Beta-Glucanase Activity and Its Impact on Beta-Glucan Molecular Weight Degradation in Cereal Products Fortified with Beta-Glucan

BetaGlucanase Activity and its Impact on BetaGlucan Molecular Weight Degradation in Cereal Products Fortified with BetaGlucan

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

βGLUCANASE ACTIVITY AND ITS IMPACT ON βGLUCAN MOLECULAR WEIGHT DEGRADATION IN CEREAL PRODUCTS FORTIFIED WITH βGLUCAN Azadeh Vatandoust Advisor: University of Guelph, 2012 Dr. Koushik Seetharaman

Health benefits of high molecular weight (MW) βglucans are well documented.

Therefore, understanding and controlling depolymerization of βglucan in baked products, would increase the effectiveness of βglucan to confer health benefits. In this study we demonstrated that endogenous βglucanase in wheat kernels are responsible for the depolymerization of βglucans. A protocol was developed based on the Megzayme procedure to detect low levels of βglucanase activity in wheat flour. This was confirmed by using HPLCCalcofluor detection to monitor molecular weight changes. The distribution of βglucanase in wheat kernels was also investigated. The effect of genotype, location, planting season and environmental factors on the level of endogenous

βglucanase in selected wheat cultivars was investigated using different wheat varieties planted under different condition and different seasons. Furthermore, kinetics of βglucan depolymerization by endogenous glucanase in two dough systems with different moisture content was investigated. The results demonstrated that enzymes with βglucanase activity are concentrated primarily in the outer layer of wheat kernels. Also genotype, environmental conditions and agronomic practice all had significant effects on the β glucanase activity in wheat flours and poor harvesting conditions can significantly increase βglucanase activity level in wheat. The kinetics results demonstrated that moisture content, incubation time and levels of endogenous βglucanase activity of the system had significant impact on the final MW of βglucan 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 βglucan.

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 βglucan molecular weight; also for her helpful inspiration in βglucan 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.

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 βglucan 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 βglucan 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 AgricultureAgri 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 FarahBakhs 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, selfcentered decisions and pushed me time after time. Knowing that they will always be there to pick up the

pieces has given me the strength and confidence to repeatedly risk getting shattered. I am grateful and fortunate to have their constant support.

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 βGlucan ...... 7

2.2 βGlucan as a Dietary Fiber ...... 8

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2.2.1 βGlucan Sources...... 8

2.2.2 Extraction of βGlucan ...... 9

2.2.2.1 Determination of βGlucan Concentration ...... 10

2.2.2.2 βGlucan Molecular Weight ...... 11

2.2.3 βGlucan Health Benefits ...... 11

2.3 βGlucanase Activity ...... 14

2.3.1. Microbial βGlucanase ...... 15

2.3.1.1 Properties of Microbial βGlucanase ...... 16

2.3.2 βGlucanase Activity in Plants ...... 17

2.3.2.1 Properties of Plant βGlucanase ...... 17

2.3.3 βGlucanase Activity in Baking Ingredients ...... 19

2.3.4 Food Processing and Depolymerization of βGlucan ...... 20

2.4 Effect of βGlucan on Technological Properties of Cereal Products ...... 22

3. Chapter Three Detection, Localization and Variability of Endogenous βGlucanase 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 βGlucanase 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 βGlucanase Activity in Wheat Kernel ...... 42

3.4.3 Effect of Variety and Location on βGlucanase Activity ...... 43

3.4.4 Effect of Planting Season on βGlucanase Activity ...... 44

3.5 Conclusion ...... 45

4. Chapter Four βGlucan 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 βGlucan ...... 58

4.3.3 βGlucanase Activity ...... 58

4.3.4 Experimental Doughs ...... 59

4.3.5 Extraction βGlucan From the Dough ...... 59

4.3.6 Physicochemical Analysis of βGlucan 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 βGlucanase Activity on Degradation of βGlucan in

Dough ...... 61

4.4.3 Effect of Moisture Content and Incubation Time on Depolymerization of

βGlucan in the Dough System ...... 62

4.5 Conclusion ...... 64

5. Chapter Five Conclusions ...... 69

6. References Cited ...... 72

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

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 βglucan, where “n” usually is either one or two but infrequently can be large ...... 26

Fig. 2.4: Schematic representation of βGlucan ...... 27

Fig. 2.5: Procedures for physiological extract of βglucan ...... 28

Fig. 2.6: Principle of the mix linkage assay for determining βglucan 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 βglucan...... 30

Fig. 2.8: Schematic representation of differences in action of B.subtillis lichenase and

A.japonicus enzyme on βglucan ...... 31

Fig. 2.9: Enzymatic hydrolysis of cell wall (1→3),(1→4)βDglucans ...... 32

Fig. 3.1: Standard curve relating the activity of malt βglucanase activity to absorbance at

590 nm upon hydrolysis of azobarley βglucan. Incubation time was 3 hr...... 47

Fig. 3.2: Molecular weight of βglucans following a 10 min incubation of wheat flours with a βglucan isolate (MW=1,200 × 10 3 g/mol) measured through highperformance sizeexclusion chromatography with Calcofluor detection...... 48

Fig. 3.3: Distribution of βglucanase activity in wheat kernel ...... 49 xii

Fig. 3.4: Effect of germination time on βglucanase activity of wheat kernels ...... 50

Fig. 3.5: Distribution of enzymes with βglucanase activity in wheat kernels after germination...... 51

Fig. 3.6: βGlucanase activity of wheat varieties/lines grown in different location...... 52

Fig. 3.7: Effect of planting season on βglucanase activity of wheat varieties ...... 53

Fig. 4.1: Degradation of βglucan during preparation of bread dough and cookie dough of using flours containing different endogenous βglucanase activity...... 67

Fig. 4.2: Effect of moisture content and endogenous βglucanase activity on depolymerization of βglucan in the systems...... 68

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1. CHAPTER ONE

INTRODUCTION

(1 −>3)(1 −>4) βDglucan (known as βglucans) 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 βglucan depend on the grain type (Wood 2010).

For instance, extracting βglucans 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 β glucans 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 βglucan 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 lowdensity lipoproteins (LDL)

(Tosh et al 2010; Wolever et al 2010). Foods containing at least 0.75g of oatderived β glucan for each serving portion are allowed to have a health claim and this regulation has been expanded to include barley as well (FDA 19972005; EFSA 2010; Health Canada

2010).

There have been controversial suggestions about mechanism of how βglucan consumption results in health benefits (Brown et al 1999). In general, most studies suggest that the potential health benefits of βglucan 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 βglucan 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 βglucan 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 βglucan 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 β glucan depolymerization in the cereal based products. The obtained result would assist in developing strategies to maintain βglucan MW and enhance the health benefits of final products.

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 βglucan fractions, by human subjects showed that βglucan can help satiety feeling which this may result in healthy weight.

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

polymers. Cellulose and starch, the most abundant of all the polysaccharides, have a backbone chain where carbon1 has been linked with carbon4 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, βglucan 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 βDglucopyranosyl 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

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)βDxylopyranosyl in which the Arabinose substitutes are linked through O2, O3, or O2, 3 residues (Fig. 2.2). It can be found in both, water extractable and water unextractable 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 βGlucan

The nonstarch unbranched mixed linkage (1 −>3)(1 −>4)βDglucan (referred as

βglucan 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

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 βcellotriosyl and βcellotetraosyl 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).

βGlucan 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 βGlucan as a Dietary Fiber

2.2.1 βGlucan 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 βglucan content in grains is due to the cultivar diversity rather than environmental or agronomic factors (Wood 1994a).

βGlucan from different sources can be identified by the ratio of 3Oβ cellotetraosylDglucose and 3OβcellotriosylDglucose, which are the major end products of βglucan after hydrolysis by lichenase, which has βglucanase 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 βglucan with enzymes having βglucanase 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

βglucan is the level of difficulty in extracting this fibre from cell walls of cereal sources

(Wood 2010). Oat and barley βglucan can be readily extracted in high yield, purity and

MW when using hot water while βglucan from whole meal rye required 1 N NaOH

(Ragaee et al 2008). Some of the βglucan in rye is difficult to solubilise because it is entrapped in an arabinoxylan matrix.

2.2.2 Extraction of βGlucan

βGlucan can be extracted from cereal seeds or products in a number of ways such as using NaOH, Na 2CO 3 or even extraction using hot water with thermostable αamylase added. Rimsten et al (2003) reported differences in molecular weight, molecular weight

distribution and yield based on different extraction methods applied to different βglucan 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 Na 2CO 3. 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 βglucan, 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 β

glucan 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 βglucan due to

high concentration of NaOH or extracting the βglucans which have lower MW.

A method simulating human digestion has also been used to extract βglucan from cereal products (Beer et al 1997a). The details of this extraction are shown in Figure 2.5.

2.2.2.1 Determination of βGlucan Concentration

There are several reliable methods for determining the content of βglucan

(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 βglucan is hydrolyzed to glucose enzymatically

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 βGlucan Molecular Weight

The weight average molecular weight (Mw) of βglucan can be determined using

high–performance sizeexclusion chromatography (HPSEC) with post column calcofluor

addition. In this method βglucan, is first separated by size exclusion chromatography

(SEC) and then detected fluorometrically with Calcofluor. Calcofluor binds selectively to

βglucan which allows analysis of unpurified and crude extracts. The presence of β

glucan increases the fluorescent intensity of calcoflour and this is used to detect peaks

(Wood et al 1991b). Purified βglucans with a known narrow molecular weight can be

used to construct a calibration curve for βglucan 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 βGlucan Health Benefits

There have not been any long term observation or clinical studies to directly

relate consuming βglucan 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 βglucan to

moderating glycaemic response to starchy foods (Tappy et al 1996; Regand et al 2009), lowering serum lipid levels, especially the lowdensity lipoproteins (LDL) (Tosh et al

2010; Wolever et al 2010), producing shortchain 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 βglucan 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 βglucan has immunepotentiation 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 βglucan which has been mentioned. Chen et al (2006) reported that in a randomized clinical trial including 110 healthy men and women aged 3065, consuming soluble dietary fiber including βglucan didn’t reduced total and LDLcholesterol. In addition, Lovegrove et al (2000) reported that 3 g/d of β glucan 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 βglucan 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 βglucan on reducing serum cholesterol levels, and attenuating postpandrial blood glucose and insulin levels led to the allowance of health claims for oat foods containing βglucan. Foods containing at least 0.75 g of oatderived βglucan for

each serving portion are allowed to have a health claim and this regulation has been expanded to include barley as well (FDA 19972005; 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 upregulating 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 βglucan 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 β glucan), MW and structure of βglucan in a food can affect its ability to generate a desirable viscosity (Wood 2010). Clinical studies have shown that reducing the MW of β glucan 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 βglucan 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 βglucan with a MW of 40,000 was without significant health effects. This was related to the comparatively lower MW of the barley

βglucan and as a result, lower viscosity. Also Kim and White (2011) reported that muffins with highMW βglucan bound more bile acid than did those with low and mediumMW βglucan.

There are a number of factors that can affect the MW of βglucan 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 βGlucanase Activity

In this study, βglucanase activity is a term for any enzymatic processes against β glucan which could result in cleaving it. As an example, since cellulases (endo(1 −>4)β glucanase, EC 3.2.1.4) are able to cleave the internal β(1 −>4)linkages in βglucan, β glucan is therefore susceptible to attack by cellulases (Tosh et al 2004; Grishutin et al

2006), and they are considered to be a βglucanase. Moreover, Lichenase, consider to be a βglucanase 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 βglucanase activity are broad. The ones capable of degrading

(1 −>3)(1 −>4)βDglucan in the Poaceae family of higher plants are (1 −>3)(1 −>4)βD glucan endohydrolases, (1 −>4)βDglucan glucohydrolases and βDglucan 14

exohydrolases (Hrmova et al 2001). These enzymes along with other enzymes with β glucanase activity can be found in microbes, plants and there have also been some reports of this group of enzymes in snails and sea urchins (LeubnerMetzger 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 βGlucanase

Enzymes such as Laminarinases (endo(1 −>3)βglucanases EC3.2.1.39, and

endo(1 −>3)(1→4)βglucanases), exo(l→3)βglucosidase (glucan 1,3βglucosidase

EC3.2.1.58), lichenase (1,31,4βDglucan 4glucanohydrolase EC3.2.1.73) and β

glucosidases (EC 3.2.1.21) are microbial enzymes that have the ability to cleave β

glucosidic 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 βglucanase

activity. Most of this type are bacterial in origin, with the Bacillus species being the most well known.

Fungi can also produce enzymes with βglucanase 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)βglucanase, 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)

and it produces different oligosaccharides resulting from βglucan hydrolysis. As mentioned, this enzyme behaves, similarly to bacterial lichenase, producing mostly tri and tetrasaccharides (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 βGlucanase

Enzymes with βglucanase 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)βglucosidic bonds while others only cleave (1−>4)βglucosidic 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)βglucanase 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 βglucanase 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 3060 ºC and a pH range from 3.57.0 (Bielecki and Galas 1991).

2.3.2 βGlucanase Activity in Plants

Generally, enzyme systems capable of hydrolyzing βglucans, which are usually endo type enzymes, are involved in a variety of key metabolic events and growthrelated responses including protection against pathogens and diseases (AnguelovaMerhar 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; LeubnerMetzger 2003). Studies indicate that environmental

factors as well as secretion of hormones by plants result in inducing βglucanase enzymes

followed by germination (LeubnerMetzger 2003; O’Brien et al 2010).

2.3.2.1 Properties of Plant βGlucanase

As was mentioned, enzymes having βglucanase activity are a family with

numerous isoforms that are different in their MW, isoelectric point, primary structure,

cellular localization and pattern of regulation (LeubnerMetzger 2003). Also it has been

reported that the (1−>3)βglucanases 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 (LeubnerMetzger et al 1995). One significant enzyme important

for cleaving the germinated grain cell wall completely is (1 −>3), (1 −>4)βDglucan

endohydrolases (EC 3.2.1.73). This has the same specificity as lichenase, hydrolyzing β

glucan as shown below:

↓ ↓ ↓ G4 G4 G3 G 4 G 4 G3 G4 G4 G4 G4 G3 G 4 G4...

Where G indicates a βDglucosyl 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)βDtri and tetrasaccharides 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 βglucan cell wall called EndoX, an enzyme for which there is little and debatable information since it has not been isolated yet. EndoX is known to release large (1 −>3)(1 −>4)βDglucan 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)βDglucan endohydrolases of the endo1, 4βcellulase 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)βD glucosyl residues. Only 10% of the total βDglucan chain contains long blocks of

(1 −>4)βDglucosyl residues. As a result limited hydrolysis will occur and large

(1 −>3)(1 −>4)βDglucan 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 βD glucan endo and exo hydrolases are group of enzymes where the anomeric configuration is retained in the product (Hrmova et al 2001).

2.3.3 βGlucanase Activity in Baking Ingredients

βGlucan 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 β glucan. Kivela et al (2009) investigated the effect of 10 mM ascorbic acid and 10 mM dehyroascorbic acid in pure 0.6% barley βglucan solutions in presence of ionic iron and found that βglucan 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 βglucan at levels similar to what would be used in industry. When extracted under physiological conditions, the β glucan viscosity was not affected. Since the viscosity of βglucan is proportional to concentration and MW (Wood et al 2000) and the concentration of βglucan in the dough remained constant, it can be concluded that the addition of yeast, premixing and gluten did not have an effect on βglucan. This finding is in contrast with those of Cleary et al

(2007) who suggested that enzymatic hydrolysis of βglucan in white bread fortified with

βglucan could be due to enzymes present in added yeast.

Courtin et al (2001) reported that water unextractable arabinoxylans have a negative effect on bread characteristics. Endo(1 −>4)βxylanase (EC 3.2.1.8) can be added as an ingredient in bread making to hydrolyze (1 −>4)βlinkages 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 βglucan 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 βGlucan

Interest in studying βglucan 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 βglucan viscosity (Wolever et al 2010), which can be negatively affected by processing techniques and storage condition (Tosh et al 2008; LanPidhainy et al 2007; Tosh et al 2010). In some studies consuming βglucan fortified foods did not result in any significant health benefits but low βglucan solubility and depolymerization of the βglucan 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 βglucan. Naumann et al (2006) showed fruit drinks fortified with βglucan (80 KDa as mean MW) could lower serum concentration of total and LDL cholesterol.

Researchers studying βglucan 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 βglucan viscosity. Aman et al (2004) studied products such as porridge made of rolled oats, breakfast cereal products, an extruded product fortified with βglucan and found no reduction in the average molecular weight of βglucan while products such as crisp bread

and a yogurtlike product showed a moderate reduction in βglucan MW. Andersson et al

(2008) studied the depolymerization of βglucan in rye crisp bread and its correlation with the rye flour falling number. They were able to show that βglucan fortified rye breads from rye flour with the lowest falling number (i.e. highest enzyme activity) will have the lowest βglucan molecular weight compared to rye breads with the same fermentation time. Beck et al (2009) reported that oat βglucan 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 βglucan 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 βglucan fortified products. Tosh et al (2010) reported that

extruding will not change or reduce the MW of βglucan 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 βglucan fortified cereals.

Tosh et al (2008) studied muffins fortified with high molecular weight βglucan, and found that peak molecular weight of added βglucan 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 βglucans 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 βglucan 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 βglucan can reduces βglucan 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 βglucan. Furthermore, Aman et al (2004) showed that boiling porridge and frying pancakes did not have any effect on the βglucan MW. Andersson et al (2008) showed that the molecular weight of βglucan is almost unchanged during oven baking.

In addition, Andersson et al (2004) showed that increasing the mixing time in hullless barley bread will result in higher depolymerization of βglucan.

2.4 Effect of βGlucan on Technological Properties of Cereal Products

There have been controversial ideas and suggestions on the effect of βglucan on cereal product characteristics. Wang et al (1998) reported that βglucan fractions in wheat bread improve the crumb structure while Gill et al (2002) had a completely different suggestion, and found that barley βglucan reduces loaf volume. Furthermore, Izydorczyk et al (2008) reported that additional of 20% barley fiberrich fractions to wheat flour for making flat breads resulted in 1018% 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 βglucan using barley coarse fraction, obtained by air classification, which

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 βglucan. They reported based on consumer data that chocolate chip cookies which were substituted with up to 50% with barley flour (0.8 g βglucan/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 β glucan/serving, had a denser texture compared to whole wheat muffins without βglucan.

This can be related to the water binding capacity of βglucan present in the oat bran muffins and because of the hydrocolloid nature of βglucan, the bound water resists evaporation during baking (Tosh et al 2008).

Fig. 2.1: Schematic representation of Cellulose (Reproduced from: BeMiller and Huber 2008).

Fig. 2.2: Schematic representation of Arabinoxylan

Fig. 2.3: Representative structure of βglucan, where “n” usually is either one or two but in frequently can be larger (Reproduced from: BeMiller and Huber 2008).

Fig. 2.4: Schematic representation of βGlucan (Reproduced from: Morgan 2000).

5 g Sample

100 mL Sodium Phosphate Buffer pH 6.9 15 min at 37ºC, slow stirring add 25o L αAmylase (5

mg/mL CaCl 2)

15 min at 37ºC, slow stirring

Adjust pH to 2.0 ± 0.1 with HCL 6 M/ 1M add 625 L pepsin (0.5

mg/mL in 0.9% NaCl) 30 min at 37ºC, stirring

Adjust pH to 6.9 ± 0.1 with NaOH 3 M add 1.25 mL pancreatin (0.5 mg/mL in Sodium Phosphate Buffer 90 min at 37ºC, stirring

Centrifuge, 10 min at 7,000 × g

Physiological Extract

Fig. 2.5: Procedures for physiological extract of βglucan (Adopted from Beer et al 1997a).

Fig. 2.6: Principle of the mix linkage assay for determining βglucan content using Mega zyme International, Bray, Ireland Kit.

A: βGlc(1 −>4)βGlc(1 −>3)βGlc

B: βGlc(1 −>4)βGlc(1 −>4)βGlc(1 −>3)βGlc

Fig. 2.7: A represents trisaccharide and B represent tetrasaccharide which are both result of lichenase and fungus enzyme on βglucan.

Fig. 2.8: Schematic representation of differences in action of B.subtillis lichenase and A.japonicus enzyme on βglucan (Reproduced from: Grishutin et al 2006). *Nonreducing ends are located on the left polymer molecules. Black dots donates the βDGlcp residue; (–) β(1 −>4)linkage; (\) β(1 −>3)linkage.

Fig. 2.9: Enzymatic hydrolysis of cell wall (1→3),(1→4)βDglucans (Reproduced from: Hrmova et al 2001). *G appoints a βDglucosyl residue; 3 are (1→3) linkages, 4 are (1→4) linkages, and red notes the reducing end.

3. CHAPTER THREE

DETECTION, LOCALIZATION AND VARIABILITY OF ENDOGENOUS Β GLUCANASE IN WHEAT KERNELS

3.1 Abstract

Clinical studies with isolates of βglucan 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 βglucan 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 βglucanase in wheat kernels and to determine factors that influence the levels of this enzyme. A modified protocol to quantify β glucanase was developed and then confirmed by using highperformance sizeexclusion chromatography (HPSEC) with calcofluor detection. Using this protocol it was shown that the concentration of βglucanase activity was the highest in the bran fraction of the kernel in nongerminated 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 βglucanase activity level in wheat kernels.

Key words: wheat flour; molecular weight of βglucan, βglucanase

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; TasleemTahir 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, alphaamylase 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 βglucanases activity, which hydrolyze βglucans containing mixed

β1, 3 and β1, 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 growthrelated responses in plants including as a protective tool against invading pathogens (Liu et al 2009). Furthermore, studies have also proposed that βglucanases 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 (LeubnerMetzger et al 1995). The importance of this enzyme is even more critical in the context of βglucan and its importance as nutrient in human foods (Aman et al 2004).

βGlucan 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 βglucan content in

grains are due to cultivar diversity rather than environmental or agronomic factors (Wood

1994a). Clinical trials where participants consumed oat βglucan 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

shortchain 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 oatderived βglucan

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 βglucan 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 βglucan 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 βglucan is proportional to concentration and molecular weight (MW) 35

(Wood et al 2000; Wood 2010). Kim and White (2011) reported that muffins with high

MW βglucan bound more bile acid than did those with low and mediumMW βglucan, but there were no differences in short chain fatty acid production among muffins with different MW. However, the MW of βglucan decreases during processing (Knuckles and

Chiu 1999; Trogh et al 2004). There are several hypotheses concerning the cause of β glucan depolymerization. One hypothesis is the possibility that the microorganisms present on cereals have enzymes that possess βglucanase activity, and as a result depolymerise βglucan. 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)βlinkage immediately following a (1 →3)βlinkage which produce series of lower MW oligomers (Wood et al 1994b). Because of the contiguous β(1 →4)

linkages, βglucan 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 βglucan. Kivela et al (2009)

investigated the effect of 10 mM ascorbic acid and 10 mM dehyroascorbic acid in pure

0.6 % barley βglucan solution in presence of ionic iron and the result was a drop in β

glucan viscosity. Finally, the last suggestion for degradation of βglucan MW in cereal based products is the possibility of presence of endogenous enzyme (Andersson et al

2004). Endogenous hydrolyzing enzymes have been reported in wheat, oat and barley

(BoskovHansen et al 2002). The objective of the present study was to develop a protocol 36

to detect very low levels of βglucanase 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 β glucanase 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

Azobarley βglucan (Remazolbrilliant Blue R labeled 1 % w/v barley βglucan), 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 βglucanase 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 βglucanase kit from Megazyme (Megazyme

International) was adapted for use on samples with very low βglucanase activity. Azo

Barley βglucan (Remazolbrilliant Blue R labeled 1 % w/v barley βglucan) 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 βglucanase 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 βGlucanase 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 βglucanase activity in selected

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 βglucanase activity detection.

βGlucanase activity was determined by incubating 0.5 mL of sample extract with

0.5 mL Azobarley βglucan 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 β glucanase and Azobarley βglucan as the substrate. Stock solutions of the malt β glucanase were prepared to contain from 6 × 10 4 to 4.4 × 10 3 units per mL. 0.5 mL of

Azobarley βglucan substrate was incubated with 0.5 mL of the different malt β glucanase 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 β glucanase activity (Fig. 3.1). βGlucanase activity of wheat flour was calculated using the following equation:

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 βglucanase 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 βglucan for 10 min.

Peak molecular weight (M p) of the standard βglucan was determined using high performance sizeexclusion 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

(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 βglucanase activity, wherein the highest absorbance obtained as after a 3 hr incubation. As a result, optimum conditions to detect very low βglucanase activity of wheat wholegrain 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 βglucanase activities in winter barley cultivars in southern China. The procedure they used for investigating the βglucanase activity was the same malt and bacterial βglucanase 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 βglucanase was further validated by incubating an isolated β

3 glucan (prepared as described in Wood et al 1989, MW = 1,200 × 10 g/mol) for 10 min

with extracted enzyme from wheat flours (Fig. 3.2). The flour with low βglucanase

activity (09HWW111, βglucanase activity = 5.33 U/kg) reduced the M W from 1,200 ×

103 to 79 × 103 g/mol, whereas the flour with higher βglucanase activity (Warthog, β glucanase activity = 10.53 U/kg) reduced the M W to 42 × 103 g/mol. The correspondence between the two data sets validated the optimized procedure and confirmed that low 41

levels of βglucanase activity found in wheat grain can have a significant effect on β glucan MW.

3.4.2 Distribution of βGlucanase Activity in Wheat Kernel

βGlucanase activity of wheat fractions (flour and bran) as compared with whole

grain flour for four wheat samples harvested in the field is presented in Figure 3.3.

Results indicated that the bran fraction had the highest βglucanase activity, whereas the

refined flour had the lowest βglucanase activity, for all varieties tested. The differences

in βglucanase activity of bran, endosperm, and whole grain varied among varieties. For

example, βglucanase 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 wholegrain flour. By

comparison, for Warthog variety, βglucanase 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

wholegrain 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 βglucanase as well. βGlucanase activity also increased when the seeds were

germinated in the laboratory (Fig. 3.4). Furthermore, unlike ungerminated seeds, in which

βglucanase activity was mainly located in bran, it was observed that after germination β

glucanase 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, βglucanase activity was predominantly located in the scutellum and embryo,

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 βGlucanase Activity

βGlucanase 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 βglucanase activity among wheat varieties and lines. For some varieties or lines, location had significant influence on βglucanase activity level, whereas other varieties were not affected by location. Among all soft wheat samples, 09SWW738 line planted in Nairn had the highest βglucanase activity (12.33 U/kg), and Becher variety planted in Nairn had the lowest βglucanase activity (4.66 U/kg). However, location did not have a significant effect on the βglucanase activity for Ava and 09SWW217

(SWW samples) or 09SRW 952 and 09SRW616 (SRW samples). βGlucanase activity of HRS wheat samples indicated significant differences among the five varieties.

Furthermore, location significantly affected the level of βglucanase activity; all varieties planted in Auburn had significantly higher βglucanase activity compared with those planted in St. Marys. In general, the results indicated that hard wheat varieties planted in

Auburn had significantly higher βglucanase 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 βglucanase activity. Wang et al (2004) also reported small differences in βglucanase activities of eight winter barley cultivars planted in seven different locations in southern China.

Spring wheats are typically harvested in the fall (midAugust 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 β

glucanase 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 β

glucanase 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 wholegrain flour of the greenhouseplanted samples had very low β

glucanase values (1.2 and 1.93 U/kg), much lower than observed in any of the fieldtested

samples. Therefore, it is likely that environment conditions trigger βglucanase activity.

3.4.4 Effect of Planting Season on βGlucanase 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

βglucanase activity. The differences in the βglucanase 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 βglucanase activity, respectively, compared with the

same varieties planted in the fall. However, no significant differences in βglucanase activity were observed for Norwell variety when planted in spring or fall. Monsalve

Gonzalez 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 αamylase.

3.5 Conclusion

This research highlights the potential for endogenous βglucanase enzymes in wheat

kernels to hydrolyze highMW βglucans. An optimized procedure that uses azobarley

βglucan as a substrate was developed as an easy and fast procedure to determine very

low (≈1 U/kg) levels of βglucanase activity in wheat flours. The results also demonstrate

that a number of factors including genotype, environment, and agronomic practice can

affect the level of βglucanase activity in the wheat flour and, as a consequence, could

affect βglucan MW when wheat products are fortified with βglucan or βglucanrich

fractions from oats or barley. As the concentration of bran increases in the wheat flour,

the βglucanase activity increases. Further research is underway to investigate the activity

levels of βglucanase enzyme during the baking process of wholegrain wheat flour

fortified with oat βglucan and to develop strategies to minimize its effect on βglucan

MW.

Table 3.1: Differences in spectrophotometer absorbance (590nm) for different samples using the original Megazyme procedure and the modified procedure.

Sample Absorbance (based Absorbance on Megazyme procedure) (based on optimized β glucanase procedure)

Ava 0.08 ± 0.01 0.49 ± 0.01

09SRW 0.09 ± 0.00 0.82 ± 0.00 952 Helious 0.18 ± 0.01 1.29 ± 0.03

Mean values ± standard deviation.

1.6

1.4 y = 0.0623x 1.2 R² = 0.9882 1 0.8 0.6

Absorbance (590 (590 nm) Absorbance 0.4 0.2 0 0 5 10 15 20 25 βGlucanase Activity (Units) × 10 4

Fig. 3.1: Standard curve relating the activity of malt βglucanase activity to absorbance at 590 nm upon hydrolysis of azobarley βglucan. Incubation time was 3 hr.

90 80 70

(g/mol) 60 3 50 40 30 20 βGlucan MW MW ×βGlucan 10 10 0 4 5 6 7 8 9 10 11 βGlucanase Activity (U/Kg)

Fig. 3.2: Molecular weight of βglucans following a 10 min incubation of wheat flours with a βglucan isolate (MW=1,200 × 10 3 g/mol) measured through high performance sizeexclusion chromatography with Calcofluor detection. Values are mean ± standard deviation of at least two replicates.

a Whole meal 20 Bran b Flour 15 b, c c

d d 10 d e 5 βGlucanase Activity(U/kg) βGlucanase f f f f 0

Fig. 3.3: Distribution of βglucanase activity in wheat kernel. Bars with different letters indicate significance at P < 0.05.Values are mean ± standard deviation of at least two replicates.

45 40 35 30 25 20 15 10 βGlucanase Activity (U/kg) βGlucanase 5 0 0 24 48 72 Germination Time (h)

Fig. 3.4: Effect of germination time on βglucanase activity of wheat kernels. Values are mean ± standard deviation of at least two replicates.

45 Whole Grain a 40 Whole Grain Whitout Germ 35 a,b a,b

30 b,c b,c 25 c 20 c 15 10

βGlucanase Activity(U/Kg) βGlucanase 5 0 0 24 48 72 Germination Time (h)

Fig. 3.5: Distribution of enzymes with βglucanase activity in wheat kernels after germina tion. Bars with different letters indicate significance at P < 0.05. Values are mean ± standard deviation of at least two replicates.

35 Soft White Winter Soft Red Winter Hard Red Spring Nairn 30 Watford 25 Auburn 20 St.Marys

* 10 * *

βGlucanase Activity (U/Kg) βGlucanase 5 *

Fig. 3.6: βGlucanase activity of wheat varieties/lines grown in different location. Asterisk (*) indicates the βglucanase 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.

5 Spring Planted a a Fall Planted 4.5 a,b 4 b,c 3.5 c,d c,d 3 2.5 2 e e 1.5 1 βGlucanase Activity (U/Kg) βGlucanase 0.5 0 Norwell Hobson Superb Sable

Fig. 3.7: Effect of planting season on βglucanase 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.

4. CHAPTER FOUR ΒGLUCAN DEGRADATION BY ENDOGENOUS ENZAYMES IN WHEAT FLOUR DOUGHS WITH DIFFERENT MOISTURE CONTENTS

4.1 Abstract

Health benefits of foods fortified with oat and barley βglucan are well documented. Clinical studies have shown that the health benefits of such foods are regulated not only by the concentration of βglucan but also by its molecular weight (MW) in the gut. Our previous study reported different levels of endogenous βglucanase activity in different wheat varieties. It was also shown that genotype and environmental factors impact the activity level of βglucanase. The objective of the present study was to investigate the kinetics of endogenous βglucanase activity on the MW of βglucan 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 β glucan were prepared to study the effect of moisture content, incubation time and β glucanase activity in the final βglucan MW. Evaluating final βglucan MW of each dough sample indicated that the moisture content played the most critical role among other factors in depolymerization of βglucan within the food system. Meanwhile, there was a positive correlation between βglucanase activity available in the system and βglucan degradation. Thus to retain high MW βglucans, which is important for delivering health benefits, it is important to manage the available water, βglucans and source of β glucanase activity.

Key words: Dough, molecular weight of βglucan, βglucanase, barley

4.2 Introduction

(1→3)(1→4)βDGlucans (known as βglucans) 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 coworkers reported that regular consumption of oat products can result in lowering blood cholesterol. This was followed by several studies reporting that consumption of βglucan 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 shortchain 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 βglucan rich products. Therefore, foods containing at least 0.75g of oat derived βglucan 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 βglucan 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

βglucan 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 βglucan. Kivela et al (2009) reported that 10 mM ascorbic acid and 10 mM dehydroascorbic acid in pure 0.6% barley βglucan solution in the presence of ionic iron are able to reduce βglucan viscosity. Tosh et al

(submitted in Journal Cereal Science) also reported that low concentration (0.11.0%) of organic acid salts (calcium propionate, potassium sorbate, and sodium benzoate) reduce the depolymerization rate of βglucan (p < 0.0001). Additionally, there is debate about the presence of βglucanase activity in yeast. Moriartey et al (2010) reported no effect of yeast or gluten on white bread doughfortified with βglucan, while Cleary et al (2007) suggested that some enzymes in yeast might be responsible for βglucan degradation.

Our previous study (Vatandoust et al in press) demonstrated different levels of endogenous βglucanase activity in wheat grains. We reported that genotype and environmental factors have an effect on endogenous βglucanase 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 βglucanase enzymes becomes even more critical in the context of the physiological performance of βglucan in the gut as influenced by the MW of βglucan.

Different processing technique with different conditions and different ingredients can either minimize or maximize the effect of endogenous βglucanase on the MW of β glucans.

Fortifying cereal products with βglucan 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 βglucan is required to meet the guidelines set by the FDA to use for the health claim. Several studies on βglucan fortified cereal products 56

investigating the effect of fermentation period or mixing time on βglucan 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 βglucan 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 βglucan 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. βGlucan 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 βglucan 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 βglucan (24%)

was used as a source of βglucan with high MW (2.5 × 10 6 g/mol). All other ingredients

were purchased locally. Thermostable αamylase and all chemicals for determining the β

glucanase 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 βGlucan

βGlucan was determined in barley flour using the Mixed linkage BetaGlucan kit

from Megzayme by the method of McCleary and GlennieHlomes (1985).

4.3.3 βGlucanase Activity

βGlucanase 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.

βGlucanase activity was determined by incubating 0.5 mL of sample extract with

0.5 mL Azobarley βglucan 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 βglucanase activity of samples was expressed as units per kg wheat flour.

4.3.4 Experimental Doughs

Bread and cookie doughs were made on a 5 g scale from whole grain using the

AACC methods 1010.03 and 1053.01, 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 βglucanase 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 βglucan as a source of βglucan 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 βGlucan From the Dough

βGlucan 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

CaCl 2 (0.28 mg/mL of water), 50 L of thermostable αamylase (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 βGlucan 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

average molecular weight (Mw) of βglucan was analyzed using highperformance size

exclusion chromatography (HPSEC) as described by Wood et al (1991a). The

chromatographic system consisted of a Perkin Elmer ISS100 autosampler and injector, a

Shimadzu model ADvt HPLC pump,a Shimadzu RF10Axl 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 βglucan molecular weight standards (ranging

from 20,000 to 1,300,00 g/mol), prepared inhouse (Wang et al 2004) or obtained

commercially (Megazyme) were used to construct a calibration curve for βglucan 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 βglucanase activity (112 U/Kg) while the

refined flour had the lowest (12 U/Kg) and the composite flour contained intermediate β

glucanase activity (56 U/Kg). Barley flour contained 24 % of high Mw (2.5 × 10 6) β glucan

4.4.2 Effect of Level of βGlucanase Activity on Degradation of βGlucan in Dough

Reduction of βglucan Mw during preparation of bread and cookie with flours containing different levels of endogenous βglucanase activity is presented in Fig. 4.1.

The results show a significant difference in βglucan Mw when using flours with different

βglucanase activity to prepare the bread doughs. A greater reduction in molecular weight

was observed with higher levels of endogenous βglucanase activity in the flour during

the 90 min incubation time. Flour exhibiting the lowest βglucanase activity (12 U/Kg)

6 resulted reduction to 1.25 × 10 in βglucan Mw after the 90 min incubation time. The

6 reduction in βglucan Mw was greater and βglucan Mw was reduced to 0.58 × 10 for bread dough when using the flour with the highest βglucanase activity (112 U/Kg),

while using flour exhibiting moderate βglucanase activity (56 U/Kg) resulted in

6 reduction of βglucan Mw to 0.89 × 10 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 βglucan 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 βglucan molecular weight. Additionally, Andersson et al (2008) reported a rapid drop in the average molecular weight of βglucan 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 βglucan. Furthermore, by drawing a logarithmic trend line based on the available data points for the bread dough, it can be observed that as the βglucanase activity of the flour increases, the slope for the

equation decreases which results in a lower value for the Y axes that presents the β glucan molecular weight.

In the case of cookie dough, flours with different βglucanase activity did not have significant effect on βglucan Mw during the 90 min incubation (Fig. 4.1). For

instance, a slight reduction was observed in βglucan Mw after 90 min when using whole

grain flour (having the highest βglucanase activity of 112 U/Kg) compared to when

using the composite flour (having an intermediate βglucanase activity i.e 56 U/Kg) or

when using the refined flour (having the lowest βglucanase activity i.e 12 U/Kg).

4.4.3 Effect of Moisture Content and Incubation Time on Depolymerization of βGlucan 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 βglucan when using flour exhibiting different levels of βglucanase activity (112,

56 and 12 U/Kg). Significant reduction in βglucan 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 βglucanase activity. This demonstrated that the available moisture in dough system plays an important role as a mobile phase for endogenous βglucanase enzymes to be more accessible to βglucan. 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.

The final βglucan MW in the cookie dough samples were in agreement with those for muffins by Tosh et al (2008). They reported preserved MW of βglucan in oat bran muffins and explained that to be due to short contact time between available water in the system, flour and βglucan as a reason that high MW βglucan was preserved. On the other hand, another study by Beer et al (1997a) reported a 50 % reduction in the peak molecular weight of βglucan in oat branfortified 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 branbased breads, wheat/hull less barley breads and rye crisp bread), having high moisture content and enough incubation time have reported reduction in β glucan 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 βglucanase activities, the reduction in βglucan Mw after 90 min was not significantly different from that after 15

min, while in the case of bread dough significant differences in βglucan Mw were

observed between the 15 min and the 90 min incubation time when using any of the

flours.

4.5 Conclusion

A combination of moisture content, incubation time and levels of endogenous β

glucanase activity of the system, all have significant effect on the MW of βglucan in the

final product. Among the three mentioned factors, the available moisture content in the

system can be considered the most critical factor. βGlucan 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 βglucan.

Furthermore, novel strategies need to be developed in dough containing high moisture

contents to maintain high MW βglucan without having negative effect on the quality of

the end product, which would promote the consumption of βglucan fortified functional products.

Table 4.1: Bread Formulation

Dough containing Dough containing Dough containing 12 U/Kg β 56 U/Kg β 112 U/Kg β glucanase activity glucanase activity glucanase activity

Refined 4.48 flour a (g)

Composite 4.48 flour a (g)

Whole 4.48 grain flour a (g)

Barley flour 0.52 0.52 0.52 b(g)

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 0.15 0.15 0.15 (g)

a Refined Flour, Composite flour and Whole grain flour was obtained using Glenn wheat. b 6 Barley Flour rich ( 24 %) in high (2.5 × 10 ) MW βglucan.

Table 4.2: Cookie Formulation

Dough containing Dough containing Dough containing 12 U/Kg β 56 U/Kg β 112 U/Kg β glucanase activity glucanase activity glucanase activity Refined 4.48 flour a (g) Composite 4.48 flour a (g) Whole grain 4.48 flour a (g) Barley flour 0.52 0.52 0.52 b(g) Sucrose (g) 2.1 2.1 2.1 NaCl (g) 0.062 0.062 0.062 Non fat dry 0.05 0.05 0.05 milk (g) Sodium bi 0 .05 0.05 0.05 carbonate (g) 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) aRefined Flour, Composite flour and Whole grain flour was obtained using Glenn wheat. b 6 Barley Flour rich ( 24 %) in high (2.5 × 10 ) MW βglucan.

3 Refined Flour Composite Flour Whole Grain Flour

2.5

2 (g/mol) 6 1.5 Cookie Bread Y=0.134ln(x) 1 + 2.0005 Y=0.174ln(x) R2 =0.8439 + 1.7026 Y=0.2ln(x) 0.5 2

βGlucan MW MW ×10 βGlucan R =0.9758 + 1.5212 R2 =0.9671 0 0 15 45 90 0 15 45 90 0 15 45 90 Incubation Time (min)

Fig. 4.1: Degradation of βglucan during preparation of bread dough and cookie dough of using flours containing different endogenous βglucanase activity.

12 (U/Kg) βglucanase activity 56 (U/Kg) βglucanase activity 112 (U/Kg) βglucanase activity 80 70 15 min 45 min 90 min 60 50 40 30 20 10 0 βGlucanase Activity (U/Kg) βGlucanase Percentage Reduction of βGlucan Reductionof Percentage 20% 60% 20% 60% 20% 60%

Moisture Content

Fig. 4.2: Effect of moisture content and endogenous βglucanase activity on depolymeriza tion of βglucan in the systems.

5. CHAPTER FIVE

CONCLUSIONS

From an industrial point of view, including barley or oat flours in wheatbased formula to enrich bread and other products with high MW/viscosity βglucan will enhance the health profile of baked products. Several studies have indicated that bioactivity in the gut is dependent, among other things, on βglucan MW and food microstructure. Recent studies revealed that oat and barley βglucan incorporated into wheat based products result in lower MW. Several researchers have suggested that there might be βglucanase enzymes present in wheat flour, but todate 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 βglucan depolymerization is important. Therefore, the objectives of this study were to understand the causes for depolymerization of βglucans in wheatbased baked products; to characterize the kinetics and properties of the factors that lead to the depolymerization of βglucan.

The only procedure available for evaluating βglucanase activity was the

Megazyme procedure for determining the Malt βglucanase activity (ranging from 130 to

760 Unit/Kg βglucanase 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 β glucanase activity by using the available Megzayme standard procedure (Malt & Bacteria

βglucanase). The reliability and accuracy of the optimized protocol was confirmed by

using an HPSECCalcofluor detector to monitor the weight average molecular weight

6 (M W) of standard βglucan (MW = 1.2 × 10 g/mol) incubated with flours exhibited

different βglucanase 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 βglucanase activity

in wheat flours. Furthermore the distribution of βglucanase in different fractions of the

wheat kernel (bran, flour) was also investigated using this protocol. The result confirmed

that endogenous enzymes with βglucanase activity are concentrated primarily in the

outer layer of wheat kernels. The effect of germination on the βglucanase activity was

studied to investigate the effect of poor harvesting conditions on postharvest glucanase

activity. The results demonstrated that not only germination increases βglucanase

activity but also it redistributed 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 βglucan by endogenous glucanase was investigated.

In this stud we used three flours exhibiting different levels of βglucanase activity (low,

6 intermediate or high) and a source of high MW βglucan (MW = 2.5 × 10 g/mol). The

results demonstrated that depolymerization of βglucan occurs whitin the first few

minutes of dough preparation (during the mixing process). Moisture content, incubation

time and levels of endogenous βglucanase activity of the system, had significant impact

on the final MW of βglucan in the dough. Among the three mentioned factors, the

available moisture content of the system (e.g bread) had the greatest impact on the β

glucan depolymerization. While in cookie dough (low moisture system) the βglucan 70

MW remained almost unchanged due to the absence of enough mobile phase. The reduction in βglucan 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

βglucan into baked products and maintain high MW.

Future work of this study could investigate the exact location where the breakdown occurs on the βglucan chain at different stages during dough preparation to

characterize the βglucanase activity. Also, develop strategies to maintain the

functionality of βglucans in the baked product systems. The results will provide a

scientific basis for the industry to incorporate βglucan in baked products under

conditions that will maintain or enhance its health benefits.

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