no clinical trials that have used dairy beverages as
an avenue for increased oat β-glucan consumption. Consequently this study will
address this by investigating the effect of iso-volumetric preloads of dairy beverages
enriched with oat β-glucan on subjective appetite, short term food intake, and
postprandial glycemic responses on male and female subjects in good health.
4.2
Materials and methods
4.2.1 Subjects
30 non-smoking participants (male and female) between the ages of 20-30, and
with a BMI between 20-24.9 kg / m2 and in good health, participated in this study. The
female subjects undertook the test between their menstrual cycles. Informed written
consent was obtained from all volunteers, and financial remuneration was provided for
their participation. The clinical trial was a randomized single-blind repeated measures
design, and sessions were separated by a one week washout period. Following a 12 hr
overnight fast, subjects arrived at the same chosen time for each session. They were
instructed to refrain from alcohol consumption and any unusual activity the night before.
Upon arrival, subjects filled out a sleep habits and stress factors questionnaire, and a
recent food intake and activity questionnaire. Visual analogue scale questionnaires
were then completed to measure motivation to eat, physical comfort, and
fatigue/energy. Each VAS consisted of a 100-mm line anchored at the beginning and
60
end by opposing statements. The subjects marked an “X” on the line to indicate their
feelings at that given moment. Scores were determined by measuring the distance (in
mm) from the left starting point of the line to the intersection of the “X.” If, and or when,
significant deviations from their usual patterns were reported, they were asked to reschedule.
Following completion of the VAS questionnaires, a baseline blood sample was
taken by finger prick to measure blood glucose. Finger-prick blood samples were
obtained with the use of a Monojector Lancet Device (Sherwood Medical, St Louis,
MO). One drop of blood was placed on an Accu-Chek test strip for immediate reading of
glucose concentration with the Accu-Chek monitor (Accu-Chek Compact and CompactPlus; Roche Diagnostics Canada, Laval, QC). Accuracy and variance of the monitors
and test strips were monitored before and after each experimental session for each
subject by comparison against a commercial human serum standard (Assayed Human
Multi-Sera; Randox Laboratories Canada Ltd, Mississauga, ON). If the blood glucose
was above 5.5 mmol / L, the participant would be asked to re-schedule. If it was below
5.5 mmol / L, the subject was then asked to immediately thereafter drink one of the five
beverage preloads from a non transparent container using a straw within 5 min.
Capillary blood glucose via finger prick was then measured and VAS motivation to eat
was also completed at 10, 20, 30, 45, 60, 75, 90, 105 and 120 min (pre-meal) and 140,
170, 200, 230, and 260 min (post-meal) after baseline.
To measure the effect of the treatments on food intake, subjects were served an
ad libitum pizza meal between the 120 and 140 min time points (they were instructed to
eat until they were comfortably full). The experiment required a maximum of 4.5 hr per
61
session. Food intake and blood collection took place at the Department of Nutritional
Sciences, University of Toronto, with approval from the Research Ethics Board of the
University of Toronto while all the treatments were prepared at the Department of Food
Science, University of Guelph.
4.2.2 Materials
These included; a β-glucan concentrate (pilot plant, University of Guelph; βglucan, protein, and starch at ~10 %, ~70 %, and ~6 %), skim milk powder (34.7 %
protein), κ-carrageenan (Danisco, Brabrand, Denmark), vanilla flavour (Food
Specialities, Mississauga, Canada), and sugar (commercial grade).
4.2.3 Preparation of beverages
The treatments were prepared by mixing the β-glucan concentrate, skim milk
powder, κ-carrageenan, and sugar with distilled water at 250C. After sufficient mixing,
the beverages were homogenized in two stages at 2500 psi and 500 psi (APV Gaulin,
Everett, Ma, U.S.A.) at 250C. The beverages were then pasteurised at 75 0C for 10 min,
flavoured with the vanilla extract, and immediately filled into previously sterilized
containers. They were then cooled and refrigerated at 40C, until they were used in the
trial. The procedure was the same for all beverages at all stages, except for the noninclusion of the β-glucan concentrate for treatment A, and non-inclusion of skim milk
powder for treatment E (Tables 4.1 and 4.2). The participants consumed 500 mL of
each treatment as a pre-load.
62
Table 4.1: Composition of the oat β-glucan fortified beverages (pre-loads)
Pre-load
Sugar
Water
A
B
C
D
E
6.0
6.0
6.0
6.0
6.0
85.035
87.339
89.270
88.477
88.685
Ingredients (%)
κCarrageenan
0.015
0.015
0.015
0.015
0.015
Skim milk
powder
8.65
4.346
0.4146
0.2073
0
β-Glucan
Flavour
0
0.2
0.4
0.5
0.5
0.3
0.3
0.3
0.3
0.3
Table 4.2: Macro-nutrient composition of the beverages
Pre-load
(500mL)
β-glucan
(g)
Protein
(g)
Fat
(g)
Carbohydrate
(g)
Energy
(Kcal)
A
B
C
D
E
0
1
2
2.5
2.5
14.7
14.4
14.7
17.8
17.5
0.4
0.2
0.02
0.01
0
52
43
34
34
34
271
228
193
205
202
4.2.4 The meal
Pepperoni, Deluxe, and 3-Cheese type pizzas (5 inch diameter, ~200kcal each; Deep „N
Delicious Minis, McCain Minis) were purchased from a local supermarket, and kept
frozen until they were required. The pizzas were prepared in an oven 10 min prior to
meal time at the 120 min mark. The subjects were told that additional identical hot tray
replacement would be presented in 6-7 min intervals. The subjects were free to choose
which type of pizza they preferred to eat, and were instructed to eat until they were
comfortably full within the allocated 20 min time period. Each variety of pizza was
weighed separately, and the energy consumed was calculated by converting the net
weight to kilocalories with information provided by the manufacturer (McCain). Water
served with the meal was weighed before and after the meal to determine intake. An
advantage of using these pizzas was the lack of an outer crust, which results in a pizza
63
with uniform energy content and eliminates the possibility that the subject will eat the
energy denser filling and leave the outside crust of the pizza.
4.3
Statistical analysis
All statistical analysis was carried out using GraphPad Prism 5.0 (GraphPad
Software, Inc. USA). Incremental area under the curve (AUC) calculations for appetite
and blood glucose responses over the pre-meal and post- meal periods were calculated
using the trapezoidal method (Brouns et al., 2005). One way repeated measures
analysis of variance (ANOVA) with Tukey‟s Multiple Comparison testing was performed
to determine differences in the determined area under the curves (AUC). Two way
repeated measures analysis of variance (ANOVA) with Bonferroni Multiple Comparison
testing was performed to determine differences in both baseline adjusted satiety, and
blood glucose, treatment effects. A significance level of P < 0.05 was used in all tests.
4.4
Results and discussion
4.4.1 Satiety
There was a decrease in appetite scores after ingestion of both the beverages
(preload graph) and the meal (Fig. 4.1). The 120-140 min time period represents the
period where subjects were eating the pizza meal, and so measurements were carried
out before and after this period. The depression in appetite scores for both pre meal and
64
I
II
5.0
10.0
Time (min)
0.0
0.0
0
10
20
30
45
60
75
90 120
120
140
170
200
230
260
-10.0
-10.0
-15.0
A
-20.0
Δ Appetite (mm)
-5.0
Δ Appetite (mm)
Time (min)
-20.0
-30.0
A
-40.0
B
-25.0
C
D
-30.0
III
C
-60.0
E
-35.0
D
E
-70.0
IV
B
C
D
0.0
-200.0
-400.0
-600.0
-800.0
-1000.0
-1200.0
-1400.0
-1600.0
-1800.0
-2000.0
A
E
B
C
D
E
-4600.0
Appetite AUC (mm.min)
A
Appetite AUC (mm.min)
B
-50.0
-4800.0
-5000.0
-5200.0
-5400.0
-5600.0
-5800.0
-6000.0
-6200.0
Fig. 4.1: Baseline adjusted changes (Δ) in the VAS ratings for I) pre-meal appetite and II) post-meal
appetite; corresponding area under the curve for III) pre-meal and IV) post-meal sessions. Values are
means ± SEM, n = 29. For each time point (I and II), significant differences in means are indicated by
different letters, (P<0.05). A = milk -, B = milk +1 g β-glucan -, C= milk +2 g β-glucan -, D= milk + 2.5 g βglucan -, E= 2.5 g β-glucan - beverage
65
post meal curves is as was expected (after having something to eat). For the pre meal
period, there were observable differences in the appetite scores for the different
beverages (preloads). However, none of these differences were significant (p>0.05), at
any time point during the measurement period. It is thought that differences in β-glucan
content may not have been high enough to illicit significant differences between the
beverages. The post meal curve shows a similar trend, with a depression in appetite
scores for all beverage types, but these also were not significantly different (p>0.05) at
any time point within the measurement period. According to data generated in the pre
meal session we see that by the 120 min time point, the subjects are about as hungry as
when they started the session (0 min) and so we probably should not have expected to
see any effects due to pre-load intake by this time.
The area under the curve (AUC) for the pre meal period shows a general
increase in area as the β-glucan content increases in the preloads (A->E), with D
showing lower than expected AUC. Satiety is expected to increase with increasing βglucan content and so the trend shows this, even though there was no significant
difference (p>0.05) between the preload AUCs. The post meal AUCs were all higher
than the pre meal values for all beverages though these also were not significantly
different (p>0.05). As mentioned earlier, by the 120 min time point, subjects were as
hungry as when they started the session, and so what is seen post meal may only be
sparingly attributed to pre-load composition.
66
4.4.2 Blood glucose
Pre meal blood glucose showed a general peak around the 30 min time point for the
beverages used in this trial. There was a significant difference (p<0.05) between
beverages A and E at the 30 min time point (Fig. 4.2). At all other time points during the
pre meal session, no significant differences were generated. For the post meal period, A
was significantly different from both D and E at the 170 min time point, while at the 200
and 230 min mark, A was significantly different from E. For the area under the curve
(AUC) graphs, there was a significant difference between A and C in the pre meal
session (Fig. 4.2iii). There was no other significant difference between the beverages
for either the pre meal or post meal sessions.
The study had hoped to show a decrease in blood glucose correlating to an increase in
the β-glucan content of the beverages. The trend shown in the graphs did suggest this
relationship but a conclusion based on the β-glucan content alone is mired by the fact
that there was a decrease in the carbohydrate content of the beverages. The decrease
resulted from a change in the formulation of the beverages that had sought to negate
any effects on satiety that could arise from significantly different protein content in the
beverages. What was then done was to lower the amount of skim milk added to the
formulations, corresponding to increasing amounts of protein that came along with the
extract used. This therefore resulted in decrease of lactose with increasing extract
added, and it is thought that this decrease possibly contributed to differences in the
blood glucose readings as well. Therefore, it was not possible to determine conclusively
whether the observed trend was due to increased β-glucan content or due to the
decreased carbohydrate content, of the preloads A, B, C, D and E, or both. Therefore a
67
II
I
3.0
A
B
D
2.0
E
b
1.0
C
b
1.5
Δ Glucose (mg/dL)
Δ Glucose (mg/dL)
C
1.5
A
a
B
a
2.5
2.0
a
b
1.0
D
a
E
b
0.5
0.5
0.0
0.0
0
10
20
30
45
60
75
90
120
120
200
230
260
-0.5
III
IV
120.0
200.0
180.0
a
160.0
a,b
80.0
b
60.0
a,b
a,b
40.0
20.0
Glucose AUC (mg.min/dl)
100.0
Glucose AUC (mg.min/dl)
170
Time (min)
Time (min)
-0.5
140
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
0.0
A
B
C
D
E
A
B
C
D
E
Fig. 4.2: Baseline adjusted changes (Δ) in the measurement ratings for I) pre-meal blood glucose and II)
post-meal blood glucose; corresponding area under the curve for III) pre-meal and IV) post-meal sessions.
Values are means ± SEM, n = 29. Significant differences in means are indicated by different letters
(P<0.05), for each time point (I, II), and for the AUCs (III). A = milk -, B = milk +1 g β-glucan -, C= milk +2 g
β-glucan -, D= milk + 2.5 g β-glucan -, E= 2.5 g β-glucan - beverage
68
conclusive statement can not be made on the effect of a fiber supplemented beverage
on pre- and post meal glycemic responses.
4.4.3 Meal intake
There was no significant difference between pre loads on the food intake, and
neither was there a significant difference shown for cumulative food intake (Fig. 4.3).
Food intake herein refers to the test meal while cumulative food intake refers to the test
meal plus preload. Given the appetite scores (satiety) by the 120 min mark, this trend
lends credence to the suggestion that future pre- and post meal studies use an earlier
time point to assess the effects of the beverage pre loads as by this time point, these
effects are in all likelihood no longer a factor at play.
1600.0
1400.0
1200.0
(Kcal)
1000.0
Food Intake
800.0
Cumulative Food Intake
600.0
400.0
200.0
0.0
A
B
C
D
E
Fig. 4.3: Food intake (lunch meal) and cumulative food intake (pre-load + lunch meal) (Kcal).
Values are means ± SEM, n = 29. Significant differences in means are indicated by different
letters (P<0.05). A = milk -, B = milk +1 g β-glucan -, C= milk +2 g β-glucan -, D= milk +2.5 g
β-glucan -, E= 2.5 g β-glucan - beverage
69
Relatively larger amounts of dietary fibre (>10 g) have been used in many short term
studies to illicit effects on postprandial appetite profile and gastrointestinal hormonal
responses (Freeland et al., 2009; American Dietetic Association, 2008; Samra and
Anderson, 2007). Such amounts would obviously affect the palatability and acceptance
of β-glucan enriched beverages, and so lesser amounts as used in this study were
deemed more practical.
Despite differing quantity and characteristics (insoluble and soluble) of dietary
fibre among test meals, Juvonen et al., (2010), reported that consumption of these
fibres did not influence gastrointestinal peptide release, appetite or subsequent energy
intake in young healthy adults. The test meals were wheat and oat bran alone and in
combination, in a semisolid food matrix (isoenergetic and isovolumic pudding).
Beck et al., (2009) suggested that subjective ratings of hunger are improved at a
minimum dose of 2.2 g of β-glucan, while appetite suppressants such as CCK are
released in response to a minimum dose of approximately 3.8 g of β-glucan. Their study
measured acute biochemical and subjective measures of satiety (followed by energy
intake from a subsequent meal) after varying doses of β-glucan in extruded breakfast
cereals were investigated. Blood was collected to measure glucose, insulin, ghrelin and
cholectocystokinin, and visual analogue scales measured subjective satiety. They found
that subsequent meal intake decreased by greater than 400 kJ with higher β-glucan
dose (>5 g), with cholecystokinin release a likely part of the mechanism (Beck et al.,
2009).
70
4.5
Conclusion
This study had hoped to show a significant correlation between the amount of β-
glucan consumed in a dairy beverage, and a decrease in both satiety and blood glucose
responses in healthy subjects. The satiety responses generated were not significantly
different between treatments even though the trend shown was similar to what the study
had expected. Results from the blood glucose response were not conclusive and this
was thought to have arisen due to the composition of the treatments. In order to
generate more significant results future studies should keep the composition of
treatments as similar as possible with the exception arising from the β-glucan content,
as well as considering shorter time periods in which to provide the test meal for post
meal investigations.
71
Chapter V
General Discussion
72
5.1
Dietary fibre
The last two decades have seen an ever increasing interest in the consumption
of dietary fibre due to its perceived health benefits. The reduction of the risk factors
associated with the metabolic syndrome and obesity are some of the benefits
associated with regular consumption of products containing oat derived β-glucan, and
the main mechanism by which β-glucan is thought to work is through increased gut
viscosity thereby affecting changes such as reduced absorption of dietary cholesterol.
Researchers such as Hipsley and Groot (Groot et al., 1963; Hipsley, 1953; Hipsley,
1952) were among the first to study the correlation between dietary fibre consumption
and the so-called civilisation diseases. An intensive research soon followed regarding
effects of foods rich in dietary fibre and of isolated fibre components. Effects of various
fibre materials on blood glucose and insulin were found in several studies, and the
influence of dietary fibre on blood cholesterol and insulin were investigated by several
groups. Active research was also performed by several groups on the effects of fibre on
the physiology of the gastrointestinal tract and on its various diseases.
On January 21, 1997, the U.S. Food and Drug Administration (FDA) approved a
health claim on food products that “a diet high in soluble fibre from whole oats (oat bran,
oatmeal and oat flour) and low in saturated fat and cholesterol may reduce the risk of
heart disease” (FDA, 1997). The FDA concluded that at least 3 g / day of β-glucan from
oats should be consumed to achieve a clinically relevant reduction in serum total
cholesterol concentrations. The whole oat containing food would therefore provide at
least 0.75 g of water-soluble fibre per serving. Following this, an official
recommendation to increase consumption of whole grain foods and cereals was
73
included in the report of the Surgeon General by the U. S. Department of Health and
Human Services. In the period thereafter, a commercial boom concentrating mainly on
the use of oats was experienced, and after some balancing it stabilized in a permanent
public interest and elevated demand for fibre containing foods in most of the
industrialized countries.
Consequently there was, and still is, an increased interest within the food
industry to produce food products that meet the above criteria and therefore provide
more avenues for consumers to meet their daily recommended dietary fibre intake. In
this study we looked at the concentration of β-glucan from oat bran and the subsequent
physiological effect of oat β-glucan supplemented beverages on satiety and blood
glucose levels, after consumption. This was done because there is a need to increase
the consumption of oat based products. The well documented, positive effects of a diet
rich in oat bran and dietary fibres from oats on blood cholesterol levels and other
diseases related to the metabolic syndrome indicate that an increased intake of oat
based products are beneficial. The low consumption of products based on oats is due
mainly to the lack of acceptable and suitable food products containing soluble fibre at
appropriate levels, and so investigating the use of a dairy beverage as a suitable
„carrier‟ of dietary fibre was one of the objectives within this study.
5.2
The extraction
The study investigated the extraction of β-glucan from oat bran using an alternate
process based upon the thermodynamic incompatibility between proteins and
polysaccharides. Linked to this extraction process was a clinical trial that would need a
74
large amount of the extracted β-glucan. Various factors such as depolymerisation of
products, flame proofing of the process, and costs that would be incurred if pure
samples were obtained commercially, not to mention ethical limitations that would arise
from the use of fibre extracted in certain processes, culminated into the need to develop
and carry out an extraction that fell into our desired parameters. These parameters
essentially were the non-utilization of either alcohol and / or enzymes in the extraction
protocol, and so based upon previous protein-polysaccharide research, an alternate βglucan concentration process was investigated.
The concentration process that was developed and used in this study was based
upon the thermodynamic incompatibility between proteins and polysaccharides.
Specifically oat β-glucan and whey protein isolate were the polymers that were used in
this processing technology. The concept was based on the separation of the
polysaccharide and the protein into two phases from an initial mixture, with each of the
phases having a higher concentration of one of the polymers than the other. The phase
containing the higher concentration of the polysaccharide would be isolated, dried, and
a high fibre containing extract achieved as the end result. What was observed for the
particular polymers in this study, though, was an incomplete phase separation as the
concentration of the β-glucan in the lower phase is believed to have led to gelation that
consequently trapped some of the protein, and eventually led to what is referred to as
an „entrapped‟ system.
Though many difficulties and challenges still remain, active research in the field
of protein-polysaccharide interactions has provided many new insights into the phase
behaviour, rheology and microstructure of mixed systems. The extensive use of
75
scattering techniques such as small angle neutron scattering (SANS), dynamic light
scattering (DLS) and static light scattering (SLS), have been used to describe phase
separation mechanisms and the kinetics of the process. Appropriate spectroscopic and
microscopic techniques have as well been coupled with these techniques to provide
further information on the overall mechanisms involved in protein polysaccharide
interactions. Therefore a much better understanding of these processes could have an
enormous impact on wide ranging processes within the food industry, from the
extraction of a desired substance, to the final taste / mouthfeel perception of a finished
product.
5.3
The clinical trial
Beverages with different concentrations of β-glucan were used in the
investigation of pre- and post meal effects on satiety and blood glucose responses in
healthy adult subjects. The results indicated a general trend towards increased satiety
with increasing β-glucan content, in both the pre- and post-meal periods. Based upon
the data generated within the study, the post meal results were thought to have less
relevance since for instance, by the meal intake period, subjects‟ ratings for appetite
had generally returned to levels that they had when they started the session. The premeal sessions showed more relevant results though these were not statistically
significant from each other for the satiety responses, and for the blood glucose
responses, were statistically significant at only one or two points in the time line. The
blood glucose responses were subsequently judged to be inconclusive since the
carbohydrate content of the pre-loads had been decreased as the β-glucan content had
76
been increased within their composition. The responses could therefore not be
conclusively attributed to either factor.
The choice to use beverages as a suitable medium was arrived at based on the
desire to work with a food item that could be consumed multiple times within the day,
thereby increasing the probability that one would meet the recommended daily dietary
fibre intake need to minimize the risk factors associated with the metabolic syndrome.
Researchers such as, Paeschke and Aimutis (2011), and Juvonen et al., (2010), have
also suggested that beverages are more suitable vehicles for the intake of dietary fibre,
as compared to semi-solid and solid foods such as puddings and muffins. They mention
that in order to be effective, dietary fibre needs to become hydrated, and given the
nature of the conditions within the stomach, it is thought that this hydration may not be
sufficient in the case of solid foods, hence non availability of the fibre leading to no
physiological benefits gained.
That said, as was realised in this study too, there is only a limited amount of
dietary fibre that can be added to beverages before palatability and therefore
acceptance, of the products is affected. One therefore has to strike a balance between
including as much fibre in the composition as possible, and not affecting the sensorial
attributes to such an extent as to make the product undesirable, which would lead to
consumers not buying the product, which defeats the purpose of the dietary fibre
inclusion in the first place.
There were a number of factors that possibly affected the results that were
achieved in the study. The composition of the beverages should have been more
specific, with differences only arising from the β-glucan content. The time the test meal
77
was served to the healthy young subjects could have probably been earlier given the
nature of their metabolism, which was reflected in the „back to pre- beverage appetite
scores‟ by the test meal time period. Some authors have also suggested that effects
may be better observed in hypercholesteromic and / or overweight individuals as
opposed to healthy young adults. Essentially these are some of the potential areas that
could be addressed moving forward, and these could therefore serve as a learning
curve in the design of related future oat β-glucan based dairy beverage clinical trials.
78
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Appendix A: Information Sheet and Consent Form
Background and Purpose of Research
In 2004, almost 60% of adult Canadians were overweight or obese. This is a serious
health problem because obesity and being overweight are related to many risk factors
of disease, including increased blood sugar. Overweight and obesity can be treated by
changing what we eat. It is important to find food-based ways to prevent and treat
overweight and obesity.
Dairy products enriched with dietary fibers, more precisely β-glucan, are types of food
that can be used to decrease feelings of hunger and control blood sugar. This study will
test whether milk drinks with added fibre can control the feeling of hunger and blood
sugar. The information obtained from this study will be used to better understand the
effects of dairy products with added fiber on health of young men and women.
The purpose of this study is to find out the effects of drinking fiber-containing dairy
beverages on blood sugar and appetite in young men and women.
This study will have 30 participants.
Invitation to Participate:
You are being invited to take part in this study. If you choose to take part, you will be
asked to drink milk beverages five times (five sessions), one week apart. At each
session, your appetite (feelings of hunger), blood sugar and salivary cortisol (a measure
of stress) will be measured after drinking the test beverage and eating a pizza lunch.
Each session will take up to 4 ½ hours of your time.
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Eligibility:
To participate in this study, you must be a healthy male or female and between 20-30
years old. You must be a nonsmoker and you cannot be taking any medications. The
study will take place in the Department of Nutritional Sciences, Rooms 305, 329A, 334,
331 and 331A, FitzGerald Building, 150 College Street, Toronto, ON (in collaboration
with the University of Guelph).
Procedure:
To find out if you can take part in this study, you will be asked to fill out questionnaires,
which will ask questions about your age, if you smoke, exercise, your health, if you are
on any medications and your eating habits. Your height and weight will be measured.
If you can take part, you will be asked to fill out questionnaires about the foods you like.
You will be scheduled to meet with us five times over five weeks. You will be asked to
fast for 12 hours prior to your session (no eating for 12 hours before coming in except
for water).
You will be asked to arrive at the FitzGerald Building between 07:50 a.m. and 10:00
a.m. You will be asked to stick to your normal routine, including exercise and to eat a
similar meal the night before each session. You can drink water up to one hour before
meeting with us.
At each session, you will be asked to drink a milk drink (500 ml), give blood samples
and to complete questionnaires at the times outlined in the table below. Fifteen times
during each session, for a total of 75 times over the whole study, you will be asked to
provide blood by finger-prick to measure blood sugar. Blood will be sampled before
105
drinking the test beverage and at 10, 20, 30, 45, 60, 75, 90, 105, 120, 140, 170, 200,
230 and 260 minutes after drinking the drink. You will be asked to fill out visual analog
scale (VAS) questionnaires measuring your appetite (hunger), physical comfort,
energy/fatigue and stress as well as the palatability (pleasantness) of the drink and
pizza throughout the study sessions. You will be served a pizza meal two hours after
you drink the test beverage. In addition, every 30 minutes, we will ask you to provide a
small portion of your saliva (you will spit it into a special container) to measure a certain
molecule called cortisol that shows us your level of stress. Each session will last up to 4
½ hours.
Example of Time and Activity Schedule for Each Session
Time
Activity
07:50
Arrive at the laboratory (fasted for 12 hours)
07:50
Fill in Sleep, Stress, and VAS questionnaires and take first blood
sample
08:00-08:05
Drink the test beverage (0 minutes)
08:00-10:00
Blood sampling and VAS questionnaires at 10, 20, 30, 45, 60, 75, 90,
105 and 120 minutes
10:00-10:20
Pizza served and eaten (120-140 minutes)
10:20-12:20
Blood sampling and VAS questionnaires at 140, 170, 200, 230 and
260 minutes
VAS: Visual Analogue Scale
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Voluntary Participation and Early Withdrawal:
It is hoped that you will finish all five sessions. However, you may choose to stop being
in the study at anytime without any problems.
Early Termination:
Not applicable.
Risks:
All of the test beverage that you will be asked to consume are prepared in the University
of Guelph, with fresh and natural food grade ingredients. In addition, the pizza that you
will be also asked to consume are prepared hygienically in the kitchen at the time of the
session and present minimal risk. You may feel dizzy following the overnight fast, but
this is rare. If this happens, you will likely feel fine once you drink the test beverage
provided to you.
The risks and discomfort will come from the blood sampling procedure. Great care will
be taken when taking your finger-prick blood samples. The investigator will be there to
help you. To make sure that you are not exposed to another person‟s needle, we will
ask you to sit away from other study participants. We will put a needle into the fingerprick gun before taking each blood sample and then put it into the safety container.
There is very little risk of infection. We will clean your finger with a new alcohol swab
before and after each finger-prick and will use a new sterile needle each time. You will
be provided with your own finger-prick gun for the entire study.
Some discomfort will be felt as a result of a sharp momentary pain caused as the
needle enters the skin. However, because the lancet needle is very small, the pain felt
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is usually less than you might feel from skin puncture during vaccination or if a blood
sample is taken by a needle inserted in a vein. There might be slight bruising under the
skin, but this will be minimized by applying pressure after the finger is pricked and blood
sugar is measured. A total of 15 finger-pricks will be conducted per one session and 75
throughout the whole study (five sessions).
You may experience flatulence (passing gas) and feelings of gastrointestinal discomfort
(bloating) from the drinks. This is rare and there is no health risk linked with these
effects.
In addition, there are no anticipated risks from taking your saliva. This is a hygienic
procedure, carried out by chewing new cotton wool swabs to obtain fluid samples.
Sampling is also painless and can be repeated without difficulty.
There is always a possibility that you will become ill following consumption of food, but
that is very unlikely. All drinks are safe to consume and the pizzas are freshly prepared
at the time of your session. The pizzas are stored frozen and cooked accordingly to the
manufacturer‟s instructions immediately before you are served.
Benefits:
You will not benefit directly from taking part in this study. You will be shown your blood
sugar results and, if they are not normal, you will be told and advised to talk to your
doctor. The foods and drinks will be provided for free.
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Confidentiality and Privacy:
Confidentiality will be respected and no information that shows your identity will be
released or published without your permission unless required by law. Your name,
personal information and signed consent form will be kept in a locked filing cabinet in
the investigator‟s office. Your results will not be kept in the same place as your name.
Your results will be recorded on data sheets and in computer records that have an ID
number for identification, but will not include your name. Your results, identified only by
an ID number, will be made available to the study sponsor if requested. Only study
investigators will have access to your individual results.
Publication of Results:
The results of the study may be presented at scientific meetings and published in a
scientific journal. If the results are published, only average and not individual values will
be reported.
Possible Commercialization of Findings:
This study is preliminary. Once these products are tested more widely in future studies,
results may lead to commercialization of a product, new product formulation, changes in
the labeling of a product and/or changes in the marketing of a product. You will not
share in any way from the possible gains or money made by commercial application of
findings.
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Alternative Treatment / Therapy:
Not applicable.
New Findings:
If anything is found during the course of this research which may change your decision
to continue, you will be told about it.
Compensation:
You will be paid $46 per session ($230 in total). Reimbursement for travel expenses ($6
per session) is included in each session total. If you withdraw from the study before
finishing or asked to withdraw, you will be paid for the sessions you have already
finished.
Injury Statement:
If you begin to feel sick following participation in the study, please seek medical advice
as soon as possible. We will provide your medical specialist with information about the
food you have consumed during the session, so take our phone number with you.
Rights of Subjects:
Before agreeing to take part in this research study, it is important that you read and
understand your role as described here in this study information sheet and consent
form. You waive no legal rights by taking part in this study. If you have any questions or
110
concerns about your rights as a participant you can contact the Ethics Review Office at
[email protected] or call 416-946-3273.
If you have any questions after you read through this information, please do not hesitate
to ask the investigators for further clarification.
Dissemination of findings:
A summary of results will be made available for you to pick up after the study is done.
Copy of informed consent for participant:
You are given a copy of this informed consent to keep for your own records.
Consent:
I acknowledge that the research study described above has been explained to me and
that any questions that I have asked have been answered to my satisfaction. I have
been informed of the alternatives to participation in this study, including the right not to
participate and the right to withdraw. As well, the potential risks, harms and discomforts
have been explained to me. I understand that I will receive compensation for my time
spent participating in the study.
As part of my participation in this study, I understand that I may come in contact with
other study participants because our session times overlap. I agree to keep anything I
learn about other participants confidential and know that other participants have agreed
to do the same for me.
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I hereby agree and give my authorized consent to participate in the study and to treat
confidential information in a restrictive manner as described above. I have been given a
copy of the consent form to keep for my own records.
___________________
___________________
Participant Name
Signature
___________________
___________________
Witness Name
Signature
_________________
Investigator Name
___________________
Signature
___________
Date
___________
Date
____________
Date
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Appendix B: Food Acceptability Form
Please indicate with a rating between 1 and 10 how much you enjoy the following foods
(1 = not at all, 10 = very much) and how often you eat them (never, daily, weekly,
monthly).
Enjoyment?
How often?
1. Pasta
__________
__________
2. Rice
__________
__________
3. Potatoes (mashed, roasted)
__________
__________
4. French fries
__________
__________
5. Pizza
__________
__________
6. Bread, bagels, dinner rolls
__________
__________
7. Sandwiches, subs
__________
__________
8. Cereal
__________
__________
9. Cake, donuts, cookies
__________
__________
10. Tomato/vegetable juice
__________
__________
11. Milk/chocolate milk
__________
__________
Will you be able to drink a milk or milk substitute (e.g. soy beverage)?
 YES
 NO
At the end of each session, you will be provided with pizza. In order to provide you with
a meal that you will enjoy, we ask that you rank the following pizzas according to your
personal preferences (i.e. 1st, 2nd, 3rd choice) in the space provided. If you do NOT
113
like a particular type of pizza, then do not rank it but instead place an “X” in the space
provided.
Pepperoni (cheese, pepperoni)
__________
Deluxe (cheese, pepperoni, peppers, mushrooms)
__________
Three-cheese (mozzarella, cheddar, parmesan)
__________
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Appendix C: Recent Food Intake and Activity Form
At what time did you have dinner? _____________________
Please describe your dinner last night (list all food and drink and give an estimate of the
portion size):
___________________________________________________________________
___________________________________________________________________
___________________________________________________________________
The following three (3) questions relate to your food intake, activity and stress over the
last 24 hours. Please rate yourself by placing a small “x” across the horizontal line at the
point which best reflects your present feelings.
How would you describe your food intake over the past 24 hours?
Much LESS
than usual
Much MORE
than usual
How would you describe your level of activity over the last 24 hours?
Much LESS
than usual
Much MORE
than usual
How would you describe your level of stress over the last 24 hours?
Much LESS
than usual
Much MORE
than usual
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Appendix D: Visual Analogue Scales - Motivation to Eat
These questions relate to your “motivation to eat” at this time. Please rate yourself by
placing a small “x” across the horizontal line at the point which best reflects your
present feelings.
1. How strong is your desire to eat?
VERY
weak
VERY
strong
2. How hungry do you feel?
NOT
hungry
at all
As hungry
as I have
ever felt
3. How full do you feel?
NOT
full
at all
VERY
full
4. How much food do you think you could eat?
NOTHING
at all
A LARGE
amount
5. How thirsty do you feel?
NOT
thirsty
at all
As thirsty
as I have
ever felt
116
Appendix E: Visual Analogue Scales - Physical Comfort
These questions relate to your “physical comfort” at this time. Please rate yourself by
placing a small “x” across the horizontal line at the point which best reflects your
present feelings.
1. Do you feel nauseous?
NOT
at all
VERY
much
2. Does your stomach hurt?
NOT
at all
VERY
much
3. How well do you feel?
NOT
well
at all
VERY
well
4. Do you feel like you have gas?
NOT
at all
VERY
much
5. Do you feel like you have diarrhea?
NOT
at all
VERY
much
117
Appendix F: Visual Analogue Scales - Energy / Fatigue and Stress
These questions relate to your “energy level and fatigue” at this time. Please rate
yourself by placing a small “x” across the horizontal line at the point which best reflects
your present feelings.
1. How energetic do you feel right now?
NOT
at all
2. How tired do you feel right now?
NOT
at all
3. How anxious do you feel right now?
NOT
at all
anxious
VERY
energetic
VERY
tired
VERY
anxious
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Appendix G: Fibre-enriched Beverage Study Session Schedule
Time (min)
Count-up
Instructions
0
-0:15 – 0:00
0
0:00
●
●
●
●
●
5
0:05
● Subject finishes drinking
10
0:10
20
0:20
30
0:30
45
0:45
60
1:00
75
1:15
90
1:30
108
1:50
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1:58
120
2:00
140
2:20
170
2:50
200
3:20
230
3:50
260
4:20
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
Subject fills out Baseline VAS forms
Measure baseline blood glucose
Take out treatment and bring it to the subject
Start count-up timer for 5 minutes and place it on the subject‟s table
Subject drinks treatment
Measure blood glucose
Subject fills out 10 minute VAS questionnaires
Measure blood glucose
Subject fills out 20 minute VAS questionnaires
Measure blood glucose
Subject fills out 30 minute VAS questionnaires
Preheat oven at 430° F
Measure blood glucose
Subject fills out 45 minute VAS questionnaires
Measure blood glucose
Subject fills out 60 minute VAS questionnaires
Measure blood glucose
Subject fills out 75 minute VAS questionnaires
Take out 12 (or 8) pizzas and arrange them onto baking sheets with 4 per sheet
Measure blood glucose
Subject fills out 90 minute VAS questionnaires
Place pizza tray 1 into oven; set timer for 8 minutes
Weigh 500 mL of water with cup and bring to feeding room with a count-down
timer set for 20 minutes
Remove pizza tray 1 from oven and place tray 2 into oven at 1:58
Cut and weigh pizzas and bring it to feeding room in a serving tray
Instruct subject to fill out 120 minute VAS forms
Measure blood glucose
Bring subject to feeding room and start count-down time for 20 minutes and
instruct subject to “eat until comfortably full” and to take their time
Serve pizza tray 2 at 2:07, bring back and weigh tray 1
Serve pizza tray 3 at 2:14, bring back and weigh tray 2
Subject finishes eating
Measure blood glucose
Bring back water and pizza tray 3 and weigh them
Measure blood glucose
Subject fills out 170 minute VAS questionnaires
Measure blood glucose
Subject fills out 200 minute VAS questionnaires
Measure blood glucose
Subject fills out 230 minute VAS questionnaires
Measure blood glucose
Subject fills out 260 minute VAS questionnaires
Make sure VAS booklet is completely filled out
119