Beta-Glucanase Activity and its Impact on Beta

Beta-Glucanase Activity and its Impact on Beta-Glucan Molecular
Weight Degradation in Cereal Products Fortified with Beta-Glucan
by
Azadeh Vatandoust
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
© Azadeh Vatandoust, January, 2012
ABSTRACT
β-GLUCANASE ACTIVITY AND ITS IMPACT ON β-GLUCAN
MOLECULAR WEIGHT DEGRADATION IN CEREAL PRODUCTS
FORTIFIED WITH β-GLUCAN
Azadeh Vatandoust
Advisor:
University of Guelph, 2012
Dr. Koushik Seetharaman
Health benefits of high molecular weight (MW) β-glucans are well documented.
Therefore, understanding and controlling depolymerization of β-glucan in baked
products, would increase the effectiveness of β-glucan to confer health benefits. In this
study we demonstrated that endogenous β-glucanase in wheat kernels are responsible for
the depolymerization of β-glucans. A protocol was developed based on the Megzayme
procedure to detect low levels of β-glucanase activity in wheat flour. This was confirmed
by using HPLC-Calcofluor detection to monitor molecular weight changes.
The
distribution of β-glucanase in wheat kernels was also investigated. The effect of
genotype, location, planting season and environmental factors on the level of endogenous
β-glucanase in selected wheat cultivars was investigated using different wheat varieties
planted under different condition and different seasons. Furthermore, kinetics of β-glucan
depolymerization by endogenous glucanase in two dough systems with different moisture
content was investigated. The results demonstrated that enzymes with β-glucanase
activity are concentrated primarily in the outer layer of wheat kernels. Also genotype,
environmental conditions and agronomic practice all had significant effects on the βglucanase activity in wheat flours and poor harvesting conditions can significantly
increase β-glucanase activity level in wheat. The kinetics results demonstrated that
moisture content, incubation time and levels of endogenous β-glucanase activity of the
system had significant impact on the final MW of β-glucan in the dough. Among all
factors investigated, moisture content had the greatest impact. This study presents
opportunities for industry to fortify baked products with high molecular weight β-glucan.
ACKNOWLEDGEMENTS
Venturing on an unknown journey of studies abroad initially seemed frightening.
There were numerous moments that I didn’t know where the journey would end, but
having faith that throughout the journey God is with me, was a constant source of
strength and inspiration.
First and foremost I would like to express my gratitude to my supervisor Dr.
Koushik Seetharaman. The generous opportunity that he provided by accepting me as his
student made my dreams of graduate studies become a reality. His advice, insightful
criticisms, and patient encouragement aided me throughout my studies in innumerable
ways. His truly scientific intuitions inspired me and enriched my life both as a student,
and an aspiring scholar. One simply could not wish for a better or friendlier supervisor.
I would like to express my sincere appreciation to Dr. Sanaa Ragaee for her
support, supervision and encouragement which made her the true backbone of this study.
Her long hours of discussion and her constant urge to learn, made me understand my
research problem in greater depth. She believed in me and taught me to believe in
myself. Her understanding and help towards me to reach beyond academic boundaries is
greatly appreciated. I learned from her not just how to research and finish my thesis but
how to inhale the downside of life while living abroad. I was continually amazed by her
willingness to help and teach me all the small details in life, and I am grateful to her in
every possible way.
I would also like to thank Dr. Susan Tosh, my committee member, whose support
for this project is greatly appreciated. She made her lab available for my experiments,
which was the reason I was able to conduct all the experiments related to β-glucan
molecular weight; also for her helpful inspiration in β-glucan which led me to read and
educate myself further.
Special thanks also go to Yolanda Brummer who thought me
how to use the HPSEC. Her useful suggestions and comments during my research, her
help in editing and correcting my literature review and finally her friendship and sense of
humor made working in Agriculture and Agri Food Canada lab more than mere fun.
iv
It has been an honor to be a student of the late Dr. Peter J. Wood. Despite his ill
health, he generously shared his time and ideas with me. He was my primary source for
getting β-glucan questions answered. His unflinching courage and conviction will always
inspire me, and I hope to continue to work with his noble thoughts. The joy and
enthusiasm he had for β-glucan was contagious and served as a source of motivation
during difficult times. I am most grateful for having known him during my studies and I
will never forget him as a remarkable individual.
I need to extend my gratitude and acknowledgement for the financial support
provided by the Ontario Ministry of Agricultural, Food and Rural Affairs and the
Department of Food Science.
I would also like to thank all my lab mates at the University of Guelph and
Agriculture-Agri Food Canada that became a part of my life. I am grateful to them for
the sleepless nights we worked together before deadlines and for all the fun we had
during our studies. My thanks go to my dear roommate and friend Mina Kaviani who put
up with my difficult times and my crazy schedule. I can’t even imagine how I could have
completed my work without her. I am indebted to Renuka Waduge for her friendship,
support and valuable advices during my research; to George Annor who helped me
understand every concept of my thesis, to Farzaneh Farah-Bakhs for her generous care
and ultimate support, to Sepideh Khalili who has been a great friend and constant support
since we were high school classmates, and to Neda Barjesteh, Mehdi Emam and Davood
Rezaie for their genuine friendship which brightened my life at Guelph.
Lastly, this thesis is dedicated to my father, Gholamreza, who taught me that
nothing can be more joyful than learning for the sake of learning. I would like to thank
him for inspiring in me the love to learn. It is also dedicated to my mother, Shayesteh,
who taught me that even the most difficult task can become easy if I have faith and
advance one step at a time. The thesis is also dedicated to my brother, Ardeshir, whose
advice lightened my journey in life and his sense of humor made life all the more
colorful. My family supported me in all my endeavors and crazy, self-centered decisions
and pushed me time after time. Knowing that they will always be there to pick up the
v
pieces has given me the strength and confidence to repeatedly risk getting shattered. I am
grateful and fortunate to have their constant support.
vi
TABLE OF CONTENTS
Acknowledgements ............................................................................................................ iv
Table of Contents .............................................................................................................. vii
List of Tables ..................................................................................................................... xi
List of Figures ................................................................................................................... xii
1. Chapter One Introduction .............................................................................................. 1
2. Chapter Two Litreature Review...................................................................................... 3
2.1 Dietary Fiber ..................................................................................................... 3
2.1.1 Fibers Health Benefits ............................................................................... 3
2.1.1.1 Role of Dietary Fiber in Obesity ........................................................ 4
2.1.1.2 Role of Fibers in Diabetes .................................................................. 5
2.1.1.3 Role of Fibers in Coronary Heart Diseases ........................................ 5
2.1.2 Structure of Dietary Fiber .......................................................................... 5
2.1.3 Examples of Dietary Fiber......................................................................... 6
2.1.3.1 Cellulose ............................................................................................. 6
2.1.3.2 Arabinoxylan ...................................................................................... 7
2.1.3.3 β-Glucan ............................................................................................. 7
2.2 β-Glucan as a Dietary Fiber .............................................................................. 8
vii
2.2.1 β-Glucan Sources....................................................................................... 8
2.2.2 Extraction of β-Glucan .............................................................................. 9
2.2.2.1 Determination of β-Glucan Concentration ....................................... 10
2.2.2.2 β-Glucan Molecular Weight ............................................................. 11
2.2.3 β-Glucan Health Benefits ........................................................................ 11
2.3 β-Glucanase Activity ...................................................................................... 14
2.3.1. Microbial β-Glucanase ........................................................................... 15
2.3.1.1 Properties of Microbial β-Glucanase ................................................ 16
2.3.2 β-Glucanase Activity in Plants ................................................................ 17
2.3.2.1 Properties of Plant β-Glucanase ....................................................... 17
2.3.3 β-Glucanase Activity in Baking Ingredients ........................................... 19
2.3.4 Food Processing and Depolymerization of β-Glucan .............................. 20
2.4 Effect of β-Glucan on Technological Properties of Cereal Products ............. 22
3. Chapter Three Detection, Localization and Variability of Endogenous β-Glucanase in
Wheat Kernels ................................................................................................................... 33
3.1 Abstract........................................................................................................... 33
3.2 Introduction .................................................................................................... 34
3.3 Materials and Methods ................................................................................... 37
3.3.1 Sample Preparation .................................................................................. 37
viii
3.3.2 Optimizing Extraction and Incubation Conditions .................................. 38
3.3.3 Determination of β-Glucanase Activity................................................... 38
3.3.4 Calibration Curve .................................................................................... 39
3.3.5 Statistics ................................................................................................... 40
3.3.6 Validation of the Procedure Using HPLC with Calcoflour Detection .... 40
3.4 Results and Discussions ................................................................................. 40
3.4.1 Optimization of the Assay ....................................................................... 40
3.4.2 Distribution of β-Glucanase Activity in Wheat Kernel ........................... 42
3.4.3 Effect of Variety and Location on β-Glucanase Activity ........................ 43
3.4.4 Effect of Planting Season on β-Glucanase Activity ................................ 44
3.5 Conclusion ...................................................................................................... 45
4. Chapter Four β-Glucan Degradation by Endogenous Enzaymes in Wheat Flour Doughs
with Different Moisture Contents ..................................................................................... 54
4.1 Abstract........................................................................................................... 54
4.2 Introduction .................................................................................................... 55
4.3 Materials and Methods ................................................................................... 57
4.3.1 Sample Preparation .................................................................................. 57
4.3.2 Determination of β-Glucan ...................................................................... 58
4.3.3 β-Glucanase Activity ............................................................................... 58
ix
4.3.4 Experimental Doughs .............................................................................. 59
4.3.5 Extraction β-Glucan From the Dough ..................................................... 59
4.3.6 Physicochemical Analysis of β-Glucan in Dough ................................... 59
4.3.7 Statistics ................................................................................................... 60
4.4 Results and Discussions ................................................................................. 60
4.4.1 Determining of Flours Characteristics..................................................... 60
4.4.2 Effect of Level of β-Glucanase Activity on Degradation of β-Glucan in
Dough ........................................................................................................................ 61
4.4.3 Effect of Moisture Content and Incubation Time on Depolymerization of
β-Glucan in the Dough System ................................................................................. 62
4.5 Conclusion ...................................................................................................... 64
5. Chapter Five Conclusions ........................................................................................... 69
6. References Cited ........................................................................................................... 72
x
LIST OF TABLES
Table 3.1: Differences in spectrophotometer absorbance (590nm) for different samples
using the original Megazyme procedure and the modified procedure. ............................. 46
Table 4.1: Bread Formulation ........................................................................................... 65
Table 4.2: Cookie Formulation ......................................................................................... 66
xi
LIST OF FIGURES
Fig. 2.1: Schematic representation of Cellulose ............................................................... 24
Fig. 2.2: Schematic representation of Arabinoxylan ........................................................ 25
Fig. 2.3: Representative structure of β-glucan, where “n” usually is either one or two but
infrequently can be large .................................................................................................. 26
Fig. 2.4: Schematic representation of β-Glucan ............................................................... 27
Fig. 2.5: Procedures for physiological extract of β-glucan .............................................. 28
Fig. 2.6: Principle of the mix linkage assay for determining β-glucan content using
Megazyme International, Bray, Ireland Kit. ..................................................................... 29
Fig. 2.7: A represents trisaccharide and B represent tetrasaccharide which are both result
of lichenase and fungus enzyme on β-glucan. .................................................................. 30
Fig. 2.8: Schematic representation of differences in action of B.subtillis lichenase and
A.japonicus enzyme on β-glucan . .................................................................................... 31
Fig. 2.9: Enzymatic hydrolysis of cell wall (1→3),(1→4)-β-D-glucans ......................... 32
Fig. 3.1: Standard curve relating the activity of malt β-glucanase activity to absorbance at
590 nm upon hydrolysis of azo-barley β-glucan. Incubation time was 3 hr. .................... 47
Fig. 3.2: Molecular weight of β-glucans following a 10 min incubation of wheat flours
with a β-glucan isolate (MW=1,200 × 103 g/mol) measured through high-performance
size-exclusion chromatography with Calcofluor detection............................................... 48
Fig. 3.3: Distribution of β-glucanase activity in wheat kernel .......................................... 49
xii
Fig. 3.4: Effect of germination time on β-glucanase activity of wheat kernels . .............. 50
Fig. 3.5: Distribution of enzymes with β-glucanase activity in wheat kernels after
germination. ...................................................................................................................... 51
Fig. 3.6: β-Glucanase activity of wheat varieties/lines grown in different location. ........ 52
Fig. 3.7: Effect of planting season on β-glucanase activity of wheat varieties................. 53
Fig. 4.1: Degradation of β-glucan during preparation of bread dough and cookie dough of
using flours containing different endogenous β-glucanase activity. ................................. 67
Fig. 4.2: Effect of moisture content and endogenous β-glucanase activity on
depolymerization of β-glucan in the systems.................................................................... 68
xiii
1. CHAPTER ONE
INTRODUCTION
(1−>3)(1−>4) β-D-glucan (known as β-glucans) is an unbranched water soluble
polysaccharide component of the endosperm and aleurone cells of Poacea family (Wood
and Fulcher 1978; BeMiller and Huber 2008). This polysaccharide can be mainly found
in oat and barley and comparatively smaller amounts in other cereals such as corn, rice,
rye, wheat and sorghum (Wood 1994a; Ragaee et al 2008; Tiwari and Cummins 2009).
The concentration and extractability of β-glucan depend on the grain type (Wood 2010).
For instance, extracting β-glucans from rye or wheat bran is much more difficult than oat
and barley due to the arabinoxylan matrix which is present in rye and wheat (Ragaee et al
2008).
Over the past few years interests in producing functional foods fortified with βglucans has become the priority response of food scientists and industries mainly because
of the health benefits which have contributed to the consumption of this dietary fiber.
Studies have related consuming products containing β-glucan to several health benefits
including moderating glycaemic response to starchy foods (Tappy et al 1996; Regand et
al 2009) and lowering serum lipid levels, specially the low-density lipoproteins (LDL)
(Tosh et al 2010; Wolever et al 2010). Foods containing at least 0.75g of oat-derived βglucan for each serving portion are allowed to have a health claim and this regulation has
been expanded to include barley as well (FDA 1997-2005; EFSA 2010; Health Canada
2010).
1
There have been controversial suggestions about mechanism of how β-glucan
consumption results in health benefits (Brown et al 1999). In general, most studies
suggest that the potential health benefits of β-glucan are due its ability to enhance the
viscosity of gut contents and as a result change the absorption rates of nutrients and bile
acid (Wood 2010). Consequently, it is important to understand that concentration,
extractability, MW and structure of β-glucan in food, can affect its ability to generate the
desirable viscosity (Wood 2010).
Furthermore, studies (Knuckles and Chiu 1999;
Nauman et al 2006; Regand et al 2009; Tiwari et al 2009) demonstrated that MW and the
consequent viscosity generated by β-glucan can be altered by food matrix, or methods of
processing and storage. This matter becomes critical when Thondre et al (2011)
demonstrated that low molecular weight β-glucan did not induce the expected health
benefits. Studies (Andersson et al 2004; Trogh et al 2004) hypothesized the presence of
endogenous glucanase in wheat flour, but so far no study has reported the actual presence
of these enzymes in the wheat.
Therefore, the goal of the present study was to identify factors that lead to βglucan depolymerization in the cereal based products. The obtained result would assist in
developing strategies to maintain β-glucan MW and enhance the health benefits of final
products.
2
2. CHAPTER TWO
LITREATURE REVIEW
2.1 Dietary Fiber
One of the earliest definitions of dietary fiber was as early as 1953 (Hipsley et al
1953; Spiller et al 1993). Today the American Association of Cereal Chemists (AACC)
(2001) defines dietary fiber as “the remnants of the edible part of plants and analogous
carbohydrates that are resistant to digestion and absorption in the human small intestine
with complete or partial fermentation in the human large intestine. It includes
polysaccharides, oligosaccharides, lignin and associated plant substances. Dietary fiber
exhibits one or more of either laxation (fecal bulking and softening; increased frequency;
and/or regularity), blood cholesterol attenuation, and/or blood glucose attenuation.” In
general the term “Dietary fiber” is more a nutrition term then a description of the physical
and chemical properties of the component even though both chemical and physical
properties are necessary for a component to be considered dietary fiber (BeMiller and
Huber 2008).
2.1.1 Fibers Health Benefits
Dietary fibers have been associated with improved human health and reduce risk
of diseases. The strength of evidence varies from one health issue to another but in
general it has been reported dietary fiber plays an important role in human health by
increasing post prandial satiety (BeMiller and Huber 2008), reducing the risk of heart
disease (Liu et al 1999), stroke (Steffen et al 2003), type 2 diabetes (Montonen et al
2003), obesity (Lairon et al 2005) and certain gastrointestinal disorders (e.g. Diverticular
3
disease) (Petruzziello et al 2006). Moreover, increasing the consumption of dietary fiber
can improve serum lipid concentration (Brown et al 1999), lower blood pressure (Keenan
et al 2002), facilitate weight loss (Birketvedt et al 2005) and potentially improve immune
functions (Watzl et al 2005). Based on the beneficial effects of consuming dietary fiber,
a recommended daily intake has been established. The recommended intake differs
according to sexes and ages, but in general it is 38 and 25 g/d respectively for men and
women between the ages of 19 and 50 years (AACC 2003).
2.1.1.1 Role of Dietary Fiber in Obesity
As was reported by the World Health Organization (WHO) in 2003, obesity and
health problems related to it are common worldwide and are on the rise. There is
therefore a lot of interest in finding ways to improve this global problem. Hlebowicz
(2009) suggested that one solution for obesity is to decrease food consumption by
increasing satiety. As FAO/WHO (1998) reported, satiety is the state after a meal, where
the feeling of hunger is reduced and there is little or no desire for consuming more food.
Different studies were able to show that foods with dietary fibers have a positive effect
on satiety and as a result could contribute to a solution for the obesity problem. As an
example, Koh Banerjee et al (2004) reported that increasing the consumption of whole
grain products can contribute to maintaining a healthy weight and reducing the risk of
weight gain in the long term. Also Beck et al (2009) have reported that consuming
breakfast meals having β-glucan fractions, by human subjects showed that β-glucan can
help satiety feeling which this may result in healthy weight.
4
2.1.1.2 Role of Fibers in Diabetes
Studies showed that the consumption of dietary fiber and whole grain products
can reduce the risk of type 2 diabetes. A study by Fung et al (2002) demonstrated that the
risk of type 2 diabetes was reduced by up to 30% for those who consumed wholegrain
products. Montonen et al (2003) further reported that cereal fibers had a great impact on
reducing the risk of type 2 diabetes.
The mechanism of how consumption of whole grains and dietary fibers result in
lowering the risk of type 2 diabetes have been suggested to include resistance to digestion
and improvement in glucose tolerance (Hallfrisch and Behall 2000; Jensen et al 2006).
2.1.1.3 Role of Fibers in Coronary Heart Diseases
Consuming dietary fibers have been associated with reducing risk of
hypertension, stroke, heart diseases and in general coronary heart diseases. Mozaffarian
et al (2003) reported that Finns consuming diets high in dietary fiber (median intake 35
g/daily) showed significantly lower risk of coronary diseases than those consumed lower
dietary fiber intake (median intake 16 g/daily). Other studies where human subjects
consumed whole grain as a source of fiber also show that consuming fibers can reduces
the risk of chronic diseases (Jacobs et al 1998; Liu et al 1999; Ludwig et al 1999; Erkkila
et al 2006).
2.1.2 Structure of Dietary Fiber
The dietary fibers that are present in plants are mostly either polysaccharides of
plant cell walls or a range of analogous extrinsic polysaccharides. They contain different
monosaccharide which can combine in a number of different ways to form different
5
polymers. Cellulose and starch, the most abundant of all the polysaccharides, have a
backbone chain where carbon-1 has been linked with carbon-4 with branches or side
chains from the hydroxyl group which is present on other carbons.
In general polysaccharides are either a homopolymers where they are composed
of one monosaccharide species or heteropolymers, which are formed from two or more
monosaccharides in either a branched or linear structure (Southgate 1995).
Polysaccharide structure is very important since it determines its susceptibility to bind to
hydrolytic enzymes, as well as its tendency to form intermolecular association with other
polysaccharides. This is an important factor for determining either solubility or/and
colloidal behavior of the polysaccharide (Southgate 1995). Furthermore, fibers are
divided into two groups: soluble or insoluble. Soluble fibers, such as pectin, β-glucan and
arabinoxylan are water soluble and can be fermented in the colon whereas insoluble
fibers, such as wheat bran are insoluble in water and have a bulking action and can only
be fermented to some extent in the colon (Anderson et al 2009). Division of dietary fibers
to soluble and insoluble can be studied in different ways. Some examples of dietary fiber
will be discussed.
2.1.3 Examples of Dietary Fiber
2.1.3.1 Cellulose
Cellulose is an insoluble, linear, high molecular weight homopolymer made up of
repeating β-D-glucopyranosyl units joined by (1−>4) glycosidic linkage (Fig. 2.1).
However, through derivatization, they can be converted into water soluble gums, such as
Carboxymethylcelluloses (CMC). As cellulose passes through the human digestive
6
system, it remains undigested with no significant nourishment or calories which make it a
dietary fiber. This polysaccharide is used in the food industry to increase the content of
dietary fiber in a food product and act as a non caloric bulking agent. Also the presence
of this dietary fiber in foods can keep foods moist and fresh for a longer period of time
(BeMiller and Huber 2008).
2.1.3.2 Arabinoxylan
Arabinoxylan is a non starch polysaccharide which can be found in tissues of
many cereals such as wheat, rye, barley oat rice and sorghum (Fincher and Stone 1986;
Ragaee et al 2001). Although this polysaccharide known as a minor component of cereal
grains, but it serves as an important constituent in the cereal cell wall (Fincher and Stone
1986; Ragaee et al 2001).
This polysaccharide has a linear backbone of (1−>4)-β-D-xylopyranosyl in which
the Arabinose substitutes are linked through O-2, O-3, or O-2, 3 residues (Fig. 2.2). It can
be found in both, water extractable and water un-extractable forms in cereal grains and
tissues and this has made this polysaccharide an important dietary fiber (Fincher and
Stone 1986; Ragaee et al 2001).
2.1.3.3 β-Glucan
The non-starch unbranched mixed linkage (1−>3)(1−>4)-β-D-glucan (referred as
β-glucan hereafter) is a polysaccharide component of the endosperm and/or aleurone cell
walls of plants (distribution is different from plant to plant) belonging to the Poaceae
family (Graminiae) and represents an important source of stored glucose for the
7
developing seedling (Wood and Fulcher 1978; Planas 2000; Autio et al 2001; Wang et al
2004; Ajthkumar et al 2005).
About 70 % of the linkages in this polysaccharide are (1−>4) and every third or
fourth of this sequence is separated by a single (1−>3) linkage (Wood 2002).
Accordingly, the molecule is made of (1−>3)-linked β-cellotriosyl and β-cellotetraosyl
units (Fig. 2.3 and Fig. 2.4) (BeMiller and Huber 2008). The 30% rest of this
polysaccharide have the single (1−>3) linkage after five or more of (1−>4) linkages
(BeMiller and Huber 2008).
β-Glucan is a water soluble polysaccharide which can be used to form viscous
solutions. This fiber has been the subject of many studies because of its rheological
properties which make it a functional food hydrocolloid (Wood et al 1978; Morgan 2000;
Konuklar et al 2004; Sudha et al 2007), and its perceived health benefits (Morgan 2000;
Wood 2010). It has also been studied because of its negative effect on energy metabolism
in poultry when it is introduced in their feed (Bhatty 1986) and the numerous problems it
causes in the brewing industry including inefficient filtration, formation of gels and hazes
and leaving residues in beer (Bielecki and Galas 1991). The functionality and nutritional
benefits of this polysaccharide are mainly dependent in its concentration and molecular
weight in the end product and these will be discussed later on.
2.2 β-Glucan as a Dietary Fiber
2.2.1 β-Glucan Sources
This polysaccharide can be found in significant amounts in oats and barley,
ranging from 2 to 7 % on a dry weight basis (dwb) (Miller et al 1993; Wood 1994a;
8
Tiwari and Cummins 2009) and in comparatively smaller amounts in other cereals
including corn (0.1 to 3.8 % dwb), rice (0.04 to 0.1 % dwb) (Wood 1994a), rye (1.3–3.11
% dwb) (Ragaee et al 2001), wheat (0.5 to 2 % dwb) (Wood 1994a; Havrlentová and
Kraic 2006; Tiwari and Cummins 2009) and sorghum (0.2 to 0.5 % dwb) (Tiwari and
Cummins 2009). The large variation in β-glucan content in grains is due to the cultivar
diversity rather than environmental or agronomic factors (Wood 1994a).
β-Glucan from different sources can be identified by the ratio of 3-O-βcellotetraosyl-D-glucose and 3-O-β-cellotriosyl-D-glucose, which are the major end
products of β-glucan after hydrolysis by lichenase, which has β-glucanase activity (Wood
2002). These ratios are known to be around 2:1 for oats, 3:1 for barley and rye and 4:1 for
wheat. Hydrolysis of oats and barleys β-glucan with enzymes having β-glucanase activity
can result in other products such as soluble oligosaccharides (up to DP 9 or 10) and
insoluble oligosaccharides (Wood 2002). Another difference between different sources of
β-glucan is the level of difficulty in extracting this fibre from cell walls of cereal sources
(Wood 2010). Oat and barley β-glucan can be readily extracted in high yield, purity and
MW when using hot water while β-glucan from whole meal rye required 1 N NaOH
(Ragaee et al 2008). Some of the β-glucan in rye is difficult to solubilise because it is
entrapped in an arabinoxylan matrix.
2.2.2 Extraction of β-Glucan
β-Glucan can be extracted from cereal seeds or products in a number of ways such
as using NaOH, Na2CO3 or even extraction using hot water with thermostable α-amylase
added. Rimsten et al (2003) reported differences in molecular weight, molecular weight
9
distribution and yield based on different extraction methods applied to different β-glucan
sources and products such as barley, barley malt, rye, wheat, oat, bread, crisp bread,
pancake batter, etc. The yield varied between 19 to 96 % for NaOH method, 7 to 52% for
hot water extraction with an exception for pancake batter, which was 75% and
significantly lower for oats and finally a range from 14 to 98% using Na2CO3. In
addition, Rimsten et al (2003) reported that there isn’t that much of a difference between
the molecular weight distribution and average molecular weight of different extraction
methods. Some studies using a similar extract procedure as Rimsten et al (2003) have
obtained different MWs for β-glucan, likely because either the concentration of extraction
solvent was different or treatment of flours to inactive the enzymes haven’t been the
same. As an example, Beer et al (1997b) used 25 times less NaOH for extracting βglucan than Rimsten et al (2003) used and as a result Rimsten et al (2003) reported lower
average molecular weights. This result can be related to degradation of β-glucan due to
high concentration of NaOH or extracting the β-glucans which have lower MW.
A method simulating human digestion has also been used to extract β-glucan from
cereal products (Beer et al 1997a). The details of this extraction are shown in Figure 2.5.
2.2.2.1 Determination of β-Glucan Concentration
There are several reliable methods for determining the content of β-glucan
(Rimesten et al 2003; Tosh et al 2008; Verardo et al 2011). The easiest to apply is an
enzymatic method, which is commercially available as a kit from Megazyme
International (Bray, Ireland). First, the β-glucan is hydrolyzed to glucose enzymatically
10
and then the quantity of glucose released is measured using glucose oxidase/peroxidase
reagents. Figure 2.6 shows the principle of this assay in more details.
2.2.2.2 β-Glucan Molecular Weight
The weight average molecular weight (Mw) of β-glucan can be determined using
high–performance size-exclusion chromatography (HPSEC) with post column calcofluor
addition. In this method β-glucan, is first separated by size exclusion chromatography
(SEC) and then detected fluorometrically with Calcofluor. Calcofluor binds selectively to
β-glucan which allows analysis of unpurified and crude extracts. The presence of βglucan increases the fluorescent intensity of calcoflour and this is used to detect peaks
(Wood et al 1991b). Purified β-glucans with a known narrow molecular weight can be
used to construct a calibration curve for β-glucan by plotting retention time versus log
peak molecular weight (Mp). The number of standards used should be enough to cover
the whole range of molecular weights contained in the sample, but unfortunately there are
not many standards available for research at this point.
2.2.3 β-Glucan Health Benefits
There have not been any long term observation or clinical studies to directly
relate consuming β-glucan with a reduced chance of any disease (Wood 2010) or disease
end point (Wood 2007), but many studies have shown that consuming this fibre will
result in health benefits. De Groot et al (1963) was the first to report that consuming
sufficient amounts of rolled oats for four weeks could lower the cholesterol content of the
blood serum significantly in rats. Since then many clinical trials and studies have been
conducted on humans. Studies have related consuming products containing β-glucan to
11
moderating glycaemic response to starchy foods (Tappy et al 1996; Regand et al 2009),
lowering serum lipid levels, especially the low-density lipoproteins (LDL) (Tosh et al
2010; Wolever et al 2010), producing short-chain fatty acids (mainly butyric acid) in
large intestines (Morgan 2000) and stimulating beneficial gut microflora (Chaplin 1998;
Brennan and Samyue 2004). Beck et al (2009) have reported that β-glucan consumption
helps satiety feeling which can be assumed as a result of cholecystokinin release, key
hormone in gastrointestinal system which is responsible in facilitating and simulating
digestion, and this can help the obesity problem. Also, Murphy et al (2004) reported that
soluble β-glucan has immune-potentiation properties but there has been no connection of
this finding to health benefits. Meanwhile there have been some studies which don’t
support some of the health benefits regarding β-glucan which has been mentioned. Chen
et al (2006) reported that in a randomized clinical trial including 110 healthy men and
women aged 30-65, consuming soluble dietary fiber including β-glucan didn’t reduced
total and LDL-cholesterol. In addition, Lovegrove et al (2000) reported that 3 g/d of βglucan didn’t reduce the total cholesterol or LDL cholesterol of 62 healthy, middle aged
men and women. A possible explanation for these inconsistent results could be because
of differences in solubility and molecular weight of the β-glucan used in the different
studies, which can result in different gut viscosities.
The numerous clinical studies that evaluated the effectiveness of consuming
products rich in β-glucan on reducing serum cholesterol levels, and attenuating
postpandrial blood glucose and insulin levels led to the allowance of health claims for oat
foods containing β-glucan. Foods containing at least 0.75 g of oat-derived β-glucan for
12
each serving portion are allowed to have a health claim and this regulation has been
expanded to include barley as well (FDA 1997-2005; EFSA 2010; Health Canada 2010).
The mechanism by which fibers lower blood cholesterol is unclear and no single
mechanism have been accepted generally by all researches, but there have been some
suggestions. One possible mechanism is that soluble fibers bind and excrete cholesterol
or/and bile acids during the intralumina formation of micelles in small intestine which is
followed by up-regulating of LDL receptors by liver cells using the cholesterol content of
the body (Brown et al 1999). Other suggestion is that dietary fibers increase the viscosity
resulting in reduction of motility in intestine (Schneeman et al 1985). Also there have
been some suggestions in relationships between production of short chain fatty acid such
as acetate, butyrate and propionate as a result of fermentation in large intestine and LDL
reduction (Nishina et al 1990).
In general, and as reported by Wood (2010), the potential health benefits can be
attributed to the ability of β-glucan to enhance the viscosity of gut contents and as a result
change the absorption rate of fat, cholesterol and bile acids. Since gut viscosity is a
significant factor for physiological efficacy, it is important to understand that
concentration, extractability (an alternative term for solubility to describe properties of βglucan), MW and structure of β-glucan in a food can affect its ability to generate a
desirable viscosity (Wood 2010). Clinical studies have shown that reducing the MW of βglucan from 1,930,000 to 251,000 g/mol resulted in a drop in viscosity from 2900 to 131
mPas (at 30 s -1) (Tosh et al 2010). Furthermore, Bioklund et al (2005) reported that
consuming fortified drinks with 5 g of β-glucan with a MW of 70,000 from oat bran
lowered total cholesterol, postprandial concentration of glucose and also insulin, while
13
the same concentration of drinks with barley β-glucan with a MW of 40,000 was without
significant health effects. This was related to the comparatively lower MW of the barley
β-glucan and as a result, lower viscosity. Also Kim and White (2011) reported that
muffins with high-MW β-glucan bound more bile acid than did those with low- and
medium-MW β-glucan.
There are a number of factors that can affect the MW of β-glucan in food
products, including genotype of the raw cereal, environmental conditions, agronomic
practices, processing, cooking, storage, and combinations of these factors (Tiwari et al
2009).
2.3 β-Glucanase Activity
In this study, β-glucanase activity is a term for any enzymatic processes against βglucan which could result in cleaving it. As an example, since cellulases (endo-(1−>4)-βglucanase, EC 3.2.1.4) are able to cleave the internal β-(1−>4)-linkages in β-glucan, βglucan is therefore susceptible to attack by cellulases (Tosh et al 2004; Grishutin et al
2006), and they are considered to be a β-glucanase. Moreover, Lichenase, consider to be
a β-glucanase enzyme too, since its capable of breaking β-(1−>4)-linkages when there are
after -(1−>3)-linkages and therefore this enzyme does not have any breakage activity
against other polysaccharide such as cellulose and carboxymethylcellulos (Grishutin et al
2006).
Enzymes with β-glucanase activity are broad. The ones capable of degrading
(1−>3)(1−>4)-β-D-glucan in the Poaceae family of higher plants are (1−>3)(1−>4)-β-Dglucan
endohydrolases,
(1−>4)-β-D-glucan
14
glucohydrolases
and
β-D-glucan
exohydrolases (Hrmova et al 2001). These enzymes along with other enzymes with βglucanase activity can be found in microbes, plants and there have also been some reports
of this group of enzymes in snails and sea urchins (Leubner-Metzger 2005). More
importantly humans do not have them. For enzymes capable of β–glucanase activity,
having an active site which matches the substrate and a highly specific spatial orientation
in the bind is necessary (Hrmova et al 2001).
2.3.1. Microbial β-Glucanase
Enzymes such as Laminarinases (endo-(1−>3)-β-glucanases EC3.2.1.39, and
endo-(1−>3)(1→4)-β-glucanases), exo-(l→3)-β-glucosidase (glucan 1,3-β-glucosidase
EC3.2.1.58), lichenase (1,3-1,4-β-D-glucan 4-glucanohydrolase EC3.2.1.73) and βglucosidases (EC 3.2.1.21) are microbial enzymes that have the ability to cleave βglucosidic bonds in glucans other than cellulose (Bielecki and Galas 1991; Grishutin et al
2006). Lichenase is an enzyme that has been significant because of its β-glucanase
activity. Most of this type are bacterial in origin, with the Bacillus species being the most
well known.
Fungi can also produce enzymes with β-glucanase activity. Orpinomyces sp.
(Chen et al 1997), Cochibolus carbonum (Gorlach et al 1998), and Talaromyces
emersonii, Phaffia rhodozyma (Bang et al 1999) are among the fungi known to have
enzyme with this activity. Additionally, Grishutin et al (2006) reported a lichenase –like
endo -(1−>4)-β-glucanase, which was isolated from Aspergillus japonicas. Interestingly,
this enzyme resembles that of bacterial lichenase, with a minor difference in that this
enzyme has a low activity toward Carboxymethyl cellulose (modified β-(1−>4)-glucan)
15
and it produces different oligosaccharides resulting from β-glucan hydrolysis. As
mentioned, this enzyme behaves, similarly to bacterial lichenase, producing mostly triand tetra-saccharides (Fig. 2.7) but the released oligosaccharides have the β-(1−>3) at the
non reducing end while the ones derived from lichenase hydrolysis have it at the reducing
end. This is because bacterial lichenases split the β-(1−>4) glycosidic linkage exactly
after the β-(1−>3) linkage while the fungal enzyme acts on the β-(1−>4) linkage before
the β-(1−>3) (Fig. 2.8) (Grishutin et al 2006).
2.3.1.1 Properties of Microbial β-Glucanase
Enzymes with β-glucanase activity are produced by different microorganism and
as a result, have different substrate and product specificities. As was mentioned earlier,
some of these enzymes have the ability to hydrolyze (1−>3)-β-glucosidic bonds while
others only cleave (1−>4)-β-glucosidic bonds. The Bacillus species produce an enzyme
capable of splitting both (1−>3)-β- and (1−>4)-β- glycosidic bonds and others, such as
(1−>3)-β-glucanase purified from a culture filtrate of Streptomyces sp. is capable of
degrading lichenan, a polysaccharide consisting of (1−>3)-β- and (1−>4)-β- glycosidic
bond, however it doesn’t have any activity against barley glucan.
Diversity in β-glucanase enzymes is mainly because of differences in number and
location of subsites, affinity for monomer residues and in general differences in the active
sites. Furthermore, these enzymes are known to generally have an optimum temperature
range of 30-60 ºC and a pH range from 3.5-7.0 (Bielecki and Galas 1991).
16
2.3.2 β-Glucanase Activity in Plants
Generally, enzyme systems capable of hydrolyzing β-glucans, which are usually
endo- type enzymes, are involved in a variety of key metabolic events and growth-related
responses including protection against pathogens and diseases (Anguelova-Merhar et al
2001; Liu et al 2009; Aggarwal et al 2011), diverse physiological and development
processes (Branco et al 2011; Rinne et al 2001) and seed germination (Luchsinger et al
1960; Ballance et al 1976; Leubner-Metzger 2003). Studies indicate that environmental
factors as well as secretion of hormones by plants result in inducing β-glucanase enzymes
followed by germination (Leubner-Metzger 2003; O’Brien et al 2010).
2.3.2.1 Properties of Plant β-Glucanase
As was mentioned, enzymes having β-glucanase activity are a family with
numerous isoforms that are different in their MW, isoelectric point, primary structure,
cellular localization and pattern of regulation (Leubner-Metzger 2003). Also it has been
reported that the (1−>3)-β-glucanases of higher plants and algae have a pH optimum
around 5 (Bielecki and Galas 1991).
The critical role of enzymes and hormones in the germination step and dormancy
breakage is well known (Leubner-Metzger et al 1995). One significant enzyme important
for cleaving the germinated grain cell wall completely is (1−>3), (1−>4)-β-D-glucan
endohydrolases (EC 3.2.1.73). This has the same specificity as lichenase, hydrolyzing βglucan as shown below:
↓
↓
↓
G4 G4 G3 G 4 G 4 G3 G4 G4 G4 G4 G3 G 4 G4...
17
Where G indicates a β-D-glucosyl residue and 3 and 4 shows the linkage, (1−>3)
and (1−>4), and the reducing end is to the right. After hydrolysis, there will be (1−>3)
(1−>4)-β-D-tri- and tetra-saccharides as major products and some oligosaccharides with
10 or more glucosyl residues (Fig. 2.9). As indicated in the Figure, there is another
enzyme involved in degrading the β-glucan cell wall called Endo-X, an enzyme for which
there is little and debatable information since it has not been isolated yet. Endo-X is
known to release large (1−>3)(1−>4)-β-D-glucan molecules from cereal cell walls
(Hrmova et al 2001). One hypothesis for explaining the action of this enzyme is that the
enzyme is the (1−>4)-β-D-glucan endohydrolases of the endo-1, 4-β-cellulase group (EC
3.2.1.4). Based on this hypothesis, if the enzyme required a bigger substrate binding site
compared to EC 3.2.1.73, it would result in the cleavage of a longer block of (1−>4)-β-Dglucosyl residues. Only 10% of the total β-D-glucan chain contains long blocks of
(1−>4)-β-D-glucosyl residues. As a result limited hydrolysis will occur and large
(1−>3)(1−>4)-β-D-glucan oligosaccharides will be the final products of the hydrolysis
(Fig. 2.9). As shown in the Figure, the oligosaccharides released by hydrolysis with EC
3.2.1.73 can be cleaved further by different enzymes (Hrmova et al 2001). When a
glycosidic linkage has been hydrolyzed, the anomeric configuration of the product can
have the same configuration as it had in the substrate or it can change. The barley β-Dglucan endo- and exo- hydrolases are group of enzymes where the anomeric
configuration is retained in the product (Hrmova et al 2001).
18
2.3.3 β-Glucanase Activity in Baking Ingredients
β-Glucan breakage observed during processing may be the result of commonly
used additives in the baking industry or bacterial components, such as phenolics, which
are present in the ingredients.
Ascorbic acid, commonly added as a flour enhancer is known to depolymerise βglucan. Kivela et al (2009) investigated the effect of 10 mM ascorbic acid and 10 mM
dehyroascorbic acid in pure 0.6% barley β-glucan solutions in presence of ionic iron and
found that β-glucan viscosity decreased.
A study by Moriartey et al (2010) investigated the effect of yeast, premixing and
gluten addition on white bread dough fortified with barley β-glucan at levels similar to
what would be used in industry. When extracted under physiological conditions, the βglucan viscosity was not affected. Since the viscosity of β-glucan is proportional to
concentration and MW (Wood et al 2000) and the concentration of β-glucan in the dough
remained constant, it can be concluded that the addition of yeast, premixing and gluten
did not have an effect on β-glucan. This finding is in contrast with those of Cleary et al
(2007) who suggested that enzymatic hydrolysis of β-glucan in white bread fortified with
β-glucan could be due to enzymes present in added yeast.
Courtin et al (2001) reported that water un-extractable arabinoxylans have a
negative effect on bread characteristics. Endo-(1−>4)-β-xylanase (EC 3.2.1.8) can be
added as an ingredient in bread making to hydrolyze (1−>4)-β-linkages of the
arabinoxylan backbone. As a result, water solubilised arabinoxylan will be released
which has positive effect on bread making. The addition of this enzyme to bread made
from wheat flour and hull less barley has showed no negative effect on either
19
extractability or MW of β-glucan but improved bread loaf volume (Aman et al 2004;
Andersson et al 2004; Trogh et al 2004).
2.3.4 Food Processing and Depolymerization of β-Glucan
Interest in studying β-glucan fortified foods ranging from muffins (Tosh et al
2008) to beverages (Temelli et al 2004) has risen recently as producers aim to formulate
products that promise cholesterol reduction and glycaemic regulation for consumers.
Health benefits may be mainly dependent on β-glucan viscosity (Wolever et al 2010),
which can be negatively affected by processing techniques and storage condition (Tosh et
al 2008; Lan-Pidhainy et al 2007; Tosh et al 2010). In some studies consuming β-glucan
fortified foods did not result in any significant health benefits but low β-glucan solubility
and depolymerization of the β-glucan as a consequence of processing conditions or
storage treatments, may compromise its physiological effectiveness (Lovegrove et al
2000; Chen et al 2006). However, there are a few studies that showed cholesterol
lowering even with a low MW β-glucan. Naumann et al (2006) showed fruit drinks
fortified with β-glucan (80 KDa as mean MW) could lower serum concentration of total
and LDL cholesterol.
Researchers studying β-glucan in bread (Andersson et al 2004; Aman et al 2004;
Trogh et al 2004; Cleary et al 2007; Flander et al 2007; Andersson et al 2008) have
reported that increasing the fermentation time results in greater decreases in β-glucan
viscosity. Aman et al (2004) studied products such as porridge made of rolled oats,
breakfast cereal products, an extruded product fortified with β-glucan and found no
reduction in the average molecular weight of β-glucan while products such as crisp bread
20
and a yogurt-like product showed a moderate reduction in β-glucan MW. Andersson et al
(2008) studied the depolymerization of β-glucan in rye crisp bread and its correlation
with the rye flour falling number. They were able to show that β-glucan fortified rye
breads from rye flour with the lowest falling number (i.e. highest enzyme activity) will
have the lowest β-glucan molecular weight compared to rye breads with the same
fermentation time. Beck et al (2009) reported that oat β-glucan used for fortified
breakfast cereals was stable through the extrusion process and it was able to maintain its
characteristics (such as high MW, solubility and viscosity) for attributed metabolic
effects. Based on these findings they were able to state that “extrusion is an acceptable
downstream processing for β-glucan products” (Beck et al 2009). Furthermore, Tosh et al
(2010) confirmed Beck et al (2009) statement that extruding is likely to increase the
expected bioactivity from β-glucan fortified products. Tosh et al (2010) reported that
extruding will not change or reduce the MW of β-glucan in fortified cereals, whereas it
can increase its solubility up to 28% which may result in a higher viscosity and likely to a
better bioactivity. This finding is despite widespread acceptance that processing will have
a negative effect on final viscosity of β-glucan fortified cereals.
Tosh et al (2008) studied muffins fortified with high molecular weight β-glucan,
and found that peak molecular weight of added β-glucan wasn’t significantly reduced in
the final product as compared to the original oat brans. This could be due to the fact that
muffins do not have any fermentation or proofing time so there is less time for enzyme
present in wheat flour, to act on β-glucans under conditions favorable for enzyme activity
(Tosh et al 2008). This result is in contrast with what Beer et al (1997a) reported. They
were able to show a 50 % reduction in the peak MW of β-glucan in baked muffins using
21
different muffins recipes with different ingredients (Beer et al 1997a). The two
contrasting results could be due to differences in the particle size, mixing time or in the
muffin preparation procedure.
Frozen storage of muffins fortified with β-glucan can reduces β-glucan solubility
up to 50%, possibly as a result of molecular organization and crystallinity (Beer et al
1997a). However, in the same study they reported that storage did not have any effect on
peak MW of β-glucan. Furthermore, Aman et al (2004) showed that boiling porridge and
frying pancakes did not have any effect on the β-glucan MW. Andersson et al (2008)
showed that the molecular weight of β-glucan is almost unchanged during oven baking.
In addition, Andersson et al (2004) showed that increasing the mixing time in hull-less
barley bread will result in higher depolymerization of β-glucan.
2.4 Effect of β-Glucan on Technological Properties of Cereal Products
There have been controversial ideas and suggestions on the effect of β-glucan on
cereal product characteristics. Wang et al (1998) reported that β-glucan fractions in wheat
bread improve the crumb structure while Gill et al (2002) had a completely different
suggestion, and found that barley β-glucan reduces loaf volume. Furthermore, Izydorczyk
et al (2008) reported that additional of 20% barley fiber-rich fractions to wheat flour for
making flat breads resulted in 10-18% more water absorption and weaker doughs, but the
appearance, diameter, layer separation, crumb and aroma of the enriched bread was
comparable to the control flat bread.
In the case of other cereal products Verardo et al (2011) reported on spaghetti
enriched in β-glucan using barley coarse fraction, obtained by air classification, which
22
was able to reach the FDA requirements for health benefits. They reported overall a good
firmness, bulkiness and stickiness values for their functional spaghetti. Frost et al (2011)
reported on chocolate chip cookies fortified with different percentage of barley flour as
the source of β-glucan. They reported based on consumer data that chocolate chip cookies
which were substituted with up to 50% with barley flour (0.8 g β-glucan/serving) were
still comparable with commercial cookies regarding physical characteristics and liking.
However, Tosh et al (2008) reported that oat bran muffins containing either 4 or 8 g of βglucan/serving, had a denser texture compared to whole wheat muffins without β-glucan.
This can be related to the water binding capacity of β-glucan present in the oat bran
muffins and because of the hydrocolloid nature of β-glucan, the bound water resists
evaporation during baking (Tosh et al 2008).
23
Fig. 2.1: Schematic representation of Cellulose (Reproduced from: BeMiller and Huber
2008).
24
Fig. 2.2: Schematic representation of Arabinoxylan
25
Fig. 2.3: Representative structure of β-glucan, where “n” usually is either one or two but infrequently can be larger (Reproduced from: BeMiller and Huber 2008).
26
Fig. 2.4: Schematic representation of β-Glucan (Reproduced from: Morgan 2000).
27
5 g Sample
100 mL Sodium Phosphate Buffer pH 6.9
15 min at 37ºC, slow stirring
add 25o µL α-Amylase (5
mg/mL CaCl2)
15 min at 37ºC, slow stirring
Adjust pH to 2.0 ± 0.1 with HCL 6M/1M
add 625 µL pepsin (0.5
mg/mL in 0.9% NaCl)
30 min at 37ºC, stirring
Adjust pH to 6.9 ± 0.1 with NaOH 3M
90 min at 37ºC, stirring
add 1.25 mL pancreatin
(0.5 mg/mL in Sodium
Phosphate Buffer
Centrifuge, 10 min at 7,000 × g
Physiological Extract
Fig. 2.5: Procedures for physiological extract of β-glucan (Adopted from Beer et al 1997a).
28
Fig. 2.6: Principle of the mix linkage assay for determining β-glucan content using Megazyme International, Bray, Ireland Kit.
29
A: β-Glc-(1−>4)-β-Glc-(1−>3)-β-Glc
B: β-Glc-(1−>4)-β-Glc-(1−>4)-β-Glc(1−>3)-β-Glc
Fig. 2.7: A represents trisaccharide and B represent tetrasaccharide which are both result of
lichenase and fungus enzyme on β-glucan.
30
Fig. 2.8: Schematic representation of differences in action of B.subtillis lichenase and
A.japonicus enzyme on β-glucan (Reproduced from: Grishutin et al 2006).
*Non-reducing ends are located on the left polymer molecules. Black dots donates
the β-D-Glcp residue; (–) β-(1−>4)-linkage; (\) β-(1−>3)-linkage.
31
Fig. 2.9: Enzymatic hydrolysis of cell wall (1→3),(1→4)-β-D-glucans (Reproduced from:
Hrmova et al 2001).
*G appoints a β-D-glucosyl residue; 3 are (1→3) linkages, 4 are (1→4) linkages,
and red notes the reducing end.
32
3. CHAPTER THREE
DETECTION, LOCALIZATION AND VARIABILITY OF ENDOGENOUS ΒGLUCANASE IN WHEAT KERNELS
3.1 Abstract
Clinical studies with isolates of β-glucan have shown that the health benefits are
regulated not only by the polysaccharide concentration but also by its molecular weight
and concentration in solution, since these health benefits are controlled, inter alia, by
viscosity in the gut. The degradation of β-glucan in baked product is likely caused by
baking ingredients, processes or by endogenous enzymes in wheat flour. The objectives
of the present study were to quantify β-glucanase in wheat kernels and to determine
factors that influence the levels of this enzyme. A modified protocol to quantify βglucanase was developed and then confirmed by using high-performance size-exclusion
chromatography (HPSEC) with calcofluor detection. Using this protocol it was shown
that the concentration of β-glucanase activity was the highest in the bran fraction of the
kernel in non-germinated wheats while it was distributed in the entire kernel following
germination. Furthermore, investigation on different wheat cultivars planted in
same/different locations showed that genotype, environment and agronomic practice all
can have an effect on β-glucanase activity level in wheat kernels.
Key words: wheat flour; molecular weight of β-glucan, β-glucanase
33
3.2 Introduction
Wheat kernels can be divided into three distinct morphological parts: endosperm,
bran layers, also called peripheral layers which are composed of aleurone layer, nucellus
tegument, testa and pericarp layer, and finally germ which include the embryo and
scultellum (Evers and Bechtel 1988; Dobraszczyk 2001; Tasleem-Tahir et al 2011). The
location of enzymes within the kernels is not uniform. Enzymes are predominantly
located in the outer aleurone and bran layers of the kernel, and in the germ (Poutanen
1997). For instance, alpha-amylase is located mainly in the pericarp with small quantities
present in the aleurone layer and the seed coat. Proteases are concentrated in the
endosperm, germ and aleurone layer. The scutellum and embryo are rich in lipoxygenase,
whereas, polyphenol oxidase and peroxidase are predominant in bran layers (Rani et al
2001).
Enzymes with β-glucanases activity, which hydrolyze β-glucans containing mixed
β-1, 3 and β-1, 4 glycoside linkages, occur ubiquitously in plants, fungi, animals, and
bacteria (Addington et al 2010). These enzymes in plants have been implicated in a
variety of key metabolic events and growth-related responses in plants including as a
protective tool against invading pathogens (Liu et al 2009). Furthermore, studies have
also proposed that β-glucanases have an important role in the physiological processes in
uninfected plants through their role in pollen maturity, fertilization, cell division and
finally mobilization of nutrients stored in endosperm (Leubner-Metzger et al 1995). The
importance of this enzyme is even more critical in the context of β-glucan and its
importance as nutrient in human foods (Aman et al 2004).
34
β-Glucan is found in cell walls of the higher plant family Poaceae, and it is
mainly rich in the endosperm cell walls (Planas 2000; Wang et al 2004). The highest
concentrations of this polysaccharide are found in oats and barley at 2 to 7 % of their dry
weight (dwb) (Miller et al 1993; Wood 1994a; Tiwari and Cummins 2009) and in smaller
amounts in corn (0.1 to 3.8 % dwb) (Wood 1994a), rice (0.04 to 0.1 % dwb) (Wood
1994a), rye (1.3–3.11% % dwb) (Ragaee et al 2008), wheat (0.5 to 2 % dwb) (Wood
1994a; Havrlentová and Kraic 2006; Tiwari and Cummins 2009) and sorghum (0.2 to 0.5
% dwb) (Tiwari and Cummins 2009). The large variations in the β-glucan content in
grains are due to cultivar diversity rather than environmental or agronomic factors (Wood
1994a). Clinical trials where participants consumed oat β-glucan have shown positive
health benefits such as moderating glycaemic response to starchy foods (Tappy et al
1996; Regand et al 2009), lowering serum lipid levels (Wolever et al 2010), producing
short-chain fatty acids (mainly butyric acid) in large intestines (Morgan 2000), and
stimulating beneficial gut microflora (Chaplin 1998; Brennan and Samyue 2004). Health
claims have been permitted for foods containing at least 0.75 g of oat-derived β-glucan
for each serving portion and has been expanded since then to include barley (FDA 1997,
2005; EFSA 2010; Health Canada 2010).
High viscosity and solubility of β-glucan are thought to be necessary for their
physiological benefits (Anderson et al 1990; Ajithkumar et al 2005; Wood 2007). Studies
show that the potential physiological benefits of β-glucan are due to its ability to increase
the viscosity of intestinal contents (Tosh et al 2008; Wolever et al 2010) and thereby
modify the rates of absorption of nutrients (Wood 2010). In addition, it has been proven
that viscosity of β-glucan is proportional to concentration and molecular weight (MW)
35
(Wood et al 2000; Wood 2010). Kim and White (2011) reported that muffins with highMW β-glucan bound more bile acid than did those with low- and medium-MW β-glucan,
but there were no differences in short chain fatty acid production among muffins with
different MW. However, the MW of β-glucan decreases during processing (Knuckles and
Chiu 1999; Trogh et al 2004). There are several hypotheses concerning the cause of βglucan depolymerization. One hypothesis is the possibility that the microorganisms
present on cereals have enzymes that possess β-glucanase activity, and as a result
depolymerise β-glucan. From the wide variety of microorganisms that are present on the
outer or inner layers of cereals, only few have hydrolyzing enzymes such as Bacillus
subtilis and B. pamilus (Bielecki and Galas 1991). The enzyme lichenase, specifically
cleaves the (1→4)-β-linkage immediately following a (1→3)-β-linkage which produce
series of lower MW oligomers (Wood et al 1994b). Because of the contiguous β-(1→4)
linkages, β-glucan is also susceptible to attack by cellulases (Tosh et al 2004). Another
hypothesis is the possibility of additives that are commonly used in the baking industry;
as well as the cereal bioactive components such as phenolics that might have some
hydrolyzing activity. Ascorbic acid, which can be categorized as an additive and is used
as flour enhancer, has been shown to have some effects on β-glucan. Kivela et al (2009)
investigated the effect of 10 mM ascorbic acid and 10 mM dehyroascorbic acid in pure
0.6 % barley β-glucan solution in presence of ionic iron and the result was a drop in βglucan viscosity. Finally, the last suggestion for degradation of β-glucan MW in cerealbased products is the possibility of presence of endogenous enzyme (Andersson et al
2004). Endogenous hydrolyzing enzymes have been reported in wheat, oat and barley
(Boskov-Hansen et al 2002). The objective of the present study was to develop a protocol
36
to detect very low levels of β-glucanase activity in wheat flours. Using this protocol,
further investigations were conducted to evaluate the effect of different factors, such as
genotype, environment and agronomic practice, on the location of endogenous βglucanase activity in kernels and to determine levels in selected wheat varieties and lines.
3.3 Materials and Methods
Twenty Four wheat varieties or lines (6 soft white winter [SWW], 7 soft red winter
[SRW], 7 hard red spring [HRS], 3 hard red winter [HRW] and one hard white winter
[HWW]) gown in different locations in Ontario (Narin, Watford, Auburn, St.Marys, and
Elora) were kindly provided by C&M Seeds (Palmerston, ON), Dow AgroScience
(Narin, ON) and Dr. Duane Falk (University of Guelph, ON).
3.3.1 Sample Preparation
Whole grain flours were obtained by using a Cyclone Sample Mill (UDY Corp.,
Fort Collins, CO) equipped with a 1.0 mm screen. Milling fractions (refined flour and
bran) were obtained using Quadrumat Jr. flour mill (Brabender GmBh Inc., Duisburg,
Germany) equipped with a 60 mesh (U.S.) reel sifter, yielding bran, shorts, and flour
fractions. All samples were stored at -20 ºC until analysis.
Seeds were germinated in the dark by wrapping them in a wet cheese cloth for 24,
48 or 72 hr with sufficient double distilled water, added periodically, to keep the seeds
moist. After germination, the seeds were cut in two with one fraction containing the germ
and the other containing most of the endosperm. Both samples were dried over night in
an oven at 35 ºC. Dried samples were treated in the same way as other samples as
mentioned above.
37
Azo-barley β-glucan (Remazolbrilliant Blue R labeled 1 % w/v barley β-glucan),
extracting buffer (800 mM sodium acetate plus 800 mM sodium phosphate) and
precipitant solution (sodium acetate, zinc acetate and methoxyethanol, pH=5) were either
included in the kit (Malt and Bacterial β-glucanase kit from Megazyme International,
Bray, Ireland) or prepared according to the procedure explained in the kit.
3.3.2 Optimizing Extraction and Incubation Conditions
The Malt and Bacterial β-glucanase kit from Megazyme (Megazyme
International) was adapted for use on samples with very low β-glucanase activity. Azo
Barley β-glucan (Remazolbrilliant Blue R labeled 1 % w/v barley β-glucan) was used as
the substrate.
Preliminary experiments using whole grain wheat flours were employed to
investigate the ratio of sample weight to extraction buffer (800 mM sodium acetate plus
800 mM sodium phosphate), extraction time, and incubation time to increase the
sensitivity of the Megazyme procedure and detect a very low β-glucanase activity in the
wheat samples. Different sample weights (g) to buffer (mL) ratios (1:1, 1:2, 1:3, 1:4, and
1:8) and different extraction times (15 min and 1, 2 or 3 hr) were also investigated.
Different incubation times (10 min and 1, 2, 3, 6, or 12 hr) between the substrate and the
sample extract were also examined.
3.3.3 Determination of β-Glucanase Activity
Based on the preliminary trials, a 15 min extraction time with continuous vortex
mixing and 3 hr incubation time between the substrate and sample extract at room
temperature (25 °C) were chosen for determination of β-glucanase activity in selected
38
wheat varieties and lines. Ratio of sample to extraction buffer of 1:4 w/v for refined flour
and whole grain flour or 1:8 w/v for bran was selected. The ground samples (1 g of flour
or whole meal or 0.5 g of bran) were mixed with extraction buffer at a ratio of 1:4 w/v
(for refined flour or whole grain flour) or 1:8 w/v (for bran), and the contents were
vortexed thoroughly for 15 min. The mixtures were centrifuged (1000xg/10 min) and the
supernatants were separated and used for β-glucanase activity detection.
β-Glucanase activity was determined by incubating 0.5 mL of sample extract with
0.5 mL Azo-barley β-glucan for 3 hr. The reaction was terminated by adding 3 mL of the
precipitant solution provided in the kit at pH=5). The extract was stirred vigorously and
centrifuged (5 min/1000xg). Absorbance of the supernatant was measured at 590 nm
against double distilled water. A reaction blank was prepared with each set.
3.3.4 Calibration Curve
The calibration curve was prepared using different concentrations of malt βglucanase and Azo-barley β-glucan as the substrate. Stock solutions of the malt βglucanase were prepared to contain from 6 × 10-4 to 4.4 × 10-3 units per mL. 0.5 mL of
Azo-barley β-glucan substrate was incubated with 0.5 mL of the different malt βglucanase solutions for 3 hr in room temperature. The reaction was terminated by adding
precipitant solution followed by centrifugation at 1000xg. Absorbance at 590 nm of the
supernatant was measured and plotted against the malt extracted enzyme with βglucanase activity (Fig. 3.1). β-Glucanase activity of wheat flour was calculated using the
following equation:
39
where A590 = absorbance at 590 nm, 623 = intercept, 4/0.5 = total volume/volume taken,
and DB = dry basis of the sample weight.
3.3.5 Statistics
All analyses were performed at least in duplicate and the mean values are
reported. Analysis of variance was performed using PASW 18 (SPSS Inc., Chicago, IL).
Significant difference (p<0.05) among means were detected using the Tukey’s multiple
range test at a fixed level of α = 0.05.
3.3.6 Validation of the Procedure Using HPLC with Calcoflour Detection
To confirm results obtained by the β-glucanase activity optimized procedure,
enzyme extracts from 3 wheat samples with different enzyme activities (measured by the
optimized procedure) were incubated with a known MW standard β-glucan for 10 min.
Peak molecular weight (Mp) of the standard β-glucan was determined using highperformance size-exclusion chromatography (HPSEC) with calcofluor detection
according to Ragaee et al (2008).
3.4 Results and Discussions
3.4.1 Optimization of the Assay
On the basis of preliminary experiments that examined different extraction ratios,
extraction times, and incubation times to increase sensitivity of the original procedure
(data not shown), it was determined that increasing the extraction time from 15 min
40
(original procedure) to 3 hr did not have a significant effect on the absorbance value,
indicating that most of the enzymes were extracted within the first 15 min. However,
increasing the incubation period resulted in significant differences in β-glucanase
activity, wherein the highest absorbance obtained as after a 3 hr incubation. As a result,
optimum conditions to detect very low β-glucanase activity of wheat whole-grain flours
were found to be 15 min extraction time with continuous vortexing at a ratio of 1:4 or 1:8
sample to extraction buffer (for flour or bran, respectively) followed by a 3 hr incubation
time. Differences in spectrophotometer absorbance readings following the original
Megazyme procedure and the modified procedure for three wheat samples are presented
in Table 3.1. The optimized method was able to better discriminate enzyme activity in the
wheat samples and was more sensitive. Wang et al (2004) also evaluated β-glucanase
activities in winter barley cultivars in southern China. The procedure they used for
investigating the β-glucanase activity was the same malt and bacterial β-glucanase kit
from Megazyme but with no further optimization, resulting in small differences between
cultivars and location. It is likely that the differences between different barley cultivars
would have been better differentiated through this optimized procedure.
The presence of β-glucanase was further validated by incubating an isolated βglucan (prepared as described in Wood et al 1989, MW = 1,200 × 103 g/mol) for 10 min
with extracted enzyme from wheat flours (Fig. 3.2). The flour with low β-glucanase
activity (09-HWW-111, β-glucanase activity = 5.33 U/kg) reduced the MW from 1,200 ×
103 to 79 × 103 g/mol, whereas the flour with higher β-glucanase activity (Warthog, βglucanase activity = 10.53 U/kg) reduced the MW to 42 × 103 g/mol. The correspondence
between the two data sets validated the optimized procedure and confirmed that low
41
levels of β-glucanase activity found in wheat grain can have a significant effect on βglucan MW.
3.4.2 Distribution of β-Glucanase Activity in Wheat Kernel
β-Glucanase activity of wheat fractions (flour and bran) as compared with wholegrain flour for four wheat samples harvested in the field is presented in Figure 3.3.
Results indicated that the bran fraction had the highest β-glucanase activity, whereas the
refined flour had the lowest β-glucanase activity, for all varieties tested. The differences
in β-glucanase activity of bran, endosperm, and whole grain varied among varieties. For
example, β-glucanase activity of bran from Emmit variety was almost 23 times higher
than that of its refined flour and two times higher than that of its whole-grain flour. By
comparison, for Warthog variety, β-glucanase activity of the bran was only 8.5 times
higher than that of its refined flour and approximately 1.1 times higher than that of its
whole-grain flour. Dornez et al (2006) reported that during milling, the microbial
endoxylanases, which are a group of enzymes associated with the outer layers of the
wheat kernel, were concentrated in the shorts and bran fractions, although small amounts
were found in the flour as contaminants. It is likely that this distributioin could be the
case for β-glucanase as well. β-Glucanase activity also increased when the seeds were
germinated in the laboratory (Fig. 3.4). Furthermore, unlike ungerminated seeds, in which
β-glucanase activity was mainly located in bran, it was observed that after germination βglucanase enzymes were distributed evenly in the kernel layers (Fig. 3.5). Similar
observations were also reported by Ballance et al (1976). They reported that in barley
seeds, β-glucanase activity was predominantly located in the scutellum and embryo,
42
whereas after germination, enzyme activity was distributed in the aleurone and
endosperm, and to some extent in the hull, embryo, and scutellum.
3.4.3 Effect of Variety and Location on β-Glucanase Activity
β-Glucanase activity of different varieties and lines of soft and hard wheats each
planted in two different locations is presented in Figure 3.6. The results demonstrated
differences in β-glucanase activity among wheat varieties and lines. For some varieties or
lines, location had significant influence on β-glucanase activity level, whereas other
varieties were not affected by location. Among all soft wheat samples, 09-SWW-738 line
planted in Nairn had the highest β-glucanase activity (12.33 U/kg), and Becher variety
planted in Nairn had the lowest β-glucanase activity (4.66 U/kg). However, location did
not have a significant effect on the β-glucanase activity for Ava and 09-SWW-217
(SWW samples) or 09-SRW- 952 and 09-SRW-616 (SRW samples). β-Glucanase
activity of HRS wheat samples indicated significant differences among the five varieties.
Furthermore, location significantly affected the level of β-glucanase activity; all varieties
planted in Auburn had significantly higher β-glucanase activity compared with those
planted in St. Marys. In general, the results indicated that hard wheat varieties planted in
Auburn had significantly higher β-glucanase activity than other wheat samples tested in
this study. The weather conditions in Auburn throughout the planting season could be one
reason for the high β-glucanase activity. Wang et al (2004) also reported small
differences in β-glucanase activities of eight winter barley cultivars planted in seven
different locations in southern China.
43
Spring wheats are typically harvested in the fall (mid-August through October).
Precipitation in August was considered to be above average (120 mm) for the region
where these varieties or lines were sampled. Furthermore, substantial rain at the end of
September followed by a very wet October in Auburn could explain the higher βglucanase activity in wheat samples from this location in 2009. Pirgozliev et al (2006)
reported that poor weather conditions immediately prior to the week of harvest resulted in
a relatively high endogenous enzyme activity and sprouting in some wheat varieties. The
same author reported that storage conditions after harvesting of grain samples can have
significant effect on activating the enzymes. As a result, it is evident that both weather
conditions before harvesting and storage conditions may have contributed to the high βglucanase activity in the HRS samples planted in Auburn. To test the hypothesis, two
wheat varieties were grown in the greenhouse with no misting after physiological
maturity. The whole-grain flour of the greenhouse-planted samples had very low βglucanase values (1.2 and 1.93 U/kg), much lower than observed in any of the field-tested
samples. Therefore, it is likely that environment conditions trigger β-glucanase activity.
3.4.4 Effect of Planting Season on β-Glucanase Activity
To further investigate the influence of environmental factors, we obtained wheat
varieties that were planted at the same location in two different seasons (spring or fall).
The results (Fig. 3.7) demonstrated that planting season significantly affected the level of
β-glucanase activity. The differences in the β-glucanase activity of the refined flours
varied among samples. For example, Hobson and Superb varieties planted in spring had
almost 1.5 and 1.6 times higher β-glucanase activity, respectively, compared with the
44
same varieties planted in the fall. However, no significant differences in β-glucanase
activity were observed for Norwell variety when planted in spring or fall. MonsalveGonzalez and Pomeranz (1993) reported no consistent differences in enzyme activities of
seven wheat varieties when the same variety was planted in spring or winter. In another
study, Smith and Gooding (1999) reported that summer temperature could have a
significantly positive effect on Hagberg falling number, which is an indication of low
levels of α-amylase.
3.5 Conclusion
This research highlights the potential for endogenous β-glucanase enzymes in wheat
kernels to hydrolyze high-MW β-glucans. An optimized procedure that uses azo-barley
β-glucan as a substrate was developed as an easy and fast procedure to determine very
low (≈1 U/kg) levels of β-glucanase activity in wheat flours. The results also demonstrate
that a number of factors including genotype, environment, and agronomic practice can
affect the level of β-glucanase activity in the wheat flour and, as a consequence, could
affect β-glucan MW when wheat products are fortified with β-glucan or β-glucan-rich
fractions from oats or barley. As the concentration of bran increases in the wheat flour,
the β-glucanase activity increases. Further research is underway to investigate the activity
levels of β-glucanase enzyme during the baking process of whole-grain wheat flour
fortified with oat β-glucan and to develop strategies to minimize its effect on β-glucan
MW.
45
Table 3.1: Differences in spectrophotometer absorbance (590nm) for different
samples using the original Megazyme procedure and the modified procedure.
Sample
Absorbance (based
on Megazyme procedure)
Absorbance
(based on optimized βglucanase procedure)
Ava
0.08 ± 0.01
0.49 ± 0.01
09-SRW-
0.09 ± 0.00
0.82 ± 0.00
Helious
0.18 ± 0.01
1.29 ± 0.03
952
Mean values ± standard deviation.
46
1.6
Absorbance (590 nm)
1.4
y = 0.0623x
R² = 0.9882
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
15
β-Glucanase Activity (Units) × 10-4
20
25
Fig. 3.1: Standard curve relating the activity of malt β-glucanase activity to absorbance at
590 nm upon hydrolysis of azo-barley β-glucan. Incubation time was 3 hr.
47
90
β-Glucan MW × 103 (g/mol)
80
70
60
50
40
30
20
10
0
4
5
6
7
8
9
β-Glucanase Activity (U/Kg)
10
11
Fig. 3.2: Molecular weight of β-glucans following a 10 min incubation of wheat
flours with a β-glucan isolate (MW=1,200 × 103 g/mol) measured through highperformance size-exclusion chromatography with Calcofluor detection. Values are mean
± standard deviation of at least two replicates.
48
25
a
Whole meal
β-Glucanase Activity (U/kg)
20
Bran
b
Flour
15
b, c
c
10
d
d
d
e
5
f
f
f
f
0
Fig. 3.3: Distribution of β-glucanase activity in wheat kernel.
Bars with different letters indicate significance at P < 0.05.Values are mean ±
standard deviation of at least two replicates.
49
45
β-Glucanase Activity (U/kg)
40
35
30
25
20
15
10
5
0
0
24
48
Germination Time (h)
72
Fig. 3.4: Effect of germination time on β-glucanase activity of wheat kernels. Values are
mean ± standard deviation of at least two replicates.
50
45
40
β-Glucanase Activity (U/Kg)
a
Whole Grain
Whole Grain Whitout Germ
35
30
b,c
25
20
a,b
a,b
b,c
c
c
15
10
5
0
0
24
48
Germination Time (h)
72
Fig. 3.5: Distribution of enzymes with β-glucanase activity in wheat kernels after germination.
Bars with different letters indicate significance at P < 0.05. Values are mean ±
standard deviation of at least two replicates.
51
35
Soft White Winter
Hard Red Spring
Soft Red Winter
Nairn
30
β-Glucanase Activity (U/Kg)
Watford
25
Auburn
20
St.Marys
15
*
10
*
*
5
*
0
Fig. 3.6: β-Glucanase activity of wheat varieties/lines grown in different location.
Asterisk (*) indicates the β-glucanase activity values for the two growing
locations were not significantly different at P<0.05. Values are mean ± standard deviation
of at least two replicates.
52
5
a
Spring Planted
Fall Planted
a
β-Glucanase Activity (U/Kg)
4.5
a,b
4
b,c
3.5
c,d
c,d
3
2.5
2
e
e
1.5
1
0.5
0
Norwell
Hobson
Superb
Sable
Fig. 3.7: Effect of planting season on β-glucanase activity of wheat varieties.
Bars with the same letter are not significantly different at P<0.05. Values are
mean ± standard deviation of at least two replicates.
53
4. CHAPTER FOUR
Β-GLUCAN DEGRADATION BY ENDOGENOUS ENZAYMES IN WHEAT FLOUR
DOUGHS WITH DIFFERENT MOISTURE CONTENTS
4.1 Abstract
Health benefits of foods fortified with oat and barley β-glucan are well
documented. Clinical studies have shown that the health benefits of such foods are
regulated not only by the concentration of β-glucan but also by its molecular weight (MW)
in the gut. Our previous study reported different levels of endogenous β-glucanase activity
in different wheat varieties. It was also shown that genotype and environmental factors
impact the activity level of β-glucanase. The objective of the present study was to
investigate the kinetics of endogenous β-glucanase activity on the MW of β-glucan in
fortified dough having different moisture contents. Bread and cookie dough (using refined
flour, composite flour or whole grain flour) fortified with barley flour rich in high MW βglucan were prepared to study the effect of moisture content, incubation time and βglucanase activity in the final β-glucan MW. Evaluating final β-glucan MW of each dough
sample indicated that the moisture content played the most critical role among other
factors in depolymerization of β-glucan within the food system. Meanwhile, there was a
positive correlation between β-glucanase activity available in the system and β-glucan
degradation. Thus to retain high MW β-glucans, which is important for delivering health
benefits, it is important to manage the available water, β-glucans and source of βglucanase activity.
Key words: Dough, molecular weight of β-glucan, β-glucanase, barley
54
4.2 Introduction
(1→3)(1→4)-β-D-Glucans (known as β-glucans) are mainly present in oat and
barley and in comparatively smaller amounts in other cereals such as corn, rice, rye, wheat
and sorghum (Wood 1994a; Ragaee et al 2008; Tiwari and Cummins 2009). In 1963, De
Groot and co-workers reported that regular consumption of oat products can result in
lowering blood cholesterol. This was followed by several studies reporting that
consumption of β-glucan would contribute to several health benefits such as moderating
glycaemic response to starchy foods (Tappy et al 1996; Regand et al 2009), lowering
serum lipid levels (Behall et al 2004; Naumann et al 2006; Wolever et al 2010), producing
short-chain fatty acids (mainly butyric acid) in large intestines (Morgan 2000), and
stimulating beneficial gut microflora (Chaplin 1998; Brennan and Samyue 2004). Human
studies (Naumann et al 2006; Tosh et al 2008; Wolever 2010) indicated positive effects of
consuming β-glucan rich products. Therefore, foods containing at least 0.75g of oatderived β-glucan for each serving portion have been allowed to have a health claim and
have been expanded to include barley as well (FDA 1997, 2005; EFSA 2010; Health
Canada 2010).
The ability of β-glucan to have the expected physiological benefits is mainly
dependent on its ability to increase viscosity of the intestinal contents and thereby
modifying the rates of nutrient absorption (Tosh et al 2008; Wood 2010). The viscosity of
β-glucan solutions is determined by its MW, solubility and concentration (Wood et al
1991b, 2000; Wood 2002), which can be altered by the food matrix, methods of
processing or storage conditions (Knuckles and Chiu 1999; Nauman et al 2006; Regand et
al 2009; Tiwari e al 2009). Moreover, some additives which are commonly used in the
55
baking industry were shown to alter the viscosity of β-glucan. Kivela et al (2009) reported
that 10 mM ascorbic acid and 10 mM dehydro-ascorbic acid in pure 0.6% barley β-glucan
solution in the presence of ionic iron are able to reduce β-glucan viscosity. Tosh et al
(submitted in Journal Cereal Science) also reported that low concentration (0.1-1.0%) of
organic acid salts (calcium propionate, potassium sorbate, and sodium benzoate) reduce
the depolymerization rate of β-glucan (p < 0.0001). Additionally, there is debate about the
presence of β-glucanase activity in yeast. Moriartey et al (2010) reported no effect of yeast
or gluten on white bread dough-fortified with β-glucan, while Cleary et al (2007)
suggested that some enzymes in yeast might be responsible for β-glucan degradation.
Our previous study (Vatandoust et al in press) demonstrated different levels of
endogenous β-glucanase activity in wheat grains. We reported that genotype and
environmental factors have an effect on endogenous β-glucanase activity of wheat. The
enzyme previously has been localized in barley (Balance et al 1976; Leah et al 1991),
maize (Cordero et al 1994) and germinated pea (Petruzzelli et al 1999). The importance of
endogenous β-glucanase enzymes becomes even more critical in the context of the
physiological performance of β-glucan in the gut as influenced by the MW of β-glucan.
Different processing technique with different conditions and different ingredients can
either minimize or maximize the effect of endogenous β-glucanase on the MW of βglucans.
Fortifying cereal products with β-glucan has been a long term interest since cereal
products are staple food for a majority of the world’s population. However, a specific
amount of barley or oat flours containing β-glucan is required to meet the guidelines set by
the FDA to use for the health claim. Several studies on β-glucan fortified cereal products
56
investigating the effect of fermentation period or mixing time on β-glucan MW
(Andersson et al 2004; Aman et al 2004; Trogh et al 2004; Cleary et al 2007; Flander et al
2007; Andersson et al 2008) reported reduction in β-glucan MW in the final product,
which might result in lower viscosity in the gut. As a consequence some researchers
suggested that fortification of bread with β-glucan should be avoided (Cavallero et al
2002).
Increased intake of low Glycaemic Index food has been recommended by the
FAO since 1998. Since then, consumers have become more aware of the relationships
between diets and diseases. β-Glucan fortified cereal products are good examples of
functional foods that have positive health benefits. The objective of this study was to
investigate the kinetics of β-glucan depolymerization during dough processing of two
cereal products (bread and cookie) that have different dough moisture contents.
4.3 Materials and Methods
Hard red spring wheat (Glenn; 15.9% protein) was kindly provided by Dow
AgroSciences (Ontario, Canada). Barley flour with high concentration of β-glucan (24%)
was used as a source of β-glucan with high MW (2.5 × 106 g/mol). All other ingredients
were purchased locally. Thermostable α-amylase and all chemicals for determining the βglucanase activity were purchased from Megazyme (Megazyme International, Ireland).
4.3.1 Sample Preparation
Refined flour was obtained using Brabender Quadmat Junior Mill (Brabender
GmBh, Duisburg, Germany) equipped with a 60 mesh (U.S.) reel sifter. Whole grain
flour was prepared by mixing milled fractions (refined flour and bran), after the bran
57
fraction was milled with a Cyclone Sample Mill (UDY Corp., Fort Collins, CO) equipped
with a 1.0 mm screen. Composite flour was prepared by mixing refined flour and whole
grain flour (50 % w/w). All samples were stored at -20 ºC until analysis.
4.3.2 Determination of β-Glucan
β-Glucan was determined in barley flour using the Mixed linkage Beta-Glucan kit
from Megzayme by the method of McCleary and Glennie-Hlomes (1985).
4.3.3 β-Glucanase Activity
β-Glucanase activity was determined used the optimized procedure described by
Vatandoust et al (in press) with some modification. Briefly, flour was mixed with
extraction buffer (800 mM sodium acetate plus 800 mM sodium phosphate) in a ratio of
1:4 w/v, respectively and was vortexed thoroughly for 15 min. The mixtures were then
centrifuged (1000 g for 10 min) and the supernatants were separated and used for the
further analysis.
β-Glucanase activity was determined by incubating 0.5 mL of sample extract with
0.5 mL Azo-barley β-glucan for 3 h at 30 ºC in water bath. The reaction was terminated
by adding 3 mL of the precipitant solution (sodium acetate, zinc acetate and industrial
methylated spirits (95% ethanol and 5 % methanol), pH 5) and stirred vigorously and
centrifuged (5 min at 1000 g). Absorbance of the supernatant was measured at 590 nm
against double distilled water. The β-glucanase activity of samples was expressed as units
per kg wheat flour.
58
4.3.4 Experimental Doughs
Bread and cookie doughs were made on a 5 g scale from whole grain using the
AACC methods 10-10.03 and 10-53.01, respectively (AACC 2011). Whole grain flour,
composite flour (50% patent flour + 50% whole grain flour) or patent flour representing
high, medium or low levels of β-glucanase activity in flour were included in the formula
of each product (Table 4.1 and Table 4.2) For each dough 10% of wheat flour was
replaced with barley flour rich in high molecular weight β-glucan as a source of β-glucan
to provide sufficient amount based on FDA guideline and Canada Health Claim (FDA
1997, 2005; EFSA 2010; Health Canada 2010). Cookie doughs were incubated at room
temperature, while bread doughs were kept at 32 ºC, and dough samples were taken
periodically after 2, 4, 15, 30, 45, 60, 90 min of incubation.
4.3.5 Extraction β-Glucan From the Dough
β-Glucan was extracted from the dough using hot water extraction method
(Rimsten et al 2003) with some modification. Briefly, 1 gram of dough sample was cut to
small pieces and incubated for 90 min in 10 mL boiling deionized water (containing
CaCl2 (0.28 mg/mL of water), 50 µL of thermostable α-amylase (EC 3. 2. 1. 1) with
0.02% sodium azide) with continuous stirring. After cooling samples were centrifuged at
15000 g for 10 min at 5 ºC. The supernatant was kept at room temperature until further
analysis.
4.3.6 Physicochemical Analysis of β-Glucan in Dough
Supernatant was heat treated at 90 ºC for 30 min, diluted 1:10 with deionized
water to reach desirable concentration to be used (0.1 mg/mL or less). Then the weight
59
average molecular weight (Mw) of β-glucan was analyzed using high-performance sizeexclusion chromatography (HPSEC) as described by Wood et al (1991a). The
chromatographic system consisted of a Perkin Elmer ISS-100 autosampler and injector, a
Shimadzu model AD-vt HPLC pump,a Shimadzu RF-10Axl fluorescence detector, a
Waters (Milford, CT) model 510 HPLC pump for postcolumn addition of calcofluor, and
the Viscotek DM 400 data manager. Data integration was performed using TriSEC 3.0
software (Viscotek, Houston, TX). Five β-glucan molecular weight standards (ranging
from 20,000 to 1,300,00 g/mol), prepared in-house (Wang et al 2004) or obtained
commercially (Megazyme) were used to construct a calibration curve for β-glucan by
plotting retention time versus log peak molecular weight (Mp).
4.3.7 Statistics
All analyses were performed in duplicate and the mean values are reported.
Analysis of variance was performed using PASW 18 (SPSS Inc., Chicago, IL).
Significant difference (p<0.05) among means were detected using the Tukey’s multiple
range test at a fixed level of α = 0.05.
4.4 Results and Discussions
4.4.1 Determining of Flours Characteristics
Whole grain flour had the highest β-glucanase activity (112 U/Kg) while the
refined flour had the lowest (12 U/Kg) and the composite flour contained intermediate βglucanase activity (56 U/Kg). Barley flour contained 24 % of high Mw (2.5 × 106) βglucan
60
4.4.2 Effect of Level of β-Glucanase Activity on Degradation of β-Glucan in Dough
Reduction of β-glucan Mw during preparation of bread and cookie with flours
containing different levels of endogenous β-glucanase activity is presented in Fig. 4.1.
The results show a significant difference in β-glucan Mw when using flours with different
β-glucanase activity to prepare the bread doughs. A greater reduction in molecular weight
was observed with higher levels of endogenous β-glucanase activity in the flour during
the 90 min incubation time. Flour exhibiting the lowest β-glucanase activity (12 U/Kg)
resulted reduction to 1.25 × 106 in β-glucan Mw after the 90 min incubation time. The
reduction in β-glucan Mw was greater and β-glucan Mw was reduced to 0.58 × 106 for
bread dough when using the flour with the highest β-glucanase activity (112 U/Kg),
while using flour exhibiting moderate β-glucanase activity (56 U/Kg) resulted in
reduction of β-glucan Mw to 0.89 × 106 after 90 min incubation for bread dough. Similar
observation in the case of bread has been reported. Andersson et al (2004) reported a
negative correlation between β-glucan molecular weight and both dough mixing time and
fermentation time in dough and bread made from composite flour (60% wheat flour and
40% whole meal or sifted barley flour). Moreover, they reported no effect of yeast or
baking on β-glucan molecular weight. Additionally, Andersson et al (2008) reported a
rapid drop in the average molecular weight of β-glucan using rye flours with different
falling numbers when making rye doughs and breads. They reported that rye flour with
the lowest enzyme activity resulted in the least effect on β-glucan. Furthermore, by
drawing a logarithmic trend line based on the available data points for the bread dough, it
can be observed that as the β-glucanase activity of the flour increases, the slope for the
61
equation decreases which results in a lower value for the Y axes that presents the βglucan molecular weight.
In the case of cookie dough, flours with different β-glucanase activity did not
have significant effect on β-glucan Mw during the 90 min incubation (Fig. 4.1). For
instance, a slight reduction was observed in β-glucan Mw after 90 min when using whole
grain flour (having the highest β-glucanase activity of 112 U/Kg) compared to when
using the composite flour (having an intermediate β-glucanase activity i.e 56 U/Kg) or
when using the refined flour (having the lowest β-glucanase activity i.e 12 U/Kg).
4.4.3 Effect of Moisture Content and Incubation Time on Depolymerization of β-Glucan in
the Dough System
Comparisons between the two dough systems (bread dough with 60% moisture
content and cookie dough with 22% moisture content) are presented in Fig. 4.2. The
results demonstrated that moisture content of the system has significant influence on the
Mw of β-glucan when using flour exhibiting different levels of β-glucanase activity (112,
56 and 12 U/Kg). Significant reduction in β-glucan Mw was observed in the case of bread
dough that contained more than three times the available moisture content than cookie
dough using different flours having different β-glucanase activity. This demonstrated that
the available moisture in dough system plays an important role as a mobile phase for
endogenous β-glucanase enzymes to be more accessible to β-glucan. A clinical study by
Casiraghi et al (2006) using 10 healthy volunteers consumed crackers or cookies made
from barley or whole wheat flour, reported better Glycaemic Index when volunteers
consumed cookies compared to when they consumed crackers.
62
The final β-glucan MW in the cookie dough samples were in agreement with
those for muffins by Tosh et al (2008). They reported preserved MW of β-glucan in oat
bran muffins and explained that to be due to short contact time between available water
in the system, flour and β-glucan as a reason that high MW β-glucan was preserved. On
the other hand, another study by Beer et al (1997a) reported a 50 % reduction in the peak
molecular weight of β-glucan in oat bran-fortified muffins. The two contrasting results
could be due to differences in the bran particle size, mixing time, or the muffin
preparation recipes used.
Several Studies (Kerckhoffs et al 2003; Andersson et al 2004; Aman et al 2004;
Trogh et al 2004; Cleary et al 2007; Flander et al 2007; Andersson et al 2008) working on
cereal products (oat bran-based breads, wheat/hull less barley breads and rye crisp bread),
having high moisture content and enough incubation time have reported reduction in βglucan MW in the final products.
Data presented in Fig. 4.2 highlighted that when moisture content of the system is
low, such in cookie dough, the incubation time is not a significant factor. As an example
in the case of cookie using flours exhibiting three different β-glucanase activities, the
reduction in β-glucan Mw after 90 min was not significantly different from that after 15
min, while in the case of bread dough significant differences in β-glucan Mw were
observed between the 15 min and the 90 min incubation time when using any of the
flours.
63
4.5 Conclusion
A combination of moisture content, incubation time and levels of endogenous βglucanase activity of the system, all have significant effect on the MW of β-glucan in the
final product. Among the three mentioned factors, the available moisture content in the
system can be considered the most critical factor. β-Glucan in the low moisture dough
system remains intact and protected from enzymatic degradation due to the absence of
available water. The present study highlights that, depending on the product type,
different commercial strategies can be developed to preserve high MW β-glucan.
Furthermore, novel strategies need to be developed in dough containing high moisture
contents to maintain high MW β-glucan without having negative effect on the quality of
the end product, which would promote the consumption of β-glucan fortified functional
products.
64
Table 4.1: Bread Formulation
Dough containing
12 U/Kg βglucanase activity
Dough containing
56 U/Kg βglucanase activity
Dough containing
112 U/Kg βglucanase activity
Refined
floura (g)
4.48
-
-
Composite
floura (g)
-
4.48
-
Whole
grain floura
(g)
-
-
4.48
Barley flour
b
(g)
0.52
0.52
0.52
Sucrose (g)
0.3
0.3
0.3
NaCl (g)
0.07
0.07
0.07
Yeast (g)
0.4
0.4
0.4
Water (mL)
3
3
3
Shortening
(g)
0.15
0.15
0.15
a
Refined Flour, Composite flour and Whole grain flour was obtained using Glenn
b
Barley Flour rich ( 24 %) in high (2.5 × 106) MW β-glucan.
wheat.
65
Table 4.2: Cookie Formulation
Refined
floura (g)
Composite
floura (g)
Whole grain
floura (g)
Barley flour
b
(g)
Sucrose (g)
NaCl (g)
Non fat dry
milk (g)
Sodium bi
carbonate
(g)
Dough containing
12 U/Kg βglucanase activity
4.48
Dough containing
56 U/Kg βglucanase activity
-
Dough containing
112 U/Kg βglucanase activity
-
-
4.48
-
-
-
4.48
0.52
0.52
0.52
2.1
0.062
0.05
2.1
0.062
0.05
2.1
0.062
0.05
0 .05
0.05
0.05
Water (mL)
1.1ml
1.1
1.1
Shortening
2
2
2
(g)
Amounium
0.025
0.025
0.025
bi carbonate
(g)
High
0.075
0.075
0.075
fructose
corn syrup
(g)
a
Refined Flour, Composite flour and Whole grain flour was obtained using Glenn
wheat.
b
Barley Flour rich ( 24 %) in high (2.5 × 106) MW β-glucan.
66
3
Refined Flour
Composite Flour
Whole Grain Flour
β-Glucan MW ×106 (g/mol)
2.5
2
Cookie
1.5
Bread
Y=-0.134ln(x)
+ 2.0005
R2 =0.8439
1
Y=-0.174ln(x)
+ 1.7026
R2 =0.9758
0.5
Y=-0.2ln(x)
+ 1.5212
R2 =0.9671
0
0
15
45
90
0
15 45 90
Incubation Time (min)
0
15
45
90
Fig. 4.1: Degradation of β-glucan during preparation of bread dough and cookie dough of
using flours containing different endogenous β-glucanase activity.
67
80
70
60
50
40
30
20
10
0
15 min
20%
60%
45 min
20%
β-Glucanase Activity (U/Kg)
Percentage Reduction of β-Glucan
12 (U/Kg) β-glucanase activity
56 (U/Kg) β-glucanase activity
112 (U/Kg) β-glucanase activity
90 min
60%
20%
60%
Moisture Content
Fig. 4.2: Effect of moisture content and endogenous β-glucanase activity on depolymerization of β-glucan in the systems.
68
5. CHAPTER FIVE
CONCLUSIONS
From an industrial point of view, including barley or oat flours in wheat-based
formula to enrich bread and other products with high MW/viscosity β-glucan will
enhance the health profile of baked products. Several studies have indicated that
bioactivity in the gut is dependent, among other things, on β-glucan MW and food
microstructure. Recent studies revealed that oat and barley β-glucan incorporated into
wheat based products result in lower MW. Several researchers have suggested that there
might be β-glucanase enzymes present in wheat flour, but to-date this has not been
directly demonstrated by identification or isolation of enzymes and determining them.
Since, producing products which can enhance health benefits has become a key priority
of food scientist and industry, identifying the causal factors of β-glucan depolymerization
is important. Therefore, the objectives of this study were to understand the causes for
depolymerization of β-glucans in wheat-based baked products; to characterize the
kinetics and properties of the factors that lead to the depolymerization of β-glucan.
The only procedure available for evaluating β-glucanase activity was the
Megazyme procedure for determining the Malt β-glucanase activity (ranging from 130 to
760 Unit/Kg β-glucanase activities). However, the level of enzyme activity in wheat
kernels is much lower, thus making the megazyme procedure ineffective. Therefore in the
first part of this study we developed and optimized a procedure to detect low levels of βglucanase activity by using the available Megzayme standard procedure (Malt & Bacteria
β-glucanase). The reliability and accuracy of the optimized protocol was confirmed by
69
using an HPSEC-Calcofluor detector to monitor the weight average molecular weight
(MW) of standard β-glucan (MW = 1.2 × 106 g/mol) incubated with flours exhibited
different β-glucanase activity. We then tested the procedure on different varieties of
wheat planted in different location/season. Our results demonstrated that genotype,
environment and agronomic practice have significant impact on the β-glucanase activity
in wheat flours. Furthermore the distribution of β-glucanase in different fractions of the
wheat kernel (bran, flour) was also investigated using this protocol. The result confirmed
that endogenous enzymes with β-glucanase activity are concentrated primarily in the
outer layer of wheat kernels. The effect of germination on the β-glucanase activity was
studied to investigate the effect of poor harvesting conditions on post-harvest glucanase
activity. The results demonstrated that not only germination increases β-glucanase
activity but also it re-distributed the enzyme within the kernel fractions including the
endosperm. This was also determined by growing wheat plants in a green house with no
misting.
The kinetics of hydrolysis of β-glucan by endogenous glucanase was investigated.
In this stud we used three flours exhibiting different levels of β-glucanase activity (low,
intermediate or high) and a source of high MW β-glucan (MW = 2.5 × 106 g/mol). The
results demonstrated that depolymerization of β-glucan occurs whitin the first few
minutes of dough preparation (during the mixing process). Moisture content, incubation
time and levels of endogenous β-glucanase activity of the system, had significant impact
on the final MW of β-glucan in the dough. Among the three mentioned factors, the
available moisture content of the system (e.g bread) had the greatest impact on the βglucan depolymerization. While in cookie dough (low moisture system) the β-glucan
70
MW remained almost unchanged due to the absence of enough mobile phase. The
reduction in β-glucan MW was greater in the bread dough compared to that in the cookie
dough. Thus opportunities exist to develop products or modify procedures to incorporate
β-glucan into baked products and maintain high MW.
Future work of this study could investigate the exact location where the
breakdown occurs on the β-glucan chain at different stages during dough preparation to
characterize the β-glucanase activity. Also, develop strategies to maintain the
functionality of β-glucans in the baked product systems. The results will provide a
scientific basis for the industry to incorporate β-glucan in baked products under
conditions that will maintain or enhance its health benefits.
71
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